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Duncan JD, Setati ME, Divol B. The cellular symphony of redox cofactor management by yeasts in wine fermentation. Int J Food Microbiol 2025; 427:110966. [PMID: 39536648 DOI: 10.1016/j.ijfoodmicro.2024.110966] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2024] [Revised: 10/21/2024] [Accepted: 11/03/2024] [Indexed: 11/16/2024]
Abstract
Redox metabolism is pivotal in anaerobic fermentative processes such as winemaking where it results in the production of many metabolites that contribute to the aroma and flavour of wine. Key to this system are NAD+ and NADP+, which play essential roles as cofactors in maintaining cellular redox balance and regulating metabolism during fermentation. This review comprehensively explores redox metabolism under winemaking conditions, highlighting the influence of factors such as oxygen availability and vitamins including B3 and B1. Recent findings underscore the rapid assimilation and recycling dynamics of these vitamins during fermentation, reinforcing their critical role in yeast performance. Despite extensive research, the roles of diverse yeast species and specific vitamins remain insufficiently explored. By consolidating current knowledge, this review emphasises the implications of redox dynamics for metabolite synthesis and overall wine quality. Understanding these metabolic intricacies offers options to enhance fermentation efficiency and refine aroma profiles. The review also identifies gaps in studies for intracellular vitamin metabolism and underlines the need for deeper insights into non-Saccharomyces yeast metabolism. Future research directions should focus on elucidating specific metabolic responses, exploring environmental influences, and harnessing the potential of diverse yeasts to innovate and diversify wine production strategies.
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Affiliation(s)
- James D Duncan
- South African Grape and Wine Research Institute, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa
| | - Mathabatha E Setati
- South African Grape and Wine Research Institute, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa
| | - Benoit Divol
- South African Grape and Wine Research Institute, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa.
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2
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Liu C, Liu Q, Mou Z. Redox signaling and oxidative stress in systemic acquired resistance. JOURNAL OF EXPERIMENTAL BOTANY 2024; 75:4535-4548. [PMID: 38693779 DOI: 10.1093/jxb/erae193] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Accepted: 04/28/2024] [Indexed: 05/03/2024]
Abstract
Plants fully depend on their immune systems to defend against pathogens. Upon pathogen attack, plants not only activate immune responses at the infection site but also trigger a defense mechanism known as systemic acquired resistance (SAR) in distal systemic tissues to prevent subsequent infections by a broad-spectrum of pathogens. SAR is induced by mobile signals produced at the infection site. Accumulating evidence suggests that reactive oxygen species (ROS) play a central role in SAR signaling. ROS burst at the infection site is one of the earliest cellular responses following pathogen infection and can spread to systemic tissues through membrane-associated NADPH oxidase-dependent relay production of ROS. It is well known that ROS ignite redox signaling and, when in excess, cause oxidative stress, damaging cellular components. In this review, we summarize current knowledge on redox regulation of several SAR signaling components. We discuss the ROS amplification loop in systemic tissues involving multiple SAR mobile signals. Moreover, we highlight the essential role of oxidative stress in generating SAR signals including azelaic acid and extracellular NAD(P) [eNAD(P)]. Finally, we propose that eNAD(P) is a damage-associated molecular pattern serving as a converging point of SAR mobile signals in systemic tissues.
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Affiliation(s)
- Cheng Liu
- Department of Microbiology and Cell Science, University of Florida, PO Box 110700, Gainesville, FL 32611, USA
| | - Qingcai Liu
- Department of Microbiology and Cell Science, University of Florida, PO Box 110700, Gainesville, FL 32611, USA
| | - Zhonglin Mou
- Department of Microbiology and Cell Science, University of Florida, PO Box 110700, Gainesville, FL 32611, USA
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3
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Audrito V, Messana VG, Brandimarte L, Deaglio S. The Extracellular NADome Modulates Immune Responses. Front Immunol 2021; 12:704779. [PMID: 34421911 PMCID: PMC8371318 DOI: 10.3389/fimmu.2021.704779] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2021] [Accepted: 07/21/2021] [Indexed: 12/30/2022] Open
Abstract
The term NADome refers to the intricate network of intracellular and extracellular enzymes that regulate the synthesis or degradation of nicotinamide adenine dinucleotide (NAD) and to the receptors that engage it. Traditionally, NAD was linked to intracellular energy production through shuffling electrons between oxidized and reduced forms. However, recent data indicate that NAD, along with its biosynthetic and degrading enzymes, has a life outside of cells, possibly linked to immuno-modulating non-enzymatic activities. Extracellular NAD can engage puriginergic receptors triggering an inflammatory response, similar - to a certain extent - to what described for adenosine triphosphate (ATP). Likewise, NAD biosynthetic and degrading enzymes have been amply reported in the extracellular space, where they possess both enzymatic and non-enzymatic functions. Modulation of these enzymes has been described in several acute and chronic conditions, including obesity, cancer, inflammatory bowel diseases and sepsis. In this review, the role of the extracellular NADome will be discussed, focusing on its proposed role in immunomodulation, together with the different strategies for its targeting and their potential therapeutic impact.
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Affiliation(s)
- Valentina Audrito
- Laboratory of Cancer Immunogenetics, Department of Medical Sciences, University of Turin, Turin, Italy
| | - Vincenzo Gianluca Messana
- Laboratory of Cancer Immunogenetics, Department of Medical Sciences, University of Turin, Turin, Italy
| | - Lorenzo Brandimarte
- Laboratory of Cancer Immunogenetics, Department of Medical Sciences, University of Turin, Turin, Italy
| | - Silvia Deaglio
- Laboratory of Cancer Immunogenetics, Department of Medical Sciences, University of Turin, Turin, Italy
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Odnoshivkina YG, Petrov AM. The Role of Neuro-Cardiac Junctions
in Sympathetic Regulation of the Heart. J EVOL BIOCHEM PHYS+ 2021. [DOI: 10.1134/s0022093021030078] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
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Sanders KM, Mutafova-Yambolieva VN. Neurotransmitters responsible for purinergic motor neurotransmission and regulation of GI motility. Auton Neurosci 2021; 234:102829. [PMID: 34146957 DOI: 10.1016/j.autneu.2021.102829] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Revised: 05/25/2021] [Accepted: 05/28/2021] [Indexed: 12/17/2022]
Abstract
Classical concepts of peripheral neurotransmission were insufficient to explain enteric inhibitory neurotransmission. Geoffrey Burnstock and colleagues developed the idea that ATP or a related purine satisfies the criteria for a neurotransmitter and serves as an enteric inhibitory neurotransmitter in GI muscles. Cloning of purinergic receptors and development of specific drugs and transgenic mice have shown that enteric inhibitory responses depend upon P2Y1 receptors in post-junctional cells. The post-junctional cells that transduce purinergic neurotransmitters in the GI tract are PDGFRα+ cells and not smooth muscle cells (SMCs). PDGFRα+ cells express P2Y1 receptors, are activated by enteric inhibitory nerve stimulation and generate Ca2+ oscillations, express small-conductance Ca2+-activated K+ channels (SK3), and generate outward currents when exposed to P2Y1 agonists. These properties are consistent with post-junctional purinergic responses, and similar responses and effectors are not functional in SMCs. Refinements in methodologies to measure purines in tissue superfusates, such as high-performance liquid chromatography (HPLC) coupled with etheno-derivatization of purines and fluorescence detection, revealed that multiple purines are released during stimulation of intrinsic nerves. β-NAD+ and other purines, better satisfy criteria for the purinergic neurotransmitter than ATP. HPLC has also allowed better detection of purine metabolites, and coupled with isolation of specific types of post-junctional cells, has provided new concepts about deactivation of purine neurotransmitters. In spite of steady progress, many unknowns about purinergic neurotransmission remain and require additional investigation to understand this important regulatory mechanism in GI motility.
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Affiliation(s)
- Kenton M Sanders
- Department of Physiology and Cell Biology, University of Nevada, School of Medicine, 1664 North Virginia Street, Reno, NV 89557, USA.
| | - Violeta N Mutafova-Yambolieva
- Department of Physiology and Cell Biology, University of Nevada, School of Medicine, 1664 North Virginia Street, Reno, NV 89557, USA
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6
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Kumar SU, Sankar S, Kumar DT, Younes S, Younes N, Siva R, Doss CGP, Zayed H. Molecular dynamics, residue network analysis, and cross-correlation matrix to characterize the deleterious missense mutations in GALE causing galactosemia III. Cell Biochem Biophys 2021; 79:201-219. [PMID: 33555556 DOI: 10.1007/s12013-020-00960-z] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/28/2020] [Indexed: 01/17/2023]
Abstract
Epimerase-deficiency galactosemia (EDG) is caused by mutations in the UDP-galactose 4'-epimerase enzyme, encoded by gene GALE. Catalyzing the last reaction in the Leloir pathway, UDP-galactose-4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose. This study aimed to use in-depth computational strategies to prioritize the pathogenic missense mutations in GALE protein and investigate the systemic behavior, conformational spaces, atomic motions, and cross-correlation matrix of the GALE protein. We searched four databases (dbSNP, ClinVar, UniProt, and HGMD) and major biological literature databases (PubMed, Science Direct, and Google Scholar), for missense mutations that are associated with EDG patients, our search yielded 190 missense mutations. We applied a systematic computational prediction pipeline, including pathogenicity, stability, biochemical, conservational, protein residue contacts, and structural analysis, to predict the pathogenicity of these mutations. We found three mutations (p.K161N, p.R239W, and p.G302D) with a severe phenotype in patients with EDG that correlated with our computational prediction analysis; thus, they were selected for further structural and simulation analyses to compute the flexibility and stability of the mutant GALE proteins. The three mutants were subjected to molecular dynamics simulation (MDS) with native protein for 200 ns using GROMACS. The MDS demonstrated that these mutations affected the beta-sheets and helical region that are responsible for the catalytic activity; subsequently, affects the stability and flexibility of the mutant proteins along with a decrease and more deviations in compactness when compared to that of a native. Also, three mutations created major variations in the combined atomic motions of the catalytic and C-terminal regions. The network analysis of the residues in the native and three mutant protein structures showed disturbed residue contacts occurred owing to the missense mutations. Our findings help to understand the structural behavior of a protein owing to mutation and are intended to serve as a platform for prioritizing mutations, which could be potential targets for drug discovery and development of targeted therapeutics.
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Affiliation(s)
- S Udhaya Kumar
- School of Biosciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, 632014, India
| | - Srivarshini Sankar
- School of Biosciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, 632014, India
| | - D Thirumal Kumar
- School of Biosciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, 632014, India
| | - Salma Younes
- Department of Biomedical Sciences, College of Health and Sciences, Qatar University, QU Health, Doha, Qatar
| | - Nadin Younes
- Department of Biomedical Sciences, College of Health and Sciences, Qatar University, QU Health, Doha, Qatar
| | - R Siva
- School of Biosciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, 632014, India
| | - C George Priya Doss
- School of Biosciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, 632014, India.
| | - Hatem Zayed
- Department of Biomedical Sciences, College of Health and Sciences, Qatar University, QU Health, Doha, Qatar.
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Guo K, Wu J, Kong Y, Zhou L, Li W, Fei Y, Ma J, Mi L. Label-free and noninvasive method for assessing the metabolic status in type 2 diabetic rats with myocardium diastolic dysfunction. BIOMEDICAL OPTICS EXPRESS 2021; 12:480-493. [PMID: 33659084 PMCID: PMC7899513 DOI: 10.1364/boe.413347] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/23/2020] [Revised: 12/10/2020] [Accepted: 12/11/2020] [Indexed: 06/12/2023]
Abstract
This study assesses the metabolic status of rat diabetic cardiomyopathy (DCM) models. Echocardiography is used to detect the diastolic dysfunction in type 2 diabetic rats, and a lower threshold for inducible atrial fibrillation is found in type 2 diabetic rats with diastolic dysfunction compared to the control. Metabolic abnormalities are detected by status changes of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), which is an essential coenzyme in cells or tissues. Fluorescence lifetime imaging microscopy (FLIM) is used to monitor changes in NAD(P)H in both myocardial tissues and blood. FLIM reveals that the protein-bound proportion of NAD(P)H in rat myocardium in the DCM group is smaller than the control group, which indicates the oxidative phosphorylation rate of the DCM group decreased. Similar results are found for blood plasma of DCM rats by the FLIM study. FLIM exhibits high potential for screening DCM as a label-free, sensitive, and noninvasive method.
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Affiliation(s)
- Kai Guo
- Department of Cardiology, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, Shanghai 200092, China
- These authors contributed equally to this work
| | - Junxin Wu
- Department of Optical Science and Engineering, Key Laboratory of Micro and Nano Photonic Structures (Ministry of Education), Shanghai Engineering Research Center of Ultra-Precision Optical Manufacturing, Green Photoelectron Platform, Fudan University, 220 Handan Road, Shanghai 200433, China
- These authors contributed equally to this work
| | - Yawei Kong
- Department of Optical Science and Engineering, Key Laboratory of Micro and Nano Photonic Structures (Ministry of Education), Shanghai Engineering Research Center of Ultra-Precision Optical Manufacturing, Green Photoelectron Platform, Fudan University, 220 Handan Road, Shanghai 200433, China
| | - Li Zhou
- Department of Optical Science and Engineering, Key Laboratory of Micro and Nano Photonic Structures (Ministry of Education), Shanghai Engineering Research Center of Ultra-Precision Optical Manufacturing, Green Photoelectron Platform, Fudan University, 220 Handan Road, Shanghai 200433, China
| | - Wei Li
- Department of Cardiology, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, Shanghai 200092, China
| | - Yiyan Fei
- Department of Optical Science and Engineering, Key Laboratory of Micro and Nano Photonic Structures (Ministry of Education), Shanghai Engineering Research Center of Ultra-Precision Optical Manufacturing, Green Photoelectron Platform, Fudan University, 220 Handan Road, Shanghai 200433, China
| | - Jiong Ma
- Department of Optical Science and Engineering, Key Laboratory of Micro and Nano Photonic Structures (Ministry of Education), Shanghai Engineering Research Center of Ultra-Precision Optical Manufacturing, Green Photoelectron Platform, Fudan University, 220 Handan Road, Shanghai 200433, China
- Institute of Biomedical Engineering and Technology, Academy for Engineer and Technology, Fudan University, 220 Handan Road, Shanghai 200433, China
- The Multiscale Research Institute of Complex Systems (MRICS), School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China
| | - Lan Mi
- Department of Optical Science and Engineering, Key Laboratory of Micro and Nano Photonic Structures (Ministry of Education), Shanghai Engineering Research Center of Ultra-Precision Optical Manufacturing, Green Photoelectron Platform, Fudan University, 220 Handan Road, Shanghai 200433, China
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8
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Strassheim D, Verin A, Batori R, Nijmeh H, Burns N, Kovacs-Kasa A, Umapathy NS, Kotamarthi J, Gokhale YS, Karoor V, Stenmark KR, Gerasimovskaya E. P2Y Purinergic Receptors, Endothelial Dysfunction, and Cardiovascular Diseases. Int J Mol Sci 2020; 21:ijms21186855. [PMID: 32962005 PMCID: PMC7555413 DOI: 10.3390/ijms21186855] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2020] [Revised: 09/11/2020] [Accepted: 09/15/2020] [Indexed: 12/13/2022] Open
Abstract
Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological importance of the purinergic signaling system in the regulation of the cardiovascular system. This review discusses the contribution of purinergic P2Y receptors to endothelial dysfunction (ED) in numerous cardiovascular diseases (CVDs). Endothelial dysfunction can be defined as a shift from a “calm” or non-activated state, characterized by low permeability, anti-thrombotic, and anti-inflammatory properties, to a “activated” state, characterized by vasoconstriction and increased permeability, pro-thrombotic, and pro-inflammatory properties. This state of ED is observed in many diseases, including atherosclerosis, diabetes, hypertension, metabolic syndrome, sepsis, and pulmonary hypertension. Herein, we review the recent advances in P2Y receptor physiology and emphasize some of their unique signaling features in pulmonary endothelial cells.
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Affiliation(s)
- Derek Strassheim
- The Department of Medicine Cardiovascular and Pulmonary Research Laboratory, University of Colorado Denver, Aurora, CO 80045, USA; (D.S.); (N.B.); (V.K.); (K.R.S.)
| | - Alexander Verin
- Vascular Biology Center, Augusta University, Augusta, GA 30912, USA; (A.V.); (R.B.); (A.K.-K.)
| | - Robert Batori
- Vascular Biology Center, Augusta University, Augusta, GA 30912, USA; (A.V.); (R.B.); (A.K.-K.)
| | - Hala Nijmeh
- The Department of Pediatrics, Division of Critical Care Medicine, University of Colorado Denver, Aurora, CO 80045, USA;
| | - Nana Burns
- The Department of Medicine Cardiovascular and Pulmonary Research Laboratory, University of Colorado Denver, Aurora, CO 80045, USA; (D.S.); (N.B.); (V.K.); (K.R.S.)
| | - Anita Kovacs-Kasa
- Vascular Biology Center, Augusta University, Augusta, GA 30912, USA; (A.V.); (R.B.); (A.K.-K.)
| | | | - Janavi Kotamarthi
- The Department of BioMedical Engineering, University of Wisconsin, Madison, WI 53706, USA; (J.K.); (Y.S.G.)
| | - Yash S. Gokhale
- The Department of BioMedical Engineering, University of Wisconsin, Madison, WI 53706, USA; (J.K.); (Y.S.G.)
| | - Vijaya Karoor
- The Department of Medicine Cardiovascular and Pulmonary Research Laboratory, University of Colorado Denver, Aurora, CO 80045, USA; (D.S.); (N.B.); (V.K.); (K.R.S.)
| | - Kurt R. Stenmark
- The Department of Medicine Cardiovascular and Pulmonary Research Laboratory, University of Colorado Denver, Aurora, CO 80045, USA; (D.S.); (N.B.); (V.K.); (K.R.S.)
- The Department of Pediatrics, Division of Critical Care Medicine, University of Colorado Denver, Aurora, CO 80045, USA;
| | - Evgenia Gerasimovskaya
- The Department of Medicine Cardiovascular and Pulmonary Research Laboratory, University of Colorado Denver, Aurora, CO 80045, USA; (D.S.); (N.B.); (V.K.); (K.R.S.)
- The Department of Pediatrics, Division of Critical Care Medicine, University of Colorado Denver, Aurora, CO 80045, USA;
- Correspondence: ; Tel.: +1-303-724-5614
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9
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Elamin M, Ruskin DN, Sacchetti P, Masino SA. A unifying mechanism of ketogenic diet action: The multiple roles of nicotinamide adenine dinucleotide. Epilepsy Res 2020; 167:106469. [PMID: 33038721 DOI: 10.1016/j.eplepsyres.2020.106469] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2020] [Revised: 08/22/2020] [Accepted: 09/09/2020] [Indexed: 01/23/2023]
Abstract
The ability of a ketogenic diet to treat seizures and render a neuronal network more resistant to strong electrical activity has been observed for a century in clinics and for decades in research laboratories. Alongside ongoing efforts to understand how this therapy works to stop seizures, metabolic health is increasingly appreciated as critical buffer to resisting and recovering from acute and chronic disease. Accordingly, links between metabolism and health, and the broader emerging impact of the ketogenic diet in improving diverse metabolic, immunological and neurological conditions, have served to intensify the search for its key and/or common mechanisms. Here we review diverse evidence for increased levels of NAD+, and thus an altered ratio of NAD+/NADH, during metabolic therapy with a ketogenic diet. We propose this as a potential unifying mechanism, and highlight some of the evidence linking altered NAD+/NADH with reduced seizures and with a range of short and long-term changes associated with the beneficial effects of a ketogenic diet. An increase in NAD+/NADH is consistent with multiple lines of evidence and hypotheses, and therefore we suggest that increased NAD+ may be a common mechanism underlying beneficial effects of ketogenic diet therapy.
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Affiliation(s)
- Marwa Elamin
- Neuroscience Department, UConn School of Medicine, Farmington CT, United States.
| | - David N Ruskin
- Neuroscience Program & Psychology Department, Trinity College, Hartford, CT, United States.
| | - Paola Sacchetti
- Neuroscience Program & Department of Biology, University of Hartford, West Hartford, CT, United States.
| | - Susan A Masino
- Neuroscience Program & Psychology Department, Trinity College, Hartford, CT, United States.
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10
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Kulikova V, Shabalin K, Nerinovski K, Yakimov A, Svetlova M, Solovjeva L, Kropotov A, Khodorkovskiy M, Migaud ME, Ziegler M, Nikiforov A. Degradation of Extracellular NAD + Intermediates in Cultures of Human HEK293 Cells. Metabolites 2019; 9:E293. [PMID: 31795381 PMCID: PMC6950141 DOI: 10.3390/metabo9120293] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2019] [Revised: 11/22/2019] [Accepted: 11/27/2019] [Indexed: 01/06/2023] Open
Abstract
Nicotinamide adenine dinucleotide (NAD) is an essential redox carrier, whereas its degradation is a key element of important signaling pathways. Human cells replenish their NAD contents through NAD biosynthesis from extracellular precursors. These precursors encompass bases nicotinamide (Nam) and nicotinic acid and their corresponding nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR), now collectively referred to as vitamin B3. In addition, extracellular NAD+ and nicotinamide mononucleotide (NMN), and potentially their deamidated counterparts, nicotinic acid adenine dinucleotide (NAAD) and nicotinic acid mononucleotide (NAMN), may serve as precursors of intracellular NAD. However, it is still debated whether nucleotides enter cells directly or whether they are converted to nucleosides and bases prior to uptake into cells. Here, we studied the metabolism of extracellular NAD+ and its derivatives in human HEK293 cells using normal and serum-free culture medium. Using medium containing 10% fetal bovine serum (FBS), mono- and dinucleotides were degraded to the corresponding nucleosides. In turn, the nucleosides were cleaved to their corresponding bases. Degradation was also observed in culture medium alone, in the absence of cells, indicating that FBS contains enzymatic activities which degrade NAD+ intermediates. Surprisingly, NR was also rather efficiently hydrolyzed to Nam in the absence of FBS. When cultivated in serum-free medium, HEK293 cells efficiently cleaved NAD+ and NAAD to NMN and NAMN. NMN exhibited rather high stability in cell culture, but was partially metabolized to NR. Using pharmacological inhibitors of plasma membrane transporters, we also showed that extracellular cleavage of NAD+ and NMN to NR is a prerequisite for using these nucleotides to maintain intracellular NAD contents. We also present evidence that, besides spontaneous hydrolysis, NR is intensively metabolized in cell culture by intracellular conversion to Nam. Our results demonstrate that both the cultured cells and the culture medium mediate a rather active conversion of NAD+ intermediates. Consequently, in studies of precursor supplementation and uptake, the culture conditions need to be carefully defined.
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Affiliation(s)
- Veronika Kulikova
- Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia; (V.K.); (M.S.); (L.S.); (A.K.)
- Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russia; (A.Y.); (M.K.)
- Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg 194223, Russia
| | - Konstantin Shabalin
- Petersburg Nuclear Physics Institute Named by B.P. Konstantinov of National Research Centre “Kurchatov Institute”, Gatchina 188300, Russia;
| | - Kirill Nerinovski
- Department of Nuclear Physics Research Methods, St. Petersburg State University, St. Petersburg 199034, Russia;
| | - Alexander Yakimov
- Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russia; (A.Y.); (M.K.)
- Petersburg Nuclear Physics Institute Named by B.P. Konstantinov of National Research Centre “Kurchatov Institute”, Gatchina 188300, Russia;
| | - Maria Svetlova
- Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia; (V.K.); (M.S.); (L.S.); (A.K.)
| | - Ljudmila Solovjeva
- Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia; (V.K.); (M.S.); (L.S.); (A.K.)
| | - Andrey Kropotov
- Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia; (V.K.); (M.S.); (L.S.); (A.K.)
| | - Mikhail Khodorkovskiy
- Peter the Great St. Petersburg Polytechnic University, St. Petersburg 195251, Russia; (A.Y.); (M.K.)
| | - Marie E. Migaud
- Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604, USA;
| | - Mathias Ziegler
- Department of Biomedicine, University of Bergen, 5020 Bergen, Norway;
| | - Andrey Nikiforov
- Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia; (V.K.); (M.S.); (L.S.); (A.K.)
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11
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Pustovit KB, Potekhina VM, Ivanova AD, Petrov AM, Abramochkin DV, Kuzmin VS. Extracellular ATP and β-NAD alter electrical properties and cholinergic effects in the rat heart in age-specific manner. Purinergic Signal 2019; 15:107-117. [PMID: 30756226 DOI: 10.1007/s11302-019-09645-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Accepted: 01/04/2019] [Indexed: 12/20/2022] Open
Abstract
Extracellular ATP and nicotinamide adenine dinucleotide (β-NAD) demonstrate properties of neurotransmitters and neuromodulators in peripheral and central nervous system. It has been shown previously that ATP and β-NAD affect cardiac functioning in adult mammals. Nevertheless, the modulation of cardiac activity by purine compounds in the early postnatal development is still not elucidated. Also, the potential influence of ATP and β-NAD on cholinergic neurotransmission in the heart has not been investigated previously. Age-dependence of electrophysiological effects produced by extracellular ATP and β-NAD was studied in the rat myocardium using sharp microelectrode technique. ATP and β-NAD could affect ventricular and supraventricular myocardium independent from autonomic influences. Both purines induced reduction of action potentials (APs) duration in tissue preparations of atrial, ventricular myocardium, and myocardial sleeves of pulmonary veins from early postnatal rats similarly to myocardium of adult animals. Both purine compounds demonstrated weak age-dependence of the effect. We have estimated the ability of ATP and β-NAD to alter cholinergic effects in the heart. Both purines suppressed inhibitory effects produced by stimulation of intracardiac parasympathetic nerve in right atria from adult animals, but not in preparations from neonates. Also, ATP and β-NAD suppressed rest and evoked release of acetylcholine (ACh) in adult animals. β-NAD suppressed effects of parasympathetic stimulation and ACh release stronger than ATP. In conclusion, ATP and β-NAD control the heart at the postsynaptic and presynaptic levels via affecting the cardiac myocytes APs and ACh release. Postsynaptic and presynaptic effects of purines may be antagonistic and the latter demonstrates age-dependence.
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Affiliation(s)
- Ksenia B Pustovit
- Department of Human and Animal Physiology, Lomonosov Moscow State University, Leninskie gory 1, building 12, Moscow, Russia, 119991.,Department of Physiology, Pirogov Russian National Research Medical University, Ostrovitjanova 1, Moscow, Russia, 117997
| | - Viktoria M Potekhina
- Department of Human and Animal Physiology, Lomonosov Moscow State University, Leninskie gory 1, building 12, Moscow, Russia, 119991
| | - Alexandra D Ivanova
- Department of Human and Animal Physiology, Lomonosov Moscow State University, Leninskie gory 1, building 12, Moscow, Russia, 119991
| | - Alexey M Petrov
- Institute of Neuroscience, Kazan State Medial University, Butlerova st. 49, Kazan, Russia, 420012.,Laboratory of Biophysics of Synaptic Processes, Kazan Institute of Biochemistry and Biophysics, Federal Research Center "Kazan Scientific Center of RAS", P. O. Box 30, Lobachevsky Str., 2/31, Kazan, Russia, 420111
| | - Denis V Abramochkin
- Department of Human and Animal Physiology, Lomonosov Moscow State University, Leninskie gory 1, building 12, Moscow, Russia, 119991.,Department of Physiology, Pirogov Russian National Research Medical University, Ostrovitjanova 1, Moscow, Russia, 117997.,Laboratory of Cardiac Physiology, Institute of Physiology, Komi Science Center, Ural Division, Russian Academy of Sciences, Pervomayskaya 50, Syktyvkar, Russia, 167982
| | - Vlad S Kuzmin
- Department of Human and Animal Physiology, Lomonosov Moscow State University, Leninskie gory 1, building 12, Moscow, Russia, 119991. .,Department of Physiology, Pirogov Russian National Research Medical University, Ostrovitjanova 1, Moscow, Russia, 117997.
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12
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Mojard Kalkhoran S, Chow SHJ, Walia JS, Gershome C, Saraev N, Kim B, Poburko D. VNUT and VMAT2 segregate within sympathetic varicosities and localize near preferred Cav2 isoforms in the rat tail artery. Am J Physiol Heart Circ Physiol 2018; 316:H89-H105. [PMID: 30311774 DOI: 10.1152/ajpheart.00560.2018] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
ATP and norepinephrine (NE) are coreleased from peripheral sympathetic nerve terminals. Whether they are stored in the same vesicles has been debated for decades. Preferential dependence of NE or ATP release on Ca2+ influx through specific voltage-gated Ca2+ channel (Cav2) isoforms suggests that NE and ATP are stored in separate vesicle pools, but simultaneous imaging of NE and ATP containing vesicles within single varicosities has not been reported. We conducted an immunohistochemical study of vesicular monoamine transporter 2 (VMAT2/SLC18A2) and vesicular nucleotide translocase (VNUT/SLC17A9) as markers of vesicles containing NE and ATP in sympathetic nerves of the rat tail artery. A large fraction of varicosities exhibited neighboring, rather than overlapping, VNUT and VMAT2 fluorescent puncta. VMAT2, but not VNUT, colocalized with synaptotagmin 1. Cav2.1, Cav2.2, and Cav2.3 are expressed in nerves in the tunica adventitia. VMAT2 preferentially localized adjacent to Cav2.2 and Cav2.3 rather than Cav2.1. VNUT preferentially localized adjacent to Cav2.3 > Cav2.2 >> Cav2.1. With the use of wire myography, inhibition of field-stimulated vasoconstriction with the Cav2.3 blocker SNX-482 (0.25 µM) mimicked the effects of the P2X inhibitor suramin (100 µM) rather than the α-adrenergic inhibitor phentolamine (10 µM). Variable sensitivity to SNX-482 and suramin between animals closely correlated with Cav2.3 staining. We concluded that a majority of ATP and NE stores localize to separate vesicle pools that use different synaptotagmin isoforms and that localize near different Cav2 isoforms to mediate vesicle release. Cav2.3 appears to play a previously unrecognized role in mediating ATP release in the rat tail artery. NEW & NOTEWORTHY Immunofluorescence imaging of vesicular nucleotide translocase and vesicular monoamine transporter 2 in rat tail arteries revealed that ATP and norepinephrine, classical cotransmitters, localize to well-segregated vesicle pools. Furthermore, vesicular nucleotide translocase and vesicular monoamine transporter 2 exhibit preferential localization with specific Cav2 isoforms. These novel observations address long-standing debates regarding the mechanism(s) of sympathetic neurotransmitter corelease.
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Affiliation(s)
- Somayeh Mojard Kalkhoran
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University , Burnaby, British Columbia , Canada.,Centre for Cell Biology, Development and Disease, Simon Fraser University , Burnaby, British Columbia , Canada
| | - Sarah Heather Jane Chow
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University , Burnaby, British Columbia , Canada
| | - Jagdeep Singh Walia
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University , Burnaby, British Columbia , Canada
| | - Cynthia Gershome
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University , Burnaby, British Columbia , Canada
| | - Nickolas Saraev
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University , Burnaby, British Columbia , Canada
| | - BaRun Kim
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University , Burnaby, British Columbia , Canada
| | - Damon Poburko
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University , Burnaby, British Columbia , Canada.,Centre for Cell Biology, Development and Disease, Simon Fraser University , Burnaby, British Columbia , Canada
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13
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Tsai CK, Yeh TS, Wu RC, Lai YC, Chiang MH, Lu KY, Hung CY, Ho HY, Cheng ML, Lin G. Metabolomic alterations and chromosomal instability status in gastric cancer. World J Gastroenterol 2018; 24:3760-3769. [PMID: 30197481 PMCID: PMC6127658 DOI: 10.3748/wjg.v24.i33.3760] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/22/2018] [Revised: 06/27/2018] [Accepted: 07/16/2018] [Indexed: 02/06/2023] Open
Abstract
AIM To explore the correlation of metabolomics profiles of gastric cancer (GC) with its chromosomal instability (CIN) status. METHODS Nineteen GC patients were classified as CIN and non-CIN type by The Cancer Genome Atlas Research Group system, based on 409 oncogenes and tumor suppressor genes sequenced. The aqueous metabolites of the GC tumor and its surrounding adjacent healthy tissues were identified through liquid chromatography-mass spectrometry. Groups were compared by defining variable importance in projection score of > 1.2, a fold change value or its reciprocal of > 1.2, and a P value of < 0.05 as a significant difference. RESULTS In total, twelve men and seven women were enrolled, with a median age of 66 years (range, 47-87 years). The numbers of gene alterations in the CIN GC group were significantly higher than those in the non-CIN GC (32-218 vs 2-17; P < 0.0005). Compared with the adjacent healthy tissues, GC tumors demonstrated significantly higher aspartic acid, citicoline, glutamic acid, oxidized glutathione, succinyladenosine, and uridine diphosphate-N-acetylglucosamine levels, but significantly lower butyrylcarnitine, glutathione hydroxyhexanoycarnitine, inosinic acid, isovalerylcarnitine, and threonine levels (all P < 0.05). CIN tumors contained significantly higher phosphocholine and uridine 5'-monophosphate levels but significantly lower beta-citryl-L-glutamic acid levels than did non-CIN tumors (all P < 0.05). CIN GC tumors demonstrated additional altered pathways involving alanine, aspartate, and glutamate metabolism, glyoxylate and dicarboxylate metabolism, histidine metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis. CONCLUSION Metabolomic profiles of GC tumors and the adjacent healthy tissue are distinct, and the CIN status is associated with downstream metabolic alterations in GC.
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Affiliation(s)
- Cheng-Kun Tsai
- Clinical Metabolomics Core Lab, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan 333, Taiwan
- Department of Medical Imaging and Intervention, Imaging Core Lab, Institute for Radiological Research, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan 333, Taiwan
| | - Ta-Sen Yeh
- Department of Surgery, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan 333, Taiwan
| | - Ren-Chin Wu
- Department of Pathology, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan 333, Taiwan
| | - Ying-Chieh Lai
- Department of Medical Imaging and Intervention, Imaging Core Lab, Institute for Radiological Research, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan 333, Taiwan
| | - Meng-Han Chiang
- Clinical Metabolomics Core Lab, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan 333, Taiwan
- Department of Medical Imaging and Intervention, Imaging Core Lab, Institute for Radiological Research, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan 333, Taiwan
| | - Kuan-Ying Lu
- Department of Medical Imaging and Intervention, Imaging Core Lab, Institute for Radiological Research, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan 333, Taiwan
| | - Cheng-Yu Hung
- Department of Medical Imaging and Intervention, Imaging Core Lab, Institute for Radiological Research, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan 333, Taiwan
| | - Hung-Yao Ho
- Clinical Metabolomics Core Lab, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan 333, Taiwan
- Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan
| | - Mei-Ling Cheng
- Clinical Metabolomics Core Lab, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan 333, Taiwan
- Department of Biomedical Science, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan
| | - Gigin Lin
- Clinical Metabolomics Core Lab, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan 333, Taiwan
- Department of Medical Imaging and Intervention, Imaging Core Lab, Institute for Radiological Research, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan 333, Taiwan
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14
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Pustovit KB, Ivanova AD, Kuz'min VS. Extracellular NAD + Suppresses Adrenergic Effects in the Atrial Myocardium of Rats during the Early Postnatal Ontogeny. Bull Exp Biol Med 2018; 165:1-4. [PMID: 29797136 DOI: 10.1007/s10517-018-4085-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2017] [Indexed: 11/28/2022]
Abstract
The effects of sympathetic cotransmitter NAD+ (10 μM) on bioelectric activity of the heart under conditions of adrenergic stimulation were studied on isolated spontaneously contracting preparations (without stimulation) of the right atrium from 2-7-day-old rats. Action potentials were recorded in the working myocardium using standard microelectrode technique. Perfusion of the right atrium with norepinephrine solution (1 μM) altered the configuration and significantly lengthened the action potentials. NAD + against the background of norepinephrine stimulation significantly decreased the duration of action potentials, in particular, at 25% repolarization. The effect of purine compounds NAD + , ATP, and adenosine on bioelectrical activity of the heart of newborn rats was studied under basal conditions (without norepinephrine stimulation). The effect of NAD + against the background of adrenergic stimulation was more pronounced than under basal conditions and was probably determined by suppression of ICaL, which can be the main mechanism of NAD + action on rat heart.
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Affiliation(s)
- K B Pustovit
- Department of Human and Animal Physiology, M. V. Lomonosov Moscow State University, Moskva, Russia. .,Department pf Physiology, N. I. Pirogov Russian National Research Medical University, Moscow, Russia.
| | - A D Ivanova
- Department of Human and Animal Physiology, M. V. Lomonosov Moscow State University, Moskva, Russia
| | - V S Kuz'min
- Department of Human and Animal Physiology, M. V. Lomonosov Moscow State University, Moskva, Russia.,Department pf Physiology, N. I. Pirogov Russian National Research Medical University, Moscow, Russia
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15
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Abstract
Nicotinic acid and nicotinamide, collectively referred to as niacin, are nutritional precursors of the bioactive molecules nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). NAD and NADP are important cofactors for most cellular redox reactions, and as such are essential to maintain cellular metabolism and respiration. NAD also serves as a cosubstrate for a large number of ADP-ribosylation enzymes with varied functions. Among the NAD-consuming enzymes identified to date are important genetic and epigenetic regulators, e.g., poly(ADP-ribose)polymerases and sirtuins. There is rapidly growing knowledge of the close connection between dietary niacin intake, NAD(P) availability, and the activity of NAD(P)-dependent epigenetic regulator enzymes. It points to an exciting role of dietary niacin intake as a central regulator of physiological processes, e.g., maintenance of genetic stability, and of epigenetic control mechanisms modulating metabolism and aging. Insight into the role of niacin and various NAD-related diseases ranging from cancer, aging, and metabolic diseases to cardiovascular problems has shifted our view of niacin as a vitamin to current views that explore its potential as a therapeutic.
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Affiliation(s)
- James B Kirkland
- Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, Canada
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16
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Abstract
SIGNIFICANCE The nicotinamide adenine dinucleotide (NAD+)/reduced NAD+ (NADH) and NADP+/reduced NADP+ (NADPH) redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders. Recent Advances: Newly identified biosynthetic enzymes and newly developed genetically encoded biosensors enable us to understand better how cells maintain compartmentalized NAD(H) and NADP(H) pools. The concept of redox stress (oxidative and reductive stress) reflected by changes in NAD(H)/NADP(H) has increasingly gained attention. The emerging roles of NAD+-consuming proteins in regulating cellular redox and metabolic homeostasis are active research topics. CRITICAL ISSUES The biosynthesis and distribution of cellular NAD(H) and NADP(H) are highly compartmentalized. It is critical to understand how cells maintain the steady levels of these redox couple pools to ensure their normal functions and simultaneously avoid inducing redox stress. In addition, it is essential to understand how NAD(H)- and NADP(H)-utilizing enzymes interact with other signaling pathways, such as those regulated by hypoxia-inducible factor, to maintain cellular redox homeostasis and energy metabolism. FUTURE DIRECTIONS Additional studies are needed to investigate the inter-relationships among compartmentalized NAD(H)/NADP(H) pools and how these two dinucleotide redox couples collaboratively regulate cellular redox states and cellular metabolism under normal and pathological conditions. Furthermore, recent studies suggest the utility of using pharmacological interventions or nutrient-based bioactive NAD+ precursors as therapeutic interventions for metabolic diseases. Thus, a better understanding of the cellular functions of NAD(H) and NADP(H) may facilitate efforts to address a host of pathological disorders effectively. Antioxid. Redox Signal. 28, 251-272.
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Affiliation(s)
- Wusheng Xiao
- Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School , Boston, Massachusetts
| | - Rui-Sheng Wang
- Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School , Boston, Massachusetts
| | - Diane E Handy
- Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School , Boston, Massachusetts
| | - Joseph Loscalzo
- Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School , Boston, Massachusetts
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17
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Behrouzfar K, Alaee M, Nourbakhsh M, Gholinejad Z, Golestani A. Extracellular NAMPT/visfatin causes p53 deacetylation via NAD production and SIRT1 activation in breast cancer cells. Cell Biochem Funct 2017; 35:327-333. [PMID: 28845527 DOI: 10.1002/cbf.3279] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2017] [Revised: 06/09/2017] [Accepted: 07/05/2017] [Indexed: 01/19/2023]
Abstract
Visfatin, which is secreted as an adipokine and cytokine, has been implicated in cancer development and progression. In this study, we investigated the NAD-producing ability of visfatin and its relationship with SIRT1 (silent information regulator 2) and p53 to clarify the role of visfatin in breast cancer. MCF-7 breast cancer cells were cultured and treated with visfatin. SIRT1 activity was assessed by measuring fluorescence intensity from fluoro-substrate peptide. To investigate the effect of visfatin on p53 acetylation, SDS-PAGE followed by western blotting was performed using specific antibodies against p53 and its acetylated form. Total NAD was measured both in cell lysate and the extracellular medium by colorimetric method. Visfatin increased both extracellular and intracellular NAD concentrations. It also induced proliferation of breast cancer cells, an effect that was abolished by inhibition of its enzymatic activity. Visfatin significantly increased SIRT1 activity, accompanied by induction of p53 deacetylation. In conclusion, the results show that extracellular visfatin produces NAD that causes upregulation of SIRT1 activity and p53 deacetylation. These findings explain the relationship between visfatin and breast cancer progression.
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Affiliation(s)
- Kiarash Behrouzfar
- Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Mohammad Alaee
- Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Mitra Nourbakhsh
- Department of Biochemistry, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Zafar Gholinejad
- Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Abolfazl Golestani
- Department of Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
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18
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Pakhomov NV, Pustovit KB, Abramochkin DV, Kuz’min VS. The role of diadenosine pentaphosphate and nicotinamide adenine dinucleotide (NAD+) as potential nucleotide comediators in the adrenergic regulation of cardiac function. NEUROCHEM J+ 2017. [DOI: 10.1134/s1819712417010111] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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19
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Fedchenko V, Medvedev A. Comparative analysis of expression of genes encoding enzymes of catecholamine catabolism and renalase in tissues of normotensive and hypertensive rats. ACTA ACUST UNITED AC 2017; 63:312-315. [DOI: 10.18097/pbmc20176304312] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Comparative analysis of expression of genes encoding enzymes of catecholamine catabolism (monoaminbe oxidases A and B (MAO A and MAO B) and catechol-O-methyl transferase (COMT)) and renalase has been carried out in tissues of normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Among investigated tissues the highest level of mRNA of genes encoding key enzymes of catecholamine catabolism (MAO A, MAO B, COMT) was found in the heart of WKY rats. In SHR the mRNA levels of these genes were lower (p<0.05-0.01), however, no similar changes were observed in the tissues studied in dependence of hypertension. The relative mRNA levels of the studied genes normalized versus actin mRNA significantly varied. In heart and kidney the relative level of COMT mRNA significantly exceeded the relative levels of both MAO A mRNA and MAO B mRNA. In the brain differences in mRNAs of MAOA, MAOB, and COMT were less pronounced. However, in all examined tissue the renalase mRNA level was much (at least 10-20-fold) lower than any other mRNA studied. Taking into consideration known correlations between mRNAs and corresponding protein products reported in the literature for many genes these results suggest that in the case of any catalytic scenarios proposed or even proved for renalase this protein cannot contribute to catecholamine degradation. It is also unlikely that the products of renalase reaction, b-NAD(P)+ and hydrogen peroxide, can exhibit a hypotensive effect due to low expression of the renalase encoding gene.
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20
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Durnin L, Hayoz S, Corrigan RD, Yanez A, Koh SD, Mutafova-Yambolieva VN. Urothelial purine release during filling of murine and primate bladders. Am J Physiol Renal Physiol 2016; 311:F708-F716. [PMID: 27465992 DOI: 10.1152/ajprenal.00387.2016] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2016] [Accepted: 07/26/2016] [Indexed: 01/15/2023] Open
Abstract
During urinary bladder filling the bladder urothelium releases chemical mediators that in turn transmit information to the nervous and muscular systems to regulate sensory sensation and detrusor muscle activity. Defects in release of urothelial mediators may cause bladder dysfunctions that are characterized with aberrant bladder sensation during bladder filling. Previous studies have demonstrated release of ATP from the bladder urothelium during bladder filling, and ATP remains the most studied purine mediator that is released from the urothelium. However, the micturition cycle is likely regulated by multiple purine mediators, since various purine receptors are found present in many cell types in the bladder wall, including urothelial cells, afferent nerves, interstitial cells in lamina propria, and detrusor smooth muscle cells. Information about the release of other biologically active purines during bladder filling is still lacking. Decentralized bladders from C57BL/6 mice and Cynomolgus monkeys (Macaca fascicularis) were filled with physiological solution at different rates. Intraluminal fluid was analyzed by high-performance liquid chromatography with fluorescence detection for simultaneous evaluation of ATP, ADP, AMP, adenosine, nicotinamide adenine dinucleotide (NAD+), ADP-ribose, and cADP-ribose content. We also measured ex vivo bladder filling pressures and performed cystometry in conscious unrestrained mice at different filling rates. ATP, ADP, AMP, NAD+, ADPR, cADPR, and adenosine were detected released intravesically at different ratios during bladder filling. Purine release increased with increased volumes and rates of filling. Our results support the concept that multiple urothelium-derived purines likely contribute to the complex regulation of bladder sensation during bladder filling.
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Affiliation(s)
- Leonie Durnin
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Sebastien Hayoz
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Robert D Corrigan
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Andrew Yanez
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Sang Don Koh
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
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21
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Kuzmin VS, Pustovit KB, Abramochkin DV. Effects of exogenous nicotinamide adenine dinucleotide (NAD+) in the rat heart are mediated by P2 purine receptors. J Biomed Sci 2016; 23:50. [PMID: 27350532 PMCID: PMC4924331 DOI: 10.1186/s12929-016-0267-y] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2016] [Accepted: 06/17/2016] [Indexed: 12/13/2022] Open
Abstract
Background Recently, NAD+ has been considered as an essential factor, participating in nerve control of physiological functions and intercellular communication. NAD+ also has been supposed as endogenous activator of P1 and P2 purinoreceptors. Effects of extracellular NAD+ remain poorly investigated in cardiac tissue. This study aims to investigate the effects of extracellular NAD+ in different types of supraventricular and ventricular working myocardium from rat and their potential mechanisms. Methods The standard technique of sharp microelectrode action potential recording in cardiac multicellular preparations was used to study the effects of NAD+. Results Extracellular NAD+ induced significant changes in bioelectrical activity of left auricle (LA), right auricle (RA), pulmonary veins (PV) and right ventricular wall (RV) myocardial preparations. 10–100 μM NAD+ produced two opposite effects in LA and RA – quickly developing and transient prolongation of action potentials (AP) and delayed sustained AP shortening, which follows the initial positive effect. In PV and RV only AP shortening was observed in response to NAD+ application. In PV preparations AP shortening induced by NAD+ may be considered as a potential proarrhythmic effect. Revealed cardiotropic effects of NAD+ are likely to be mediated by P2 purine receptors, since P1 blocker DPCPX failed to affect them and P2 antagonist suramin abolished NAD + −induced alterations of electrical activity. P2X receptors may be responsible for NAD + −induced short-lasting AP prolongation, while P2Y receptors mediate persistent AP shortening. The latter effect is partially removed by PLC inhibitor U73122 showing the potential involvement of phosphoinositide signaling pathway in mediation of NAD+ cardiotropic effects. Conclusions Extracellular NAD+ is supposed to be a novel regulator of cardiac electrical activity. P2 receptors represent the main target of NAD+ at least in the rat heart.
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Affiliation(s)
- Vladislav S Kuzmin
- Department of Human and Animal Physiology, Lomonosov Moscow State University, Leninskie gory 1, building 12, Moscow, 119991, Russia.,Department of Physiology, Pirogov Russian National Research Medical University, Ostrovitjanova 1, Moscow, 117997, Russia
| | - Ksenia B Pustovit
- Department of Human and Animal Physiology, Lomonosov Moscow State University, Leninskie gory 1, building 12, Moscow, 119991, Russia.,Department of Physiology, Pirogov Russian National Research Medical University, Ostrovitjanova 1, Moscow, 117997, Russia
| | - Denis V Abramochkin
- Department of Human and Animal Physiology, Lomonosov Moscow State University, Leninskie gory 1, building 12, Moscow, 119991, Russia. .,Department of Physiology, Pirogov Russian National Research Medical University, Ostrovitjanova 1, Moscow, 117997, Russia.
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22
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Severina IS, Fedchenko VI, Veselovsky AV, Medvedev AE. [The history of renalase from amine oxidase to a a-NAD(P)H-oxidase/anomerase]. BIOMEDIT︠S︡INSKAI︠A︡ KHIMII︠A︡ 2016; 61:667-79. [PMID: 26716738 DOI: 10.18097/pbmc20156106667] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Renalase is a recently discovered secretory protein, which plays a certain (still poorly understood) role in regulation of blood pressure. The review summarizes own and literature data accumulated since the first publication on relanase (2005). Initial reports on FAD-dependent amine oxidase activity of this protein were not confirmed in independent experiments performed in different laboratories. In addition, proposed amine oxidase activity of circulating extracellular renalase requires the presence of FAD, which has not been detected either in blood or urinary renalase. Moreover, renalase excreted into urine lacks its N-terminal peptide, which is ultimately needed for accommodation of the FAD cofactor. Results of the Aliverti's group on NAD(P)H binding by renalase and weak diaphorase activity of this protein stimulated further studies of renalase as NAD(P)H oxidase catalyzing reaction of catecholamine co-oxidation. However, physiological importance of such extracellular catecholamine-metabolizing activity (demonstrated in one laboratory and not detected in another laboratory) remains unclear due to existence of much more active enzymatic systems (e.g. neutrophil NAD(P)H oxidase, xanthine oxidase/xanthine) in circulation, which can perform such co-oxidation reactions. Recently a-NAD(P)H oxidase/anomerase activity of renalase, which also pomotes oxidative conversion of b-NADH isomers inhibiting activity of NAD-dependent dehydrogenases, has been described. However, its possible contribution to the antihypertensive effect of renalase remains unclear. Thus, the antihypertensive effect of renalase still remains a phenomenon with unclear biochemical mechanim(s) and functions of intracellular and extracellular (circulating) renalases obviously differ.
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Affiliation(s)
- I S Severina
- Institute of Biomedical Chemistry, Moscow, Russia
| | | | | | - A E Medvedev
- Institute of Biomedical Chemistry, Moscow, Russia
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23
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Detection of Epistatic and Gene-Environment Interactions Underlying Three Quality Traits in Rice Using High-Throughput Genome-Wide Data. BIOMED RESEARCH INTERNATIONAL 2015; 2015:135782. [PMID: 26345334 PMCID: PMC4539430 DOI: 10.1155/2015/135782] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 12/03/2014] [Revised: 03/20/2015] [Accepted: 03/24/2015] [Indexed: 11/18/2022]
Abstract
With development of sequencing technology, dense single nucleotide polymorphisms (SNPs) have been available, enabling uncovering genetic architecture of complex traits by genome-wide association study (GWAS). However, the current GWAS strategy usually ignores epistatic and gene-environment interactions due to absence of appropriate methodology and heavy computational burden. This study proposed a new GWAS strategy by combining the graphics processing unit- (GPU-) based generalized multifactor dimensionality reduction (GMDR) algorithm with mixed linear model approach. The reliability and efficiency of the analytical methods were verified through Monte Carlo simulations, suggesting that a population size of nearly 150 recombinant inbred lines (RILs) had a reasonable resolution for the scenarios considered. Further, a GWAS was conducted with the above two-step strategy to investigate the additive, epistatic, and gene-environment associations between 701,867 SNPs and three important quality traits, gelatinization temperature, amylose content, and gel consistency, in a RIL population with 138 individuals derived from super-hybrid rice Xieyou9308 in two environments. Four significant SNPs were identified with additive, epistatic, and gene-environment interaction effects. Our study showed that the mixed linear model approach combining with the GPU-based GMDR algorithm is a feasible strategy for implementing GWAS to uncover genetic architecture of crop complex traits.
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Sangamithirai D, Narayanan V, Muthuraaman B, Stephen A. Investigations on the performance of poly(o-anisidine)/graphene nanocomposites for the electrochemical detection of NADH. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2015; 55:579-91. [PMID: 26117792 DOI: 10.1016/j.msec.2015.05.066] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 01/23/2015] [Revised: 04/15/2015] [Accepted: 05/25/2015] [Indexed: 11/25/2022]
Abstract
The electrocatalytic oxidation of dihydronicotinamide adenine dinucleotide (NADH) based on poly(o-anisidine)/graphene (POA/GR) nanocomposites modified glassy carbon electrode (GCE) was explored for the first time. POA/GR nanocomposites were synthesized via chemical oxidative polymerization method. X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), high resolution transmission electron microscopy (HRTEM), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and UV-Vis spectroscopy results demonstrate that nanocomposites are successfully synthesized. An intriguing composite structure was observed using different ratios of o-anisidine monomer and graphene. The electrical properties and electrochemical properties of these nanocomposites are investigated by impedance spectroscopy technique and cyclic voltammetric (CV) method, respectively. The synthesized nanocomposites were used to modify glassy carbon electrode (GCE), and the modified electrodes were found to exhibit electrocatalytic activity for oxidation of NADH at low potential range of +0.045 V in a neutral environment. The fabricated sensor based on POA/GR31-modified GCE exhibited enhanced current response with very high sensitivity of 47.1 μA μM(-1) for the detection of NADH. The developed POA/GR-modified GCE exhibited excellent reproducibility, stability, and selectivity for the determination of NADH. The practical analytical utility of the proposed method was demonstrated by NADH spiked ascorbic acid (AA) and the results confirmed that the proposed method is suitable for the determination of NADH in the presence of AA. This can open up new opportunities for simple and selective detection of NADH and provide a promising platform for biosensor designs.
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Affiliation(s)
- D Sangamithirai
- Department of Nuclear Physics, University of Madras, Guindy Campus, Chennai 600025, India
| | - V Narayanan
- Department of Inorganic Chemistry, University of Madras, Guindy Campus, Chennai 600025, India
| | - B Muthuraaman
- Department of Energy, University of Madras, Guindy Campus, Chennai 600025, India
| | - A Stephen
- Department of Nuclear Physics, University of Madras, Guindy Campus, Chennai 600025, India.
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Nikiforov A, Kulikova V, Ziegler M. The human NAD metabolome: Functions, metabolism and compartmentalization. Crit Rev Biochem Mol Biol 2015; 50:284-97. [PMID: 25837229 PMCID: PMC4673589 DOI: 10.3109/10409238.2015.1028612] [Citation(s) in RCA: 175] [Impact Index Per Article: 17.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
The metabolism of NAD has emerged as a key regulator of cellular and organismal homeostasis. Being a major component of both bioenergetic and signaling pathways, the molecule is ideally suited to regulate metabolism and major cellular events. In humans, NAD is synthesized from vitamin B3 precursors, most prominently from nicotinamide, which is the degradation product of all NAD-dependent signaling reactions. The scope of NAD-mediated regulatory processes is wide including enzyme regulation, control of gene expression and health span, DNA repair, cell cycle regulation and calcium signaling. In these processes, nicotinamide is cleaved from NAD+ and the remaining ADP-ribosyl moiety used to modify proteins (deacetylation by sirtuins or ADP-ribosylation) or to generate calcium-mobilizing agents such as cyclic ADP-ribose. This review will also emphasize the role of the intermediates in the NAD metabolome, their intra- and extra-cellular conversions and potential contributions to subcellular compartmentalization of NAD pools.
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Affiliation(s)
- Andrey Nikiforov
- a Institute of Nanobiotechnologies, St. Petersburg State Polytechnical University , St. Petersburg , Russia .,b Institute of Cytology, Russian Academy of Sciences , St. Petersburg , Russia , and
| | - Veronika Kulikova
- a Institute of Nanobiotechnologies, St. Petersburg State Polytechnical University , St. Petersburg , Russia
| | - Mathias Ziegler
- c Department of Molecular Biology , University of Bergen , Bergen , Norway
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Mutafova-Yambolieva VN, Durnin L. The purinergic neurotransmitter revisited: a single substance or multiple players? Pharmacol Ther 2014; 144:162-91. [PMID: 24887688 PMCID: PMC4185222 DOI: 10.1016/j.pharmthera.2014.05.012] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2014] [Accepted: 05/23/2014] [Indexed: 12/20/2022]
Abstract
The past half century has witnessed tremendous advances in our understanding of extracellular purinergic signaling pathways. Purinergic neurotransmission, in particular, has emerged as a key contributor in the efficient control mechanisms in the nervous system. The identity of the purine neurotransmitter, however, remains controversial. Identifying it is difficult because purines are present in all cell types, have a large variety of cell sources, and are released via numerous pathways. Moreover, studies on purinergic neurotransmission have relied heavily on indirect measurements of integrated postjunctional responses that do not provide direct information for neurotransmitter identity. This paper discusses experimental support for adenosine 5'-triphosphate (ATP) as a neurotransmitter and recent evidence for possible contribution of other purines, in addition to or instead of ATP, in chemical neurotransmission in the peripheral, enteric and central nervous systems. Sites of release and action of purines in model systems such as vas deferens, blood vessels, urinary bladder and chromaffin cells are discussed. This is preceded by a brief discussion of studies demonstrating storage of purines in synaptic vesicles. We examine recent evidence for cell type targets (e.g., smooth muscle cells, interstitial cells, neurons and glia) for purine neurotransmitters in different systems. This is followed by brief discussion of mechanisms of terminating the action of purine neurotransmitters, including extracellular nucleotide hydrolysis and possible salvage and reuptake in the cell. The significance of direct neurotransmitter release measurements is highlighted. Possibilities for involvement of multiple purines (e.g., ATP, ADP, NAD(+), ADP-ribose, adenosine, and diadenosine polyphosphates) in neurotransmission are considered throughout.
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Affiliation(s)
| | - Leonie Durnin
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, United States
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Uridine adenosine tetraphosphate is a novel neurogenic P2Y1 receptor activator in the gut. Proc Natl Acad Sci U S A 2014; 111:15821-6. [PMID: 25341729 DOI: 10.1073/pnas.1409078111] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Enteric purinergic motor neurotransmission, acting through P2Y1 receptors (P2Y1R), mediates inhibitory neural control of the intestines. Recent studies have shown that NAD(+) and ADP ribose better meet criteria for enteric inhibitory neurotransmitters in colon than ATP or ADP. Here we report that human and murine colon muscles also release uridine adenosine tetraphosphate (Up4A) spontaneously and upon stimulation of enteric neurons. Release of Up4A was reduced by tetrodotoxin, suggesting that at least a portion of Up4A is of neural origin. Up4A caused relaxation (human and murine colons) and hyperpolarization (murine colon) that was blocked by the P2Y1R antagonist, MRS 2500, and by apamin, an inhibitor of Ca(2+)-activated small-conductance K(+) (SK) channels. Up4A responses were greatly reduced or absent in colons of P2ry1(-/-) mice. Up4A induced P2Y1R-SK-channel-mediated hyperpolarization in isolated PDGFRα(+) cells, which are postjunctional targets for purinergic neurotransmission. Up4A caused MRS 2500-sensitive Ca(2+) transients in human 1321N1 astrocytoma cells expressing human P2Y1R. Up4A was more potent than ATP, ADP, NAD(+), or ADP ribose in colonic muscles. In murine distal colon Up4A elicited transient P2Y1R-mediated relaxation followed by a suramin-sensitive contraction. HPLC analysis of Up4A degradation suggests that exogenous Up4A first forms UMP and ATP in the human colon and UDP and ADP in the murine colon. Adenosine then is generated by extracellular catabolism of ATP and ADP. However, the relaxation and hyperpolarization responses to Up4A are not mediated by its metabolites. This study shows that Up4A is a potent native agonist for P2Y1R and SK-channel activation in human and mouse colon.
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Alefishat E, Alexander SPH, Ralevic V. Effects of NAD at purine receptors in isolated blood vessels. Purinergic Signal 2014; 11:47-57. [PMID: 25315718 DOI: 10.1007/s11302-014-9428-1] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2014] [Accepted: 09/23/2014] [Indexed: 11/25/2022] Open
Abstract
Nicotinamide adenine dinucleotide (NAD) belongs to the family of naturally occurring adenine dinucleotides, best known for their various intracellular roles. However, there is evidence that they can also be released from cells to act as novel extracellular signalling molecules. Relatively little is known about the extracellular actions of NAD, especially in the cardiovascular system. The present study investigated the actions of NAD in the rat thoracic aorta, porcine coronary artery and porcine mesenteric arteries, mounted in organ baths for isometric tension recording. In the rat thoracic aorta and porcine coronary artery, NAD caused endothelium-independent concentration-dependent vasorelaxations which were unaffected by palmitoylCoA, a P2Y1 receptor antagonist, but which were blocked by CGS15943, a non-selective adenosine receptor antagonist. In the porcine coronary artery, NAD-evoked relaxations were abolished by SCH58261, a selective A2A receptor antagonist. In the rat thoracic aorta, NAD-evoked relaxations were attenuated by A2A receptor antagonism with SCH58261 but were unaffected by an A2B receptor antagonist, MRS1754. In contrast, in the porcine mesenteric artery, NAD-evoked endothelium-independent contractions, which were unaffected by a P2 receptor antagonist, suramin, or by NF449, a P2X1 receptor antagonist, but were attenuated following P2X receptor desensitisation with αβ-meATP. In conclusion, the present results show that NAD can alter vascular tone through actions at purine receptors in three different arteries from two species; its molecular targets differ according to the type of blood vessel.
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Affiliation(s)
- E Alefishat
- Department of Biopharmaceutics and Clinical Pharmacy Faculty of Pharmacy, University of Jordan, Amman, 11942, Jordan
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Gotoh K, Nemoto E, Kanaya S, Shimauchi H. Extracellular β-NAD(+) inhibits interleukin-1-induced matrix metalloproteinase-1 and -3 expression on human gingival fibroblasts. Connect Tissue Res 2013; 54:204-9. [PMID: 23509928 DOI: 10.3109/03008207.2013.782013] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
There is increasing evidence to show that extracellular β-nicotinamide adenine dinucleotide (β-NAD(+)) modulates various biological functions in inflammatory/immune regions. The aim of this study was to determine the effect of β-NAD(+) on matrix metalloproteinase (MMP) expression on human gingival fibroblasts (hGF), the excess production of which leads to the matrix degradation associated with the pathological processes of periodontitis. The expression of MMP-1 and MMP-3 on hGF was determined by real-time polymerase chain reaction (PCR) and an enzyme-linked immunosorbent assay. The phosphorylated status of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1/2 (ERK), c-Jun N-terminal kinase (JNK), and p38 and the expression of inhibitor κB (IκB)α were determined by Western blotting. β-NAD(+) inhibited the expression of MMP-1 and MMP-3 triggered by IL-1α at gene and protein levels. β-NAD(+) had no significant effect on the IL-1α-induced phosphorylation of ERK1/2, JNK, and p38 and also had no effect on the IL-1α-induced degradation of IκBα relative to the control, suggesting that inhibition by β-NAD(+) was independent of the MAP kinase and the nuclear factor-κB signaling pathways. Transcripts of NAD(+)-metabolizing enzymes, such as NAD(+)-glycohydrolase, adenosine diphosphate (ADP)-ribosylcyclase, and ADP-ribosyltransferase, were expressed by hGF as assessed by RT-PCR. Experiments using α-NAD(+), which is not a substrate for ADP-ribosylcyclase or ADP-ribosyltransferase, revealed the possible contribution of NAD(+)-glycohydrolase to the inhibition of MMP. This is consistent with the finding that ADP-ribose, an NAD(+)-metabolite by NAD(+)-glycohydrolase, exhibited MMP inhibition similar to β-NAD(+). The present findings may provide an additional viewpoint to clarify a natural feedback mechanism during the inflammatory process in periodontal tissue.
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Affiliation(s)
- Kazuhiro Gotoh
- Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai, Japan
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30
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Sangsiri S, Dong H, Swain GM, Galligan JJ, Xu H. Impaired function of prejunctional adenosine A1 receptors expressed by perivascular sympathetic nerves in DOCA-salt hypertensive rats. J Pharmacol Exp Ther 2013; 345:32-40. [PMID: 23397055 PMCID: PMC3608448 DOI: 10.1124/jpet.112.199612] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2012] [Accepted: 02/07/2013] [Indexed: 01/07/2023] Open
Abstract
Increased sympathetic nervous system activity contributes to deoxycorticosterone acetate (DOCA)-salt hypertension in rats. ATP and norepinephrine (NE) are coreleased from perivascular sympathetic nerves. NE acts at prejunctional α2-adrenergic receptors (α2ARs) to inhibit NE release, and α2AR function is impaired in DOCA-salt rats. Adenosine, an enzymatic ATP degradation product, acts at prejunctional A1 adenosine receptors (A1Rs) to inhibit NE release. We tested the hypothesis that prejunctional A1R function is impaired in sympathetic nerves supplying mesenteric arteries (MAs) and veins (MVs) of DOCA-salt rats. Electrically evoked NE release and constrictions of blood vessels were studied in vitro with use of amperometry to measure NE oxidation currents and video microscopy, respectively. Immunohistochemical methods were used to localize tyrosine hydroxylase (TH) and A1Rs in perivascular sympathetic nerves. TH and A1Rs colocalized to perivascular sympathetic nerves. Adenosine and N(6)-cyclopentyl-adenosine (CPA, A1R agonist) constricted MVs but not MAs. Adenosine and CPA (0.001-10 µM) inhibited neurogenic constrictions and NE release in MAs and MVs. DOCA-salt arteries were resistant to adenosine and CPA-mediated inhibition of NE release and constriction. The A2A adenosine receptor agonist CGS21680 (C23H29N7O6.HCl.xH2O) (0.001-0.1 μM) did not alter NE oxidation currents. We conclude that there are prejunctional A1Rs in arteries and both pre- and postjunctional A1Rs in veins; thus, adenosine selectively constricts the veins. Prejunctional A1R function is impaired in arteries, but not veins, from DOCA-salt rats. Sympathetic autoreceptor dysfunction is not specific to α2ARs, but there is a more general disruption of prejunctional mechanisms controlling sympathetic neurotransmitter release in DOCA-salt hypertension.
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Affiliation(s)
- Sutheera Sangsiri
- Departments of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan, USA
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31
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Durnin L, Sanders KM, Mutafova-Yambolieva VN. Differential release of β-NAD(+) and ATP upon activation of enteric motor neurons in primate and murine colons. Neurogastroenterol Motil 2013; 25:e194-204. [PMID: 23279315 PMCID: PMC3578016 DOI: 10.1111/nmo.12069] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
BACKGROUND The purinergic component of enteric inhibitory neurotransmission is important for normal motility in the gastrointestinal (GI) tract. Controversies exist about the purine(s) responsible for inhibitory responses in GI muscles: ATP has been assumed to be the purinergic neurotransmitter released from enteric inhibitory motor neurons; however, recent studies demonstrate that β-nicotinamide adenine dinucleotide (β-NAD(+)) and ADP-ribose mimic the inhibitory neurotransmitter better than ATP in primate and murine colons. The study was designed to clarify the sources of purines in colons of Cynomolgus monkeys and C57BL/6 mice. METHODS High-performance liquid chromatography with fluorescence detection was used to analyze purines released by stimulation of nicotinic acetylcholine receptors (nAChR) and serotonergic 5-HT(3) receptors (5-HT(3)R), known to be present on cell bodies and dendrites of neurons within the myenteric plexus. KEY RESULTS Nicotinic acetylcholine receptor or 5-HT(3)R agonists increased overflow of ATP and β-NAD(+) from tunica muscularis of monkey and murine colon. The agonists did not release purines from circular muscles of monkey colon lacking myenteric ganglia. Agonist-evoked overflow of β-NAD(+), but not ATP, was inhibited by tetrodotoxin (0.5 μmol L(-1)) or ω-conotoxin GVIA (50 nmol L(-1)), suggesting that β-NAD(+) release requires nerve action potentials and junctional mechanisms known to be critical for neurotransmission. ATP was likely released from nerve cell bodies in myenteric ganglia and not from nerve terminals of motor neurons. CONCLUSIONS & INFERENCES These results support the conclusion that ATP is not a motor neurotransmitter in the colon and are consistent with the hypothesis that β-NAD(+), or its metabolites, serve as the purinergic inhibitory neurotransmitter.
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Affiliation(s)
- L Durnin
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557-0575, USA
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Umapathy NS, Gonzales J, Fulzele S, Kim KM, Lucas R, Verin AD. β-Nicotinamide adenine dinucleotide attenuates lipopolysaccharide-induced inflammatory effects in a murine model of acute lung injury. Exp Lung Res 2012; 38:223-32. [PMID: 22563684 DOI: 10.3109/01902148.2012.673049] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) occur in approximately 200,000 patients per year. Studies indicate that lung endothelium plays a significant role in ALI. The authors' recent in vitro studies demonstrate a novel mechanism of β-nicotinamide adenine dinucleotide (β-NAD)-induced protection against gram-positive (pneumolysin, PLY) and gram-negative (lipopolysaccharide, LPS) toxin-induced lung endothelial cell (EC) barrier dysfunction. The objective of the current study was to evaluate the protective effect of β-NAD against LPS-induced ALI in mice. C57BL/6J mice were randomly divided into 4 groups: vehicle, β-NAD, LPS, and LPS/β-NAD. After surgery, mice were allowed to recover for 24 hours. Evans blue dye-albumin (EBA) was given through the internal jugular vein 2 hours prior to the termination of the experiments. Upon sacrificing the animals, bronchoalveolar lavage fluid (BALF) was collected and the lungs were harvested. β-NAD treatment significantly attenuated the inflammatory response by means of reducing the accumulation of cells and protein in BALF, blunting the parenchymal neutrophil infiltration, and preventing capillary leak. In addition, the histological examination demonstrated decreased interstitial edema in the LPS/β-NAD specimens, as compared to the LPS-only specimens. The mRNA levels of the anti-inflammatory cytokines were up-regulated in the LPS group treated with β-NAD compared to the LPS-only-treated group. β-NAD treatment down-regulated the mRNA levels of the proinflammatory cytokines. These findings suggest that β-NAD could be investigated as a therapeutic option against bacterial toxin-induced lung inflammation and ALI in mice.
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Affiliation(s)
- Nagavedi Siddaramappa Umapathy
- Vascular Biology Center and Section of Pulmonary and Critical Care Medicine, Georgia Health Sciences University, Augusta, Georgia 30912, USA.
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Mutafova-Yambolieva VN. Neuronal and extraneuronal release of ATP and NAD(+) in smooth muscle. IUBMB Life 2012; 64:817-24. [PMID: 22941916 DOI: 10.1002/iub.1076] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2012] [Accepted: 07/05/2012] [Indexed: 12/24/2022]
Abstract
Adenosine 5'-triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD(+) ) are key intracellular constituents involved in energy transfer and redox homeostasis in the cell. ATP is also released in the extracellular space and in the past half century it has been assumed to be the purinergic neurotransmitter in many systems including smooth muscle. In some smooth muscles (i.e., the human urinary bladder detrusor muscle), ATP does appear to be primarily released from nerves upon action potential firings, but in other smooth muscles (i.e., the human large intestine), ATP does not mimic the endogenous purine neurotransmitter. It was recently found that NAD(+) , another ubiquitous intracellular adenine nucleotide, also follows a regulated release in neurosecretory cells, vascular and visceral smooth muscles, and the brain. In some cases, NAD(+) fulfills presynaptic and postsynaptic criteria for a neurotransmitter better than ATP. Therefore, the purine hypothesis of neural regulation in smooth muscle is in need of reevaluation. This article will briefly review the current understanding of neuronal and extraneuronal release of purines in smooth muscle with emphasis on the roles of extracellular ATP and NAD(+) and, further, will discuss more recent information about the likely involvement of multiple purines in smooth muscle neurotransmission.
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Zhang X, Mou Z. Expression of the human NAD(P)-metabolizing ectoenzyme CD38 compromises systemic acquired resistance in Arabidopsis. MOLECULAR PLANT-MICROBE INTERACTIONS : MPMI 2012; 25:1209-18. [PMID: 22670756 DOI: 10.1094/mpmi-10-11-0278] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/25/2023]
Abstract
Plant systemic acquired resistance (SAR) is a long-lasting, broad-spectrum immune response that is mounted after primary pathogen infection. Although SAR has been extensively researched, the molecular mechanisms underlying its activation have not been completely understood. We have previously shown that the electron carrier NAD(P) leaks into the plant extracellular compartment upon pathogen attack and that exogenous NAD(P) activates defense gene expression and disease resistance in local treated leaves, suggesting that extracellular NAD(P) [eNAD(P)] might function as a signal molecule activating plant immune responses. To further establish the function of eNAD(P) in plant immunity, we tested the effect of exogenous NAD(P) on resistance gene-mediated hypersensitive response (HR) and SAR. We found that exogenous NAD(P) completely suppresses HR-mediated cell death but does not affect HR-mediated disease resistance. Local application of exogenous NAD(P) is unable to induce SAR in distal tissues, indicating that eNAD(P) is not a sufficient signal for SAR activation. Using transgenic Arabidopsis plants expressing the human NAD(P)-metabolizing ectoenzyme CD38, we demonstrated that altering eNAD(P) concentration or signaling compromises biological induction of SAR. This result suggests that eNAD(P) may play a critical signaling role in activation of SAR.
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Affiliation(s)
- Xudong Zhang
- Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA
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Durnin L, Hwang SJ, Ward SM, Sanders KM, Mutafova-Yambolieva VN. Adenosine 5-diphosphate-ribose is a neural regulator in primate and murine large intestine along with β-NAD(+). J Physiol 2012; 590:1921-41. [PMID: 22351627 DOI: 10.1113/jphysiol.2011.222414] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Adenosine 5′-triphosphate (ATP) has long been considered to be the purine inhibitory neurotransmitter in gastrointestinal (GI) muscles, but recent studies indicate that another purine nucleotide, β-nicotinamide adenine dinucleotide (β-NAD(+)), meets pre- and postsynaptic criteria for a neurotransmitter better than ATP in primate and murine colons. Using a small-volume superfusion assay and HPLC with fluorescence detection and intracellular microelectrode techniques we compared β-NAD(+) and ATP metabolism and postjunctional effects of the primary extracellular metabolites of β-NAD(+) and ATP, namely ADP-ribose (ADPR) and ADP in colonic muscles from cynomolgus monkeys and wild-type (CD38(+/+)) and CD38(−/−) mice. ADPR and ADP caused membrane hyperpolarization that, like nerve-evoked inhibitory junctional potentials (IJPs), were inhibited by apamin. IJPs and hyperpolarization responses to ADPR, but not ADP, were inhibited by the P2Y1 receptor antagonist (1R,2S,4S,5S)-4-[2-iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (MRS2500). Degradation of β-NAD(+) and ADPR was greater per unit mass in muscles containing only nerve processes than in muscles also containing myenteric ganglia. Thus, mechanisms for generation of ADPR from β-NAD(+) and for termination of the action of ADPR are likely to be present near sites of neurotransmitter release. Degradation of β-NAD(+) to ADPR and other metabolites appears to be mediated by pathways besides CD38, the main NAD-glycohydrolase in mammals. Degradation of β-NAD(+) and ATP were equal in colon. ADPR like its precursor, β-NAD(+), mimicked the effects of the endogenous purine neurotransmitter in primate and murine colons. Taken together, our observations support a novel hypothesis in which multiple purines contribute to enteric inhibitory regulation of gastrointestinal motility.
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Affiliation(s)
- Leonie Durnin
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557-0575, USA
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The high-resolution crystal structure of periplasmic Haemophilus influenzae NAD nucleotidase reveals a novel enzymatic function of human CD73 related to NAD metabolism. Biochem J 2012; 441:131-41. [PMID: 21933152 DOI: 10.1042/bj20111263] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Haemophilus influenzae is a major pathogen of the respiratory tract in humans that has developed the capability to exploit host NAD(P) for its nicotinamide dinucleotide requirement. This strategy is organized around a periplasmic enzyme termed NadN (NAD nucleotidase), which plays a central role by degrading NAD into adenosine and NR (nicotinamide riboside), the latter being subsequently internalized by a specific permease. We performed a biochemical and structural investigation on H. influenzae NadN which determined that the enzyme is a Zn2+-dependent 5'-nucleotidase also endowed with NAD(P) pyrophosphatase activity. A 1.3 Å resolution structural analysis revealed a remarkable conformational change that occurs during catalysis between the open and closed forms of the enzyme. NadN showed a broad substrate specificity, recognizing either mono- or di-nucleotide nicotinamides and different adenosine phosphates with a maximal activity on 5'-adenosine monophosphate. Sequence and structural analysis of H. influenzae NadN led us to discover that human CD73 is capable of processing both NAD and NMN, therefore disclosing a possible novel function of human CD73 in systemic NAD metabolism. Our data may prove to be useful for inhibitor design and disclosed unanticipated fascinating evolutionary relationships.
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Durnin L, Dai Y, Aiba I, Shuttleworth CW, Yamboliev IA, Mutafova-Yambolieva VN. Release, neuronal effects and removal of extracellular β-nicotinamide adenine dinucleotide (β-NAD⁺) in the rat brain. Eur J Neurosci 2012; 35:423-35. [PMID: 22276961 DOI: 10.1111/j.1460-9568.2011.07957.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
Recent evidence supports an emerging role of β-nicotinamide adenine dinucleotide (β-NAD(+) ) as a novel neurotransmitter and neuromodulator in the peripheral nervous system -β-NAD(+) is released in nerve-smooth muscle preparations and adrenal chromaffin cells in a manner characteristic of a neurotransmitter. It is currently unclear whether this holds true for the CNS. Using a small-chamber superfusion assay and high-sensitivity high-pressure liquid chromatography techniques, we demonstrate that high-K(+) stimulation of rat forebrain synaptosomes evokes overflow of β-NAD(+) , adenosine 5'-triphosphate, and their metabolites adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate, adenosine, ADP-ribose (ADPR) and cyclic ADPR. The high-K(+) -evoked overflow of β-NAD(+) is attenuated by cleavage of SNAP-25 with botulinum neurotoxin A, by inhibition of N-type voltage-dependent Ca(2+) channels with ω-conotoxin GVIA, and by inhibition of the proton gradient of synaptic vesicles with bafilomycin A1, suggesting that β-NAD(+) is likely released via vesicle exocytosis. Western analysis demonstrates that CD38, a multifunctional protein that metabolizes β-NAD(+) , is present on synaptosomal membranes and in the cytosol. Intact synaptosomes degrade β-NAD(+) . 1,N (6) -etheno-NAD, a fluorescent analog of β-NAD(+) , is taken by synaptosomes and this uptake is attenuated by authentic β-NAD(+) , but not by the connexin 43 inhibitor Gap 27. In cortical neurons local applications of β-NAD(+) cause rapid Ca(2+) transients, likely due to influx of extracellular Ca(2+) . Therefore, rat brain synaptosomes can actively release, degrade and uptake β-NAD(+) , and β-NAD(+) can stimulate postsynaptic neurons, all criteria needed for a substance to be considered a candidate neurotransmitter in the brain.
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Affiliation(s)
- Leonie Durnin
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
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Durnin L, Mutafova-Yambolieva VN. Cyclic ADP-ribose requires CD38 to regulate the release of ATP in visceral smooth muscle. FEBS J 2011; 278:3095-108. [PMID: 21740519 PMCID: PMC4838287 DOI: 10.1111/j.1742-4658.2011.08233.x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
It is well established that the intracellular second messenger cADP-ribose (cADPR) activates Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors. CD38 is a multifunctional enzyme involved in the formation of cADPR in mammals. CD38 has also been reported to transport cADPR in several cell lines. Here, we demonstrate a role for extracellular cADPR and CD38 in modulating the spontaneous, but not the electrical field stimulation-evoked, release of ATP in visceral smooth muscle. Using a small-volume superfusion assay and an HPLC technique with fluorescence detection, we measured the spontaneous and evoked release of ATP in bladder detrusor smooth muscles isolated from CD38(+/+) and CD38(-/-) mice. cADPR (1 nM) enhanced the spontaneous overflow of ATP in bladders isolated from CD38(+/+) mice. This effect was abolished by the inhibitor of cADPR receptors on sarcoplasmic reticulum 8-bromo-cADPR (80 μM) and by ryanodine (50 μm), but not by the nonselective P2 purinergic receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulfonate (30 μM). cADPR failed to facilitate the spontaneous ATP overflow in bladders isolated from CD38(-/-) mice, indicating that CD38 is crucial for the enhancing effects of extracellular cADPR on spontaneous ATP release. Contractile responses to ATP were potentiated by cADPR, suggesting that the two adenine nucleotides may work in synergy to maintain the resting tone of the bladder. In conclusion, extracellular cADPR enhances the spontaneous release of ATP in the bladder by influx via CD38 and subsequent activation of intracellular cADPR receptors, probably causing an increase in intracellular Ca(2+) in neuronal cells.
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Affiliation(s)
- Leonie Durnin
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557-0575, USA
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Zemskov E, Lucas R, Verin AD, Umapathy NS. P2Y receptors as regulators of lung endothelial barrier integrity. J Cardiovasc Dis Res 2011; 2:14-22. [PMID: 21716747 PMCID: PMC3120267 DOI: 10.4103/0975-3583.78582] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Endothelial cells (ECs), forming a semi-permeable barrier between the interior space of blood vessels and underlying tissues, control such diverse processes as vascular tone, homeostasis, adhesion of platelets, and leukocytes to the vascular wall and permeability of vascular wall for cells and fluids. Mechanisms which govern the highly clinically relevant process of increased EC permeability are under intense investigation. It is well known that loss of this barrier (permeability increase) results in tissue inflammation, the hall mark of inflammatory diseases such as acute lung injury and its severe form, acute respiratory distress syndrome. Little is known about processes which determine the endothelial barrier enhancement or protection against permeability increase. It is now well accepted that extracellular purines and pyrimidines are promising and physiologically relevant barrier-protective agents and their effects are mediated by interaction with cell surface P2Y receptors which belong to the superfamily of G-protein-coupled receptors. The therapeutic potential of P2Y receptors is rapidly expanding field in pharmacology and some selective agonists became recently available. Here, we present an overview of recently identified P2Y receptor agonists that enhance the pulmonary endothelial barrier and inhibit and/or reverse endothelial barrier disruption.
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Affiliation(s)
- Evgeny Zemskov
- Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912, USA
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40
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Vaisitti T, Audrito V, Serra S, Bologna C, Brusa D, Malavasi F, Deaglio S. NAD+-metabolizing ecto-enzymes shape tumor-host interactions: the chronic lymphocytic leukemia model. FEBS Lett 2011; 585:1514-20. [PMID: 21514298 DOI: 10.1016/j.febslet.2011.04.036] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2011] [Revised: 04/13/2011] [Accepted: 04/14/2011] [Indexed: 11/28/2022]
Abstract
Nicotinamide adenine dinucleotide (NAD(+)) is an essential co-enzyme that can be released in the extracellular milieu. Here, it may elicit signals through binding purinergic receptors. Alternatively, NAD(+) may be dismantled to adenosine, up-taken by cells and transformed to reconstitute the intracellular nucleotide pool. An articulated ecto-enzyme network is responsible for the nucleotide-nucleoside conversion. CD38 is the main mammalian enzyme that hydrolyzes NAD(+), generating Ca(2+)-active metabolites. Evidence suggests that this extracellular network may be altered or used by tumor cells to (i) nestle in protected areas, and (ii) evade the immune response. We have exploited chronic lymphocytic leukemia as a model to test the role of the ecto-enzyme network, starting by analyzing the individual elements that make up the whole picture.
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Affiliation(s)
- Tiziana Vaisitti
- Department of Genetics, Biology and Biochemistry, University of Turin, Turin, Italy
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Hwang SJ, Durnin L, Dwyer L, Rhee PL, Ward SM, Koh SD, Sanders KM, Mutafova-Yambolieva VN. β-nicotinamide adenine dinucleotide is an enteric inhibitory neurotransmitter in human and nonhuman primate colons. Gastroenterology 2011; 140:608-617.e6. [PMID: 20875415 PMCID: PMC3031738 DOI: 10.1053/j.gastro.2010.09.039] [Citation(s) in RCA: 74] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/10/2010] [Revised: 08/03/2010] [Accepted: 09/17/2010] [Indexed: 12/25/2022]
Abstract
BACKGROUND & AIMS An important component of enteric inhibitory neurotransmission is mediated by a purine neurotransmitter, such as adenosine 5'-triphosphate (ATP), binding to P2Y1 receptors and activating small conductance K(+) channels. In murine colon β-nicotinamide adenine dinucleotide (β-NAD) is released with ATP and mimics the pharmacology of inhibitory neurotransmission better than ATP. Here β-NAD and ATP were compared as possible inhibitory neurotransmitters in human and monkey colons. METHODS A small-volume superfusion assay and high-pressure liquid chromatography with fluorescence detection were used to evaluate spontaneous and nerve-evoked overflow of β-NAD, ATP, and metabolites. Postjunctional responses to nerve stimulation, β-NAD and ATP were compared using intracellular membrane potential and force measurements. Effects of β-NAD on smooth muscle cells (SMCs) were recorded by patch clamp. P2Y receptor transcripts were assayed by reverse transcription polymerase chain reaction. RESULTS In contrast to ATP, overflow of β-NAD evoked by electrical field stimulation correlated with stimulation frequency and was diminished by the neurotoxins, tetrodotoxin, and ω-conotoxin GVIA. Inhibitory junction potentials and responses to exogenous β-NAD, but not ATP, were blocked by P2Y receptor antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS 2179), and (1R,2S,4S,5S)-4-[2-Iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (MRS 2500). β-NAD activated nonselective cation currents in SMCs, but failed to activate outward currents. CONCLUSIONS β-NAD meets the criteria for a neurotransmitter better than ATP in human and monkey colons and therefore may contribute to neural regulation of colonic motility. SMCs are unlikely targets for inhibitory purine neurotransmitters because dominant responses of SMCs were activation of net inward, rather than outward, current.
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Affiliation(s)
- Sung Jin Hwang
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557
| | - Leonie Durnin
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557
| | - Laura Dwyer
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557
| | - Poong-Lyul Rhee
- Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, South Korea
| | - Sean M. Ward
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557
| | - Sang Don Koh
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557
| | - Kenton M. Sanders
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557
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Lecka J, Molski S, Komoszynski M. Extracellular-purine metabolism in blood vessels (part I). Extracellular-purine level in blood of patients with abdominal aortic aneurysm. NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS 2010; 29:647-57. [PMID: 20706956 DOI: 10.1080/15257770.2010.502164] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
Adenosine and adenosine derivatives are the main regulators of purinoceptors (P1 and P2) mediated hemostasis and blood pressure. Since impaired hemostasis and high blood pressure lead to atherosclerosis and to the development of aneurysm, in this study we tested and compared the concentration of extracellular purines (e-purines) in the blood in of patients having abdominal aortic aneurysm with that from healthy volunteers. Whereas adenine nucleosides and nucleotides level in human blood plasma was analysed using reverse phase high performance liquid chromatography (HPLC), cholesterol concentration was estimated by an enzymatic assay. We did not find any correlation between e-purines concentration and the age of healthy volunteers. Furthermore, the sum level of e-purines (ATP, ADP, AMP, adenosine, and inosine) in the control group did not exceed 70 microM, while it was nearly two-fold higher in the blood of patients having abdominal aortic aneurysm, (123 microM). In a special case of people with Leriche Syndrome, a disease characterized by deep atherosclerotic changes, the e-purines level had further increased. Additionally, we also report typical atherosclerotic changes in the aorta using histological assays as well as total cholesterol rise. The significant rise in cholesterol concentration in the blood of the patients with abdominal aortas aneurysm, compared with the control groups, was not unique since 23% of the healthy people also exceeded the normal level of cholesterol. Therefore, our results strongly indicate that the estimation of e-purines concentration in the blood may serve as another indicator of atherosclerosis and warrant further consideration as a futuristic diagnostic tool.
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Affiliation(s)
- Joanna Lecka
- Department of Biochemistry Collegium Medicum Bydgoszcz, N. Copernicus University, Torun, Poland.
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Grahnert A, Grahnert A, Klein C, Schilling E, Wehrhahn J, Hauschildt S. Review: NAD +: a modulator of immune functions. Innate Immun 2010; 17:212-33. [PMID: 20388721 DOI: 10.1177/1753425910361989] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Latterly, nicotinamide adenine dinucleotide (NAD+) has emerged as a molecule with versatile functions and of enormous impact on the maintenance of cell integrity. Besides playing key roles in almost all major aspects of energy metabolism, there is mounting evidence that NAD+ and its degradation products affect various biological activities including calcium homeostasis, gene transcription, DNA repair, and intercellular communication. This review is aimed at giving a brief insight into the life cycle of NAD+ in the cell, referring to synthesis, action and degradation aspects. With respect to their immunological relevance, the importance and function of the major NAD+ metabolizing enzymes, namely CD38/CD157, ADP-ribosyltransferases (ARTs), poly-ADP-ribose-polymerases (PARPs), and sirtuins are summarized and roles of NAD+ and its main degradation product adenosine 5'-diphosphoribose (ADPR) in cell signaling are discussed. In addition, an outline of the variety of immunological processes depending on the activity of nicotinamide phosphoribosyltransferase (Nampt), the key enzyme of the salvage pathway of NAD+ synthesis, is presented. Taken together, an efficient supply of NAD+ seems to be a crucial need for a multitude of cell functions, underlining the yet only partly revealed potency of this small molecule to influence cell fate.
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Affiliation(s)
- Andreas Grahnert
- Department of Immunobiology, Institute of Biology, University of Leipzig, Talstrasse 33, Leipzig, Germany
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Shi M, Liu D, Duan H, Qian L, Wang L, Niu L, Zhang H, Yong Z, Gong Z, Song L, Yu M, Hu M, Xia Q, Shen B, Guo N. The β2-adrenergic receptor and Her2 comprise a positive feedback loop in human breast cancer cells. Breast Cancer Res Treat 2010; 125:351-62. [PMID: 20237834 DOI: 10.1007/s10549-010-0822-2] [Citation(s) in RCA: 96] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2010] [Accepted: 02/24/2010] [Indexed: 01/16/2023]
Abstract
In this study, β2-AR level was found to be up-regulated in MCF-7 cells overexpressing Her2 (MCF-7/Her2). Correlation of β2-AR level with Her2 status was demonstrated in breast cancer tissue samples. Constitutive phosphorylation of ERK, mRNA expression up-regulation of catecholamine-synthesis enzymes, and increased epinephrine release were detected in MCF-7/Her2 cells. β2-AR expression induced by epinephrine and involvement of ERK signaling were validated. The data indicate that Her2 overexpression and excessive phosphorylation of ERK cause epinephrine autocrine release from breast cancer cells, resulting in up-regulation of β2-AR expression. The data also showed that catecholamine prominently stimulated Her2 mRNA expression and promoter activity. The activation and nuclear translocation of STAT3 triggered by isoproterenol were observed. Enhanced binding activities of STAT3 to the Her2 promoter after isoproterenol stimulation were verified. Using STAT3 shRNA and dominant negative STAT3 mutant, the role of STAT3 in isoproterenol-induced Her2 expression was further confirmed. The data support a model where β2-AR and Her2 comprise a positive feedback loop in human breast cancer cells.
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Affiliation(s)
- Ming Shi
- Department of Molecular Immunology, Institute of Basic Medical Sciences, Beijing, People's Republic of China
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Yamboliev IA, Smyth LM, Durnin L, Dai Y, Mutafova-Yambolieva VN. Storage and secretion of beta-NAD, ATP and dopamine in NGF-differentiated rat pheochromocytoma PC12 cells. Eur J Neurosci 2009; 30:756-68. [PMID: 19712094 DOI: 10.1111/j.1460-9568.2009.06869.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
In nerve-smooth muscle preparations beta-nicotinamide adenine dinucleotide (beta-NAD) has emerged as a novel extracellular substance with putative neurotransmitter and neuromodulator functions. beta-NAD is released, along with noradrenaline and adenosine 5'-triphosphate (ATP), upon firing of action potentials in blood vessels, urinary bladder and large intestine. At present it is unclear whether noradrenaline, ATP and beta-NAD are stored in and released from common populations of synaptic vesicles. The answer is unattainable in complex systems such as nerve-smooth muscle preparations. Adrenal chromaffin cells are thus used here as a single-cell model to examine mechanisms of concomitant neurosecretion. Using high-performance liquid chromatography techniques with electrochemical and fluorescence detection we simultaneously evaluated secretion of dopamine (DA), ATP, adenosine 5'-diphosphate, adenosine 5'-monophosphate, adenosine, beta-NAD and its immediate metabolites ADP-ribose and cyclic ADP-ribose in superfused nerve growth factor-differentiated rat pheochromocytoma PC12 cells. beta-NAD, DA and ATP were released constitutively and upon stimulation with high-K(+) solution or nicotine. Botulinum neurotoxin A tended to increase the spontaneous secretion of all substances and abolished the high-K(+)-evoked release of beta-NAD and DA but not of ATP. Subcellular fractionation by continuous glycerol and sucrose gradients along with immunoblot analysis of the vesicular marker proteins synaptophysin and secretogranin II revealed that beta-NAD, ATP and DA are stored in both small synaptic-like vesicles and large dense-core-like vesicles. However, the three substances appear to have different preferential sites of release upon membrane depolarization including sites associated with SNAP-25 and sites not associated with SNAP-25.
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Affiliation(s)
- Ilia A Yamboliev
- Department of Pharmacology, University of Nevada School of Medicine, Reno, NV 89557, USA
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Zhang X, Mou Z. Extracellular pyridine nucleotides induce PR gene expression and disease resistance in Arabidopsis. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2009; 57:302-12. [PMID: 18798871 DOI: 10.1111/j.1365-313x.2008.03687.x] [Citation(s) in RCA: 80] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/20/2023]
Abstract
Although it is well known that the pyridine nucleotides NAD and NADP function inside the cell to regulate intracellular signaling processes, recent evidence from animal studies suggests that NAD(P) also functions in the extracellular compartment (ECC). Extracellular NAD(P) [eNAD(P)] can either directly bind to plasma membrane receptors or be metabolized by ecto-enzymes to produce cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate, and/or may ADP-ribosylate cell-surface receptors, resulting in activation of transmembrane signaling. In this study, we report that, in plants, exogenous NAD(P) induces the expression of pathogenesis-related (PR) genes and resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. Chelation of Ca(2+) by EGTA significantly inhibits the induction of PR genes by exogenous NAD(P), suggesting that exogenous NAD(P) may induce PR genes through a pathway that involves Ca(2+) signaling. We show that exogenous application of NAD(P) causes accumulation of the defense signal molecule salicylic acid (SA), and induces both SA/NPR1-dependent and -independent PR gene expression and disease resistance. Furthermore, we demonstrate that NAD(P) leaks into the plant ECC after mechanical wounding and pathogen infection, and that the amount of NAD(P) leaking into the ECC after P. syringae pv. tobacco DC3000/avrRpt2 infection is sufficient for induction of both PR gene expression and disease resistance. We propose that NAD(P) leakage from cells losing membrane integrity upon environmental stress may function as an elicitor to activate plant defense responses. Our data provide evidence that eNAD(P) functions in plant signaling, and illustrate the potential importance of eNAD(P) in plant innate immunity.
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Affiliation(s)
- Xudong Zhang
- Department of Microbiology and Cell Science, University of Florida, PO Box 110700, Gainesville, FL 32611, USA
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Smyth LM, Yamboliev IA, Mutafova-Yambolieva VN. N-type and P/Q-type calcium channels regulate differentially the release of noradrenaline, ATP and beta-NAD in blood vessels. Neuropharmacology 2008; 56:368-78. [PMID: 18824011 DOI: 10.1016/j.neuropharm.2008.09.007] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2007] [Revised: 08/08/2008] [Accepted: 09/09/2008] [Indexed: 01/08/2023]
Abstract
Using HPLC techniques we evaluated the electrical field stimulation-evoked overflow of noradrenaline (NA), adenosine 5'-triphosphate (ATP), and beta-nicotinamide adenine dinucleotide (beta-NAD) in the presence of low nanomolar concentrations of omega-conotoxin GVIA or omega-agatoxin IVA in the canine mesenteric arteries and veins. omega-conotoxin GVIA abolished the evoked overflow of NA and beta-NAD in artery and vein, whereas the evoked overflow of ATP remained unchanged in the presence of omega-conotoxin GVIA. omega-agatoxin IVA significantly reduced the evoked overflow of ATP and beta-NAD. The overflow of NA remained largely unaffected by omega-agatoxin IVA, except at 16Hz in the vein where the overflow of NA was reduced by about 50%. Artery and vein exhibited similar expression levels of the alpha(1B) (CaV2.2, N-type) subunit, whereas the vein showed greater levels of the alpha(1A) (CaV2.1, P/Q-type) subunit than artery. Therefore, there are at least two release sites for NA, beta-NAD and ATP in the canine mesenteric artery and vein: an N-type-associated site releasing primarily NA, beta-NAD and some ATP, and a P/Q-type-associated site releasing ATP, beta-NAD and some NA. The N-type-mediated mechanisms are equally expressed in artery and vein, whereas the P/Q-type-mediated mechanisms are more pronounced in the vein and may ensure additional neurotransmitter release at higher levels of neural activity. In artery, beta-NAD caused a dual effect consisting of vasodilatation or vasoconstriction depending on concentrations, whereas vein responded with vasodilatation only. In contrast, ATP caused vasoconstriction in both vessels. beta-NAD and ATP may mediate disparate functions in the canine mesenteric resistive and capacitative circulations.
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Affiliation(s)
- Lisa M Smyth
- Department of Physiology and Cell Biology, Anderson Medical Sciences Building, MS 352, University of Nevada School of Medicine, 1664 N. Virginia Street, Reno, NV 89557-0352, USA
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Billington RA, Travelli C, Ercolano E, Galli U, Roman CB, Grolla AA, Canonico PL, Condorelli F, Genazzani AA. Characterization of NAD uptake in mammalian cells. J Biol Chem 2008; 283:6367-74. [PMID: 18180302 DOI: 10.1074/jbc.m706204200] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Recent evidence has shown that NAD(P) plays a variety of roles in cell-signaling processes. Surprisingly, the presence of NAD(P) utilizing ectoenzymes suggests that NAD(P) is present extracellularly. Indeed, nanomolar concentrations of NAD have been found in plasma and other body fluids. Although very high concentrations of NAD have been shown to enter cells, it is not known whether lower, more physiological concentrations are able to be taken up. Here we show that two mammalian cell types are able to transport low NAD concentrations effectively. Furthermore, extracellular application of NAD was able to counteract FK866-induced cell death and restore intracellular NAD(P) levels. We propose that NAD uptake may play a role in physiological NAD homeostasis.
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Affiliation(s)
- Richard A Billington
- Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche and the Drug and Food Biotechnology Center, Università del Piemonte Orientale, Via Bovio 6, Novara, Italy.
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Extracellular NAD is a regulator for FcgammaR-mediated phagocytosis in murine macrophages. Biochem Biophys Res Commun 2007; 367:156-61. [PMID: 18166151 DOI: 10.1016/j.bbrc.2007.12.131] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2007] [Accepted: 12/17/2007] [Indexed: 11/23/2022]
Abstract
NAD is available in the extracellular environment and elicits immune modulation such as T cell apoptosis by being used as the substrate of cell surface ADP-ribosyl transferase. However, it is unclear whether extracellular NAD affects function of macrophages expressing cell surface ADP-ribosyl transferase. Here we show that extracellular NAD enhances Fcgamma receptor (FcgammaR)-mediated phagocytosis in J774A.1 macrophages via the conversion into cyclic ADP-ribose (cADPR), a potent calcium mobilizer, by CD38, an ADP-ribosyl cyclase. Extracellular NAD increased the phagocytosis of IgG-coated sheep red blood cells (IgG-SRBC) in J774A.1 macrophages, which was completely abolished by pretreatment of 8-bromo-cADPR, an antagonist of cADPR, or CD38 knockdown. Extracellular NAD increased basal intracellular Ca(2+) concentration, which also was abolished by pretreatment of 8-bromo-cADPR or CD38 knockdown. Moreover, the chelation of intracellular calcium abolished NAD-induced enhancement of phagocytosis of IgG-SRBC. Our results suggest that extracellular NAD act as a regulator for FcgammaR-mediated phagocytosis in macrophages.
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50
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Mutafova-Yambolieva VN, Hwang SJ, Hao X, Chen H, Zhu MX, Wood JD, Ward SM, Sanders KM. Beta-nicotinamide adenine dinucleotide is an inhibitory neurotransmitter in visceral smooth muscle. Proc Natl Acad Sci U S A 2007; 104:16359-64. [PMID: 17913880 PMCID: PMC2042211 DOI: 10.1073/pnas.0705510104] [Citation(s) in RCA: 137] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Peripheral inhibitory nerves are physiological regulators of the contractile behavior of visceral smooth muscles. One of the transmitters responsible for inhibitory neurotransmission has been reputed to be a purine, possibly ATP. However, the exact identity of this substance has never been verified. Here we show that beta-nicotinamide adenine dinucleotide (beta-NAD), an inhibitory neurotransmitter candidate, is released by stimulation of enteric nerves in gastrointestinal muscles, and the pharmacological profile of beta-NAD mimics the endogenous neurotransmitter better than ATP. Levels of beta-NAD in superfusates of muscles after nerve stimulation exceed ATP by at least 30-fold; unlike ATP, the release of beta-NAD depends on the frequency of nerve stimulation. beta-NAD is released from enteric neurons, and release was blocked by tetrodotoxin or omega-conotoxin GVIA. beta-NAD is an agonist for P2Y1 receptors, as demonstrated by receptor-mediated responses in HEK293 cells expressing P2Y1 receptors. Exogenous beta-NAD mimics the effects of the enteric inhibitory neurotransmitter. Responses to beta-NAD and inhibitory junction potentials are blocked by the P2Y1-selective antagonist, MRS2179, and the nonselective P2 receptor antagonists, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid and suramin. Responses to ATP are not blocked by these P2Y receptor inhibitors. The expression of CD38 in gastrointestinal muscles, and specifically in interstitial cells of Cajal, provides a means of transmitter disposal after stimulation. beta-NAD meets the traditional criteria for a neurotransmitter that contributes to enteric inhibitory regulation of visceral smooth muscles.
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Affiliation(s)
| | - Sung Jin Hwang
- *Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557; and
| | - Xuemei Hao
- Department of Neuroscience, Center for Molecular Neurobiology, and
| | - Hui Chen
- *Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557; and
| | - Michael X. Zhu
- Department of Neuroscience, Center for Molecular Neurobiology, and
| | - Jackie D. Wood
- Department of Physiology and Cell Biology, Ohio State University, Columbus, OH 43210
| | - Sean M. Ward
- *Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557; and
| | - Kenton M. Sanders
- *Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557; and
- To whom correspondence should be addressed. E-mail:
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