|
1
|
Natural statin derivatives as potential therapy to reduce intestinal fluid loss in cholera. PLoS Negl Trop Dis 2022; 16:e0010989. [PMID: 36490300 PMCID: PMC9770395 DOI: 10.1371/journal.pntd.0010989] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2022] [Revised: 12/21/2022] [Accepted: 11/28/2022] [Indexed: 12/14/2022] Open
Abstract
As a leading cause of death in children under 5 years old, secretory diarrheas including cholera are characterized by excessive intestinal fluid secretion driven by enterotoxin-induced cAMP-dependent intestinal chloride transport. This study aimed to identify fungal bioactive metabolites possessing anti-secretory effects against cAMP-dependent chloride secretion in intestinal epithelial cells. Using electrophysiological analyses in human intestinal epithelial (T84) cells, five fungus-derived statin derivatives including α,β-dehydrolovastatin (DHLV), α,β-dehydrodihydromonacolin K, lovastatin, mevastatin and simvastatin were found to inhibit the cAMP-dependent chloride secretion with IC50 values of 1.8, 8.9, 11.9, 11.4 and 5 μM, respectively. Being the most potent statin derivatives, DHLV was evaluated for its pharmacological properties including cellular toxicity, mechanism of action, target specificity and in vivo efficacy. DHLV at concentrations up to 20 μM did not affect cell viability and barrier integrity of T84 cells. Electrophysiological analyses indicated that DHLV inhibited cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent apical chloride channel, via mechanisms not involving alteration of intracellular cAMP levels or its negative regulators including AMP-activated protein kinases and protein phosphatases. DHLV had no effect on Na+-K+ ATPase activities but inhibited Ca2+-dependent chloride secretion without affecting intracellular Ca2+ levels. Importantly, intraperitoneal (2 mg/kg) and intraluminal (20 μM) injections of DHLV reduced cholera toxin-induced intestinal fluid secretion in mice by 59% and 65%, respectively without affecting baseline intestinal fluid transport. This study identifies natural statin derivatives as novel natural product-derived CFTR inhibitors, which may be beneficial in the treatment of enterotoxin-induced secretory diarrheas including cholera.
Collapse
|
|
2
|
Tharabenjasin P, Ferraris RP, Choowongkomon K, Pongkorpsakol P, Worakajit N, Sawasvirojwong S, Pabalan N, Na-Bangchang K, Muanprasat C. β-eudesmol but not atractylodin exerts an inhibitory effect on CFTR-mediated chloride transport in human intestinal epithelial cells. Biomed Pharmacother 2021; 142:112030. [PMID: 34426253 DOI: 10.1016/j.biopha.2021.112030] [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: 02/15/2021] [Revised: 07/13/2021] [Accepted: 08/07/2021] [Indexed: 11/19/2022] Open
Abstract
Oriental herbal medicine with the two bioactive constituents, β-eudesmol (BE) and atractylodin (AT), has been used as a remedy for gastrointestinal disorders. There was no scientific evidence reporting their antidiarrheal effect and underpinning mechanisms. Therefore, we aimed to investigate the anti-secretory activity of these two compounds in vitro. The inhibitory effect of BE and AT on cAMP-induced Cl- secretion was evaluated by Ussing chamber in human intestinal epithelial (T84) cells. Short-circuit current (ISC) and apical Cl- current (ICl-) were measured after adding indirect and direct cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activator. MTT assay was used to determine cellular cytotoxicity. Protein-ligand interaction was investigated by in silico molecular docking analysis. BE, but not AT concentration-dependently (IC50 of ~1.05 µM) reduced cAMP-mediated, CFTRinh-172 inhibitable Cl- secretion as determined by transepithelial ISC across a monolayer of T84 cells. Potency of CFTR-mediated ICl- inhibition by BE did not change with the use of different CFTR activators suggesting a direct blockage of the channel active site(s). Pretreatment with BE completely prevented cAMP-induced ICl-. Furthermore, BE at concentrations up to 200 µM (24 h) had no effect on T84 cell viability. In silico studies indicated that BE could best dock onto dephosphorylated structure of CFTR at ATP-binding pockets in nucleotide-binding domain (NBD) 2 region. These findings provide the first evidence for the anti-secretory effect of BE involving inhibition of CFTR function. BE represents a promising candidate for the therapeutic or prophylactic intervention of diarrhea resulted from intestinal hypersecretion of Cl.
Collapse
Affiliation(s)
- Phuntila Tharabenjasin
- Chulabhorn International College of Medicine, Thammasat University (Rangsit Campus), Klongnung, Klongluang, Pathum Thani 10120, Thailand
| | - Ronaldo P Ferraris
- Department of Pharmacology, Physiology and Neuroscience, New Jersey Medical School, Rutgers University, Newark, NJ 07946, USA
| | - Kiattawee Choowongkomon
- Department of Biochemistry, Faculty of Science, Kasetsart University, Ngam Wong Wan Rd, Ladyaow, Chatuchak, Bangkok 10900, Thailand
| | - Pawin Pongkorpsakol
- Faculty of Medicine and Public Health, HRH Princess Chulabhorn College of Medical Science, Chulabhorn Royal Academy, Bangkok 10210, Thailand
| | - Nichakorn Worakajit
- Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bang Pla, Bang Phli, Samut Prakan 10540, Thailand
| | - Sutthipong Sawasvirojwong
- Department of Pathology, Faculty of Medicine, Chulalongkorn University, Phayathai Rd, Pathumwan, Bangkok 10330, Thailand
| | - Noel Pabalan
- Chulabhorn International College of Medicine, Thammasat University (Rangsit Campus), Klongnung, Klongluang, Pathum Thani 10120, Thailand
| | - Kesara Na-Bangchang
- Chulabhorn International College of Medicine, Thammasat University (Rangsit Campus), Klongnung, Klongluang, Pathum Thani 10120, Thailand; Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma, Chulabhorn International College of Medicine, Rangsit Center, Thammasat University (Rangsit Campus), Klongnung, Klongluang, Pathum Thani 10120, Thailand
| | - Chatchai Muanprasat
- Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bang Pla, Bang Phli, Samut Prakan 10540, Thailand.
| |
Collapse
|
|
3
|
Kiefer MC, Motyka NI, Clements JD, Bitoun JP. Enterotoxigenic Escherichia coli Heat-Stable Toxin Increases the Rate of Zinc Release from Metallothionein and Is a Zinc- and Iron-Binding Peptide. mSphere 2020; 5:e00146-20. [PMID: 32238569 PMCID: PMC7113584 DOI: 10.1128/msphere.00146-20] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2020] [Accepted: 03/12/2020] [Indexed: 12/16/2022] Open
Abstract
Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies have identified heat-stable enterotoxin (ST)-producing ETEC as one of the major diarrhea-causing pathogens in children younger than five years. In this study, we examined iron and zinc binding by both human and porcine ST variants and determined how host metallothionein could detoxify ST. We found that ST purified from ETEC culture supernatants eluted as a doublet during C18 reverse-phase chromatography. Leading edge fractions of the ST doublet were found to be devoid of iron, while trailing edge fractions of the ST doublet were found to contain measurable iron. Next, we found that purified ST could be reconstituted with iron under reducing and anaerobic conditions, and iron-bound ST attenuated the induction of cGMP in T84 epithelial cells. Moreover, we demonstrated that supernatants of ETEC 214-4 grown under increasing iron concentrations were only able to induce cGMP at iron concentrations greater than 5 μM. In vitro studies also demonstrated that ST binds zinc, and once bound, zinc removal from ST required denaturing conditions. Zinc-bound ST also failed to induce cGMP. We found that ST contributes disulfide bonds to the perceived oxidized glutathione pool, increases the rate of zinc release from metallothionein, and can be detoxified by metallothionein. Lastly, we showed ST induces transcriptional changes in genes previously shown to be regulated by deferoxamine. These studies demonstrate ST ETEC pathogenesis may be tied intimately to host mucosal metal status.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries, deployed military personnel, and travelers to regions of endemicity. The heat-stable toxin (ST) is a small nonimmunogenic secreted peptide with 3 disulfide bonds. It has been appreciated that dietary disulfides modulate intestinal redox potential and that ST could be detoxified using exogenous reductants. Using biochemical and spectroscopic approaches, we demonstrated that ST can separately bind iron and zinc under reducing conditions, thereby reducing ST toxicity. Moreover, we demonstrated that ST modulates the glutathione (GSH)/oxidized glutathione (GSSG) ratio and that ST should be considered a toxin oxidant. ST can be detoxified by oxidizing zinc-loaded metallothionine, causing free zinc to be released. These studies help lay a foundation to understand how diarrheal pathogens modulate intestinal redox potential and may impact how we design therapeutics and/or vaccines for the pathogens that produce them.
Collapse
Affiliation(s)
- Mallory C Kiefer
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
| | - Natalya I Motyka
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
| | - John D Clements
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
| | - Jacob P Bitoun
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
| |
Collapse
|
|
4
|
Wongdee K, Rodrat M, Teerapornpuntakit J, Krishnamra N, Charoenphandhu N. Factors inhibiting intestinal calcium absorption: hormones and luminal factors that prevent excessive calcium uptake. J Physiol Sci 2019; 69:683-696. [PMID: 31222614 PMCID: PMC10717634 DOI: 10.1007/s12576-019-00688-3] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2019] [Accepted: 06/09/2019] [Indexed: 12/11/2022]
Abstract
Besides the two canonical calciotropic hormones, namely parathyroid hormone and 1,25-dihydroxyvitamin D [1,25(OH)2D3], there are several other endocrine and paracrine factors, such as prolactin, estrogen, and insulin-like growth factor that have been known to directly stimulate intestinal calcium absorption. Generally, to maintain an optimal plasma calcium level, these positive regulators enhance calcium absorption, which is indirectly counterbalanced by a long-loop negative feedback mechanism, i.e., through calcium-sensing receptor in the parathyroid chief cells. However, several lines of recent evidence have revealed the presence of calcium absorption inhibitors present in the intestinal lumen and extracellular fluid in close vicinity to enterocytes, which could also directly compromise calcium absorption. For example, luminal iron, circulating fibroblast growth factor (FGF)-23, and stanniocalcin can decrease calcium absorption, thereby preventing excessive calcium uptake under certain conditions. Interestingly, the intestinal epithelial cells themselves could lower their rate of calcium uptake after exposure to high luminal calcium concentration, suggesting a presence of an ultra-short negative feedback loop independent of systemic hormones. The existence of neural regulation is also plausible but this requires more supporting evidence. In the present review, we elaborate on the physiological significance of these negative feedback regulators of calcium absorption, and provide evidence to show how our body can efficiently restrict a flood of calcium influx in order to maintain calcium homeostasis.
Collapse
Affiliation(s)
- Kannikar Wongdee
- Faculty of Allied Health Sciences, Burapha University, Chonburi, Thailand
- Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok, Thailand
| | - Mayuree Rodrat
- Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok, Thailand
- Department of Physiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok, 10400, Thailand
| | - Jarinthorn Teerapornpuntakit
- Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok, Thailand
- Department of Physiology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand
| | - Nateetip Krishnamra
- Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok, Thailand
- Department of Physiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok, 10400, Thailand
| | - Narattaphol Charoenphandhu
- Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok, Thailand.
- Department of Physiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok, 10400, Thailand.
- Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand.
- The Academy of Science, The Royal Society of Thailand, Dusit, Bangkok, Thailand.
| |
Collapse
|
|
5
|
Romero-Calvo I, Ocón B, Gámez-Belmonte R, Hernández-Chirlaque C, de Jonge HR, Bijvelds MJ, Martínez-Augustin O, Sánchez de Medina F. Adenylyl cyclase 6 is involved in the hyposecretory status of experimental colitis. Pflugers Arch 2018; 470:1705-1717. [PMID: 30094477 DOI: 10.1007/s00424-018-2187-z] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2018] [Revised: 07/12/2018] [Accepted: 07/24/2018] [Indexed: 12/12/2022]
Abstract
One of the cardinal symptoms of intestinal inflammation is diarrhea. Acute intestinal inflammation is associated with inhibition of ion absorption and increased secretion, along with fluid leakage due to epithelial injury and changes in permeability. However, in the chronic situation, a downregulation of both absorptive and secretory transport has been reported. We investigated how experimental colitis reduces cAMP levels in intestinal epithelial cells through modulation of adenylyl cyclases (AC). Primary colonic epithelial cells obtained from rats with trinitrobenzenesulfonic acid colitis and non-colitic controls were analyzed for AC expression by RT-qPCR and Western blot, following a preliminary microarray analysis. AC6 and AC5 were found to be expressed in colonocytes, and downregulated by inflammation, with the former exhibiting considerably higher mRNA levels in both cases. To test the hypothesis that inflammatory cytokines may account for this effect, Caco 2 cells were treated with IL-1β, TNF-α, or IFN-γ. All three cytokines inhibited forskolin evoked short-circuit currents in Ussing chambers and lowered intracellular cAMP, but failed to alter AC6 mRNA levels. AC5/AC6 expression was however inhibited in mouse jejunal organoids treated with IFN-γ and TNF-α, but not IL-1β. Gene knockdown of AC6 resulted in a significant decrease of ion secretion in T84 cells. We conclude that the disturbances in ion secretion observed in rat TNBS colitis are associated with low intracellular levels of cAMP in the epithelium, which may be explained in part by the downregulation of AC5/AC6 expression by proinflammatory cytokines.
Collapse
Affiliation(s)
- Isabel Romero-Calvo
- Department of Biochemistry and Molecular Biology II, School of Pharmacy, Campus de Cartuja s/n, 18071, Granada, Spain
| | - Borja Ocón
- Department of Pharmacology, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, Granada, Spain
| | - Reyes Gámez-Belmonte
- Department of Pharmacology, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, Granada, Spain
| | - Cristina Hernández-Chirlaque
- Department of Biochemistry and Molecular Biology II, School of Pharmacy, Campus de Cartuja s/n, 18071, Granada, Spain
| | - Hugo R de Jonge
- Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
| | - Marcel J Bijvelds
- Department of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
| | - Olga Martínez-Augustin
- Department of Biochemistry and Molecular Biology II, School of Pharmacy, Campus de Cartuja s/n, 18071, Granada, Spain.
| | - Fermín Sánchez de Medina
- Department of Pharmacology, CIBERehd, School of Pharmacy, Instituto de Investigación Biosanitaria ibs.GRANADA, University of Granada, Granada, Spain
| |
Collapse
|
|
6
|
Pauwelyn V, Lefebvre RA. cAMP Catalyzing Phosphodiesterases Control Cholinergic Muscular Activity But Their Inhibition Does Not Enhance 5-HT 4 Receptor-Mediated Facilitation of Cholinergic Contractions in the Murine Gastrointestinal Tract. Front Pharmacol 2018; 9:171. [PMID: 29568269 PMCID: PMC5852062 DOI: 10.3389/fphar.2018.00171] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2017] [Accepted: 02/15/2018] [Indexed: 12/11/2022] Open
Abstract
Background: As the signal transduction of 5-HT4 receptors on cholinergic neurons innervating smooth muscle is controlled by phosphodiesterase (PDE) 4 in porcine stomach and colon, and human large intestine, the in vivo gastroprokinetic effects of a 5-HT4 receptor agonist might be enhanced by combination with a selective PDE4 inhibitor. The presence of 5-HT4 receptors on cholinergic neurons towards murine gastrointestinal circular muscle was recently shown. If the control of this receptor pathway by PDE4 is also present in mice, this might be a good model for in vivo testing of the combination therapy. Therefore this study investigates the role of cAMP catalyzing PDEs in smooth muscle cell activity and in the intraneuronal signal transduction of the 5-HT4 receptors in the gastrointestinal tract of C57Bl/6J mice. Methods: In circular smooth muscle strips from murine fundus, jejunum, and colon, submaximal cholinergic contractions were induced by either electrical field stimulation (EFS) or by carbachol (muscarinic receptor agonist). The influence of the PDE inhibitors IBMX (non-selective), vinpocetine (PDE1), EHNA (PDE2), cilostamide (PDE3), and rolipram (PDE4) was tested on these contractions and on the facilitating effect of a submaximal concentration of prucalopride (5-HT4 receptor agonist) on EFS-induced contractions. Results: In the three gastrointestinal regions, IBMX and cilostamide concentration-dependently decreased carbachol- as well as EFS-induced contractions. Some inhibitory effect was also observed with rolipram. In the fundus a non-significant trend for an enhancement of the facilitating effect of prucalopride on EFS-induced contractions was observed with IBMX, but none of the selective PDE inhibitors enhanced the facilitating effect of prucalopride in fundus, jejunum or colon. Conclusion: In analogy with the porcine gastrointestinal tract, in murine fundus, jejunum, and colon circular smooth muscle PDE3 is the main regulator of the cAMP turnover, with some contribution of PDE4. In contrast to the porcine gastrointestinal tract, the in vitro facilitation of electrically induced cholinergic contractions by 5-HT4 receptor stimulation could not be enhanced by specific PDE inhibition. The C57Bl/6J murine model is thus not suitable for in vivo testing of a 5-HT4 receptor agonist combined with a selective PDE4 inhibitor.
Collapse
Affiliation(s)
- Vicky Pauwelyn
- Department of Pharmacology, Heymans Institute, Ghent University, Ghent, Belgium
| | - Romain A Lefebvre
- Department of Pharmacology, Heymans Institute, Ghent University, Ghent, Belgium
| |
Collapse
|
|
7
|
Pauwelyn V, Ceelen W, Lefebvre RA. Synergy between 5-HT 4 receptor stimulation and phosphodiesterase 4 inhibition in facilitating acetylcholine release in human large intestinal circular muscle. Neurogastroenterol Motil 2018; 30. [PMID: 28799255 DOI: 10.1111/nmo.13162] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/10/2017] [Accepted: 06/20/2017] [Indexed: 02/08/2023]
Abstract
BACKGROUND Gastroprokinetic properties of 5-HT4 receptor agonists, such as prucalopride, are attributed to activation of 5-HT4 receptors on cholinergic nerves innervating smooth muscle in the gastrointestinal smooth muscle layer, increasing acetylcholine release and muscle contraction. In porcine stomach and colon, phosphodiesterase (PDE) 4 has been shown to control the signaling pathway of these 5-HT4 receptors. The aim of this study was to investigate the PDE-mediated control of these 5-HT4 receptors in human large intestine. METHODS Circular smooth muscle strips were prepared from human large intestine; after incubation with [³H]-choline, electrically induced tritium outflow was determined as a measure for acetylcholine release. The influence of PDE inhibition on the facilitating effect of prucalopride on electrically induced acetylcholine release was studied. KEY RESULTS The non-selective PDE inhibitor IBMX enhanced the facilitating effect of prucalopride on electrically induced acetylcholine release. The selective inhibitors vinpocetine (PDE1), EHNA (PDE2) and cilostamide (PDE3) did not influence, while rolipram and roflumilast (PDE4) enhanced the prucalopride-induced facilitation to the same extent as IBMX. CONCLUSIONS & INFERENCES In human large intestinal circular muscle, the intracellular pathway of 5-HT4 receptors facilitating cholinergic neurotransmission to large intestinal circular smooth muscle is controlled by PDE4. If the synergy between 5-HT4 receptor agonism and PDE4 inhibition is confirmed in a functional assay with electrically induced cholinergic contractions of human large intestinal circular smooth muscle strips, combination of a selective 5-HT4 receptor agonist with a selective PDE4 inhibitor might enhance the in vivo prokinetic effect of the 5-HT4 receptor agonist in the large intestine.
Collapse
Affiliation(s)
- V Pauwelyn
- Department of Pharmacology - Heymans Institute, Ghent University, Ghent, Belgium
| | - W Ceelen
- Department of Gastrointestinal Surgery, Ghent University Hospital, Ghent, Belgium
| | - R A Lefebvre
- Department of Pharmacology - Heymans Institute, Ghent University, Ghent, Belgium
| |
Collapse
|
|
8
|
Prolonged exposure to 1,25(OH) 2D 3 and high ionized calcium induces FGF-23 production in intestinal epithelium-like Caco-2 monolayer: A local negative feedback for preventing excessive calcium transport. Arch Biochem Biophys 2018; 640:10-16. [PMID: 29317227 DOI: 10.1016/j.abb.2017.12.022] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2017] [Revised: 11/19/2017] [Accepted: 12/07/2017] [Indexed: 12/13/2022]
Abstract
Overdose of oral calcium supplement and excessive intestinal calcium absorption can contribute pathophysiological conditions, e.g., nephrolithiasis, vascular calcification, dementia, and cardiovascular accident. Since our previous investigation has indicated that fibroblast growth factor (FGF)-23 could abolish the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-enhanced calcium absorption, we further hypothesized that FGF-23 produced locally in the enterocytes might be part of a local negative feedback loop to regulate calcium absorption. Herein, 1,25(OH)2D3 was found to enhance the transcellular calcium transport across the epithelium-like Caco-2 monolayer, and this stimulatory effect was diminished by preceding prolonged exposure to high-dose 1,25(OH)2D3 or high concentration of apical ionized calcium. Pretreatment with a neutralizing antibody for FGF-23 prevented this negative feedback regulation of calcium hyperabsorption induced by 1,25(OH)2D3. FGF-23 exposure completely abolished the 1,25(OH)2D3-enhanced calcium transport. Western blot analysis revealed that FGF-23 expression was upregulated in a dose-dependent manner by 1,25(OH)2D3 or apical calcium exposure. Finally, calcium-sensing receptor (CaSR) inhibitors were found to prevent the apical calcium-induced suppression of calcium transport. In conclusion, prolonged exposure to high apical calcium and calcium hyperabsorption were sensed by CaSR, which, in turn, increased FGF-23 expression to suppress calcium transport. This local negative feedback loop can help prevent unnecessary calcium uptake and its detrimental consequences.
Collapse
|
|
9
|
Raju SV, Rasmussen L, Sloane PA, Tang LP, Libby EF, Rowe SM. Roflumilast reverses CFTR-mediated ion transport dysfunction in cigarette smoke-exposed mice. Respir Res 2017; 18:173. [PMID: 28923049 PMCID: PMC5604356 DOI: 10.1186/s12931-017-0656-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2017] [Accepted: 09/12/2017] [Indexed: 01/12/2023] Open
Abstract
BACKGROUND Dysfunction in cystic fibrosis transmembrane conductance regulator (CFTR) can be elicited by cigarette smoke and is observed in patients with chronic bronchitis. We have previously demonstrated in human airway epithelial cell monolayers that roflumilast, a clinically approved phosphodiesterase 4 inhibitor that reduces the risk of exacerbations in chronic obstructive pulmonary disease patients with chronic bronchitis and a history of exacerbations, activates CFTR-dependent chloride secretion via a cAMP-mediated pathway, partially restores the detrimental effects of cigarette smoke on CFTR-mediated ion transport, and increases CFTR-dependent gastrointestinal fluid secretion in isolated murine intestine segments. Based on these findings, we hypothesized that roflumilast could improve CFTR-mediated chloride transport and induce secretory diarrhea in mice exhibiting cigarette smoke-induced CFTR dysfunction. METHODS A/J mice expressing wild type CFTR (+/+) were exposed to cigarette smoke or air with or without roflumilast and the effect of treatment on CFTR-dependent chloride transport was quantified using nasal potential difference (NPD) measurements in vivo and short-circuit current (Isc) analysis of trachea ex vivo. Stool specimen were collected and the wet/dry ratio measured to assess the effect of roflumilast on secretory diarrhea. RESULTS Acute roflumilast treatment increased CFTR-dependent chloride transport in both smoke- and air-exposed mice (smoke, -2.0 ± 0.4 mV, 131.3 ± 29.3 μA/cm2, P < 0.01 and air, 3.9 ± 0.8 mV, 147.7 ± 38.0 μA/cm2, P < 0.01 vs. vehicle -0.3 ± 0.7 mV, 10.4 ± 7.0 μA/cm2). Oral administration of roflumilast over five weeks completely reversed the deleterious effects of cigarette smoke on CFTR function in smoke-exposed animals, in which CFTR-dependent chloride transport was 64% that of air controls (roflumilast, -15.22 ± 2.7 mV vs. air, -14.45 ± 1.4 mV, P < 0.05). Smoke exposure increased the wet/dry ratio of stool specimen to a level beyond which roflumilast had little additional effect. CONCLUSIONS Roflumilast effectively rescues CFTR-mediated chloride transport in vivo, further implicating CFTR activation as a mechanism through which roflumilast benefits patients with bronchitis.
Collapse
Affiliation(s)
- S Vamsee Raju
- Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.,Department of Cell, Integrative, and Developmental Biology, University of Alabama at Birmingham, Birmingham, AL, USA.,Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Lawrence Rasmussen
- Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.,Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Peter A Sloane
- Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Li Ping Tang
- Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.,Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Emily Falk Libby
- Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Steven M Rowe
- Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA. .,Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL, USA. .,Department of Cell, Integrative, and Developmental Biology, University of Alabama at Birmingham, Birmingham, AL, USA. .,UAB Lung Health Center, University of Alabama at Birmingham, Birmingham, AL, USA. .,Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA. .,MCLM 702, 1918 University Blvd, Birmingham, AL, 35294-0006, USA.
| |
Collapse
|
|
10
|
Kangawa Y, Yoshida T, Abe H, Seto Y, Miyashita T, Nakamura M, Kihara T, Hayashi SM, Shibutani M. Anti-inflammatory effects of the selective phosphodiesterase 3 inhibitor, cilostazol, and antioxidants, enzymatically-modified isoquercitrin and α-lipoic acid, reduce dextran sulphate sodium-induced colorectal mucosal injury in mice. ACTA ACUST UNITED AC 2017; 69:179-186. [PMID: 28089463 DOI: 10.1016/j.etp.2016.12.004] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2016] [Revised: 11/25/2016] [Accepted: 12/06/2016] [Indexed: 12/13/2022]
|
|
11
|
D'Andrea MR, Qiu Y, Haynes-Johnson D, Bhattacharjee S, Kraft P, Lundeen S. Expression of PDE11A in Normal and Malignant Human Tissues. J Histochem Cytochem 2016; 53:895-903. [PMID: 15995148 DOI: 10.1369/jhc.5a6625.2005] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Cyclic nucleotide phosphodiesterase 11A (PDE11A) is the newest member in the PDE family. Although the tissue distribution of PDE11A mRNA has been shown, its protein expression pattern has not been well studied. The goal of this report is to investigate the distribution of PDE11A proteins in a wide range of normal and malignant human tissues. We utilized a polyclonal antibody that recognized all four PDE11A isoforms. Its specificity was demonstrated by Western blot analysis on a recombinant human PDE11A protein and native PDE11A proteins in various human tissues. Immunohistochemistry showed that PDE11A is widely expressed. Various degrees of immunoreactivity were observed in the epithelial cells, endothelial cells, and smooth muscle cells of all tissues examined. The highest expression was in the epithelial, endothelial, and smooth muscle cells of the prostate, Leydig, and spermatogenic cells of the testis, the tubule epithelial cells in the kidney, the epithelial and endothelial cells in the adrenal, the epithelial cells and macrophages in the colon, and the epidermis in the skin. Furthermore, PDE11A expression was also detected in several human carcinomas. Our results suggest that PDE11A might be involved in multiple physiological processes in various organs via its ability to modulate intracellular cAMP and cGMP levels.
Collapse
Affiliation(s)
- Michael R D'Andrea
- Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, LLC, 1000 Route 202, Raritan, NJ 08869, USA
| | | | | | | | | | | |
Collapse
|
|
12
|
Tang L, Cheng CY, Sun X, Pedicone AJ, Mohamadzadeh M, Cheng SX. The Extracellular Calcium-Sensing Receptor in the Intestine: Evidence for Regulation of Colonic Absorption, Secretion, Motility, and Immunity. Front Physiol 2016; 7:245. [PMID: 27458380 PMCID: PMC4914593 DOI: 10.3389/fphys.2016.00245] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2016] [Accepted: 06/03/2016] [Indexed: 12/14/2022] Open
Abstract
Different from other epithelia, the intestinal epithelium has the complex task of providing a barrier impeding the entry of toxins, food antigens, and microbes, while at the same time allowing for the transfer of nutrients, electrolytes, water, and microbial metabolites. These molecules/organisms are transported either transcellularly, crossing the apical and basolateral membranes of enterocytes, or paracellularly, passing through the space between enterocytes. Accordingly, the intestinal epithelium can affect energy metabolism, fluid balance, as well as immune response and tolerance. To help accomplish these complex tasks, the intestinal epithelium has evolved many sensing receptor mechanisms. Yet, their roles and functions are only now beginning to be elucidated. This article explores one such sensing receptor mechanism, carried out by the extracellular calcium-sensing receptor (CaSR). In addition to its established function as a nutrient sensor, coordinating food digestion, nutrient absorption, and regulating energy metabolism, we present evidence for the emerging role of CaSR in the control of intestinal fluid homeostasis and immune balance. An additional role in the modulation of the enteric nerve activity and motility is also discussed. Clearly, CaSR has profound effects on many aspects of intestinal function. Nevertheless, more work is needed to fully understand all functions of CaSR in the intestine, including detailed mechanisms of action and specific pathways involved. Considering the essential roles CaSR plays in gastrointestinal physiology and immunology, research may lead to a translational opportunity for the development of novel therapies that are based on CaSR's unique property of using simple nutrients such as calcium, polyamines, and certain amino acids/oligopeptides as activators. It is possible that, through targeting of intestinal CaSR with a combination of specific nutrients, oral solutions that are both inexpensive and practical may be developed to help in conditioning the gut microenvironment and in maintaining digestive health.
Collapse
Affiliation(s)
- Lieqi Tang
- Department of Pediatrics, Gastroenterology, Hepatology, and Nutrition, University of Florida Gainesville, FL, USA
| | - Catherine Y Cheng
- Department of Pediatrics, Gastroenterology, Hepatology, and Nutrition, University of Florida Gainesville, FL, USA
| | - Xiangrong Sun
- Department of Pediatrics, Gastroenterology, Hepatology, and Nutrition, University of Florida Gainesville, FL, USA
| | - Alexandra J Pedicone
- Department of Pediatrics, Gastroenterology, Hepatology, and Nutrition, University of Florida Gainesville, FL, USA
| | - Mansour Mohamadzadeh
- Department of Medicine, Center for Inflammation and Mucosal Immunology, University of Florida Gainesville, FL, USA
| | - Sam X Cheng
- Department of Pediatrics, Gastroenterology, Hepatology, and Nutrition, University of Florida Gainesville, FL, USA
| |
Collapse
|
|
13
|
Moon C, Zhang W, Sundaram N, Yarlagadda S, Reddy VS, Arora K, Helmrath MA, Naren AP. Drug-induced secretory diarrhea: A role for CFTR. Pharmacol Res 2015; 102:107-112. [PMID: 26429773 DOI: 10.1016/j.phrs.2015.08.024] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/31/2015] [Accepted: 08/31/2015] [Indexed: 12/29/2022]
Abstract
Many medications induce diarrhea as a side effect, which can be a major obstacle to therapeutic efficacy and also a life-threatening condition. Secretory diarrhea can be caused by excessive fluid secretion in the intestine under pathological conditions. The cAMP/cGMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) is the primary chloride channel at the apical membrane of intestinal epithelial cells and plays a major role in intestinal fluid secretion and homeostasis. CFTR forms macromolecular complexes at discreet microdomains at the plasma membrane, and its chloride channel function is regulated spatiotemporally through protein-protein interactions and cAMP/cGMP-mediated signaling. Drugs that perturb CFTR-containing macromolecular complexes in the intestinal epithelium and upregulate intracellular cAMP and/or cGMP levels can hyperactivate the CFTR channel, causing excessive fluid secretion and secretory diarrhea. Inhibition of CFTR chloride-channel activity may represent a novel approach to the management of drug-induced secretory diarrhea.
Collapse
Affiliation(s)
- Changsuk Moon
- Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Weiqiang Zhang
- Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA.,Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN 38163, USA
| | - Nambirajan Sundaram
- Department of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Sunitha Yarlagadda
- Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Vadde Sudhakar Reddy
- Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Kavisha Arora
- Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Michael A Helmrath
- Department of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.,Department of General Surgery, University of Cincinnati, Cincinnati, OH 45229, USA
| | - Anjaparavanda P Naren
- Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| |
Collapse
|
|
14
|
Simultaneous exposure to Escherichia coli heat-labile and heat-stable enterotoxins increases fluid secretion and alters cyclic nucleotide and cytokine production by intestinal epithelial cells. Infect Immun 2014; 82:5308-16. [PMID: 25287923 DOI: 10.1128/iai.02496-14] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Enterotoxigenic Escherichia coli (ETEC) is a significant cause of diarrheal disease and death, especially in children in developing countries. ETEC causes disease by colonizing the small intestine and producing heat-labile toxin (LT), heat-stable toxin (ST), or both LT and ST (LT+ST). The majority of ETEC strains produce both ST and LT. Despite the prevalence of LT+ST-producing organisms, few studies have examined the physiologic or immunologic consequences of simultaneous exposure to these two potent enterotoxins. In the current report, we demonstrate that when LT and ST are both present, they increase water movement into the intestinal lumen over and above the levels observed with either toxin alone. As expected, cultured intestinal epithelial cells increased their expression of intracellular cyclic GMP (cGMP) when treated with ST and their expression of intracellular cyclic AMP (cAMP) when treated with LT. When both toxins were present, cGMP levels but not cAMP levels were synergistically elevated compared with the levels of expression caused by the corresponding single-toxin treatment. Our data also demonstrate that the levels of inflammatory cytokines produced by intestinal epithelial cells in response to LT are significantly reduced in animals exposed to both enterotoxins. These findings suggest that there may be complex differences between the epithelial cell intoxication and, potentially, secretory outcomes induced by ETEC strains expressing LT+ST compared with strains that express LT or ST only. Our results also reveal a novel mechanism wherein ST production may reduce the hosts' ability to mount an effective innate or adaptive immune response to infecting organisms.
Collapse
|
|
15
|
Lambert JA, Raju SV, Tang LP, McNicholas CM, Li Y, Courville CA, Farris RF, Coricor GE, Smoot LH, Mazur MM, Dransfield MT, Bolger GB, Rowe SM. Cystic fibrosis transmembrane conductance regulator activation by roflumilast contributes to therapeutic benefit in chronic bronchitis. Am J Respir Cell Mol Biol 2014; 50:549-58. [PMID: 24106801 DOI: 10.1165/rcmb.2013-0228oc] [Citation(s) in RCA: 67] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Cigarette smoking causes acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction and is associated with delayed mucociliary clearance and chronic bronchitis. Roflumilast is a clinically approved phosphodiesterase 4 inhibitor that improves lung function in patients with chronic bronchitis. We hypothesized that its therapeutic benefit was related in part to activation of CFTR. Primary human bronchial epithelial (HBE) cells, Calu-3, and T84 monolayers were exposed to whole cigarette smoke (WCS) or air with or without roflumilast treatment. CFTR-dependent ion transport was measured in modified Ussing chambers. Airway surface liquid (ASL) was determined by confocal microscopy. Intestinal fluid secretion of ligated murine intestine was monitored ex vivo. Roflumilast activated CFTR-dependent anion transport in normal HBE cells with a half maximal effective concentration of 2.9 nM. Roflumilast partially restored CFTR activity in WCS-exposed HBE cells (5.3 ± 1.1 μA/cm(2) vs. 1.2 ± 0.2 μA/cm(2) [control]; P < 0.05) and was additive with ivacaftor, a specific CFTR potentiator approved for the treatment of CF. Roflumilast improved the depleted ASL depth of HBE monolayers exposed to WCS (9.0 ± 3.1 μm vs. 5.6 ± 2.0 μm [control]; P < 0.05), achieving 79% of that observed in air controls. CFTR activation by roflumilast also induced CFTR-dependent fluid secretion in murine intestine, increasing the wet:dry ratio and the diameter of ligated murine segments. Roflumilast activates CFTR-mediated anion transport in airway and intestinal epithelia via a cyclic adenosine monophosphate-dependent pathway and partially reverses the deleterious effects of WCS, resulting in augmented ASL depth. Roflumilast may benefit patients with chronic obstructive pulmonary disease with chronic bronchitis by activating CFTR, which may also underlie noninfectious diarrhea caused by roflumilast.
Collapse
|
|
16
|
Lima AAM, Fonteles MC. From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones. ACTA ACUST UNITED AC 2014; 47:179-91. [PMID: 24652326 PMCID: PMC3982939 DOI: 10.1590/1414-431x20133063] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2013] [Accepted: 10/15/2013] [Indexed: 12/16/2022]
Abstract
The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia.
Collapse
Affiliation(s)
- A A M Lima
- Unidade de Pesquisas Clinicas, Instituto de Biomedicina, Departamento de Fisiologia e Farmacologia, Escola de Medicina, Universidade Federal do Ceara, Fortaleza, CE, Brasil
| | - M C Fonteles
- Unidade de Pesquisas Clinicas, Instituto de Biomedicina, Departamento de Fisiologia e Farmacologia, Escola de Medicina, Universidade Federal do Ceara, Fortaleza, CE, Brasil
| |
Collapse
|
|
17
|
Pondugula SR, Kampalli SB, Wu T, De Lisle RC, Raveendran NN, Harbidge DG, Marcus DC. cAMP-stimulated Cl- secretion is increased by glucocorticoids and inhibited by bumetanide in semicircular canal duct epithelium. BMC PHYSIOLOGY 2013; 13:6. [PMID: 23537040 PMCID: PMC3622586 DOI: 10.1186/1472-6793-13-6] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/14/2012] [Accepted: 03/11/2013] [Indexed: 12/13/2022]
Abstract
Background The vestibular system controls the ion composition of its luminal fluid through several epithelial cell transport mechanisms under hormonal regulation. The semicircular canal duct (SCCD) epithelium has been shown to secrete Cl- under β2-adrenergic stimulation. In the current study, we sought to determine the ion transporters involved in Cl- secretion and whether secretion is regulated by PKA and glucocorticoids. Results Short circuit current (Isc) from rat SCCD epithelia demonstrated stimulation by forskolin (EC50: 0.8 μM), 8-Br-cAMP (EC50: 180 μM), 8-pCPT-cAMP (100 μM), IBMX (250 μM), and RO-20-1724 (100 μM). The PKA activator N6-BNZ-cAMP (0.1, 0.3 & 1 mM) also stimulated Isc. Partial inhibition of stimulated Isc individually by bumetanide (10 & 50 μM), and [(dihydroindenyl)oxy]alkanoic acid (DIOA, 100 μM) were additive and complete. Stimulated Isc was also partially inhibited by CFTRinh-172 (5 & 30 μM), flufenamic acid (5 μM) and diphenylamine-2,2′-dicarboxylic acid (DPC; 1 mM). Native canals of CFTR+/− mice showed a stimulation of Isc from isoproterenol and forskolin+IBMX but not in the presence of both bumetanide and DIOA, while canals from CFTR−/− mice had no responses. Nonetheless, CFTR−/− mice showed no difference from CFTR+/− mice in their ability to balance (rota-rod). Stimulated Isc was greater after chronic incubation (24 hr) with the glucocorticoids dexamethasone (0.1 & 0.3 μM), prednisolone (0.3, 1 & 3 μM), hydrocortisone (0.01, 0.1 & 1 μM), and corticosterone (0.1 & 1 μM) and mineralocorticoid aldosterone (1 μM). Steroid action was blocked by mifepristone but not by spironolactone, indicating all the steroids activated the glucocorticoid, but not mineralocorticoid, receptor. Expression of transcripts for CFTR; for KCC1, KCC3a, KCC3b and KCC4, but not KCC2; for NKCC1 but not NKCC2 and for WNK1 but only very low WNK4 was determined. Conclusions These results are consistent with a model of Cl- secretion whereby Cl- is taken up across the basolateral membrane by a Na+-K+-2Cl- cotransporter (NKCC) and potentially another transporter, is secreted across the apical membrane via a Cl- channel, likely CFTR, and demonstrate the regulation of Cl- secretion by protein kinase A and glucocorticoids.
Collapse
|
|
18
|
Noel S, Dhooghe B, Leal T. PDE5 Inhibitors as Potential Tools in the Treatment of Cystic Fibrosis. Front Pharmacol 2012; 3:167. [PMID: 23024633 PMCID: PMC3444771 DOI: 10.3389/fphar.2012.00167] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2012] [Accepted: 08/24/2012] [Indexed: 12/31/2022] Open
Abstract
Despite great advances in the understanding of the genetics and pathophysiology of cystic fibrosis (CF), there is still no cure for the disease. Using phosphodiesterase type 5 (PDE5) inhibitors, we and others have provided evidence of rescued F508del-CFTR trafficking and corrected deficient chloride transport activity. Studies using PDE5 inhibitors in mice homozygous for the clinically relevant F508del mutation have been conducted with the aim of restoring F508del-CFTR protein function. We demonstrated, by measuring transepithelial nasal potential difference in F508del mice following intraperitoneal injection of sildenafil, vardenafil, or taladafil at clinical doses are able to restore the decreased CFTR-dependent chloride transport across the nasal mucosa. Moreover, vardenafil, but not sildenafil, stimulates chloride transport through the normal CFTR protein. We developed a specific nebulizer setup for mice, with which we demonstrated, through a single inhalation of PDE5 inhibitors, local activation of CFTR protein in CF. Significant potential advantages of inhalation drug therapy over oral or intravenous routes include rapid onset of pharmacological action, reduced systemic secondary effects, and reduced effective drug doses compared to the drug delivered orally; this underlines the relevance and impact of our work for translational science. More recently, we analyzed the bronchoalveolar lavage of CF and wild-type mice for cell infiltrates and expression of pro-inflammatory cytokines and chemokines; we found that the CFTR activating effect of vardenafil, selected as a representative long-lasting PDE5 inhibitor, breaks the vicious circle of lung inflammation which plays a major role in morbi-mortality in CF. Our data highlight the potential use of PDE5 inhibitors in CF. Therapeutic approaches using clinically approved PDE5 inhibitors to address F508del-CFTR defects could speed up the development of new therapies for CF.
Collapse
Affiliation(s)
- Sabrina Noel
- Louvain Centre for Toxicology and Applied Pharmacology, Institut de Recherche Expérimentale et Clinique, Secteur des Sciences de la Santé, Université Catholique de Louvain Brussels, Belgium
| | | | | |
Collapse
|
|
19
|
Kleberg K, Jensen GM, Christensen DP, Lundh M, Grunnet LG, Knuhtsen S, Poulsen SS, Hansen MB, Bindslev N. Transporter function and cyclic AMP turnover in normal colonic mucosa from patients with and without colorectal neoplasia. BMC Gastroenterol 2012; 12:78. [PMID: 22734885 PMCID: PMC3457850 DOI: 10.1186/1471-230x-12-78] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/23/2012] [Accepted: 06/19/2012] [Indexed: 01/18/2023] Open
Abstract
BACKGROUND The pathogenesis of colorectal neoplasia is still unresolved but has been associated with alterations in epithelial clearance of xenobiotics and metabolic waste products. The aim of this study was to functionally characterize the transport of cyclic nucleotides in colonic biopsies from patients with and without colorectal neoplasia. METHODS Cyclic nucleotides were used as model substrates shared by some OATP- and ABC-transporters, which in part are responsible for clearance of metabolites and xenobiotics from the colonic epithelium. On colonic biopsies from patients with and without colorectal neoplasia, molecular transport was electrophysiologically registered in Ussing-chamber set-ups, mRNA level of selected transporters was quantified by rt-PCR, and subcellular location of transporters was determined by immunohistochemistry. RESULTS Of four cyclic nucleotides, dibuturyl-cAMP induced the largest short circuit current in both patient groups. The induced short circuit current was significantly lower in neoplasia-patients (p = 0.024). The observed altered transport of dibuturyl-cAMP in neoplasia-patients could not be directly translated to an observed increased mRNA expression of OATP4A1 and OATP2B1 in neoplasia patients. All other examined transporters were expressed to similar extents in both patient groups. CONCLUSIONS OATP1C1, OATP4A1, OATP4C1 seem to be involved in the excretory system of human colon. ABCC4 is likely to be involved from an endoplasmic-Golgi complex and basolateral location in goblet cells. ABCC5 might be directly involved in the turnover of intracellular cAMP at the basolateral membrane of columnar epithelial cells, while OATP2B1 is indirectly related to the excretory system. Colorectal neoplasia is associated with lower transport or sensitivity to cyclic nucleotides and increased expression of OATP2B1 and OATP4A1 transporters, known to transport PGE(2).
Collapse
Affiliation(s)
- Karen Kleberg
- Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
| | | | | | | | | | | | | | | | | |
Collapse
|
|
20
|
Toriano R, Ozu M, Politi MT, Dorr RA, Curto MA, Capurro C. Uroguanylin Regulates Net Fluid Secretion via the NHE2 Isoform of the Na +/H + Exchanger in an Intestinal Cellular Model. Cell Physiol Biochem 2011; 28:733-42. [DOI: 10.1159/000335767] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/26/2011] [Indexed: 12/31/2022] Open
|
|
21
|
Takeuchi K, Kita K, Hayashi S, Aihara E. Regulatory mechanism of duodenal bicarbonate secretion: Roles of endogenous prostaglandins and nitric oxide. Pharmacol Ther 2010; 130:59-70. [PMID: 21185865 DOI: 10.1016/j.pharmthera.2010.12.006] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2010] [Accepted: 12/13/2010] [Indexed: 11/27/2022]
Abstract
The secretion of HCO(3)(-) in the duodenum is increased by exogenous prostaglandin (PG) E(2) and mucosal acidification, the latter being accompanied by a rise in mucosal PGE(2) content and nitric oxide (NO) release. The stimulatory effect of PGE(2) is mediated intracellularly by both Ca(2+) and 3',5'-adenosine cyclic adenosine monophosphate (cAMP), and this action is inhibited by EP3 and EP4 antagonists. The secretion is also increased by NOR3 (NO donor), and this response is mimicked by dibutyryl 3',5'-cyclic guanosine monophosphate (dbcGMP) and attenuated by indomethacin. Mucosal acidification stimulates HCO(3)(-) secretion with concomitant increases in mucosal PGE(2) production and NO release. The effects on HCO(3)(-) secretion and PGE(2) production are inhibited by indomethacin [nonselective cyclooxygenase (COX) inhibitor] and SC-560 (selective COX-1 inhibitor) but not rofecoxib (selective COX-2 inhibitor). N(G)-nitro-l-arginine methyl ester [l-NAME: nonselective NO synthase (NOS) inhibitor], but not aminoguanidine [selective inducible NOS inhibitor], attenuates the acid-induced HCO(3)(-) secretion and NO release in an l-arginine-sensitive manner. In addition, the response to PGE(2) is potentiated by vinpocetine [phosphodiesterase (PDE) 1 inhibitor] and cilostamide (PDE3 inhibitor), while the response to NOR3 is increased by vinpocetine. We conclude that endogenous PGs and NO are both involved in the local regulation of acid-induced duodenal HCO(3)(-) secretion; COX-1 and constitutive NOS are key enzymes responsible for the production of PGs and NO, respectively; NO stimulates HCO(3)(-) secretion by increasing PG production; PGE(2) stimulates HCO(3)(-) secretion via activation of EP3/EP4 receptors; and both PDE1 and PDE3 are involved in the regulation of duodenal HCO(3)(-) secretion.
Collapse
Affiliation(s)
- Koji Takeuchi
- Division of Pathological Sciences, Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto 607-8414, Japan.
| | | | | | | |
Collapse
|
|
22
|
Abstract
Chlorine is considered a chemical threat agent to which humans may be exposed as a result of accidental or intentional release. Chlorine is highly reactive, and inhalation of the gas causes cellular damage to the respiratory tract, inflammation, pulmonary edema, and airway hyperreactivity. Drugs that increase intracellular levels of the signaling molecule cyclic AMP (cAMP) may be useful for treatment of acute lung injury through effects on alveolar fluid clearance, inflammation, and airway reactivity. This article describes mechanisms by which cAMP regulates cellular processes affecting lung injury and discusses the basis for investigating drugs that increase cAMP levels as potential treatments for chlorine-induced lung injury. The effects of beta(2)-adrenergic agonists, which stimulate cAMP synthesis, and phosphodiesterase inhibitors, which inhibit cAMP degradation, on acute lung injury are reviewed, and the relative advantages of these approaches are compared.
Collapse
|
|
23
|
Penmatsa H, Zhang W, Yarlagadda S, Li C, Conoley VG, Yue J, Bahouth SW, Buddington RK, Zhang G, Nelson DJ, Sonecha MD, Manganiello V, Wine JJ, Naren AP. Compartmentalized cyclic adenosine 3',5'-monophosphate at the plasma membrane clusters PDE3A and cystic fibrosis transmembrane conductance regulator into microdomains. Mol Biol Cell 2010; 21:1097-1110. [PMID: 20089840 PMCID: PMC2836961 DOI: 10.1091/mbc.e09-08-0655] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2009] [Revised: 01/06/2010] [Accepted: 01/11/2010] [Indexed: 12/21/2022] Open
Abstract
Formation of multiple-protein macromolecular complexes at specialized subcellular microdomains increases the specificity and efficiency of signaling in cells. In this study, we demonstrate that phosphodiesterase type 3A (PDE3A) physically and functionally interacts with cystic fibrosis transmembrane conductance regulator (CFTR) channel. PDE3A inhibition generates compartmentalized cyclic adenosine 3',5'-monophosphate (cAMP), which further clusters PDE3A and CFTR into microdomains at the plasma membrane and potentiates CFTR channel function. Actin skeleton disruption reduces PDE3A-CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A-containing macromolecular complex. Consequently, compartmentalized cAMP signaling is lost. PDE3A inhibition no longer activates CFTR channel function in a compartmentalized manner. The physiological relevance of PDE3A-CFTR interaction was investigated using pig trachea submucosal gland secretion model. Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion.
Collapse
Affiliation(s)
- Himabindu Penmatsa
- *Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163
| | - Weiqiang Zhang
- *Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163
| | - Sunitha Yarlagadda
- *Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163
| | - Chunying Li
- Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI 48201
| | - Veronica G. Conoley
- *Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163
| | - Junming Yue
- *Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163
| | - Suleiman W. Bahouth
- Department of Pharmacology, University of Tennessee Health Science Center, Memphis, TN 38163
| | - Randal K. Buddington
- Department of Health and Sports Sciences, University of Memphis, Memphis, TN 38152
| | - Guangping Zhang
- Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, IL 60637
| | - Deborah J. Nelson
- Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, IL 60637
| | - Monal D. Sonecha
- Cystic Fibrosis Research Laboratory, Stanford University, Stanford, CA 94305; and
| | - Vincent Manganiello
- Division of Intramural Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892
| | - Jeffrey J. Wine
- Cystic Fibrosis Research Laboratory, Stanford University, Stanford, CA 94305; and
| | - Anjaparavanda P. Naren
- *Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163
| |
Collapse
|
|
24
|
Basu N, Arshad N, Visweswariah SS. Receptor guanylyl cyclase C (GC-C): regulation and signal transduction. Mol Cell Biochem 2009; 334:67-80. [PMID: 19960363 DOI: 10.1007/s11010-009-0324-x] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2009] [Accepted: 11/04/2009] [Indexed: 12/27/2022]
Abstract
Receptor guanylyl cyclase C (GC-C) is the target for the gastrointestinal hormones, guanylin, and uroguanylin as well as the bacterial heat-stable enterotoxins. The major site of expression of GC-C is in the gastrointestinal tract, although this receptor and its ligands play a role in ion secretion in other tissues as well. GC-C shares the domain organization seen in other members of the family of receptor guanylyl cyclases, though subtle differences highlight some of the unique features of GC-C. Gene knock outs in mice for GC-C or its ligands do not lead to embryonic lethality, but modulate responses of these mice to stable toxin peptides, dietary intake of salts, and development and differentiation of intestinal cells. It is clear that there is much to learn in future about the role of this evolutionarily conserved receptor, and its properties in intestinal and extra-intestinal tissues.
Collapse
Affiliation(s)
- Nirmalya Basu
- Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India
| | | | | |
Collapse
|
|
25
|
Chao PC, Hamilton KL. Genistein stimulates electrogenic Cl− secretion via phosphodiesterase modulation in the mouse jejunum. Am J Physiol Cell Physiol 2009; 297:C688-98. [DOI: 10.1152/ajpcell.00152.2009] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Previously, we demonstrated that genistein stimulated Cl− secretion in the mouse jejunum (Baker MJ and Hamilton KL, Am J Physiol Cell Physiol 287: C1636–C1645, 2004); however, the mode of action of genistein still remains unclear. Here, we examined the activation of Cl− secretion by the modulation of phosphodiesterases (PDEs) by genistein (75 μM) in the mouse jejunum with the Ussing short-circuit current ( Isc) technique. Drugs tested included theophylline (10 mM), a nonspecific PDE inhibitor; 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MM-IBMX; 100 μM), erythro-9-(2-hydroxyl-3-nonyl)-adenine (EHNA; 40 μM), milrinone (100 μM), and rolipram (40 and 100 μM), which are specific inhibitors of PDE1–PDE4, respectively. Theophylline stimulated a bumetanide-sensitive Isc, indicative of Cl− secretion, and abolished genistein's stimulatory action on Isc. Neither 8-MM-IBMX nor EHNA altered the basal Isc nor did these PDE inhibitors affect the stimulatory action of genistein on the Isc of the mouse jejunum. Rolipram had no effect on basal Isc, but it reduced the genistein-stimulated Isc compared with time-matched control tissues. Milrinone stimulated a concentration-dependent increase in Isc. Bumetanide (10 μM) inhibited 60 ± 4% of milrinone-induced Isc. Pretreating tissues with milrinone prevented genistein from stimulating Isc, and pretreatment with genistein reduced the effect of milrinone on Isc. H89 (50 μM), a PKA inhibitor, reduced the milrinone-stimulated Isc. Likewise, H89 reduced the genistein-stimulated Isc. Here, we demonstrate, for the first time, that genistein activates Cl− secretion of the mouse jejunum via inhibition of a PDE3-dependent pathway.
Collapse
Affiliation(s)
- Pin-Chun Chao
- Department of Physiology, School of Medical Sciences, University of Otago, Dunedin, New Zealand
| | - Kirk L. Hamilton
- Department of Physiology, School of Medical Sciences, University of Otago, Dunedin, New Zealand
| |
Collapse
|
|
26
|
Johnson M, Allen L, Dobbs L. Characteristics of Cl- uptake in rat alveolar type I cells. Am J Physiol Lung Cell Mol Physiol 2009; 297:L816-27. [PMID: 19684200 DOI: 10.1152/ajplung.90466.2008] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Although Cl- transport in fetal lung is important for fluid secretion and normal lung development, the role of Cl- transport in adult lung is not well understood. In physiological studies, the cystic fibrosis transmembrane regulator (CFTR) plays a role in fluid absorption in the distal air spaces of adult lung, and alveolar type II cells cultured for 5 days have the capacity to transport Cl-. Although both alveolar type I and type II cells express CFTR, it has previously not been known whether type I cells transport Cl-. We studied Cl- uptake in isolated type I cells directly, using either radioisotopic tracers or halide-sensitive fluorescent indicators. By both methods, type I cells take up Cl-. In the presence of beta-adrenergic agonist stimulation, Cl- uptake can be inhibited by CFTR antagonists. Type I cells express both the Cl-/HCO3- anion exchanger AE2 and the voltage-gated Cl- channels CLC5 and CLC2. Inhibitors of AE2 also block Cl- uptake in type I cells. Together, these results demonstrate that type I cells are capable of Cl- uptake and suggest that the effects seen in whole lung studies establishing the importance of Cl- movement in alveolar fluid clearance may be, in part, the result of Cl- transport across type I cells.
Collapse
Affiliation(s)
- Meshell Johnson
- Department of Medicine, University of California, San Francisco, 3333 California St., Suite 150, Box 1245, San Francisco, CA 94118, USA.
| | | | | |
Collapse
|
|
27
|
Gee HY, Kim YW, Jo MJ, Namkung W, Kim JY, Park HW, Kim KS, Kim H, Baba A, Yang J, Kim E, Kim KH, Lee MG. Synaptic scaffolding molecule binds to and regulates vasoactive intestinal polypeptide type-1 receptor in epithelial cells. Gastroenterology 2009; 137:607-17, 617.e1-4. [PMID: 19642226 DOI: 10.1053/j.gastro.2009.01.065] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/02/2022]
Abstract
BACKGROUND & AIMS Vasoactive intestinal polypeptide (VIP) is a principal regulator of fluid and electrolyte secretion in the gastrointestinal system. The VIP type-1 receptor (VPAC1), a class II G-protein-coupled receptor, contains a putative C-terminal PDZ-binding motif. A yeast 2-hybrid screen indicated that the C-terminus of VPAC1 bound to the PDZ domain of synaptic scaffolding molecule (S-SCAM, also known as membrane-associated guanylate kinase inverted-2 [MAGI-2]). We analyzed the association between S-SCAM and VPAC1. METHODS The biochemical properties and physiologic significance of the interaction between VPAC1 and S-SCAM were examined in heterologous expression systems, T84 colonic epithelial cells, and human pancreas and colon tissues using an integrated molecular and physiologic approach. RESULTS The physical interaction between VPAC1 and S-SCAM was confirmed by immunoprecipitation in HEK 293 mammalian cells and human pancreatic and colonic tissues. Immunocytochemical analysis indicated that S-SCAM recruited VPAC1 to the junctional area near the apical end of the lateral membrane in T84 cells. Several lines of evidence revealed that S-SCAM inhibits VPAC1 activation. Overexpression of S-SCAM inhibited VPAC1-mediated cAMP production and agonist-induced VPAC1 internalization in HEK 293 and HeLa cells. In addition, S-SCAM decreased the VPAC1-mediated current through the cystic fibrosis transmembrane conductance regulator in Xenopus oocytes, especially at low concentrations of VIP. Importantly, loss of S-SCAM increased VIP-induced short-circuit currents in T84 monolayers, which endogenously express VPAC1 and S-SCAM. CONCLUSIONS S-SCAM/MAGI-2 interacts with and regulates VPAC1 intracellular localization in epithelial cells and inhibits VPAC1 agonist-induced activation and internalization.
Collapse
Affiliation(s)
- Heon Yung Gee
- Department of Pharmacology and Brain Korea 21 Project for Medical Science, Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, Korea
| | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
28
|
Abstract
Different inhibitors of the Ca(2+)/calmodulin-stimulated phosphodiesterase 1 family have been described and used for the examination of phosphodiesterase 1 in cellular, organ or animal models. However, the inhibitors described differ in potency and selectivity for the different phosphodiesterase family enzymes, and in part exhibit additional pharmacodynamic actions. In this study, we demonstrate that phosphodiesterase 1C is expressed in the human glioblastoma cell line A172 with regard to mRNA, protein and activity level, and that lower activities of phosphodiesterase 2, phosphodiesterase 3, phosphodiesterase 4 and phosphodiesterase 5 are also present. The identity of the phosphodiesterase 1C activity detected was verified by downregulation of the mRNA and protein through human phosphodiesterase 1C specific small interfering RNA. In addition, the measured K(m) values (cAMP, 1.7 microm; cGMP, 1.3 microm) are characteristic of phosphodiesterase 1C. We demonstrate that treatment with the Ca(2+) ionophore ionomycin increases intracellular Ca(2+) in a concentration-dependent way without affecting cell viability. Under conditions of enhanced intracellular Ca(2+) concentration, a rapid increase in cAMP levels caused by the adenylyl cyclase activator forskolin was abolished, indicating the involvement of Ca(2+)-activated phosphodiesterase 1C. The reduction of forskolin-stimulated cAMP levels was reversed by phosphodiesterase 1 inhibitors in a concentration-dependent way. Using this cellular system, we compared the cellular potency of published phosphodiesterase 1 inhibitors, including 8-methoxymethyl-3-isobutyl-1-methylxanthine, vinpocetine, SCH51866, and two established phosphodiesterase 1 inhibitors developed by Schering-Plough (named compounds 31 and 30). We demonstrate that up to 10 microm 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine had no effect on the reduction of forskolin-stimulated cAMP levels by ionomycin, whereas the more selective and up to 10 000 times more potent phosphodiesterase 1 inhibitors SCH51866, compound 31 and compound 30 inhibited the ionomycin-induced decline of forskolin-induced cAMP at nanomolar concentrations. Thus, our data indicate that SCH51866 and compounds 31 and 30 are effective phosphodiesterase 1 inhibitors in a cellular context, in contrast to the weakly selective and low-potency phosphodiesterase inhibitors 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine. A172 cells therefore represent a suitable system in which to study the cellular effect of phosphodiesterase 1 inhibitors. 8-Methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine seem not to be suitable for the study of phosphodiesterase 1-mediated functions.
Collapse
Affiliation(s)
- Torsten R Dunkern
- Biochemistry 2 Inflammation, ALTANA Pharma AG, Member of the Nycomed Group, Konstanz, Germany.
| | | |
Collapse
|
|
29
|
Hayashi M, Kita K, Ohashi Y, Aihara E, Takeuchi K. Phosphodiesterase isozymes involved in regulation of HCO3- secretion in isolated mouse duodenum in vitro. Biochem Pharmacol 2007; 74:1507-13. [PMID: 17719564 DOI: 10.1016/j.bcp.2007.07.029] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2007] [Revised: 07/20/2007] [Accepted: 07/20/2007] [Indexed: 11/28/2022]
Abstract
We examined the effects of various isozyme-selective PDE inhibitors on HCO(3)(-) secretion in the mouse duodenum in vitro and investigated which type(s) of phosphodiesterase (PDE) isozymes are involved in the response to PGE(2) and NO. The duodenal mucosa of male DDY mice was stripped of the muscle layer and mounted on an Ussing chamber, and HCO(3)(-) secretion was measured at pH 7.0 by a pH-stat method using 2mM HCl. Both PGE(2) and NOR-3 (NO donor) increased HCO(3)(-) secretion in the mouse duodenum in vitro, and the response to PGE(2) was inhibited by both EP3 and EP4 antagonists but not EP1 antagonist, while that to NOR-3 was inhibited by methylene blue. IBMX, a nonselective PDE inhibitor, significantly increased basal HCO(3)(-) secretion and potentiated the responses to both PGE(2) and NOR-3. Likewise, vinpocetine (PDE1 inhibitor) and cilostamide (PDE3 inhibitor) also increased the basal secretion at high doses and potentiated the HCO(3)(-) response to PGE(2) at doses that had no effect by themselves on the basal secretion. By contrast, the HCO(3)(-) stimulatory action of NOR-3 was significantly potentiated by vinpocetine but not cilostamide. Inhibitors of other PDE subtypes had no effect on the HCO(3)(-) secretion under basal or stimulated conditions. Both PDE1 and PDE3 mRNAs were expressed in the duodenal mucosa. These results suggested that PDE1 and PDE3 are involved in the regulation of duodenal HCO(3)(-) secretion and that the response to PGE(2) is associated with both PDE1 and PDE3, while the response to NO is mainly modulated by PDE1.
Collapse
Affiliation(s)
- Msamune Hayashi
- Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto 607-8414, Japan
| | | | | | | | | |
Collapse
|
|
30
|
Geibel J, Sritharan K, Geibel R, Geibel P, Persing JS, Seeger A, Roepke TK, Deichstetter M, Prinz C, Cheng SX, Martin D, Hebert SC. Calcium-sensing receptor abrogates secretagogue- induced increases in intestinal net fluid secretion by enhancing cyclic nucleotide destruction. Proc Natl Acad Sci U S A 2006; 103:9390-7. [PMID: 16760252 PMCID: PMC1475505 DOI: 10.1073/pnas.0602996103] [Citation(s) in RCA: 98] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The calcium-sensing receptor (CaSR) provides a fundamental mechanism for diverse cells to detect and respond to modulations in the ionic and nutrient compositions of their extracellular milieu. The roles for this receptor are largely unknown in the intestinal tract, where epithelial cells are normally exposed to large variations in extracellular solutes. Here, we show that colonic CaSR signaling stimulates the degradation of cyclic nucleotides by phosphodiesterases and describe the ability of receptor activation to reverse the fluid and electrolyte secretory actions of cAMP- and cGMP-generating secretagogues, including cholera toxin and heat stable Escherichia coli enterotoxin STa. Our results suggest a paradigm for regulation of intestinal fluid transport where fine tuning is accomplished by the counterbalancing effects of solute activation of the CaSR on neuronal and hormonal secretagogue actions. The reversal of cholera toxin- and STa endotoxin-induced fluid secretion by a small-molecule CaSR agonist suggests that these compounds may provide a unique therapy for secretory diarrheas.
Collapse
Affiliation(s)
- John Geibel
- Departments of *Cellular and Molecular Physiology and
- Surgery, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520
- To whom correspondence may be addressed. E-mail:
or
| | | | - Rainer Geibel
- Departments of *Cellular and Molecular Physiology and
| | - Peter Geibel
- Departments of *Cellular and Molecular Physiology and
| | | | - Achim Seeger
- Departments of *Cellular and Molecular Physiology and
| | | | - Markus Deichstetter
- Department of Medicine II, Technical University, Ismaningerstrasse 22, 81675 Munich, Germany; and
| | - Christian Prinz
- Department of Medicine II, Technical University, Ismaningerstrasse 22, 81675 Munich, Germany; and
| | - Sam X. Cheng
- Departments of *Cellular and Molecular Physiology and
| | - David Martin
- Department of Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA 91320
| | - Steven C. Hebert
- Departments of *Cellular and Molecular Physiology and
- To whom correspondence may be addressed. E-mail:
or
| |
Collapse
|
|
31
|
Liu S, Veilleux A, Zhang L, Young A, Kwok E, Laliberté F, Chung C, Tota MR, Dubé D, Friesen RW, Huang Z. Dynamic activation of cystic fibrosis transmembrane conductance regulator by type 3 and type 4D phosphodiesterase inhibitors. J Pharmacol Exp Ther 2005; 314:846-54. [PMID: 15901792 DOI: 10.1124/jpet.105.083519] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The diseases of cystic fibrosis, chronic obstructive pulmonary disease (COPD), and chronic bronchitis are characterized by mucus-congested and inflamed airways. Anti-inflammatory agents that can simultaneously restore or enhance mucociliary clearance through cystic fibrosis transmembrane conductance regulator (CFTR) activation may represent new therapeutics in their treatment. Herein, we report the activation of CFTR-mediated chloride secretion by phosphodiesterase (PDE) 4 inhibitors in T84 monolayer using (125)I anion as tracer. In the absence of forskolin, the iodide secretion was insensitive to PDE4 inhibitor L-826,141 [4-[2-(3,4-bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-ethyl]-3-methylpyridine-1-oxide], roflumilast, or to PDE3 inhibitor trequinsin. However, these inhibitors potently augmented iodide secretion after forskolin stimulation, with efficacy coupled to the activation states of adenylyl cyclase. The iodide secretion from PDE3 or PDE4 inhibition was characterized at first by a prolonged efflux duration, followed by progressively elevated peak efflux rates at higher inhibitor concentrations. Paralleled with an increased phosphor-cAMP response element-binding protein formation, the CFTR activation dissociated from a global cAMP elevation and was blocked by H89 [N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide]. 2-(4-Fluorophenoxy)-N-[(1S)-1-(4-methoxyphenyl)ethyl]nicotinamide, a stereoselective PDE4D inhibitor, augmented iodide efflux more efficiently than its less potent (R)-isomer. The peak efflux from maximal PDE4 and PDE3 inhibition matched that from full adenylyl cyclase activation. These data suggest that PDE3 and PDE4 (mainly PDE4D) form the major cAMP diffusion barrier in T84 cells to ensure a compartmentalized CFTR signaling. Together with their potent anti-inflammatory properties, the potentially enhanced airway mucociliary clearance from CFTR activation may have contributed to the efficacy of PDE4 inhibitors in COPD and asthmatic patients. PDE4 inhibitors may represent new opportunities to combat cystic fibrosis and other respiratory diseases in future.
Collapse
Affiliation(s)
- Susana Liu
- Merck Frosst Center for Therapeutic Research, P.O. Box 1005, Pointe Claire-Dorval, Quebec H9R 4P8, Canada
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
32
|
He Q, Zhu JX, Xing Y, Tsang LL, Yang N, Rowlands DK, Chung YW, Chan HC. Tetramethylpyrazine stimulates cystic fibrosis transmembrane conductance regulator-mediated anion secretion in distal colon of rodents. World J Gastroenterol 2005; 11:4173-9. [PMID: 16015685 PMCID: PMC4615438 DOI: 10.3748/wjg.v11.i27.4173] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the effect of tetramethylpyrazine (TMP), an active compound from Ligustium Wollichii Franchat, on electrolyte transport across the distal colon of rodents and the mechanism involved
METHODS: The short-circuit current (ISC) technique in conjunction with pharmacological agents and specific inhibitors were used in analyzing the electrolyte transport across the distal colon of rodents. The underlying cellular signaling mechanism was investigated by radioimmunoassay analysis (RIA) and a special mouse model of cystic fibrosis.
RESULTS: TMP stimulated a concentration-dependent rise in ISC, which was dependent on both Cl- and HCO3-, and inhibited by apical application of diphenylamine-2,2’-dicarboxylic acid (DPC) and glibenclamide, but resistant to 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid disodium salt hydrate (DIDS). Removal of Na+ from basolateral solution almost completely abolished the ISC response to TMP, but it was insensitive to apical Na+ replacement or apical Na+ channel blocker, amiloride. Pretreatment of colonic mucosa with BAPTA-AM, a membrane-permeable selective Ca2+ chelator, did not significantly alter the TMP-induced ISC. No additive effect of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was observed on the TMP-induced ISC, but it was significantly reduced by a protein kinase A inhibitor, H89. RIA results showed that TMP (1 mmol/L) elicited a significant increase in cellular cAMP production, which was similar to that elicited by the adenylate cyclase activator, forskolin (10 µmol/L). The TMP-elicited ISC as well as forskolin- or IBMX-induced ISC were abolished in mice with homozygous mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) presenting defective CFTR functions and secretions.
CONCLUSION: TMP may stimulate cAMP-dependent and CFTR-mediated Cl- and HCO3- secretion. This may have implications in the future development of alternative treatment for constipation.
Collapse
Affiliation(s)
- Qiong He
- Department of Physiology, Zhengzhou University, Henan Province, China
| | | | | | | | | | | | | | | |
Collapse
|
|
33
|
Sopory S, Kaur T, Visweswariah SS. The cGMP-binding, cGMP-specific phosphodiesterase (PDE5): intestinal cell expression, regulation and role in fluid secretion. Cell Signal 2005; 16:681-92. [PMID: 15093609 DOI: 10.1016/j.cellsig.2003.11.004] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2003] [Revised: 11/12/2003] [Accepted: 11/12/2003] [Indexed: 11/26/2022]
Abstract
The expression and regulation of the cGMP-binding, cGMP-specific phosphodiesterase, PDE5, was studied in intestinal cells. Both PDE5A1 and PDE5A2 splice forms were cloned from the cDNA prepared from human colonic T84 cells, and PDE5 activity was dependent on increases in intracellular cGMP levels which correlated with increased phosphorylation of the enzyme. PDE5 expression was monitored in different regions of the gastrointestinal tract and nearly 50% of the phosphodiesterase activity in the duodenum, jejunum, ileum and colon was inhibited by sildenafil citrate. Administration of the stable toxin to intestinal loops resulted in activation of PDE5. Inhibition of PDE5 by sildenafil citrate led to fluid accumulation in loops, suggesting a possible explanation for the side effect of diarrhoea observed in individuals administered sildenafil citrate. Our results therefore represent the first study on the expression and regulation of PDE5 in intestinal tissue, and indicate that mechanisms to control its activity may have important consequences in intestinal physiology.
Collapse
Affiliation(s)
- Shailaja Sopory
- Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Malleswaram, Bangalore 560012, India
| | | | | |
Collapse
|
|
34
|
Barnes AP, Livera G, Huang P, Sun C, O'Neal WK, Conti M, Stutts MJ, Milgram SL. Phosphodiesterase 4D Forms a cAMP Diffusion Barrier at the Apical Membrane of the Airway Epithelium. J Biol Chem 2005; 280:7997-8003. [PMID: 15611099 DOI: 10.1074/jbc.m407521200] [Citation(s) in RCA: 89] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
We demonstrated previously that Calu-3 airway epithelial cells sense adenosine on their luminal surface through adenosine A2B receptors coupled to adenylyl cyclase. Occupancy of these receptors leads to activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel through protein kinase A (PKA) anchored at the apical membrane. Because luminal A2B receptor activation does not raise total cellular cAMP levels, we hypothesized that activation of phosphodiesterases (PDEs) confines cAMP generated by apical A2B receptors to a microdomain that includes the CFTR channel. Using reverse transcription-PCR, Western blotting, and activity measurements, PDE4D was identified as the major PDE species in airway epithelia. Consistent with these results, inhibitors of PDE4, but not PDE3, selectively abolished the lateral confinement of cAMP signaling in apical membrane patches during cell-attached recordings. Furthermore, stimulation of the CFTR in excised apical patches by rolipram and RS25344 indicated that PDE4 is localized in close proximity to the CFTR channel. Indeed, immunohistochemistry of human airway sections revealed that PDE4D is localized in the apical domain of the cell. PDE4 was activated after luminal adenosine exposure in a PKA-dependent manner. Because PDE4 activity is positively regulated by PKA, our results support a model whereby the PDE diffusion barrier is proportional to the degree of receptor stimulation. These findings underscore the concept that subcellular localization of individual PDE isozymes is an important mechanism for confining cAMP signaling to functional domains within cells.
Collapse
Affiliation(s)
- Anthony P Barnes
- Department of Cell and Developmental Biology and Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
35
|
Snyder PB, Esselstyn JM, Loughney K, Wolda SL, Florio VA. The role of cyclic nucleotide phosphodiesterases in the regulation of adipocyte lipolysis. J Lipid Res 2005; 46:494-503. [PMID: 15604523 DOI: 10.1194/jlr.m400362-jlr200] [Citation(s) in RCA: 79] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
This study assessed the effects of selective inhibitors of 3',5'-cyclic nucleotide phosphodiesterases (PDEs) on adipocyte lipolysis. IC224, a selective inhibitor of type 1 phosphodiesterase (PDE1), suppressed lipolysis in murine 3T3-L1 adipocytes (69.6 +/- 5.4% of vehicle control) but had no effect in human adipocytes. IC933, a selective inhibitor of PDE2, had no effect on lipolysis in either cultured murine 3T3-L1 adipocytes or human adipocytes. Inhibition of PDE3 with cilostamide moderately stimulated lipolysis in murine 3T3-L1 and rat adipocytes (397 +/- 25% and 235 +/- 26% of control, respectively) and markedly stimulated lipolysis in human adipocytes (932 +/- 7.6% of control). Inhibition of PDE4 with rolipram moderately stimulated lipolysis in murine 3T3-L1 adipocytes (291 +/- 13% of control) and weakly stimulated lipolysis in rat adipocytes (149 +/- 7.0% of control) but had no effect on lipolysis in human adipocytes. Cultured adipocytes also responded differently to a combination of PDE3 and PDE4 inhibitors. Simultaneous exposure to cilostamide and rolipram had a synergistic effect on lipolysis in murine 3T3-L1 and rat adipocytes but not in human adipocytes. Hence, the relative importance of PDE3 and PDE4 in regulating lipolysis differed in cultured murine, rat, and human adipocytes.
Collapse
|
|
36
|
Abstract
1. 5-Hydroxytryptamine (5-HT) modulates the motility and secretion of the gastrointestinal tract. To examine the direct effect of 5-HT on the secretions of colonic epithelial cells, a short-circuit current was used to measure electrolyte transport in the rat stripped distal colon. A neuronal Na+ channel blocker and a cyclo-oxygenase inhibitor were routinely added in experiments to abolish the effects of the enteric nervous system and endogenous prostaglandin, respectively. 2. Basolateral application of 5-HT (10 micromol/L) induced an increase in the short circuit current (ISC). Removal of extracellular Cl-, HCO3- or both resulted in a 59.6, 76.4 and 90% reduction of 5-HT-elicited responses, respectively. The Ca(2+)-dependent Cl- channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) had no effect on the 5-HT-induced increase in ISC, but the selective cystic fibrosis transmembrane conductance regulator (CFTR) channel blocker glibenclamide (1 mmol/L) inhibited 5-HT-induced increases in ISC by approximately 92.9%. Removal of apical Na+ reduced the 5-HT-induced increase in ISC by 33.3%. 3. Basolateral pretreatment with 100 micromol/L bumetanide (an inhibitor of the Na(+)-K(+)-2Cl- cotransporter), 200 micromol/L DIDS (an inhibitor of the Na(+)-HCO3- transporter or the Cl-/HCO3- exchanger) or both decreased the DeltaISC induced by 5-HT by approximately 75.5, 59.0 and 86.3%, respectively. Removal of basolateral Na+ also reduced the current evoked by 5-HT. 4. The selective 5-HT4 antagonist GR113808 (1 micromol/L) totally abolished the 5-HT-induced increase in ISC, whereas 2-methyl-5-HT (100 micromol/L) induced a weak ISC response. 5. In conclusion, the present study has demonstrated that 5-HT can elicit Cl(-)- and HCO3- anion secretion and Na+ absorption by acting directly on colonic epithelial cells via 5-HT4 receptors.
Collapse
Affiliation(s)
- Yang Ning
- Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong
| | | | | |
Collapse
|
|
37
|
Zhu JX, Yang N, He Q, Tsang LL, Zhao WC, Chung YW, Chan HC. Differential Cl - and HCO 3- mediated anion secretion by different colonic cell types in response to tetromethylpyrazine. World J Gastroenterol 2004; 10:1763-8. [PMID: 15188502 PMCID: PMC4572265 DOI: 10.3748/wjg.v10.i12.1763] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: Colonic epithelium is known to secrete both Cl- and HCO3-, but the secretory mechanisms of different colonic cell types are not fully understood. The present study aimed to investigate the differential activation of Cl- and HCO3-secretion by tetramethylpyrazine (TMP) in human crypt-like cell line, T84, and villus-like cell line, Caco-2, in comparison to the TMP-induced secretory response in freshly isolated rat colonic mucosa.
METHODS: Colonic epithelial anion secretion was studied by using the short circuit current (ISC) technique. RT-PCR was used to examine the expression of Na+-HCO3-- cotranspoter in different epithelial cell types.
RESULTS: TMP produced a concentration-dependent ISC which was increase in both T84 and Caco-2 cells. When extracellular Cl- was removed, TMP-induced ISC was abolished by 76.6% in T84 cells, but not in Caco-2 cells. However, after both Cl- and HCO3- were removed, TMP-induced ISC in Caco-2 cells was reduced to 10%. Bumetanide, an inhibitor of Na+-K+-Cl--cotranspoter, inhibited the TMP-induced ISC by 96.7% in T84 cells, but only 47.9% in Caco-2 cells. In the presence of bumetanide and 4, 4’-diisothiocyanostilbene-2, 2’-disulfonic acid, an inhibitor of Na+-HCO3- cotransporter, inhibited the TMP-induced current in Caco-2 cells by 93.3%. In freshly isolated rat colonic mucosa, TMP stimulated distinct ISC responses similar to that observed in T84 and Caco-2 cells depending on the concentration used. RT-PCR revealed that the expression of Na+-HCO3- cotransporter in Caco-2 cells was 4-fold more greater than that in T84 cells.
CONCLUSION: TMP exerts concentration-dependent differential effects on different colonic cell types with stimulation of predominant Cl- secretion by crypt cells at a lower concentration, but predominant HCO3- secretion by villus cells at a higher concentration, suggesting different roles of these cells in colonic Cl- and HCO3- secretion.
Collapse
Affiliation(s)
- Jin-Xia Zhu
- Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China
| | | | | | | | | | | | | |
Collapse
|