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Joyce W, Warwicker J, Shiels HA, Perry SF. Evolution and divergence of teleost adrenergic receptors: why sometimes 'the drugs don't work' in fish. J Exp Biol 2023; 226:jeb245859. [PMID: 37823524 DOI: 10.1242/jeb.245859] [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] [Indexed: 10/13/2023]
Abstract
Adrenaline and noradrenaline, released as hormones and/or neurotransmitters, exert diverse physiological functions in vertebrates, and teleost fishes are widely used as model organisms to study adrenergic regulation; however, such investigations often rely on receptor subtype-specific pharmacological agents (agonists and antagonists; see Glossary) developed and validated in mammals. Meanwhile, evolutionary (phylogenetic and comparative genomic) studies have begun to unravel the diversification of adrenergic receptors (ARs) and reveal that whole-genome duplications and pseudogenization events in fishes results in notable distinctions from mammals in their genomic repertoire of ARs, while lineage-specific gene losses within teleosts have generated significant interspecific variability. In this Review, we visit the evolutionary history of ARs (including α1-, α2- and β-ARs) to highlight the prominent interspecific differences in teleosts, as well as between teleosts and other vertebrates. We also show that structural modelling of teleost ARs predicts differences in ligand binding affinity compared with mammalian orthologs. To emphasize the difficulty of studying the roles of different AR subtypes in fish, we collate examples from the literature of fish ARs behaving atypically compared with standard mammalian pharmacology. Thereafter, we focus on specific case studies of the liver, heart and red blood cells, where our understanding of AR expression has benefited from combining pharmacological approaches with molecular genetics. Finally, we briefly discuss the ongoing advances in 'omics' technologies that, alongside classical pharmacology, will provide abundant opportunities to further explore adrenergic signalling in teleosts.
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Affiliation(s)
- William Joyce
- Department of Biology - Zoophysiology, Aarhus University, 8000 Aarhus C, Denmark
| | - Jim Warwicker
- Division of Molecular and Cellular Function, Faculty of Biology, Medicine and Health, Manchester Institute of Biotechnology, The University of Manchester, Manchester, M1 7DN, UK
| | - Holly A Shiels
- Division of Cardiovascular Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, M13 9PL, UK
| | - Steve F Perry
- Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON, Canada, K1N 6N5
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Kimura DC, Nagaoka MR, Borges DR, Kouyoumdjian M. Angiotensin II or epinephrine hemodynamic and metabolic responses in the liver of L-NAME induced hypertension and spontaneous hypertensive rats. World J Hepatol 2017; 9:781-790. [PMID: 28660012 PMCID: PMC5474724 DOI: 10.4254/wjh.v9.i17.781] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/15/2016] [Revised: 04/28/2017] [Accepted: 05/19/2017] [Indexed: 02/06/2023] Open
Abstract
AIM To study hepatic vasoconstriction and glucose release induced by angiotensin (Ang)II or Epi in rats with pharmacological hypertension and spontaneously hypertensive rat (SHR).
METHODS Isolated liver perfusion was performed following portal vein and vena cava cannulation; AngII or epinephrine (Epi) was injected in bolus and portal pressure monitored; glucose release was measured in perfusate aliquots.
RESULTS The portal hypertensive response (PHR) and the glucose release induced by AngII of L-NAME were similar to normal rats (WIS). On the other hand, the PHR induced by Epi in L-NAME was higher whereas the glucose release was lower compared to WIS. Despite the similar glycogen content, glucose release induced by AngII was lower in SHR compared to Wistar-Kyoto rats although both PHR and glucose release induced by Epi in were similar.
CONCLUSION AngII and Epi responses are altered in different ways in these hypertension models. Our results suggest that inhibition of NO production seems to be involved in the hepatic effects induced by Epi but not by AngII; the diminished glucose release induced by AngII in SHR is not related to glycogen content.
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Fabbri E, Moon TW. Adrenergic signaling in teleost fish liver, a challenging path. Comp Biochem Physiol B Biochem Mol Biol 2015; 199:74-86. [PMID: 26482086 DOI: 10.1016/j.cbpb.2015.10.002] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2015] [Revised: 09/09/2015] [Accepted: 10/12/2015] [Indexed: 01/15/2023]
Abstract
Adrenergic receptors or adrenoceptors (ARs) belong to the huge family of G-protein coupled receptors (GPCRs) that have been well characterized in mammals primarily because of their importance as therapeutic drug targets. ARs are found across vertebrates and this review examines the path to identify and characterize these receptors in fish with emphasis on hepatic metabolism. The absence of reliable and specific pharmacological agents led investigators to define the fish hepatic AR system as relying solely on a β2-AR, cAMP-dependent signaling transduction pathway. The use of calcium-radiometric imaging, purified membranes for ligand-binding studies, and perifused rather than static cultured fish hepatocytes, unequivocally demonstrated that both α1- and β2-AR signaling systems existed in the fish liver consistent with studies in mammals. Additionally, the use of molecular tools and phylogenetic analysis clearly demonstrated the existence of multiple AR-types and -subtypes in hepatic and other tissues of a number of fish species. This review also examines the use of β-blockers as pharmaceuticals and how these drugs that are now in the aquatic environment may be impacting aquatic species including fish and some invertebrates. Clearly there is a large conservation of structure and function within the AR system of vertebrates but there remain a number of key questions that need to be addressed before a clear understanding of these systems can be resolved.
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Affiliation(s)
- Elena Fabbri
- University of Bologna, Department of Biological, Geological and Environmental Sciences Unit of Ravenna, via S. Alberto 163, 48124 Ravenna, Italy.
| | - Thomas W Moon
- University of Ottawa, Department of Biology and the Centre for Advance Research in Environmental Genomics, 30 Marie Curie, K1N 6N5 Ottawa, Canada
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Antiulcerogenic Activity and Toxicity of Bauhinia holophylla Hydroalcoholic Extract. EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE 2015; 2015:439506. [PMID: 25954316 PMCID: PMC4410539 DOI: 10.1155/2015/439506] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/27/2014] [Revised: 02/12/2015] [Accepted: 03/10/2015] [Indexed: 12/14/2022]
Abstract
Several species of Bauhinia are used in traditional medicine for the treatment of gastrointestinal diseases, diabetes, and inflammation, among other conditions. The aim of this study was to investigate the antiulcer effect of a hydroalcoholic extract from the leaves of B. holophylla. The chemical profile of the extract was determined by HPLC-PAD-ESI-IT-MS. A dose-effect relation was constructed using the ethanol-induced gastric ulcer model in male Wistar rats. Histological analyses and studies of antioxidant and anti-inflammatory activities were performed in stomach samples. The involvement of SH compounds, NO, K+ATP channels, and α2-adrenergic receptors in the gastroprotective effect was evaluated. A toxicity study was performed with a single oral dose of 5000 mg/kg. The extract was composed mainly of cyanoglucoside and flavonol-O-glycosides derivatives of quercetin and myricetin. SH compounds, NO release, K+ATP channel activation, and presynaptic α2-adrenergic receptor stimulation each proved to be involved in the antiulcer effect. The levels of GSH and activity of GR and GPx were increased, and the levels of TNF-α, IL-6 and IL-10 were modulated. There was an antidiarrheal effect and there were no signs of toxicity. B. holophylla presents antiulcer activity mainly by decreasing oxidative stress and attenuating the inflammatory response, without inducing side effects.
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Wang J, Yuan L, Cheng B, Li W, Xiao C, Wang Y, Liu X. Antioxidant capacity and antitumor activity of Fructus Kochiae extracts. QUALITY ASSURANCE AND SAFETY OF CROPS & FOODS 2014. [DOI: 10.3920/qas2012.0218] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Affiliation(s)
- J. Wang
- College of Food Science and Engineering, Northwest A&F University, Taicheng Road 3, Yangling, 0086-712100 Shaanxi, China P.R
| | - L. Yuan
- College of Food Science and Engineering, Northwest A&F University, Taicheng Road 3, Yangling, 0086-712100 Shaanxi, China P.R
| | - B. Cheng
- College of Food Science and Engineering, Northwest A&F University, Taicheng Road 3, Yangling, 0086-712100 Shaanxi, China P.R
| | - W. Li
- College of Food Science and Engineering, Northwest A&F University, Taicheng Road 3, Yangling, 0086-712100 Shaanxi, China P.R
| | - C. Xiao
- College of Food Science and Engineering, Northwest A&F University, Taicheng Road 3, Yangling, 0086-712100 Shaanxi, China P.R
| | - Y. Wang
- College of Food Science and Engineering, Northwest A&F University, Taicheng Road 3, Yangling, 0086-712100 Shaanxi, China P.R
| | - X. Liu
- College of Food Science and Engineering, Northwest A&F University, Taicheng Road 3, Yangling, 0086-712100 Shaanxi, China P.R
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Gastroprotective activity of violacein isolated from Chromobacterium violaceum on indomethacin-induced gastric lesions in rats: investigation of potential mechanisms of action. ScientificWorldJournal 2014; 2014:616432. [PMID: 25162059 PMCID: PMC4138890 DOI: 10.1155/2014/616432] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2014] [Revised: 06/18/2014] [Accepted: 06/26/2014] [Indexed: 12/21/2022] Open
Abstract
Chromobacterium violaceum, Gram-negative bacteria species found in tropical regions of the world, produces a distinct deep violet-colored pigment called violacein. In the present study, we investigated whether violacein can promote a gastroprotective effect and verified the possible mechanisms involved in this action. For this study, an indomethacin-induced gastric ulcer rat model was used. The roles of biomolecules such as MPO, PGE2, pro- and anti-inflammatory cytokines, growth factors, caspase-3, NO, K+ATP channels, and α2-receptors were investigated. Violacein exhibited significant gastroprotective effect against indomethacin-induced lesions, while pretreatment with L-NAME and glibenclamide (but not with NEM or yohimbine) was able to reverse this action. Pretreatment with violacein also restored cNOS level to normal and led to attenuation of enhanced apoptosis and gastric microvascular permeability. Our results suggest that violacein provides a significant gastroprotective effect in an indomethacin-induced ulcer model through the maintenance of some vital protein molecules, and this effect appears to be mediated, at least in part, by endogenous prostaglandins, NOS, K+ATP channel opening, and inhibition of apoptosis and gastric microvascular permeability.
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Lennquist A, Asker N, Kristiansson E, Brenthel A, Björnsson BT, Kling P, Hultman M, Larsson DGJ, Förlin L. Physiology and mRNA expression in rainbow trout (Oncorhynchus mykiss) after long-term exposure to the new antifoulant medetomidine. Comp Biochem Physiol C Toxicol Pharmacol 2011; 154:234-41. [PMID: 21703361 DOI: 10.1016/j.cbpc.2011.06.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/24/2011] [Revised: 06/06/2011] [Accepted: 06/08/2011] [Indexed: 02/04/2023]
Abstract
Medetomidine is under evaluation for use as an antifouling agent, and its effects on non-target aquatic organisms are therefore of interest. In this study, rainbow trout was exposed to low (0.5 and 5.0nM) concentrations of medetomidine for up to 54 days. Recently we have reported on effects on paleness and melanophore aggregation of medetomidine in these fish. Here, specific growth rates were investigated together with a broad set of physiological parameters including plasma levels of growth hormone (GH), insulin-like growth factor-I (IGF-I) and leptin, glucose and haemoglobin (Hb), hematocrit (Ht), condition factor, liver and heart somatic indexes (LSI, HSI). Hepatic enzyme activities of CYP1A (EROD activity), glutathione S-transferases (GST) and glutathione reductase (GR) were also measured. Additionally, hepatic mRNA expression was analysed through microarray and quantitative PCR in fish sampled after 31 days of exposure. Medetomidine at both concentrations significantly lowered blood glucose levels and the higher concentration significantly reduced the LSI. The mRNA expression analysis revealed few differentially expressed genes in the liver and the false discovery rate was high. Taken together, the results suggest that medetomidine at investigated concentrations could interfere with carbohydrate metabolism of exposed fish but without any clear consequences for growth.
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Affiliation(s)
- Anna Lennquist
- Department of Zoology/Zoophysiology, University of Gothenburg, Sweden.
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Santin JR, Lemos M, Klein-Júnior LC, Machado ID, Costa P, de Oliveira AP, Tilia C, de Souza JP, de Sousa JPB, Bastos JK, de Andrade SF. Gastroprotective activity of essential oil of the Syzygium aromaticum and its major component eugenol in different animal models. Naunyn Schmiedebergs Arch Pharmacol 2010; 383:149-58. [PMID: 21140134 DOI: 10.1007/s00210-010-0582-x] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2010] [Accepted: 11/15/2010] [Indexed: 10/18/2022]
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Fabbri E, Chen X, Capuzzo A, Moon TW. Binding kinetics and sequencing of hepatic alpha1-adrenergic receptors in two marine teleosts, mackerel (Scomber scombrus) and anchovy (Engraulis encrasicolus). ACTA ACUST UNITED AC 2008; 309:157-65. [PMID: 18273865 DOI: 10.1002/jez.441] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Liver alpha(1)-adrenoceptors (ARs) are demonstrated, or at least hypothesized, in freshwater and brackish-water teleosts, whereas no data are available for marine teleosts. This study evaluates the presence of alpha(1)-ARs in the liver of two marine teleosts, the anchovy Engraulis encrasicolus and the mackerel Scomber scombrus, and examines on a broad scale the possibility that habitats posing different challenges also influence phenotypic trait selection. Binding assays were performed also on liver membranes from the carp Cyprinus carpio as a direct comparison with a freshwater species. Scatchard analysis of [(3)H]prazosin binding to purified liver membranes from anchovy, mackerel and carp resulted in K(d) values of 1.51+/-0.085, 1.26+/-0.098, and 2.61+/-0.22 nM, and B(max) values of 87.4+/-9.12, 77+/-8.29, and 115.22+/-3.31 fmol/mg protein, respectively. Thus, alpha(1)-ARs of the two marine teleosts showed higher [(3)H]prazosin affinity compared with those of the freshwater/brackish-water fish studied thus far, whereas the number of liver binding sites did not differ significantly from that of carp, eel or trout. A preliminary phylogeny based on amino acid sequence analysis indicated the presence of at least an alpha(1A)-AR in mackerel and an alpha(1D)-AR in both anchovy and mackerel. This is the first indication of alpha(1)-AR subtypes in any marine species, but further studies are needed to ascertain the physiological role of these alpha(1)-ARs in these two marine species.
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Affiliation(s)
- Elena Fabbri
- Interdepartment Centre for Environmental Science Research, University of Bologna, Ravenna, Italy.
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10
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Regulation of the black bullhead hepatic β-adrenoceptors. Comp Biochem Physiol B Biochem Mol Biol 2008; 149:265-74. [DOI: 10.1016/j.cbpb.2007.09.016] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2007] [Revised: 09/19/2007] [Accepted: 09/20/2007] [Indexed: 11/21/2022]
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Chen X, Perry SF, Aris-Brosou S, Selva C, Moon TW. Characterization and functional divergence of the alpha 1-adrenoceptor gene family: insights from rainbow trout (Oncorhynchus mykiss). Physiol Genomics 2007; 32:142-53. [PMID: 17940201 DOI: 10.1152/physiolgenomics.00258.2006] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Presently, three alpha(1)-adrenoceptor (AR) types are recognized in vertebrates: alpha(1A)-, alpha(1B)-, and alpha(1D)-ARs. These alpha(1)-subtypes have distinct pharmacology and molecular profiles, play crucial roles in metabolic and vascular control, and are the targets for numerous pharmaceuticals, especially those affecting blood pressure and vascular resistance. To better understand the functional divergence within the alpha(1)-AR gene family, we sequenced these alpha(1)-AR paralogs in the rainbow trout and performed an extensive phylogenetic analysis. We show that these AR genes evolved by duplication events just before the origin of the jawed vertebrates. Our computational analyses suggest that the differences between the three alpha(1)-AR subtypes may affect their tissue specificity, ligand specificity, and possibly signal transduction processes and desensitization. We also show that, within each subtype, differences exist between fish and mammalian receptors, both at the transcriptional and at the physiological level. These differences, however, suggest that the role of alpha(1)-ARs in fish is more complex than previously thought. Our integrated analysis of the alpha(1)-AR gene family suggests that these receptors evolved these distinct features very early within vertebrates.
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Affiliation(s)
- Xi Chen
- Department of Biology and Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, Canada
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Bagnaresi P, Rodrigues MT, Garcia CRS. Calcium signaling in lizard red blood cells. Comp Biochem Physiol A Mol Integr Physiol 2007; 147:779-787. [PMID: 17095273 DOI: 10.1016/j.cbpa.2006.09.015] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2006] [Revised: 09/21/2006] [Accepted: 09/25/2006] [Indexed: 11/16/2022]
Abstract
The ion calcium is a ubiquitous second messenger, present in all eukaryotic cells. It modulates a vast number of cellular events, such as cell division and differentiation, fertilization, cell volume, decodification of external stimuli. To process this variety of information, the cells display a number of calcium pools, which are capable of mobilization for signaling purposes. Here we review the calcium signaling on lizards red blood cells, an interesting model that has been receiving an increasing notice recently. These cells possess a complex machinery to regulate calcium, and display calcium responses to extracellular agonists. Interestingly, the pattern of calcium handling and response are divergent in different lizard families, which enforces the morphological data to their phylogenetic classification, and suggest the radiation of different calcium signaling models in lizards evolution.
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Affiliation(s)
- Piero Bagnaresi
- Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil
| | - Miguel T Rodrigues
- Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil
| | - Célia R S Garcia
- Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
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van Heeswijk JCF, Vianen GJ, van den Thillart GEEJM, Zaagsma J. Beta-adrenergic control of plasma glucose and free fatty acid levels in the air-breathing African catfishClarias gariepinusBurchell 1822. J Exp Biol 2005; 208:2217-25. [PMID: 15939765 DOI: 10.1242/jeb.01621] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
SUMMARYIn several water-breathing fish species, β-adrenergic receptor stimulation by noradrenaline leads to a decrease in plasma free fatty acid(FFA) levels, as opposed to an increase in air-breathing mammals. We hypothesised that this change in adrenergic control is related to the mode of breathing. Therefore, cannulated air-breathing African catfish were infused for 90 min with noradrenaline or with the nonselective β-agonist,isoprenaline. To identify the receptor type involved, a bolus of either a selective β1-antagonist (atenolol) or a selectiveβ 2-antagonist (ICI 118,551) was injected 15 min prior to the isoprenaline infusion. Both noradrenaline and isoprenaline led to an expected rise in glucose concentration. Isoprenaline combined with both theβ 1- and β2-antagonist led to higher glucose concentrations than isoprenaline alone. This could indicate the presence of a stimulatory β-adrenoceptor different from β1 andβ 2-adrenoceptors; these two receptors thus seemed to mediate a reduction in plasma glucose concentration. Both noradrenaline and isoprenaline led to a significant decrease in FFA concentration. Whereas theβ 1-antagonist had no effect, the β2-antagonist reduced the decrease in FFA concentration, indicating the involvement ofβ 2-adrenoceptors. It is concluded that the air-breathing African catfish reflects water-breathing fish in the adrenergic control of plasma FFA and glucose levels.
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Moon TW. Hormones and fish hepatocyte metabolism: “the good, the bad and the ugly!”. Comp Biochem Physiol B Biochem Mol Biol 2004; 139:335-45. [PMID: 15544959 DOI: 10.1016/j.cbpc.2004.06.003] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2004] [Revised: 05/31/2004] [Accepted: 06/10/2004] [Indexed: 11/18/2022]
Abstract
This short review examines some of my personal experiences with Dr. Peter Hochachka, as a mentor and friend, and how his encouragement led to the research undertaken in my laboratory over the past three decades. Specifically, our work using the fish hepatocyte preparation as a model cell system is reviewed. The hepatocyte is an ideal cellular system that can be used to probe hepatic physiology and biochemistry. The impact of insulin, glucagon and related peptides, and catecholamines is discussed from the perspective of core and diverse functions of these key vertebrate metabolic hormones. Each hormone that operates in fish species was studied in manners similar to that of mammals, but it appears that the role of glucagon-like peptide-1 (GLP-1) in particular differs substantially from that in mammals. The receptors for each of these fish hormones seem structurally and in some cases functionally quite distinct from those in mammals. Few fish hormone receptor sequences are available, but fish genomists are rapidly adding new sequence information to the existing databases, so our view of the evolution of vertebrate hormone receptors will become clearer very quickly.
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Affiliation(s)
- Thomas W Moon
- Department of Biology, University of Ottawa, PO Box 450, Stn A, Ottawa, ON, Canada K1N 6N5.
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Fabbri E, Caselli F, Piano A, Sartor G, Capuzzo A. Cd2+ and Hg2+ affect glucose release and cAMP-dependent transduction pathway in isolated eel hepatocytes. AQUATIC TOXICOLOGY (AMSTERDAM, NETHERLANDS) 2003; 62:55-65. [PMID: 12413794 DOI: 10.1016/s0166-445x(02)00063-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
Isolated hepatocytes of the European eel (Anguilla anguilla) have been used as experimental model to characterize the effects of Cd(2+) and Hg(2+) on either basal or epinephrine-stimulated glucose release. Cd(2+) strongly reduced glucose output from cells perifused in BioGel P4 columns and challenged with epinephrine, with a maximum inhibition of 95% reached at 10 microM (IC(50) 0.04 microM). The epinephrine-stimulated glucose output was also reduced by Hg(2+), although a significant inhibition of about 60% was achieved only at 10 microM (IC(50) 5 microM). The possible influence of Cd(2+) and Hg(2+) on adenylyl cyclase/cAMP transduction pathway has been investigated, since this system is known to play a pivotal role in the regulation of fish liver glycogen breakdown and consequent glucose release. Micromolar concentrations of both heavy metals significantly reduced the epinephrine-modulated cAMP levels in isolated eel hepatocytes, in good agreement with the reduction of glucose output. Cd(2+) and Hg(2+) also significantly reduced basal and epinephrine-stimulated adenylyl cyclase activity in liver membrane preparations. A competitive inhibition with respect to Mg(2+) was shown by Cd(2+) and Hg(2+), which significantly reduced the affinity of the allosteric activator for the adenylyl cyclase system. Apparent Km for Mg(2+) was 4.35 mM in basal conditions, and increased to 9.1 and 7.1 mM in the presence of 10 microM Cd(2+) and Hg(2+), respectively. These results indicate that Cd(2+) and Hg(2+) may impair a crucial intracellular transduction pathway involved in the adrenergic control of glucose metabolism, but also in several other routes of hormonal regulation of liver functions.
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Affiliation(s)
- Elena Fabbri
- Interdepartment Centre for Research in Environmental Science, University of Bologna, via Tombesi dall'Ova 55, 48 100, Ravenna, Italy.
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Caselli F, Capuzzo A, Piano A, Valbonesi P, Fabbri E. G proteins immunodetection and adrenergic transduction pathways in the liver of Anguilla anguilla. Physiol Biochem Zool 2002; 75:609-16. [PMID: 12601617 DOI: 10.1086/345483] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/24/2002] [Indexed: 11/03/2022]
Abstract
G proteins are members of a highly conserved superfamily of GTPases, which includes heterotrimeric (alpha, beta, gamma) proteins acting as critical control points for transmembrane signaling. In ectothermal vertebrates, knowledge about these proteins is scarce, and our work provides the first demonstration that G(s), G(q), and G(i) proteins are all present in the liver of a fish. G(q)alpha subunits of about 42 kDa have been identified in European eel (Anguilla anguilla) liver membranes, supporting previous reports about the existence of hormone transduction pathways coupled to inositol 1,4,5-trisphosphate/Ca(2+) enhancement in fish hepatocytes. Although two G(s)alpha proteins of about 45 and 52 kDa have been reported in mammals, a single isoform of approximately 45 kDa has been recognized in eel liver. G(s)alpha and G(q)alpha proteins are involved in the epinephrine transduction pathway, leading to cAMP and Ca(2+) intracellular increments, respectively. Interestingly, both messengers significantly stimulated glucose release from eel hepatocytes but with a different time course. In fact, the Ca(2+)-dependent glucose output preceded the cAMP-mediated release by about 7 min. G(i)alpha subunits of about 40 kDa were also immunodetected, suggesting the presence of hormone receptors leading to adenylyl cyclase inhibition in eel liver; however, alpha(2)- adrenoreceptor ligands were ineffective on both enzyme activity and glucose release.
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Affiliation(s)
- Federico Caselli
- Interdepartment Centre for Research on Environmental Sciences, University of Bologna, via Tombesi dall'Ova 55, 48100 Ravenna, Italy
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Fabbri E, Selva C, Piano A, Caselli F, Capuzzo A. Identification and properties of a Gs protein in catfish liver membranes. Gen Comp Endocrinol 2002; 125:340-8. [PMID: 11884079 DOI: 10.1006/gcen.2001.7762] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The presence of G proteins and their involvement in adrenergic signaling has been investigated in catfish (Ictalurus melas) liver membranes. Adenylyl cyclase activity was potently stimulated by the nonhydrolyzable analog of GTP, [35S]guanosine 5'-O-(gamma-thiotriphosphate) (GTPgammaS) (maximal activation of about eightfold at 10(-5) M; half-maximal activation at 1.31 x 10(-7) M), and reduced by the competitive inhibitor of GTP, GDPbetaS (70% maximal inhibition at 10(-4) M; half-maximal inhibition at 1.98 x 10(-7) M). Forskolin dramatically enhanced enzyme activity (up to about 3500% at 100 microM), and its action was not affected by guanine nucleotides, confirming that the diterpene effect occurred only at targets downstream of the G proteins. Receptor-dependent G protein activity was evaluated by a [(35)S]GTPgammaS binding assay. At 100 microM GDP, 100 mM NaCl, and 5 mM MgCl2, after an incubation of 90 min at 20 degrees, a Kd of 18.6 nM and a Bmax of 105.7 pmol/mg protein for [35S]GTPgammaS binding to catfish liver membranes were determined. The binding of the tracer was enhanced by 1 microM epinephrine, up to a maximum of 158%, and inhibited by NF 449, a G(s)alpha-selective antagonist with half-maximal effect in the micromolar range. Immunoblotting analysis with a specific anti-G(s)alpha antibody revealed a single band of about 45 kDa mass. This result represents the first demonstration of the presence of G protein alpha(s) subunits in the liver of an ectothermal vertebrate.
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Affiliation(s)
- Elena Fabbri
- Department of Biology, University of Bologna, 40100 Bologna, Italy.
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Manzl C, Schubert M, Schwarzbaum PJ, Krumschnabel G. Effects of chemical anoxia on adrenergic responses of goldfish hepatocytes and the contribution of ?- and ?-adrenoceptors. ACTA ACUST UNITED AC 2002. [DOI: 10.1002/jez.10048] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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Fabbri E, Selva C, Moon TW, Capuzzo A. Characterization of [3H]CGP 12177 binding to beta-adrenergic receptors in intact eel hepatocytes. Gen Comp Endocrinol 2001; 121:223-31. [PMID: 11254364 DOI: 10.1006/gcen.2000.7591] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The aim of this study was to characterize [3H]CGP 12177 (CGP) binding to beta-adrenergic receptors in isolated hepatocytes of the European eel (Anguilla anguilla), in which the involvement of cAMP in epinephrine-induced glucose release has been previously observed. Specific binding of CGP was saturable, reversible, and linear as a function of cell number. Analysis of binding data suggested a single class of binding sites, with a Kd of 1.31 nM and a number of approximately 7000 beta-adrenergic receptors per cell. The potency order of specific inhibition of [3H]CGP binding was CGP > propranolol > or = alprenolol >> butoxamine > or = atenolol, while phentolamine and prazosin failed to significantly displace the tracer at concentrations up to 100 microM. The binding kinetics of CGP were closely related to its biological effect. In fact, the drug dose-dependently counteracted the enhancement of intracellular cAMP levels induced by epinephrine in isolated hepatocytes with a Kd of 1.06 nM. Moreover, it antagonized the hormone-induced stimulation of adenylyl cyclase activity in hepatic membranes as well as of glucose release from cells. These data clearly show that beta-adrenergic receptors are coupled to the adenylyl cyclase/cAMP transduction pathway in eel liver.
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Affiliation(s)
- E Fabbri
- Department of Biology, University of Bologna, Bologna, 40100, Italy
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Krumschnabel G, Manzl C, Schwarzbaum PJ. Metabolic responses to epinephrine stimulation in goldfish hepatocytes: evidence for the presence of alpha-adrenoceptors. Gen Comp Endocrinol 2001; 121:205-13. [PMID: 11178886 DOI: 10.1006/gcen.2000.7587] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The effect of epinephrine on various aspects of cellular metabolism was studied in hepatocytes from the goldfish Carassius auratus. Epinephrine increased cytosolic free calcium ([Ca2+](i)) from a baseline value of 108 +/- 22 nM to a peak value of 577 +/- 127 nM in suspensions of hepatocytes. Responses of single cells ranged from a single spike (66% of hepatocytes) to variable oscillatory patterns (34%). The increase in [Ca(2+)](i) was independent of the presence of extracellular Ca2+ and was prevented by the alpha-adrenergic antagonist phentolamine. Cellular glucose release induced by epinephrine (1.7- to 3.2-fold) was significantly reduced in Ca2+-depleted cells and in the presence of phentolamine, providing evidence for the co-occurrence of alpha-adrenoceptors and a Ca2+-independent, presumably beta-adrenergic, system in these cells. Furthermore, epinephrine stimulated oxygen consumption in a Ca2+-dependent manner, which was not due to stimulated Na(+) pump activity. An increased rate of acid secretion of 50%, evoked by epinephrine, appears to be mediated by enhanced Na(+)/H(+) exchange but did not result in intracellular alkalization.
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Affiliation(s)
- G Krumschnabel
- Institut für Zoologie und Limnologie, Abteilung für Okophysiologie, Universität Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria.
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