Abstract
Chromaffin cells were isolated from the posterior cardinal vein of rainbow trout (Oncorhynchus mykiss) to assess their suitability as a model system for studying mechanisms of catecholamine secretion in fish and to evaluate intracellular calcium changes associated with cholinoreceptor stimulation. Immunocytochemistry in concert with fluorescence microscopy was employed to identify characteristic chromaffin cell proteins and thus to confirm the presence of these specific cells in suspensions and cultures. Dopamine-β-hydroxylase, an enzyme of the catecholamine-synthesising Blaschko pathway, was identified in cytoplasmic vesicles of the isolated chromaffin cells. The actin filament-severing protein, scinderin, was co-localized with actin in the sub-plasmalemmal membrane of these chromaffin cells. Intracellular calcium [Ca2+]i was measured in single chromaffin cells by microspectrofluorometry using the fluorescent dye Fura-2. Significant increases in [Ca2+]i were observed in chromaffin cells in response to depolarisation of the cell membrane by high concentrations of K+ or by the stimulation of the cell by the cholinergic receptor agonists, nicotine, acetylcholine or carbachol. The response to the reversible agonist, nicotine, was attenuated following addition of the nicotinic receptor blocker hexamethonium. Such attenuation, however, did not occur when hexamethonium was added after stimulation with the non-specific irreversible cholinergic agonist, carbachol. These results demonstrate the presence of functional cholinoreceptors, linked to intracellular calcium signalling, on isolated trout chromaffin cells and reveal the potential of these cells as a model system for studying aspects of catecholamine secretion in fish.
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Abbreviations
- BSA :
-
bovine serum albumin
- [Ca 2+]i :
-
intracellular free calcium
- DβH :
-
dopamine-β-hydroxylase
- FBS :
-
foetal bovine serum
- FITC :
-
fluorescein-5-isothiocyanate
- IP 3 :
-
inositol triphosphate
- PBS :
-
phosphate-buffered saline
- PNMT :
-
phenylethanolamine-N-methyltransferase
References
Augustine GJ, Neher E (1992) Calcium requirements for secretion in bovine chromaffin cells. J Physiol (Lond) 450: 247–271
Blair JB, Miller MR, Pack D, Barnes R, Teh SJ, Hinton DE (1990) Isolated trout liver cells: establishing short-term primary cultures exhibiting cell-to-cell interactions. In Vitro Cell Dev Biol 26: 237–249
Burgoyne RD, Morgan A, O'Sullivan AJ (1989) The control of cytoskeletal actin and exocytosis in intact and permeabilized adrenal chromaffin cells: role of calcium and protein kinase C. Cell Signal 1: 323–334
Burgoyne RD, Morgan A, Robinson I, Pender N, Cheek TR (1993) Exocytosis in adrenal chromaffin cells. J Anat 183: 309–314
Castro E, Tomé AR, Miras-Portugal MT, Rosario LM (1994) Single-cell fura-2 microfluorometry reveals different purinoceptor subtypes coupled to Ca2+ influx and intracellular Ca2+ release in bovine adrenal chromaffin and endothelial cells. Pflügers Arch 426: 524–533
Cheek TR, Burgoyne RD (1985) Effect of activation of muscarinic receptors on intracellular free calcium and secretion in bovine adrenal chromaffin cells. Biochim Biophys Acta 846: 167–173
Fritsche R, Reid SG, Thomas S, Perry SF (1993) Serotonin-mediated release of catecholamines in the rainbow troutOncorhynchus mykiss. J Exp Biol 178: 191–204
Gamperl AK, Vijayan MM, Boutilier RG (1994) Experimental control of stress hormone levels in fishes: techniques and applications. Rev Fish Biol Fish 4: 215–255
Gfell B, Kloas W, Hanke W (1995) Regulation of adrenal secretion of the common carp (Cyprinus carpio) by cholinergic receptors. Verh Dtsch Zool Ges 88: 102
Kao L-S, Schneider AS (1985) Muscarinic receptors on bovine chromaffin cells mediate a rise in intracellular calcium that is independent of extracellular calcium. J Biol Chem 260: 2019–2022
Knight DE, Baker PF (1986) Observations on the muscarinic activation of catecholamine secretion in the chicken adrenal. Neuroscience 19: 357–366
Mastrolia L, Gallo VP, La Marca A (1984) The adrenal chromaffin cells ofSalmo gairdneri Richardson (Teleostei, Salmonidae). J Anat 138:503–511
Nakano T, Tomlinson N (1967) Catecholamine and carbohydrate concentrations in rainbow trout (Salmo gairdneri) in relation to physical disturbance. J Fish Res Bd Canada 24:1701–1715
Nandi J (1961) New arrangement of the interrenal and chromaffin tissues of teleost fishes. Science 134: 389–390
Nilsson S, Abrahamsson T, Grove DJ (1976) Sympathetic nervous control of adrenaline release from the head kidney of the cod,Gadus morhua. Comp Biochem Physiol 55C: 123–127
Nilsson S (1983) Autonomic nerve function in the vertebrates. Zoophysiology vol 13. Springer, Berlin Heidelberg New York
Nilsson S (1984) Adrenergic control systems in fish. Mar Biol Lett 5: 127–146
O'Sullivan AJ, Cheek TR, Moreton RB, Berridge MJ, Burgoyne RD (1989) Localization and heterogeneity of agonist-induced calcium concentration in single bovine adrenal chromaffin cells from video imaging of fura-2. EMBO 8: 401–411
Perry SF, Kinkead R (1989) The role of catecholamines in regulating arterial oxygen content during acute hypercapnic acidosis in rainbow trout (Salmo gairdneri). Respir Physiol 77: 365–378
Perry SF, Reid SG (1994) The effects of acclimation temperature on the dynamics of catecholamine release during acute hypoxia in the rainbow troutOncorhynchus mykiss. J Exp Biol 186: 289–307
Perry SF, Kinkead R, Gallaugher P, Randall DJ (1989) Evidence that hypoxemia promotes catecholamine release during hypercapnic acidosis in rainbow trout (Salmo gairdneri). Respir Physiol 77: 2961–2970
Perry SF, Fritsche R, Kinkead R, Nilsson S (1991) Control of catecholamine release in vivo and in situ in the Atlantic cod (Gadus morhua) during hypoxia. J Exp Biol 155: 549–566
Randall DJ, Perry SF (1992) Catecholamines. In: Randall DJ, Hoar WS (eds) Fish physiology, vol XIIB. The cardiovascular system. Academic Press, New York, pp 255–300
Reid SG, Perry SF (1994) Storage and differential release of catecholamines in rainbow trout (Oncorhynchus mykiss) and American eel (Anguilla rostrata). Physiol Zool 67: 216–237
Reid SG, Perry SF (1995) Cholinoceptor-mediated control of catecholamine release from chromaffin cells in the American eel,Anguilla rostrata. J Comp Physiol B 165: 464–470
Reid SG, Fritsche R, Jönsson A-C (1995) Immunohistochemical localisation of bioactive peptides and amines associated with the chromaffin tissue of five species of fish. Cell Tissue Res 280: 499–512
Ristori MT, Laurent P (1989) Plasma catecholamines in rainbow trout (Salmo gairdneri) during hypoxia. Exp Biol 48: 285–290
Santer, RM (1994) Chromaffin systems. In: Nilsson S, Holmgren S (eds) Comparative physiology and evolution of the autonomic nervous system. Harwood, USA, pp 97–117
Schreck, CB (1990) Physiological, behavioral, and performance indicators of stress. Am Fish Soc Symp 8: 29–37
Trifaro J-M (1982) The cultured chromaffin cell: a model for the study of biology and pharmacology of paraneurons. Trends Pharmacol Sci 3: 389–392
Trifaro J-M, Vitale ML, Rodriguez del Castillo A (1993) Scinderin and chromaffin cell actin network dynamics during neurotransmitter release. J Physiol (Paris) 87: 89–106
Xu Y, Duarte EP, Forsberg EK (1991) Calcium dependency of muscarinic and nicotinic agonist-induced ATP and catecholamine secretion from porcine adrenal chromaffin cells. J Neurochem 56: 1889–1896
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Communicated by H. Huddart
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Furimsky, M., Moon, T.W. & Perry, S.F. Calcium signalling in isolated single chromaffin cells of the rainbow trout (Oncorhynchus mykiss). J Comp Physiol B 166, 396–404 (1996). https://doi.org/10.1007/BF02336923
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DOI: https://doi.org/10.1007/BF02336923