187 Adrenergic regulation of catecholamine secretion from trout (Oncorhynchus mykiss) chromaffin cells C J Montpetit and S F Perry Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario K1N 6N5, Canada (Requests for offprints should be addressed to S F Perry; Email: [email protected]) Abstract The interaction between extracellular catecholamines and without effect on baseline catecholamine secretion or catecholamine secretion from chromaffin cells was assessed stimulus-evoked secretion. In contrast, pre-treatment with in rainbow trout (Oncorhynchus mykiss) using an in situ salbutamol significantly inhibited catecholamine secretion saline-perfused posterior cardinal vein preparation. This in response to carbachol or electrical stimulation. Pre- was accomplished by comparing the effects of adrenergic treatment with clonidine did not affect carbachol-evoked receptor agonists and antagonists on stimulus-evoked secretion but did reduce catecholamine secretion during secretion. An acute bolus injection or extended perfusion electrical stimulation. The significance of this adrenergic with saline containing high levels of either noradrenaline mechanism of regulating stimulus-evoked catecholamine or adrenaline did not affect the baseline secretion of secretion was further established using the adrenergic catecholamines. However, catecholamine secretion in receptor antagonists nadolol () or phentolamine (). response to a bolus injection of the general cholinergic Catecholamine secretion in response to cholinergic stimu- receptor agonist carbachol or electrical stimulation of the lation was significantly enhanced in preparations perfused nerves innervating the chromaffin cells was abolished or with saline containing nadolol. Furthermore, pre- reduced respectively, in preparations perfused with saline treatment with phentolamine significantly enhanced containing either catecholamine. adrenaline secretion in response to neuronal stimulation. To characterize the catecholaminergic inhibition of These results suggest that the mechanisms of adrenergic catecholamine release, secretion in response to carbachol inhibition of catecholamine secretion from trout and electrical stimulation was compared in preparations chromaffin cells include activation of chromaffin cell perfused with the adrenergic receptor agonists dobutamine membrane 2-receptors and presynaptic 2-adrenergic (1), salbutamol (2), phenylephrine (1) or clonidine (2). receptors. Prior treatment with dobutamine or phenylephrine was Journal of Endocrinology (2002) 173, 187–197 Introduction investigated the impact of catecholamines themselves on the release of catecholamines from chromaffin cells. In teleost fish, the catecholamines (adrenaline and Chromaffin cells, like adrenergic neurons, are derived noradrenaline) that enter the circulation are synthesized, from the neural crest during development and share stored and released from chromaffin tissue that lines the several properties, including synthesis, storage and release walls of the posterior cardinal vein (PCV) (Reid et al. of catecholamines (Nilsson 1983). In the adrenergic 1998). Under adverse conditions including exhaustive neurons of peripheral and central nerves, specific pre- exercise, hypoxia, hypercarbia and physical disturbances synaptic inhibition of neurotransmitter release has been (Randall & Perry 1992), the rise in plasma catecholamine well documented in mammals. For example, it has been levels modulates various physiological responses that serve reported frequently that -adrenoceptor agonists and to enhance cardio-respiratory and metabolic functions antagonists exert powerful influences on stimulus-evoked (Wendelaar Bonga 1997, Reid et al. 1998, Perry & release of noradrenaline from nerve terminals (Starke et al. Gilmour 1999). The control of chromaffin cell activity and 1974b, Langer et al. 1977, Nilsson 1983). Although catecholamine secretion involves neuronal signals derived controversial, similar interactions have been described in from cholinergic and non-cholinergic neurotransmitters, mammalian chromaffin cells leading investigators to sug- and humoral signals of endocrine origin (Reid et al. 1998). gest an autocrine/paracrine regulation of catecholamine While numerous investigations have studied the regulation release via activation of chromaffin cell adrenergic recep- of the adrenergic stress response in fish, few studies have tors (Gutman & Boonyaviroj 1977, Boonyaviroj et al. Journal of Endocrinology (2002) 173, 187–197 Online version via http://www.endocrinology.org 0022–0795/02/0173–187 2002 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/30/2021 02:00:39AM via free access 188 C J MONTPETIT and S F PERRY · Adrenergic control of catecholamine release in trout 1979, Cohen et al. 1980, Greenberg & Zinder 1982, Wada ventricle was cannulated (Clay-Adams PE160 poly- et al. 1982, Powis & Baker 1985, Sharma et al. 1986, Orts ethylene tubing; internal diameter=1·14 mm, outer et al. 1987). diameter=1·57 mm; VWR, CanLab, Mississauga, ON, Studies designed to investigate the effects of catechol- Canada) by making an incision in the bulbus and inserting amines on catecholamine release from piscine chromaffin the tube through the bulbus into the ventricle. The cells have produced conflicting results. In lamprey (Petro- cannula was secured with a ligature between the two myzon marinus) and American eel (Anguilla anguilla), chambers. This cannula served as the outflow in the administration of adrenaline or noradrenaline in vivo causes perfusion system. The PCV was also cannulated (PE160 the elevation of plasma catecholamines (both adrenaline tubing) in the anterograde direction and served as the and noradrenaline) (Dashow & Epple 1983, Hathaway inflow cannula (Fritsche et al. 1993). Each preparation was et al. 1989). Furthermore, pre-treatment with -or perfused for 20 min with aerated control saline -adrenergic receptor antagonists decreases catecholamine (125 mmol/l NaCl, 2·0 mmol/l KCl, 2·0 mmol/l MgSO4, release in the eel in response to neuronal or cholinergic 5·0 mmol/l NaHCO3, 7·5 mmol/l glucose, 2·0 mmol/l ffi stimulation of the chroma n tissue in situ or in vitro CaCl2 and 1·25 mmol/l KH2PO4, final pH 7·8) to allow (Al-Kharrat et al. 1997, Abele et al. 1998). In contrast to stabilization of catecholamine secretion. Perfusion was eels and lampreys, continuous infusion of adrenaline in vivo accomplished by siphon resulting from a positive pressure in rainbow trout (Oncorhynchus mykiss)doesnotaffect the difference (14·7 mmHg; 1·96 kPa) between the surface of levels of plasma noradrenaline (Perry & Vermette 1987). the saline and the outflow cannula, which resulted in a Moreover, in Atlantic cod (Gadus morhua), the addition of flow rate of approximately 1·5 ml/min. high levels of a particular catecholamine to the inflowing After the stabilization period, preparations were pro- perfusion fluid of an in situ preparation inhibits the cessed according to the protocols described below. cholinergic-elicited release of that particular catechol- Agonists were administered by bolus injection through amine (Perry et al. 1991). Thus, there appear to be a three-way valve connected to the inflow catheter. species-dependent differences in the response of piscine Alternatively, agonists and antagonists were delivered chromaffin cells to extracellular catecholamines. To date, a continuously via the perfusion fluid by acutely switching thorough analysis of the response of trout chromaffin cells the perfusion media from one container (control saline) to to catecholamines has not yet been performed. Thus, the another container (control saline or saline containing goal of this investigation was to first assess the impact of adrenergic agents) using a three-way valve. extracellular catecholamines on catecholamine secretion For neuronal stimulation, fish were electrically stimu- from rainbow trout chromaffin cells and, secondly, to use lated using a previously validated field stimulation tech- a pharmacological approach to identify the underlying nique (Montpetit & Perry 1999). The specificity of the mechanisms. field stimulation technique was confirmed (Montpetit & Perry 1999) by demonstrating that the electrically evoked secretion of catecholamines was reduced in the presence of Materials and Methods the nicotinic receptor antagonist hexamethonium, and prolonged in the presence of the cholinesterase inhibitor Experimental animals neostigmine. Further, the absence of a response during field stimulation in preparations perfused with Na+-free Rainbow trout (Oncorhynchus mykiss) of either sex were saline or from which the spinal cord was removed indi- obtained from Linwood Acres Trout Farm (Campbellcroft, cated that the electrical current was unable to elicit Ontario, Canada) and held in large fiberglass tanks sup- sufficient chromaffin cell membrane depolarization to plied with dechlorinated city of Ottawa tap water main- initiate catecholamine secretion. Together, these results tained at 13 C. They were allowed to acclimate to the indicated that catecholamine secretion elicited by field aquaria for at least 3 weeks before experimentation. Fish stimulation was neuronally mediated. As such, a pair of were maintained on a 12 h light:12 h darkness photo- electrodes, connected to a Grass SD-9 stimulator (Quincy, period and fed daily to satiation with a commercial fish MA, USA), was sutured to the body wall on either side of diet. the fish in the anterior region of the PCV. Electrical stimulation was carried out at 60 V, 1 ms in