The fish endocrine pancreas: review, new data, and future research directions in ontogeny and phylogeny.

The literature on the ontogeny and phylogeny of the endocrine pancreas of ray-finned fishes is summarized since the latest review in fish [Youson, J.H., Al-Mahrouki, A.A., 1999. Review. Ontogenetic and phylogenetic development of the endocrine pancreas (islet organ) in fishes. Gen. Comp. Endocrinol. 116, 303-335]. A basic description and a demonstration of the diversity of the fish islet organ is provided through new immunohistochemical data on islet tissue from a basal teleost, an osteoglossomorph, and a more derived teleost, a perciforme. Unlike the previous review, the present report provides a review and discussion of the utility of sequence data of insulin, somatostatin, and NPY- and glucagon-family peptides in phylogenetic analyses of jawed and jawless fishes. The present study also provides the first comparative analysis of sequences of preprohormones of endocrine peptides from closely related basal teleost species. Some nucleotide and deduced amino acid sequence data for preprosomatostatins (PPSS-I and/or -II) are compared for four species of bonytongues, Osteoglossomorpha, and with PPSSs of the white sucker, Catostomus commersoni, representing Cypriniformes, a more generalized teleost order. Phylogenetic analysis of deduced amino acid sequences of the PPSSs of these species and others from databases indicates good support for the monophyly of Osteoglossomorpha and some support for the present taxonomic grouping of the osteoglossomorphs examined, and also the white sucker. However, PPSS may have limited phylogenetic utility due to the relative short sequence, particularly in resolving relationships among lineages that diverged over a short period of time. Since in the few fish species examined we have just touched the surface in describing the diversity of structure of the islet organ, and likely the nature of the products of its cells, this report promotes the continued study of this organ.

Adrenergic regulation of catecholamine secretion from trout (Oncorhynchus mykiss) chromaffin cells.

The interaction between extracellular catecholamines and catecholamine secretion from chromaffin cells was assessed in rainbow trout (Oncorhynchus mykiss) using an in situ saline-perfused posterior cardinal vein preparation. This was accomplished by comparing the effects of adrenergic receptor agonists and antagonists on stimulus-evoked secretion. An acute bolus injection or extended perfusion with saline containing high levels of either noradrenaline or adrenaline did not affect the baseline secretion of catecholamines. However, catecholamine secretion in response to a bolus injection of the general cholinergic receptor agonist carbachol or electrical stimulation of the nerves innervating the chromaffin cells was abolished or reduced respectively, in preparations perfused with saline containing either catecholamine. To characterize the catecholaminergic inhibition of catecholamine release, secretion in response to carbachol and electrical stimulation was compared in preparations perfused with the adrenergic receptor agonists dobutamine (beta(1)), salbutamol (beta(2)), phenylephrine (alpha(1)) or clonidine (alpha(2)). Prior treatment with dobutamine or phenylephrine was without effect on baseline catecholamine secretion or stimulus-evoked secretion. In contrast, pre-treatment with salbutamol significantly inhibited catecholamine secretion in response to carbachol or electrical stimulation. Pre-treatment with clonidine did not affect carbachol-evoked secretion but did reduce catecholamine secretion during electrical stimulation. The significance of this adrenergic mechanism of regulating stimulus-evoked catecholamine secretion was further established using the adrenergic receptor antagonists nadolol (beta) or phentolamine (alpha). Catecholamine secretion in response to cholinergic stimulation was significantly enhanced in preparations perfused with saline containing nadolol. Furthermore, pre-treatment with phentolamine significantly enhanced adrenaline secretion in response to neuronal stimulation. These results suggest that the mechanisms of adrenergic inhibition of catecholamine secretion from trout chromaffin cells include activation of chromaffin cell membrane beta(2)-receptors and presynaptic alpha(2)-adrenergic receptors.

The effects of C-type natriuretic peptide on catecholamine release in the pacific spiny dogfish (Squalus acanthias).

The interaction between homologous C-type natriuretic peptide (dfCNP) and catecholamine release in cardiovascular control was assessed in the marine dogfish (Squalus acanthias). This was accomplished by evaluation of the dynamics of the dfCNP-elicited secretion of catecholamines in situ and in vivo. With an in situ saline-perfused postcardinal sinus preparation, it was demonstrated that perfusion with saline containing dfCNP (10(-9) mol x L(-1)) did not affect the secretion of either noradrenaline or adrenaline. However, the presence of dfCNP in the perfusate significantly enhanced carbachol-evoked secretion of noradrenaline. In vivo, intravascular injection of dfCNP (10(-9) mol x kg(-1)) caused a biphasic pressor-depressor response consisting of a brief increase in caudal artery blood pressure (P(CA)) followed by a prolonged reduction in P(CA). Furthermore, although systemic resistance initially increased, it was subsequently maintained at baseline values in the face of persistent decreases in both P(CA) and cardiac output. Bolus injection of dfCNP elicited significant increases in plasma noradrenaline levels that peaked within 10 min; plasma adrenaline levels were unaffected. The release of noradrenaline elicited by dfCNP was unaffected by prior blockade of the renin-angiotensin system (RAS) (with the angiotensin converting enzyme inhibitor lisinopril) or by pretreatment with the nicotinic receptor blocker hexamethonium. The delayed decrease in P(CA) was not observed in the hexamethonium-treated fish. Prior blockade of beta-adrenoreceptors (with sotalol) or alpha-adrenoreceptors (with prazosin) either significantly reduced (sotalol) or abolished (prazosin) the increase in plasma noradrenaline levels after dfCNP injection. The results of this investigation demonstrate that the elevation of plasma noradrenaline levels observed in vivo following dfCNP injection is not caused by a direct effect of dfCNP on catecholamine secretion from axillary body chromaffin cells. Furthermore, the dfCNP-mediated increase of plasma noradrenaline appears to be unrelated to changes in P(CA) and is insensitive to blockade of the RAS or nicotinic receptors. However, stimulation of adrenergic receptors, in particular the alpha-adrenoreceptors, appears to be a key mechanism underlying the dfCNP-elicited secretion of noradrenaline.

The effects of endothelin-1 on the cardiorespiratory physiology of the freshwater trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias).

The aim of the present study was to evaluate the effects of endothelin-l-elicited cardiovascular events on respiratory gas transfer in the freshwater rainbow trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias). In both species, endothelin-1 (666 pmol kg(-1)) caused a rapid (within 4 min) reduction (ca. 30-50 mmHg) in arterial blood partial pressure of O2. The effects of endothelin-1 on arterial blood partial pressure of CO2 were not synchronised with the changes in O2 partial pressure and the responses were markedly different in trout and dogfish. In trout, arterial CO2 partial pressure was increased transiently by approximately 1.0 mmHg but the onset of the response was delayed and occurred 12 min after endothelin-1 injection. In contrast, CO2 partial pressure remained more-or-less constant in dogfish after injection of endothelin-1 and was increased only slightly (approximately 0.1 mmHg) after 60 min. Pre-treatment of trout with bovine carbonic anhydrase (5 mg ml(-1)) eliminated the increase in CO2 partial pressure that was normally observed after endothelin-1 injection. In both species, endothelin-1 injection caused a decrease in arterial blood pH that mirrored the changes in CO2 partial pressure. Endothelin-1 injection was associated with transient (trout) or persistent (dogfish) hyperventilation as indicated by pronounced increases in breathing frequency and amplitude. In trout, arterial blood pressure remained constant or was decreased slightly and was accompanied by a transient increase in systemic resistance, and a temporary reduction in cardiac output. The decrease in cardiac output was caused solely by a reduction in cardiac frequency; cardiac stroke volume was unaffected. In dogfish, arterial blood pressure was lowered by approximately 10 mmHg at 6-10 min after endothelin-1 injection but then was rapidly restored to pre-injection levels. The decrease in arterial blood pressure reflected an increase in branchial vascular resistance (as determined using in situ perfused gill preparations) that was accompanied by simultaneous decreases in systemic resistance and cardiac output. Cardiac frequency and stroke volume were reduced by endothelin-1 injection and thus both variables contributed to the changes in cardiac output. We conclude that the net consequences of endothelin-1 on arterial blood gases result from the opposing effects of reduced gill functional surface area (caused by vasoconstriction) and an increase in blood residence time within the gill (caused by decreased cardiac output.

Vasoactive intestinal polypeptide- and pituitary adenylate cyclase activating polypeptide-mediated control of catecholamine release from chromaffin tissue in the rainbow trout, Oncorhynchus mykiss.

The aim of the present investigation was to assess the relative contributions of cholinergic (acetylcholine) and non-cholinergic vasoactive intestinal polypeptide (VIP), and pituitary adenylate cyclase activating polypeptide (PACAP) neurotransmitters in the neuronal control of catecholamine secretion from the chromaffin tissue lining the posterior cardinal vein of the rainbow trout (Oncorhynchus mykiss). Using an in situ saline-perfused posterior cardinal vein preparation, it was demonstrated that exogenous administration of chicken VIP or human PACAP-27 caused a dose-dependent increase in adrenaline secretion; noradrenaline secretion was unaffected. Analysis of dose-response curves indicated that VIP and PACAP stimulated the secretion of adrenaline with a similar degree of potency (ED(50) for VIP=1.90x10(-11) mol/kg; ED(50) for PACAP=1.03x10(-11) mol/kg). The VIP/PACAP-elicited secretion was diminished in the presence of the VIP receptor antagonist, VIP 6-28, but was unaffected by the PACAP receptor antagonist, PACAP 6-27, or the cholinergic antagonists, hexamethonium and atropine. Thus, this is the first study to demonstrate a direct stimulatory role for VIP or PACAP in catecholamine secretion from piscine chromaffin cells. The relative contribution of cholinergic and non-cholinergic neurotransmitters in the neuronal control of catecholamine secretion from the chromaffin tissue was evaluated using an in situ nerve-stimulating technique previously validated by us in the rainbow trout. This was accomplished by comparing catecholamine secretion in the presence or absence of cholinergic and the VIP and PACAP receptor antagonists during different levels of electrical stimulation. The results demonstrated that cholinergic stimulation predominated during high frequency of electrical stimulation (20 Hz) while the non-cholinergic component prevailed at low frequency (1 Hz). Overall, the results of the present investigation demonstrate that VIP and/or PACAP may directly stimulate adrenaline secretion from trout chromaffin cells at low levels of neuronal activity. Therefore, the neuronal control of catecholamine secretion in teleosts may not be confined to cholinergic-evoked events.

Desensitisation of chromaffin cell nicotinic receptors does not impede catecholamine secretion during acute hypoxia in rainbow trout (Oncorhynchus mykiss).

Experiments were performed on adult rainbow trout (Oncorhynchus mykiss) in vivo using chronically cannulated fish and in situ using a perfused posterior cardinal vein preparation (i) to characterise the desensitisation of chromaffin cell nicotinic receptors and (ii) to assess the ability of fish to secrete catecholamines during acute hypoxia with or without functional nicotinic receptors. Intra-arterial injection of nicotine (6.0x10(-)(7 )mol kg(-)(1)) caused a rapid increase in plasma adrenaline and noradrenaline levels; the magnitude of this response was unaffected by an injection of nicotine given 60 min earlier. Evidence for nicotinic receptor desensitisation, however, was provided during continuous intravenous infusion of nicotine (1.3x10(-)(5 )mol kg(-)(1 )h(-)(1)) in which plasma catecholamine levels increased initially but then returned to baseline levels. To ensure that the decline in circulating catecholamine concentrations during continuous nicotine infusion was not related to changes in storage levels or altered rates of degradation/clearance, in situ posterior cardinal vein preparations were derived from fish previously experiencing 60 min of saline or nicotine infusion. Confirmation of nicotinic receptor desensitisation was provided by demonstrating that the preparations derived from nicotine-infused fish were unresponsive to nicotine (10(-)(5 )mol l(-)(1)), yet remained responsive to angiotensin II (500 pmol kg(-)(1)). The in situ experiments demonstrated that desensitisation of the nicotinic receptor occurred within 5 min of receptor stimulation and that resensitisation was established 40 min later. The ability to elevate plasma catecholamine levels during acute hypoxia (40-45 mmHg; 5.3-6.0 kPa) was not impaired in fish experiencing nicotinic receptor desensitisation. Indeed, peak plasma adrenaline levels were significantly higher in the desensitised fish during hypoxia than in controls (263+/-86 versus 69+/-26 nmol l(-)(1); means +/- s.e.m., N=6-9). Thus, the results of the present study demonstrate that activation of preganglionic sympathetic cholinergic nerve fibres and the resultant stimulation of nicotinic receptors is not the sole mechanism for eliciting catecholamine secretion during hypoxia.

The effects of acute hypoxia on chemically or neuronally induced catecholamine secretion in rainbow trout (Oncorhynchus mykiss) in situ and in vivo.

The potential direct and modulating effects of acute hypoxia on catecholamine secretion in rainbow trout (Oncorhynchus mykiss) were assessed in situ, using a perfused cardinal vein preparation, and in vivo, using chronically cannulated fish. Acute (10 min) perfusion with hypoxic (P(O2)<10 mmHg) saline or homologous hypoxic blood did not have a statistically significant effect on basal (non-stimulated) catecholamine secretion. A field stimulation technique was used to excite the sympathetic nerves innervating the chromaffin cells electrically in situ under conditions of high-P(O2) (saline P(O2)=152 mmHg; 1 mmHg=0.133 kPa) or low-P(O2) (saline P(O2)<10 mmHg) perfusion at constant P(CO2) (2.3 mmHg). The results demonstrated that neuronally evoked catecholamine secretion was significantly lowered by 50 % during perfusion with hypoxic saline. To assess whether the inhibitory effect of hypoxia during neuronal stimulation in situ resulted from modulation of nicotinic and/or muscarinic receptor-linked pathways, perfused posterior cardinal vein preparations were injected with selective nicotinic (10(-)(7) or 10(-)(6 )mol kg(-)(1) nicotine) or muscarinic (10(-)(3 )mol kg(-)(1) methacholine) receptor agonists. For both doses of nicotine, catecholamine secretion was significantly lowered during hypoxia by 55 %. During muscarinic receptor stimulation, perfusion with hypoxic saline caused a 42 % reduction in the rate of catecholamine secretion. In contrast, catecholamine secretion elicited by depolarising levels of KCl (60 mmol l(-)(1)) was unaffected by the oxygen status of the perfusate. In vivo, intra-arterial injections of nicotine (300-600 nmol kg(-)(1)) into normoxic (water P(O2)=155 mmHg) or moderately hypoxic fish (water P(O2)=80 mmHg) caused a dose-dependent elevation of circulating catecholamine levels. However, despite the inhibitory influence of localised hypoxia on chromaffin cell responsiveness previously demonstrated in situ, the increase in plasma catecholamine levels after intra-arterial injection of nicotine was significantly enhanced in the hypoxic fish. The differences between the results from the in vivo and in situ experiments may reflect the contribution of higher control centres and modulating factors in vivo that are absent in situ.

Neuronal control of catecholamine secretion from chromaffin cells in the rainbow trout (Oncorhynchus mykiss).

The goal of the present investigation was to assess the relative involvement of nicotinic and muscarinic cholinergic receptors in the neuronal control of catecholamine secretion from the chromaffin tissue of rainbow trout (Oncorhynchus mykiss). This was accomplished by first developing and validating a nerve-stimulating technique able specifically to activate the nerve fibres innervating the chromaffin cells in order to elicit secretion of catecholamines. Using an in situ saline-perfused posterior cardinal vein preparation, it was demonstrated that whole-body field stimulation caused specific voltage-dependent neuronal stimulation of adrenaline and noradrenaline secretion. The contribution of non-specific depolarization was negligible. Several experimental results confirmed the specificity of the field stimulation technique. First, pre-treatment with neostigmine (an anticholinesterase) prolonged and more than doubled the amount of adrenaline secreted in response to electrical stimulation. Second, pre-treatment with the nicotinic receptor antagonist hexamethonium inhibited the electrically evoked secretion of adrenaline and noradrenaline. Third, perfusion with Na+-free saline or removal of the spinal cord abolished secretion of both catecholamines in response to the electrical stimulus. By using the field stimulation technique, this study is the first to demonstrate conclusively a role for muscarinic receptors in catecholamine secretion from trout chromaffin cells. Specifically, muscarinic cholinergic stimulation enhances nicotinic-evoked secretion of catecholamines and, under intense stimulation, may directly cause secretion. The results of the present study suggest the presence of muscarinic receptors on rainbow trout chromaffin cells with a functional role in the cholinergic control of catecholamine secretion.

The effects of chronic hypoxia on the acute adrenergic stress response in the rainbow trout (Oncorhynchus mykiss).

We have investigated the effects of chronic hypoxia on the acute adrenergic stress response of adult rainbow trout (Oncorhynchus mykiss). The goal of this study was to determine whether a prior 5-d exposure of fish to lowered environmental oxygen levels (60 or 80 Torr) would influence the nature of catecholamine secretion from chromaffin tissue in situ. Using a saline-perfused posterior cardinal vein preparation, it was demonstrated that the basal (unstimulated) secretion of noradrenaline and adrenaline was increased at 60-Torr hypoxia. In response to cholinergic (carbachol-elicited) stimulation, noradrenaline and adrenaline secretion were significantly affected by prior exposure to hypoxia. The construction of dose response curves revealed that noradrenaline secretion was enhanced at the lowest doses of carbachol (1 - 5 x 10(-7) mol kg(-1)) and that this was reflected by an approximate 10-fold reduction in the ED50 (the dose of carbachol eliciting half-maximal noradrenaline secretion). The effect of chronic hypoxia on in situ carbachol-evoked adrenaline secretion was similar but less pronounced. The results of this study suggest that during chronic moderate hypoxia, increased basal catecholamine secretion and enhanced responsiveness of chromaffin cells to cholinergic stimulation, as well aiding the ongoing stress, may assist the physiological adaptations to subsequent bouts of more severe acute stress.