Effects of chronic dietary salt loading on the renin angiotensin and adrenergic systems of rainbow trout (Oncorhynchus mykiss).

Previous studies have demonstrated that chronic dietary salt loading causes hypertension and a decreased sensitivity of the systemic vasculature to α-adrenergic stimulation and other hypertensive stimuli (e.g. hypercapnia) in rainbow trout (Oncorhynchus mykiss). This reduced sensitivity to hypertensive stimuli is consistent with a possible blunting of homeostatic responses normally aimed at raising blood pressure. To test this idea, we examined the consequences of long-term salt feeding and the associated hypertension on the interactive capacities of the renin angiotensin system (RAS) and adrenergic systems to elevate blood pressure in trout. Secretion of catecholamines in response to a range of doses of homologous ANG II in vivo and in situ (using a perfused posterior cardinal vein preparation) was reduced in the salt-fed fish. The reduced sensitivity to ANG II could not be explained by alterations in stored catecholamine (adrenaline or noradrenaline) levels or the general responsiveness of the chromaffin cells to depolarizing stimuli (60 mmol/l KCl). Despite the decreased responsiveness of the chromaffin cells to ANG II, plasma catecholamines were increased to a greater extent in the salt-fed fish during acute hypoxia (a condition that activates the RAS). Interestingly, the pressor effects of ANG II in vivo were actually heightened in the salt-fed fish. The increased pressor response to exogenous ANG II was likely attributable to its direct interaction with vascular ANG II receptors because the effect persisted even after blockade of α-adrenergic receptors. Treating fish with the vascular smooth muscle relaxant papaverine caused similar reductions in blood pressure and increases in plasma ANG II levels regardless of diet. Similarly, inhibition of angiotensin converting enzyme with lisinopril reduced blood pressure equally in control and salt-fed fish. These results indicate that, while long-term dietary salt loading blunts the response of trout chromaffin cells to ANG II, the RAS itself appears to be unaffected. Indeed, the capacity of ANG II to elevate blood pressure is not compromised nor do fish exhibit a reduced capacity to mount an acute humoral adrenergic stress response during acute hypoxia.

Regulation of the black bullhead hepatic beta-adrenoceptors.

Black bullhead catfish (Ameiurus melas) were exposed to air for 1 h to examine the effect of an acute stress on the distribution and function of the hepatic beta-adrenoceptors (beta-ARs). Air exposure significantly reduced both adrenaline (ADR)- and noradrenaline (NADR)-stimulated glucose production in isolated hepatocytes with no effect on either receptor affinity (K(d)) or number of binding sites (B(max)). A 24 h exposure of isolated hepatocytes to the beta-agonist isoproterenol also had no significant impact on either binding parameter. Competition studies using selective agonists and antagonists suggest that the hepatic beta-AR in this species is pharmacologically beta(2)-like. However in addition to the beta(2)-AR, molecular evidence provides support for the existence of hepatic beta-ARs that phylogenetically group with the beta(3)-ARs and the beta(1)-ARs. Despite the presence of several potential phosphorylation sites in the third intracellular loop and cytoplasmic tail of the bullhead beta(2)-AR, no significant changes were observed in the binding parameters. While physiological data supports the presence of only a single subtype, molecular data supports the existence of multiple beta-AR subtypes in this species. The mechanisms thought to regulate mammalian beta-ARs exist in the bullhead ARs reported here but these mechanisms are not as effective in this fish system as in mammals.

Expression of neuropeptide Y family peptides in the brain and gut during stages of the life cycle of a parasitic lamprey (Petromyzon marinus) and a nonparasitic lamprey (Ichthyomyzon gagei).

The long-term objective of our research is to show that internal factors may be key to triggering metamorphosis and directing the life history types in lampreys (parasitism versus nonparasitism). Since neuropeptide Y family peptides are key players in the endocrine-mediated feeding and reproductive events in mammals, a role for these peptides in the control of feeding behavior and development can be predicted for lampreys. We have investigated the expression pattern of these peptides in the brain and in the gut during different stages of the life cycle of the parasitic lamprey, Petromyzon marinus, and the nonparasitic lamprey, Ichthyomyzon gagei. We provide a description of the cloning and sequencing of P. marinus and I. gagei cDNA for neuropeptide Y (NPY), peptide tyrosine-tyrosine (PYY), and peptide methionine-tyrosine (PMY). Using sequence-specific primers, the mRNA expression patterns for these peptides in brain and gut of larval (ammocoete) and adult (juvenile, prespawning) lampreys were examined by semiquantitative RT-PCR. The observations extend a potential role of neuropeptide Y family peptides in the modulation of feeding behavior and reproductive maturity in lampreys.

Catecholamine secretion in trout chromaffin cells experiencing nicotinic receptor desensitization is maintained by non-cholinergic neurotransmission.

The goal of the present study was to assess the catecholamine secretory capabilities of rainbow trout Oncorhynchus mykiss chromaffin cells experiencing desensitization of the nicotinic receptor. It was hypothesized that the potential to secrete catecholamines could be maintained under conditions of nicotinic receptor desensitization owing to activation of non-cholinergic release pathways. An in situ model for chromaffin cell nicotinic receptor desensitization was developed by perfusing a posterior cardinal vein preparation with saline containing 10(-5) mol l(-1) nicotine. Under such conditions of desensitization, the chromaffin cells were largely unresponsive to high-frequency (20 Hz) electrical stimulation; the minimal remaining secretory response was abolished by addition of the nicotinic receptor antagonist hexamethonium (10(-3) mol l(-1)). In marked contrast, however, the capacity to secrete catecholamines in response to low-frequency (1 Hz) electrical stimulation was unaffected by nicotinic receptor desensitization or by cholinergic receptor blockade (hexamethonium plus atropine). In preparations experiencing nicotinic receptor desensitization, the stimulatory effect of low-frequency (1 Hz) stimulation on catecholamine secretion was reduced by 43% in the presence of the VPAC receptor antagonist, VIP(6-28). The stimulatory effect of high-frequency (20 Hz) stimulation was unaffected by VIP(6-28). Catecholamine secretion evoked by cod VIP (10(-11) mol kg(-1)) and homologous angiotensin II ([Asn(1), Val(5)] Ang II; 5 x 10(-7) mol kg(-1)) was markedly enhanced (107 and 97%, respectively) in desensitized preparations. However, the secretory response to the muscarinic receptor agonist methylcholine (1 x 10(-3) mol kg(-1)) was unchanged by desensitization. The results of this study demonstrate that exploitation of non-cholinergic mechanisms, including peptidergic pathways activated during low-frequency neuronal stimulation, is a potential strategy whereby catecholamine secretion from trout chromaffin cells can be maintained during periods of nicotinic receptor desensitization.

Localisation of VIP-binding sites exhibiting properties of VPAC receptors in chromaffin cells of rainbow trout (Oncorhynchus mykiss).

The current model for the neuronal control of catecholamine release from piscine chromaffin cells advocates that the neurotransmitters vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are co-released with acetylcholine from preganglionic fibres upon nerve stimulation. Both VIP and PACAP elicit the secretion of exclusively adrenaline from rainbow trout chromaffin cells, which presumably arises from the activation of VPAC type receptors. Thus, the goals of the present study were (1) to localise VPAC receptors in the chromaffin cell fraction of the posterior cardinal vein (PCV) of trout and (2) to test the hypothesis that the selective secretion of adrenaline elicited by VIP could be explained by the absence of the VPAC receptors from the noradrenaline-containing cells. Fluorescent labelling of chromaffin cells using aldehyde-induced fluorescence of catecholamines and antisera raised against dopamine beta-hydroxylase (DbetaH) revealed a distinct layer of chromaffin cells lining the walls of the PCV. Furthermore, specific VIP-binding sites were demonstrated on chromaffin cells using a biotinylated VIP that was previously established as being bioactive. Although multiple labelling experiments revealed that a number of DbetaH-positive cells were immunonegative for phenylethanolamine N-methyl transferase (PNMT; noradrenaline-containing cells versus adrenaline-containing cells, respectively), labelling of VIP-binding sites was similar to that of DbetaH labelling, suggesting that all chromaffin cells possess VIP-binding sites. Pharmacological assessment of the VIP-binding sites indicated that they exhibited characteristics of VPAC receptors. Specifically, the labelling of VIP-binding sites was prevented after pre-treatment of PCV tissue sections with unlabelled VIP, PACAP or the specific VPAC receptor antagonist VIP 6-28. By contrast, sections pre-treated with the PAC(1) receptor blocker PACAP 6-27 displayed normal labelling of VIP-binding sites. Finally, partial cDNA clones for the trout VPAC(1) and VPAC(2) receptor were obtained and sequenced. Tissue distribution experiments using RT-PCR revealed the presence of VPAC(1) receptor mRNA but not that of the VPAC(2) receptor in the PCV tissue. The results provide direct evidence that VIP and PACAP can elicit the secretion of adrenaline from the chromaffin tissue via specific VIP-binding sites that exhibit properties of VPAC receptors. However, the selective secretion of adrenaline by VIP or PACAP cannot be explained by a lack of VIP-binding sites on the noradrenaline-containing cells.

Modulation of catecholamine release and cortisol secretion by social interactions in the rainbow trout, Oncorhynchus mykiss.

The objective of the present study was to investigate the effect of social status on the ability of rainbow trout to secrete the stress hormones, cortisol, and catecholamines. Rainbow trout were confined in pairs for six days to permit the formation of dominance hierarchies. An in situ saline-perfused posterior cardinal vein (PCV) preparation was then used to assess cortisol secretion or release of the catecholamine hormones, adrenaline and noradrenaline, in response to the inclusion of appropriate secretagogues in the perfusate. Fish identified as subordinate on the basis of their behaviour showed a characteristic elevation of circulating plasma cortisol concentrations when compared with dominant fish. When the interrenal cells were stimulated in situ with adrenocorticotropic hormone, subordinate fish displayed a significantly lower rate of cortisol secretion than dominant fish. However, social status had no significant effect on either adrenaline or noradrenaline secretion rates upon stimulation of the chromaffin cells in situ with acetylcholine. These results suggest that the chronic elevation of plasma cortisol associated with subordinate social status in rainbow trout reduces the sensitivity of the cortisol-secreting interrenal cells, presumably through negative feedback mechanisms.