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

Abstract

SUMMARY 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 dopamineβ-hydroxylase (DβH) 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 DβH-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 DβH 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 PAC1 receptor blocker PACAP 6-27 displayed normal labelling of VIP-binding sites. Finally, partial cDNA clones for the trout VPAC1 and VPAC2 receptor were obtained and sequenced. Tissue distribution experiments using RT-PCR revealed the presence of VPAC1 receptor mRNA but not that of the VPAC2 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.

Expression and in vitro regulation of pituitary adenylate cyclase-activating polypeptide (pacap38) and its type I receptor (pac1-r) in the gonads of tilapia (Oreochromis mossambicus)

“…The alignment of the amino acid sequences of tilapia PACAP 38 and PAC 1 -r show a high degree of evolutionary conservation with mammals, birds, other fish, and amphibians. Vip and PACAP type I receptor family members (90-100% similarities and 82-100% identities; Tables 1 and 2) show similar results and are reported in previous studies on carp, catfish, fugu, goldfish, rainbow trout, salmon, tilapia, and zebrafish Expression of pacap 38 and its type I receptor in tilapia gonads Expression of pacap 38 and its type I receptor in tilapia gonads (McRory et al 1995, Parker et al 1997, Wong et al 1998, 2000, Fradinger & Sherwood 2000, Small & Nonneman 2001, Montpetit et al 2003, Wang et al 2003, Cardoso et al 2004, Kwok et al 2006, Lo et al 2007, Sze et al 2007). This indicates a close relationship between teleosts and other vertebrates.…”

Differential expression pattern of pituitary adenylate cyclase-activating polypeptide (PACAP) alternative splicing variants and its receptors in the immune system of rainbow trout (Oncorhynchus mykiss)

“…To date, PACAP receptor expression in the brain of vertebrates is well documented (reviewed in [5,40]) and in fish, in concordance with our results, the expression of PAC1 receptor has been detected in the brain of goldfish (Carassius auratus) [41], zebrafish (Danio rerio) [42], sea bream (Sparus auratus) [43] and tilapia (Oreochromisis mossambicus) [44]. In addition, the expression of the VPAC1 and VPAC2-like transcripts in the brain of the rainbow trout (O. mykiss) was recently reported [22].…”

“…Recently, we have reported for the first time PACAP immunoreactivity and mRNA expression in isolated peripheral blood leucocytes from African catfish (Clarias gariepinus) and Nile tilapia (Orechromis niloticus) [21]. In rainbow trout (Oncorhynchus mykiss), the expression of VPAC1 and VPAC2 had been verified by conventional PCR in brain, spleen, liver and gut [22], although not in relation to PAC1. Taking into account all this information, the aim of this study is to characterize the expression of both PACAP alternative splicing variants (PRP/PACAP and PACAP) and its receptors in different lymphoid organs of the rainbow trout.…”

Ventilatory and cardiovascular actions of centrally and peripherally administered trout pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in the unanaesthetized trout

“…In mammals, PACAP and VIP generally act on one of three different G-proteincoupled receptors -the PACAP-preferring PAC1 receptor or the VPAC1 and VPAC2 receptors that exhibit a similar high affinity for PACAP and VIP (Laburthe et al, 2007). PAC1 and VPAC receptors were cloned from brain and peripheral tissues of goldfish (Wong et al, 1998) and rainbow trout (Montpetit et al, 2003). In these two species, the receptors share relatively high sequence similarity with their mammalian counterparts.…”

“…PACAP and VIP appeared very early during evolution and the primary structure of these peptides and their receptors has been remarkably well conserved from fish to mammals (Wong et al, 1998;Sherwood et al, 2000;Montpetit et al, 2003). Within the central nervous system of teleosts, PACAP-and VIP-like immunoreactivity are localized mainly in neuronal perikarya of the diencephalon at the level of the preoptic nucleus (NPO) with their fibers projecting both into the adenohypophysis (Matsuda et al, 1997;Montero et al, 1998;Wong et al, 1998;Mathieu et al, 2001) and towards many other hypothalamo-hypophysial areas (Montero et al, 1998;Mathieu et al, 2001;Matsuda et al, 2005).…”