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

Abstract

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.