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

Colin J. Montpetit, Arash Shahsavarani and Steve F. Perry*

Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario K1N 6N5, Canada



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  		Fig. 1. Identification and localisation of chromaffin cells within the posterior cardinal vein (PCV) tissue of rainbow trout based on the double labelling of (A) nucleic acids and (B) catecholamines or the double labelling of (D) dopamine ß-hydroxylase (DßH) and (E) catecholamines. Panel C is an overlay image of the sections shown in A and B (nucleic acids and catecholamines appear in red and green/yellow, respectively). Panel F is an overlay image of the sections shown in D and E (DßH and catecholamines appear in red and green, respectively; yellow indicates co-localisation of catecholamines and DßH). Note that the labelling patterns for DßH and catecholamines are identical. Scale bars D–F, 100 µm; scale bars A–C, 50 µm.

 


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  		Fig. 2. Mean tissue concentrations (µg catecholamine g–1 tissue) ± 1 S.E.M. of noradrenaline (open columns) and adrenaline (filled columns) in rainbow trout (N=6). Abbreviations: APCV, anterior posterior cardinal vein; MPCV, middle posterior cardinal vein; PPCV, posterior posterior cardinal vein; AK, anterior kidney; MK, middle kidney; PK, posterior kidney.

 


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  		Fig. 3. Localisation of vasoactive intestinal polypeptide (VIP)-binding sites to the chromaffin cell subtypes in the posterior cardinal vein of rainbow trout using triple labelling. (A) Dopamine ß-hydroxylase (DßH)-immunoreactive cells (mouse anti-DßH). (B) Phenylethanolamine N-methyl transferase (PNMT)-immunoreactive cells (rabbit anti-bovine PNMT). (C) Labelling of VIP-binding sites. (D) Overlay image of the sections shown in A and B (DßH- and PNMT-positive cells); the PNMT-negative cells are indicated by arrows. (E) Overlay image of the sections shown in A and C (DßH and VIP labelling). (F) Overlay image of the sections shown in B and C (PNMT and VIP labelling); the PNMT-negative cells are indicated by arrows. Yellow/orange colour indicates co-localisation. Note that only a few of the DßH-labelled cells were negative for PNMT, thus indicating only a small number of noradrenaline-containing cells. Moreover, the pattern of VIP binding was identical to that of DßH, indicating that all chromaffin cells possess VIP-binding sites. Scale bars A–F, 150 µm.

 


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  		Fig. 4. Secretion rates of adrenaline (filled columns) and noradrenaline (open columns) in response to a bolus injection of saline (inset; N=6) or biotinylated vasoactive intestinal polypeptide (VIP) (10–9 mol kg–1; N=6) using an in situ posterior cardinal vein preparation of rainbow trout. Values are shown as means ± 1 S.E.M. Asterisks denote a significant difference from the pre-stimulation (Pre) value (P<0.05).

 


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  		Fig. 5. Pharmacological assessment of the vasoactive intestinal polypeptide (VIP)-binding sites. Labelling of VIP-binding sites following treatment with different VPAC and PAC receptor agonists and antagonists. (A) Control, labelling of VIP-binding sites. (B) Labelling of VIP-binding sites following treatment with ckVIP. (C) Labelling of VIP-binding sites following treatment with the VIP receptor antagonist VIP 6-28. (D) Labelling of VIP-binding sites following treatment with the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor agonist PACAP-27. (E) Labelling of VIP-binding sites following treatment with the PAC1 receptor antagonist PACAP 6-27. Scale bars A–E, 150 µm.

 


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  		Fig. 6. Comparison of deduced partial amino acid sequences of the putative trout VPAC1 (A) and VPAC2 (B) receptors with those of selected vertebrates. Conserved amino acids (100% identity) among taxa are highlighted. Sequences were aligned using CLUSTAL W and the alignment was prepared from DNAMAN. The putative trans-membrane domains (TMDs) are denoted by horizonatal arrows and were deduced by visual inspection of published reports and by hydrophobocity analysis. The Gsa-protein coupling motif (basic-L/A L/A/V/S-basic) is shown by a plus. The RLA K/R motif is common to members of the G-protein-coupled receptor family. The PD I/V motif common to vasoactive intestinal polypeptide (VIP)-binding receptors is shown by an asterisk. Finally, the conserved cysteine preceding the IIRIL motif, common only to VPAC1 receptors, is shown by a dagger. Sequences were edited to include only the region spanning the primer sites used to obtain the trout partial cDNA clones.

 


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  		Fig. 7. Tissue distributions of the putative trout VPAC1 and VPAC2 receptors as revealed by RT-PCR. PCR was performed using the sequence-specific primers deduced from the partial cDNA clones for each receptor. Using the sequence-specific primers for trout VPAC1 receptors, an expected 313-bp product was observed in all tissues with the exception of blood. Prominent products were observed in the liver and intestine. Using sequence-specific primers for trout VPAC2 receptors, an expected 363-bp product was observed in brain, spleen, heart, liver and intestine. No products were observed in posterior cardinal vein (PCV), kidney, gill and blood. As an internal control, RT-PCR for trout ß-actin was performed. In all tissues, PCR products specific for trout ß-actin were consistently observed. P{phi}X, DNA molecular size ladder.

 

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