Pergamon 0306-4522(93)E006 1 -T Copyright 0 1994 IBRO Printed in Great Britain. All rights reserved 0306-4522/94 $6.00 + 0.00 VASOACTIVE INTESTINAL PEPTIDE IN THE HAMSTER SEMINAL VESICLE: DISTRIBUTION, BINDING SITES AND POSSIBLE FUNCTIONS zyxwvutsrqponmlkjihgfedcbaZYX M. S. PINHO,* 2 A. M. SEBASTI~O,~ G. RODRIGUES,*§C. P. BARROSO,*J. d. RIBEIRO,~ L. R. MATA* and S. GULBENKIAN*/] Laboratories of *Cell Biology and tPharmacology, Gulbenkian Institute of Science, 2781 Oeiras Codex, Portugal $Department of Morphology and Clinics, Faculty of Veterinary Medicine, Lisbon, Portugal §Department of Zoology and Anthropology, Faculty of Sciences, Lisbon, Portugal Abstract-The presence and functional role of vasoactive intestinal peptide in the hamster seminal vesicle were studied by a combination of structural and functional approaches. The use of an immuno- fluorescence staining technique in both cryostat sections and whole-mount preparations revealed that vasoactive intestinal peptide-immunoreactive nerve fibres were mainly localized in the lamina propria of the mucosal layer. In double-stained preparations, vasoactive intestinal peptide immunoreactivity was found to be localized in nerves also containing acetylcholinesterase activity. At the ultrastructural level, the use of an immunogold staining method showed that vasoactive intestinal peptide immunoreactivity occurred in large granular vesicles (8&l 50 nm in diameter) in nerve varicosities which also contained small pleomorphic agranular vesicles. In order to evaluate the anatomical distribution of vasoactive intestinal peptide binding sites in the seminal vesicle, we have utilized an in oitro receptor autoradiographic technique. Vasoactive intestinal peptide binding sites tiere localized in the basal region of the secretory epithelium, in the muscle layer and in the wall of blood vessels. In virro incorporation of [3H]r_-leucine into protein by tissue slices revealed that vasoactive intestinal peptide (1 FM) significantly increases the amount of released protein. Vasoactive intestinal peptide (0.1-I Jo M) did not affect the resting tension of the muscle but significantly inhibited the increase in muscle tension induced by carbachol. Atropine prevented the effect of carbachol, indicating that the latter is mediated by muscarinic receptors. Our results suggest that in the hamster seminal vesicle, vasoactive intestinal peptide is involved in the modulation of muscarinic function and in the control of secretion. The seminal vesicle is one of the male sex accessory glands, maintaining an intense secretory function under the influence of androgens. In most mam- malian species, castration induces an involution of the gland and a dramatic decline in its secretory activity. In the hamster, secretion persists for a long time after castration,‘9s26 suggesting that other fac- tors, besides androgens, might be involved in the regulation of seminal vesicle secretory activity. Several studies have demonstrated that the seminal vesicle is under both hormonal and neuronal regu- lation.‘0J6,23.34,3J It is now recognized that in addition to classical sympathetic (noradrenergic) and para- sympathetic (~holinergi~) transmitters, the popu- lations of nerve fibres supplying the seminal vesicle also contain other putative transmitters, including neuropeptides. These neuropeptides often exert potent pharmacological effects on the tissues in which /{To whom correspondence should be addressed. Abbreviations: AChE, acetyichohnesterase; BSA, bovine serum albumin; CAMP, adenosine 3’,5’-cyclic mono- phosphate; EDTA, ethylenediaminetetra-acetate; PBS, phosphate-buffered saline; TCA, trichloroacetic acid; VIP, vasoactive intestinal peptide. they occur, acting directly via specific receptors and/or indirectly by in~uen~ing the release of other transmitters.s Vasoactive intestinal peptide (VIP) is foremost among the neuropeptides found in the male accessory sex glands.2 The presence of VIP binding sites in rat seminal vesicle membrane preparations was recently demonstrated and it has been suggested that VIP participates in the regulation of the gland function, namely modulating its secretory activity.6,29 However, the precise role of this neuropeptide in the seminal vesicle remains to be established. In the present study we have employed histochem- ical, immunohistochemical, autoradiographic, bio- chemical and in vitro pha~acolo~~al techniques in order to get further insight into the possible role of VIP in the hamster seminal vesicle. EXPERIMENTAL PROCEDURES Tissues were obtained from adult male hamsters (1~IS0 g body wt) (Mesocricetus uMrat~; Charles River Laboratories) killed by decapitation under ether anaesthesia. Light microscope immunohistochemistry Seminal vesicles were collected and fixed by immersion in Zamboni’s fixative” for 16-24 h at 4°C. Following NSC 59’61; 1083