IDENTIFICATION OF VASOACTIVE-INTESTINAL-PEPTIDE RECEPTOR SUBTYPES IN THE LACRIMAL GLAND AND THEIR SIGNAL-TRANSDUCING COMPONENTS

Purpose. To determine the presence of vasoactive intestinal peptide (V IP) receptor (VIPR) subtypes in the lacrimal gland and to determine if the components of the VIP signaling pathway for protein secretion als o are present. Methods. Immunofluorescence studies using conventional fluorescence microscopy or confocal microscopy were performed on fixed sections from rat lacrimal glands using antibodies raised against VIP Rs types I and II, and four antibodies against five isoforms of adenyl yl cyclase (AC) (II, III, IV, V/VI). Guanine nucleotide binding (G) pr oteins were detected by Western blotting. Changes in intracellular [Ga 2+] ([Ca2+](i)) were measured on fura-2-loaded acini in response to VI P. The effect of a myristoylated peptide corresponding to the pseudosu bstrate sequence of protein kinase inhibitor (myr-PKI), the endogenous inhibitor of cyclic AMO (cAMO)-dependent protein kinase (PKA), was te sted on VIP-stimulated peroxidase secretion. Results. The VIPRs, types I and II, were found on the basolateral membranes of acinar and ducta l cells and on myoepithelial cells. Western blotting showed the presen ce of alpha subunits of G(s), G(13), G(0) and G(beta). The AC II was f ound exclusively on myoepithelial cells; AC IV was located intracellul arly in all cells; AC III was found on ducts and possibly nerves; no A C V/VI was detected. The VIP (10(-8) M) caused a small but significant increase in [Ca2+](i) of 26 +/- 9 nM. The VIP-stimulated protein secr etion was inhibited 71% by myr-PKI. Conclusions. All components of the VIP signals transduction pathway in the lacrimal gland were present. There findings are consistent with a pathway where VIP released from p arasympathetic nerves binds to VIPRs types I and II, activating G prot eins, which in turn stimulate AC present on myoepithelial and acinar c ells. The AC increases in the intracellular cAMP concentration, which activates PKA to stimulate protein secretion. The VIP also stimulated Ca2+ influx, which could play a role in secretion.