Immunolocalization of muscarinic and VIP receptor subtypes and their role in stimulating goblet cell secretion

PURPOSE. TO determine the subtypes of cholinergic muscarinic receptors and receptors for vasoactive intestinal peptide (VIP) present in rat conjunctiv al goblet cells and whether cholinergic agonists and VIP stimulate goblet c ell secretion. METHODS. Immunofluorescence studies were performed using antibodies against the m(1), m(2), and m(3) muscarinic receptor subtypes and VIP receptors 1 and 2 (VIPR1 and VIPR2). The lectin Ulex europeus agglutinin I was used to measure glycoconjugate secretion, the index of secretion, from goblet cells in an enzyme-linked lectin assay. In this assay, pieces of conjunctiva wer e placed on filter paper and incubated for 15 to 120 minutes, with or witho ut increasing concentrations of the cholinergic agonist carbachol or VIP. T he muscarinic antagonist atropine and the muscarinic receptor-subtype-selec tive antagonists pirenzepine (M-1), gallamine (M-2), and 4-4-diphenylacetox y-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP mustard; M-3) were inc ubated with carbachol to determine specificity of receptor activation. RESULTS. Immunoreactivity to M-2 and M-3 receptors was found on goblet cell membranes subjacent to the secretory granules. Immunoreactivity to M-1 rec eptor was not on goblet cells but was on the stratified squamous cells. Imm unoreactivity to VIPR2 was found on goblet cells with a localization simila r to that of the M-2 and M-3 receptors. VIPR1 was not found on goblet cells or on the stratified squamous cells. Carbachol and VIP induced a time- and concentration-dependent stimulation of glycoconjugate secretion. Carbachol , at 10(-4) M, induced a threefold increase in glycoconjugate secretion, wh ich was completely inhibited by atropine (10(-5) M). Carbachol-induced secr etion was inhibited 54% +/- 8% by pirenzepine (10(-5) M), 69% +/- 14% by ga llamine (10(-5) M), and 72% +/- 11% by 4-DAMP mustard (10(-5) M). A twofold increase in glycoconjugate secretion was obtained with VIP at 10(-8) M. CONCLUSIONS. Cholinergic agonists, through M-2 and/or M-3 muscarinic recept ors, and VIP, through VIPR2, regulate conjunctival goblet cell secretion, s uggesting that goblet cell secretion in vivo is under the control of parasy mpathetic nerves.