G(i)/G(o) couple met-enkephalin to inhibition of cholinergic and beta-adrenergic stimulation of lacrimal secretion

Purpose. To determine whether G-protein-mediated inhibition of secretion by met-enkephalin involves cyclic adenosine monophosphate (cAMP)-dependent ev ents and to identify the G proteins that couple met-enkephalin to inhibitio n of lacrimal secretion. Methods, Secretion of protein was measured in 3-da y primary cultures of rabbit lacrimal acini exposed to vehicle, the choline rgic agonist carbachol (Cch), the beta-adrenegic agonist isoproterenol (Iso p), vasoactive intestinal peptide (VIP). or forskolin (FSK) with or without the enkephalin analog D-ala(2)-met-enkephalinamide (DALA). In separate exp eriments, cells were pretreated with pertussis toxin or polyclonal antibodi es against the alpha subunits of G(i)/G(o) to determine the physiologic rol e of G proteins in met-enkephalin inhibition of the release of lacrimal pro tein. Adenylyl cyclase (AC) activity was measured by a cAMP-dependent prote in kinase binding assay in lacrimal membranes in response to the same agoni sts used in the secretion studies. Results, Cch resulted in a significant i ncrease in protein release from cultured lacrimal acini. Increased secretio n also occurred with Isop, VIP, and FSK. Cch- and Isop-stimulated secretion was inhibited by DALA to near-basal values. However, DALA did not inhibit VIP- or FSK-stimulated secretion. The inhibitory effect of DALA on Cch and Isop stimulation of secretion was reversed by pertussis toxin. Inhibition o f Cch-stimulated secretion was blocked by antibody specific to a common pep tide sequence of G(i alpha 1) and G(i alpha 2) but was not blocked by anti- G(i alpha 1) antibody. The inhibitory effect on Cch-stimulated secretion wa s also blocked by antiG(i alpha 3) and anti-G(o alpha). Similar experiments resulted in a reversal of DALA inhibition of beta-adrenergic stimulation o f secretion by immunoneutralization of G(i alpha 1/2) and G(o alpha) but no t by immunoneutralization of G(i alpha 1) or G(i alpha 3). VIP, Isop, and F SK significantly stimulated AC. However, Cch had no effect on the activity of the enzyme. In addition, DALA had no effect on AC activity under any con ditions. Conclusions. These results show that enkephalin inhibition of chol inergic and beta-adrenergic stimulation of secretion is mediated by G(i2), G(i3), and G(o). The effector coupled by the G proteins is not AC. However, we suggest a role for met-enkephalin in G-protein-coupled modulation of io n channels important for cholinergic and beta-adrenergic stimulation of lac rimal secretion.