OPIOID MU-RECEPTOR-INDUCED, DELTA-RECEPTOR-INDUCED, AND KAPPA-RECEPTOR-INDUCED ACTIVATION OF PHOSPHOLIPASE C-BETA-3 AND INHIBITION OF ADENYLYL-CYCLASE IS MEDIATED BY G(12) AND G(0) IN SMOOTH-MUSCLE

In neurons and transformed cell lines, opioid receptors are coupled to various signaling mechanisms involved in Ca2+ mobilization, including inhibition or activation of Ca2+ channels and phospholipase C-beta (P LC-beta), the enzyme responsible for generation of the Ca2+ mobilizing messenger inositol-1,4,5-trisphosphate [Ins(1,4,5)P-3]. In the curren t study, we used selective PLC-beta and G protein antibodies to identi fy the PLC-beta isozyme activated by opioid receptors in intestinal sm ooth muscle and the G proteins to which the PLC-beta isozyme and adeny lyl cyclase are coupled. [D-Pen(2),D-Pen(5)]Enkephalin, a delta recept or agonist, stimulated Ins(1,4,5)P-3 formation, Ca2+ release, and cont raction; inhibited forskalin-stimulated cAMP formation in dispersed mu scle cells; and stimulated phosphoinositide hydrolysis in plasma membr anes; all of the effects were blocked by pertussis toxin. [D-Pen(2),D- Pen(5)]Enkephalin-stimulated Ins(1,4,5)P-3 formation, Ca2+ release, an d contraction in permeabilized muscle cells and phosphoinositide hydro lysis in plasma membranes were selectively blocked by G(beta) antibody and PLC-beta 3 antibody; contractions stimulated by [D-Ala(2),N-MePhe (4),Gly-ol(5)]enkephalin, a mu receptor agonist, and U-69,593, a kappa receptor agonist, were also blocked by G(beta) and PLC-beta 3 antibod ies. Inhibition of forskolin-stimulated cAMP formation by delta, mu, a nd kappa receptor agonists was partially blocked by G(alpha i2) and G( alpha 0) antibodies and additively blocked by a combination of the ant ibodies. The delta, mu, and kappa receptor agonists stimulated the bin ding of guanosine-5'-O-(3-thio)triphosphate to the alpha subunits of G (i2) and G(o) but not to the or subunits of other G proteins. We concl ude that opioid mu, delta, and kappa receptors are selectively coupled to G(i2) and G(o) in intestinal smooth muscle. The beta gamma subunit s of both G proteins activate PLC-beta 3, thereby stimulating Ins(1,4, 5)P-3-dependent Ca2+ release and smooth muscle contraction, whereas th e alpha subunits inhibit adenylyl cyclase activity.