G(I2) AND G(I3) PROTEINS MEDIATE THE INHIBITION OF ADENYLYL-CYCLASE BY GALANIN IN THE RINM5F CELL

Inhibition of adenylyl cyclase activity is one of at least four mechan isms by which the neuropeptide galanin inhibits insulin secretion from pancreatic beta-cells. In a membrane preparation of the insulin-secre ting cell Line RINm5F, a maximally effective concentration of galanin inhibited forskolin-stimulated adenylyl cyclase activity by 30%. Pretr eatment of the cells with pertussis toxin abolished the inhibitory eff ect of galanin, indicating the involvement of G(i) or G(o) guanine nuc leotide binding proteins (G-proteins). Because Galanin receptors inter act with four G-proteins (G(i1),G(i2),G(i3), and G(ol)), any or all of these may inhibit adenylyl cyclase. Therefore, to identify the G-prot ein(s) involved, antibodies raised against various G-protein alpha-sub units were used to block the inhibition of forskolin-stimulated adenyl yl cyclase activity by galanin in RINm5F membrane preparations. Antise ra AS/7 and EC/2, specific for G alpha(i1)/alpha(i2) and G alpha(i3), respectively, were able to significantly attenuate the inhibitory effe ct of Galanin, whereas antisera specific for G(o) proteins were not. T he use of additional antisera specific for the various subtypes of G(i ) proteins indicated that G(i2) and G(i3), but not G(i1), are involved . Simultaneous application of antisera AS/7 and EC/2 resulted in a gre ater attenuation of the effect of galanin than application of either a ntiserum alone. Thus, galanin inhibition of adenylyl cyclase activity in these cells is selectively mediated by two inhibitory G-proteins, G (i2) and G(i3).