G_i2 and G_i3 Proteins Mediate the Inhibition of Adenylyl Cyclase by Galanin in the RINm5F Cell Free

Inhibition of adenylyl cyclase activity is one of at least four mechanisms by which the neuropeptide galanin inhibits insulin secretion from pancreatic β-cells. In a membrane preparation of the insulin-secreting cell line RINm5F, a maximally effective concentration of galanin inhibited forskolin-stimulated adenylyl cyclase activity by 30%. Pretreatment of the cells with pertussis toxin abolished the inhibitory effect of galanin, indicating the involvement of G_i or G_o guanine nucleotide binding proteins (G-proteins). Because galanin receptors interact with four G-proteins (G_i1, G_i2, G_i3, and G_o1), any or all of these may inhibit adenylyl cyclase. Therefore, to identify the G-protein(s) involved, antibodies raised against various G-protein alpha-subunits were used to block the inhibition of forskolin-stimulated adenylyl cyclase activity by galanin in RINm5F membrane preparations. Antisera AS/7 and EC/2, specific for Gɑ_i1/ɑ_i2 and Gɑ_i3, respectively, were able to significantly attenuate the inhibitory effect of galanin, whereas antisera specific for G_o proteins were not. The 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 greater attenuation of the effect of galanin than application of either antiserum alone. Thus, galanin inhibition of adenylyl cyclase activity in these cells is selectively mediated by two inhibitory G-proteins, G_i2 and G_i3.