Gi2 and Gi3 proteins mediate the inhibition of adenylyl cyclase by galanin in the RINm5F cell.
Inhibition of adenylyl cyclase activity is one of at least four mechanisms by which the neuropeptide galanin inhibits insulin secretion from pancreatic beta-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 Gi or Go guanine nucleotide binding proteins (G-proteins). Because galanin receptors interact with four G-proteins (Gi1, Gi2, Gi3, and Go1), 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 G alpha i1/alpha i2 and G alpha i3, respectively, were able to significantly attenuate the inhibitory effect of galanin, whereas antisera specific for Go proteins were not. The use of additional antisera specific for the various subtypes of Gi proteins indicated that Gi2 and Gi3, but not Gi1, 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, Gi2 and Gi3.
McDermott
A M
AM
Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York.
Sharp
G W
GW
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D005576:Colforsin / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D019004:Galanin; D066298:In Vitro Techniques; D009479:Neuropeptides / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology*; D037342:Pertussis Toxin; D051381:Rats; D015398:Signal Transduction; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D009479:Neuropeptides; D010455:Peptides; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D019004:Galanin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins