INHIBITION OF ADENYLATE-CYCLASE ACTIVITY BY GALANIN IN RAT INSULINOMA CELLS IS MEDIATED BY THE G-PROTEIN G(I3)

Galanin inhibits adenylate cyclase activity and insulin secretion and modulates ion channels in pancreatic beta-cells through pertussis-toxin-sensitive G-protein(s). Antibodies directed against the C-terminal region of specific G-protein alpha-subunits were used to determine which G-protein(s) couple galanin receptors to inhibition of adenylate cyclase in the rat insulinoma cell line RINm5F. Preincubation of membranes with EC antibody (anti-alpha(i3)) decreased the inhibition of forskolin-stimulated adenylate cyclase activity by galanin (100 nM) by 45% compared with control IgG (P < 0.05) whereas preincubation with AS (anti-alpha(i1), alpha(i2)) or GO (anti-alpha(o)) antibodies had no significant effect. To confirm these results, RINm5F cells were exposed intermittently over a 4-day period to phosphorothioate oligodeoxynucleotides that were either sense or antisense to alpha(i1), alpha(i2), alpha(i3) or alpha(o). Oligodeoxynucleotides antisense to alpha i2, alpha i3 and alpha(o) specifically decreased the levels of the targeted alpha-subunit in membranes. alpha(i1) was undetectable in these cells. Inhibition of adenylate cyclase activity by galanin was largely abolished in membranes from cells exposed to the oligodeoxynucleotide antisense to alpha(i3), whereas all other oligodeoxynucleotides had no significant effect on this pathway. Indirect immunofluorescence and immunoblotting of specific membrane fractions with EC antibody show significant localization of alpha(i3) to intracellular membrane compartments. These results suggest that G(i3) is the G protein that couples galanin receptors to inhibition of adenylate cyclase activity in RINm5F cells.