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 Gi3 is the
G protein that couples
galanin receptors to inhibition of
adenylate cyclase activity in RINm5F cells.