Inhibition of adenylate cyclase activity by galanin in rat insulinoma cells is mediated by the G-protein Gi3.
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.
de Mazancourt
P
P
Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Goldsmith
P K
PK
Weinstein
L S
LS
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D005576:Colforsin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D019004:Galanin; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007340:Insulinoma / Q000201:enzymology*; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010190:Pancreatic Neoplasms / Q000201:enzymology*; D010455:Peptides / Q000494:pharmacology*; D037342:Pertussis Toxin; D010710:Phosphates / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000235:genetics; D051381:Rats; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D007328:Insulin; D009479:Neuropeptides; D016376:Oligonucleotides, Antisense; D010455:Peptides; D010710:Phosphates; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone; D010566:Virulence Factors, Bordetella; D005576:Colforsin; C083120:phosphorodithioic acid; D019004:Galanin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D016650:Fluorescein-5-isothiocyanate