Involvement of pertussis toxin-sensitive G-proteins in the hormonal inhibition of dihydropyridine-sensitive Ca2+ currents in an insulin-secreting cell line (RINm5F).
Adrenaline inhibits insulin secretion via pertussis toxin-sensitive mechanisms. Since voltage-dependent Ca2+ currents play a key role in insulin secretion, we examined whether adrenaline modulates voltage-dependent Ca2+ currents of the rat insulinoma cell line, RINm5F. In the whole-cell configuration of the patch-clamp technique, dihydropyridine- but not omega-conotoxin-sensitive Ca2+ currents were identified. Adrenaline via alpha 2-adrenoceptors inhibited the Ca2+ currents by about 50%. Somatostatin which also inhibits insulin secretion was less efficient (inhibition by 20%). The hormonal inhibition of Ca2+ currents was not affected by intracellularly applied cAMP but blocked by the intracellularly applied GDP analog guanosine 5'-O-(2-thiodiphosphate) and by pretreatment of cells with pertussis toxin. In contrast to adrenaline and somatostatin, galanin, another inhibitor of insulin secretion, reduced Ca2+ currents by about 40% in a pertussis toxin-insensitive manner. Immunoblot experiments performed with antibodies generated against synthetic peptides revealed that membranes of RINm5F cells possess four pertussis toxin-sensitive G-proteins including Gi1, Gi2, Go2, and another Go subtype, most likely representing Go1. In membranes of control but not of pertussis toxin-treated cells, adrenaline via alpha 2-adrenoceptors stimulated incorporation of the photo-reactive GTP analog [alpha-32P]GTP azidoanilide into pertussis toxin substrates comigrating with the alpha-subunits of Gi2, Go2, and the not further identified Go subtype. The present findings indicate that activated alpha 2-adrenoceptors of RINm5F cells interact with multiple G-proteins, i.e. two forms of Go and with Gi2. These G-proteins are likely to be involved in the adrenaline-induced inhibition of dihydropyridine-sensitive Ca2+ currents and in other signal transduction pathways contributing to the adrenaline-induced inhibition of insulin secretion.
Schmidt
A
A
Institut für Pharmakologie, Freie Universität Berlin, Federal Republic of Germany.
Hescheler
J
J
Offermanns
S
S
Spicher
K
K
Hinsch
K D
KD
Klinz
F J
FJ
Codina
J
J
Birnbaumer
L
L
Gausepohl
H
H
Frank
R
R
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003000:Clonidine / Q000494:pharmacology; D004095:Dihydropyridines / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004837:Epinephrine / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006728:Hormones / Q000502:physiology*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma / Q000378:metabolism* / Q000473:pathology; D008564:Membrane Potentials; D037342:Pertussis Toxin; D051381:Rats; D013004:Somatostatin / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D004095:Dihydropyridines; D006728:Hormones; D007328:Insulin; D010566:Virulence Factors, Bordetella; D013004:Somatostatin; C038806:1,4-dihydropyridine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D003000:Clonidine; D002118:Calcium; D004837:Epinephrine