Vasopressin rapidly stimulates protein kinase C in digitonin-permeabilized Swiss 3T3 cells: involvement of a pertussis toxin-insensitive guanine nucleotide binding protein.
Guanine nucleotides and pertussis toxin were used to test for the involvement of a guanine nucleotide binding protein in the vasopressin V1 receptor-mediated stimulation of protein kinase C activity in Swiss 3T3 cells. Addition of vasopressin in the presence of [gamma-32P]ATP and digitonin caused a marked and rapid increase (8 +/- 1-fold after 1 min) in the phosphorylation of an Mr = 80,000 cellular protein (80K), a specific marker for protein kinase C activation. This phosphorylation was selectively blocked by the V1 receptor antagonist Pmp1-0-Me-Tyr2 [Arg8] vasopressin, indicating that the effect was mediated through the vasopressin V1 receptor. Down regulation of protein kinase C by prior prolonged pretreatment of intact cells with phorbol 12,13-dibutyrate (PBt2) blocked the ability of vasopressin to stimulate the phosphorylation of 80K in digitonin-permeabilized cells. Addition of a submaximal concentration of vasopressin together with the GTP analogue GTP-gamma-S caused a synergistic stimulation of 80K phosphorylation. The GDP analogue GDP-beta-S caused a 50% inhibition of the phosphorylation of 80K induced by a saturating concentration of vasopressin and shifted the vasopressin dose-response curve to the right. GDP-beta-S had no effect on the dose-response for the stimulation of 80K phosphorylation induced by PBt2. Prior incubation of intact quiescent cultures of Swiss 3T3 cells with pertussis toxin did not impair either vasopressin-induced increase in cytosolic [Ca2+] or activation of protein kinase C. These findings provide functional evidence for the involvement of a pertussis toxin-insesitive G protein in the vasopressin V1 receptor-mediated stimulation of protein kinase C in Swiss 3T3 cells.
Erusalimsky
J D
JD
Imperial Cancer Research Fund, Lincoln's Inn Fields, England.
Rozengurt
E
E
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004072:Digitonin / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000502:physiology*; D006150:Guanine Nucleotides / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D037342:Pertussis Toxin; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism* / Q000502:physiology; D011945:Receptors, Angiotensin / Q000378:metabolism / Q000502:physiology; D017483:Receptors, Vasopressin; D014667:Vasopressins / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D006150:Guanine Nucleotides; D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; D010566:Virulence Factors, Bordetella; D014667:Vasopressins; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D004072:Digitonin; D002118:Calcium