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 [γ‐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‐γ‐S caused a synergistic stimulation of 80K phosphorylation. The GDP analogue GDP‐β‐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‐β‐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.