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Vasopressin rapidly stimulates protein kinase C in digitonin‐permeabilized swiss 3T3 cells: Involvement of a pertussis toxin‐insensitive guanine nucleotide binding protein
Author(s) -
Erusalimsky Jorge D.,
Rozengurt Enrique
Publication year - 1989
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041410204
Subject(s) - pertussis toxin , digitonin , vasopressin , biology , phosphorylation , protein kinase c , protein kinase a , g protein , kinase , gtp' , autophosphorylation , medicine , endocrinology , microbiology and biotechnology , biochemistry , receptor , enzyme
Guanine nucleotides and pertussis toxin were used to test for the involvement of a guanine nucleotide binding protein in the vasopressin V 1 receptor‐mediated stimulation of protein kinase C activity in Swiss 3T3 cells. Addition of vasopressin in the presence of [γ‐ 32 P]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 V 1 receptor antagonist Pmp 1 ‐0‐Me‐Tyr 2 [Arg 8 ]vasopressin, indicating that the effect was mediated through the vasopressin V 1 receptor. Down regulation of protein kinase C by prior prolonged pretreatment of intact cells with phorbol 12, 13‐dibutyrate (PBt 2 ) 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 PBt 2 . Prior incubation of intact quiescent cultures of Swiss 3T3 cells with pertussis toxin did not impair either vasopressin‐induced increase in cytosolic [Ca 2+ ] or activation of protein kinase C. These findings provide functional evidence for the involvement of a pertussis toxin‐insesitive G protein in the vasopressin V 1 receptor‐mediated stimulation of protein kinase C in Swiss 3T3 cells.