Vasopressin rapidly stimulates protein kinase C in digitonin-permeabilized swiss 3T3 cells: Involvement of a pertussis toxin-insensitive guanine nucleotide binding protein
✍ Scribed by Jorge D. Erusalimsky; Enrique Rozengurt
- Publisher
- John Wiley and Sons
- Year
- 1989
- Tongue
- English
- Weight
- 953 KB
- Volume
- 141
- Category
- Article
- ISSN
- 0021-9541
No coin nor oath required. For personal study only.
✦ Synopsis
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 [y-32P]ATP and digitonin caused a marked and rapid increase (8 ? I-fold after 1 min) in the phosphorylation of an M r = 8O10OO cellular protein (DOK), a specific marker for protein kinase C activation. This phosphorylation was selectively blocked by the Vl receptor antagonist Pmpl-0-Me-ryr' [Arg']vasopressin, indicating that the effect was mediated through the vasopressin V l receptor. Down regulation of protein kinase C by prior prolonged pretreatment of intact cells with phorbol 12,1%dibutyrate (PBtZ) 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 y-S caused a synergistic stimulation of 80K phosphorylation. The GDP analogue GDP-P-S cau5ed 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-P-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 [Ca"] or activation of protein kinase C. These findings provide funclional evidence for the involvement of a pertussis toxin-insesitive G protein in the vasopressin V, receptor-mediated stimulation of protein kinase C in Swiss 3T3 cells.