Properties of membranous phospholipase C from WRK1 cell: sensitivity to guanylnucleotides and bacterial toxins.

As previously described, WRK1 plasma membrane possesses a vasopressin-sensitive phospholipase C [G. Guillon et al., 1986, FEBS Lett. 196, 155-159]. In the present study, we examined the sensitivity of this enzyme to guanylnucleotides. GTP gamma S induces a time- and dose-dependent stimulation of Ins(1,4,5)P3 and Ins(1,4)P2 accumulation. No accumulation of InsP1, Ins(1,3,4)P3 or Ins(1,3,4,5)P4 occurred under similar conditions. Gpp(NH)p produced the same effect but was less potent. GTP and a nonhydrolyzable analogue of ATP, App(NH)p, were without effect. Calcium also stimulated the phospholipase C activity in a time- and dose-dependent manner. In the absence of calcium, the activity of GTP gamma S was considerably reduced. Physiological calcium concentrations (between 10(-8) and 10(-7) M), allowed maximal GTP gamma S stimulation of phospholipase C activity. In this system, the presence of vasopressin alone did not generate inositol phosphate accumulation. However, this hormone: (i) reduced the lag-time observed during GTP gamma S stimulation, (ii) increased the sensitivity of phospholipase C to GTP and to GTP gamma S, and (iii) did not modify the stimulation of phospholipase C induced by maximal doses of GTP gamma S. Unlike sodium fluoride, GTP gamma S elicited an irreversible activation of phospholipase C. Calcium, GTP gamma S and sodium fluoride stimulated the phospholipase C activity via mechanisms sharing a common step, since their maximal effects were not additive. Cholera toxin treatment, known to produce complete ADP-ribosylation of 'alpha s' subunits, partially reduced the basal and the maximal GTP gamma S-mediated stimulation of phospholipase C activity as well as that caused by vasopressin. This inhibition was not mimicked by treatment with either forskolin or pertussis toxin.