Properties of membranous phospholipase C from WRK 1 cell: Sensitivity to guanylnucleotides and bacterial toxins

Abstract

As previously described, WRK 1 plasma membrane possesses a vasopressin-sensitive phospholipase C [G. Guillon et al., FEBS Lett. 196, 155–159]. In the present study, we examined the sensitivity of this enzyme to guanylnucleotides. GTPγS induced a time- and dose-dependent stimulation of Ins(1,4,5)P 3 and Ins(1,4)P 2 accumulation. No accumulation of InsP 1, Ins(1,3,4)P 3 or Ins(1,3,4,5)P 4 occured 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 GTPγS was considerably reduced. Physiological calcium concentrations (between 10 −8 and 10 −7M), allowed maximal GTPγS 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 GTPγS stimulation, (ii) increased the sensitivity of phospholipase C to GTPγS, and (iii) did not modify the stimulation of phospholipase C induced by maximal doses of GTPγS. Unlike sodium fluoride, GTPγS elicited an irreversible activation of phospholipace C. Calcium, GTPγS 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 ‘αs’ subunits, partially reduced the basal and the maximal GTPγS-mediated stimulation of phospholipase C activity as well as that caused by vasopressin. This inhibition was not mimicked by treatment with either forskolin or pertussi toxin.