1 We sought to reconstitute and characterize G-protein linked phosphatidyl-D-inositol 4,5-bisphosphate (PIP2)-directed phospholipase C (PLC) isoform activity in pig aortic vascular smooth muscle. 2 Six soluble PLC isoforms, namely gamma(1), delta(1) and beta(1) to beta(4) were partially separated by heparin affinity chromatography and were identified by Western blotting using specific antibodies. 3 In separate experiments, PLC activity was measured in the eluted fractions. Four of the partially resolved PLC isoforms gamma(1), beta(4), beta(2) and beta(1), showed corresponding activity using exogenous [H-3]-PIP2 as substrate. 4 The isolated soluble PLC isoforms were reconstituted with receptors and guanyl nucleotide regulatory proteins (G-proteins) by addition of plasma membranes, the phospholipids which had been prelabelled with [H-3]-myo-inositol. When so reconstituted PLC beta(2), beta(3) and beta(4) were inhibited (40 +/- 9, 47 +/- 12 and 40 +/- 5% respectively n = 12, +/- s.e.mean and each P < 0.05) by the addition of 1 mM guanosine 5'[beta gamma-imido]triphosphate (p[NH]ppG). 5 By contrast, when plasma membranes were preincubated with pertussis toxin to inhibit the activity of G-protein subunits G alpha(i)/alpha(0) the activities of PLC beta(2), beta(3) and beta(4) were stimulated (46 +/- 11, 31 +/- 9 and 37 +/- 8% respectively, n = 12, +/- s.e.mean and each P < 0.05) by the addition of p[NH]ppG. 6 Using well resolved fractions containing only PLC beta(3), time-dependent activity in the presence of p[NH]ppG was measurable only with membranes pretreated with pertussis toxin. 7 PLC beta(3) activity, measured with pertussis pretreated membranes, showed a dose-dependent increase in the presence of p[NH]ppG or guanosine 5'-[gamma-thio]triphosphate (GTP[S]). This increase with 10 mu M p[NH]ppG or GTP[S] 10% +/- 4 and 12% +/- 5 respectively (both P < 0.05 vs control without GTP analogue +/- s.e.mean, n = 10) was abolished by 50 mu M guanosine 5'-[beta-thio]diphosphate (GDP[S]) which also reduced constitutive PLC beta(3) activity by 9% +/- 4. 8 G-protein antibodies were used to neutralize PLC activity. Antibody to G alpha(q)/alpha(11), added to membrane fractions pretreated with pertussis toxin and assayed with GTP[S], reduced PLC beta(3) activity by 21% +/- 6 P < 0.02, n = 6, but was without effect on non-pertussis pretreated membranes. Antibodies to G alpha(i1)/alpha(i2) had no effect. Antibodies to G-protein beta subunits had no effect on PLC beta(3) activity with pertussis pretreated preparations but activity without pertussis pretreatment was increased by 30% +/- 10, P < 0.03, n = 6. All results were expressed as % change from controls containing rabbit IgG. 9 In conclusion, pig aortic vascular smooth muscle contains six PLC isoforms. Activation of pertussis sensitive G-protein by GTP analogues results in inhibition of PLC beta 3 activity from liberated G-protein beta gamma subunits. Stimulation of PLC beta(3) activity is associated with a G-protein of the G alpha(q) family acting through the a subunit. The results suggest that the G-protein linked PLC beta isoforms in vascular smooth muscle demonstrate dual regulation by an inhibitory pertussis-sensitive pathway and a stimulatory G-protein of the G alpha(q) family, which is the case for PLC beta(3). This dual regulation is analogous to that of adenyl cyclase.
