Phospholipase C isoforms in vascular smooth muscle and their regulation by G‐proteins

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 γ1, δ1 and β1 to β4 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 γ1, β4, β2 and β1, showed corresponding activity using exogenous [3H]‐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 [3H]‐myo‐inositol. When so reconstituted PLC β2, β3 and β4 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′[βγ‐imido]triphosphate (p[NH]ppG). 5 By contrast, when plasma membranes were preincubated with pertussis toxin to inhibit the activity of G‐protein subunits Gαi/αo the activities of PLC β2, β3 and β4 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 β3, time‐dependent activity in the presence of p[NH]ppG was measurable only with membranes pretreated with pertussis toxin. 7 PLC β3 activity, measured with pertussis pretreated membranes, showed a dose‐dependent increase in the presence of p[NH]ppG or guanosine 5′‐[γ‐thio]triphosphate (GTP[S]). This increase with 10 μm 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 μm guanosine 5′‐[β‐thio]diphosphate (GDP[S]) which also reduced constitutive PLC β3 activity by 9% ± 4. 8 G‐protein antibodies were used to neutralize PLC activity. Antibody to Gαq/α11, added to membrane fractions pretreated with pertussis toxin and assayed with GTP[S], reduced PLC β3 activity by 21% ± 6 P < 0.02, n = 6, but was without effect on non‐pertussis pretreated membranes. Antibodies to Gαi1/αi2 had no effect. Antibodies to G‐protein β subunits had no effect on PLC β3 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 β3 activity from liberated G‐protein βγ subunits. Stimulation of PLC β3 activity is associated with a G‐protein of the Gαq family acting through the α subunit. The results suggest that the G‐protein linked PLC β isoforms in vascular smooth muscle demonstrate dual regulation by an inhibitory pertussis‐sensitive pathway and a stimulatory G‐protein of the Gαq family, which is the case for PLC β3. This dual regulation is analogous to that of adenyl cyclase.