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Phospholipase C isoforms in vascular smooth muscle and their regulation by G‐proteins
Author(s) -
Blayney Lynda M.,
Gapper Peter W.,
Newby Andrew C.
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb15499.x
Subject(s) - pertussis toxin , phospholipase c , gene isoform , inositol , g protein , chemistry , biochemistry , vascular smooth muscle , western blot , blot , membrane , phospholipase , guanosine , receptor , microbiology and biotechnology , biology , enzyme , endocrinology , smooth muscle , gene
1 We sought to reconstitute and characterize G‐protein linked phosphatidyl‐D‐inositol 4, 5‐bisphosphate (PIP 2 )‐directed phospholipase C (PLC) isoform activity in pig aortic vascular smooth muscle. 2 Six soluble PLC isoforms, namely γ 1 , δ 1 and β 1 to β 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 γ 1 , β 4 , β 2 and β 1 , showed corresponding activity using exogenous [ 3 H]‐PIP 2 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 [ 3 H]‐ myo ‐inositol. When so reconstituted PLC β 2 , β 3 and β 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′[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 Gα i /α o the activities of PLC β 2 , β 3 and β 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 β 3 , time‐dependent activity in the presence of p[NH]ppG was measurable only with membranes pretreated with pertussis toxin. 7 PLC β 3 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 μ 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 μ m guanosine 5′‐[β‐thio]diphosphate (GDP[S]) which also reduced constitutive PLC β 3 activity by 9% ± 4. 8 G‐protein antibodies were used to neutralize PLC activity. Antibody to Gα q /α 11 , added to membrane fractions pretreated with pertussis toxin and assayed with GTP[S], reduced PLC β 3 activity by 21% ± 6 P < 0.02, n = 6, but was without effect on non‐pertussis pretreated membranes. Antibodies to Gα i1 /α i2 had no effect. Antibodies to G‐protein β subunits had no effect on PLC β 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 β 3 activity from liberated G‐protein βγ subunits. Stimulation of PLC β 3 activity is associated with a G‐protein of the Gα q 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 Gα q family, which is the case for PLC β 3 . This dual regulation is analogous to that of adenyl cyclase.