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 [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 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 o 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 microM
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 microM
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 alpha 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.