Functional characterization of phosphoinositide-specific
phospholipase c-[beta]1 and -[beta]3 in intestinal smooth muscle.
Murthy, K. S., and G. M. Makhlouf.
Departments of Physiology and Medicine, Medical College, of
Virginia, Richmond, Virginia 23298-0711
APStracts 2:0158C, 1995.
Soluble and membrane phosphoinositide-specific phospholipases (PLC)
obtained separately from dispersed circular and longitudinal
intestinal muscle cells were characterized for substrate specificity
and G protein dependence using selective antibodies to various
isoforms of PLC and G protein subunits. Western blot analysis
disclosed the presence of the main PLC isozymes, PLC-_1, PLC-[delta]1
and PLC-[beta]1. Soluble PLC from circular and longitudinal muscle
was stimulated by GTP_S and inhibited by PLC-[beta]1 antibody (80
-90%) and PLC-[beta]3 antibody (25%) but not by G protein antibodies.
Membrane PLC from circular and longitudinal muscle was stimulated by
CCK-8 and inhibited selectively by PLC-[beta]1 antibody (85%), PLC
-[beta]3 antibody (15%) and G[alpha]q/11 antibody (90%). CCK-induced
contraction in permeabilized circular muscle cells was also
selectively inhibited by PLC-[beta]1 antibody (76%), PLC-[beta]3
antibody (24%) and G[alpha]q/11 antibody (86%). The combined effects
of PLC-[beta]1 and PLC-[beta]3 antibodies on PLC activity and muscle
contraction were additive causing complete inhibition. Soluble and
membrane PLC from circular and longitudinal muscle were
immunologically similar but functionally different. The enzymes from
circular muscle preferentially hydrolyzed endogenous and exogenous
PIP2, confirming previous findings of preferential hydrolysis of PIP2
in dispersed intestinal circular muscle cells.
Received 7 October 1994; accepted in final form 31 March 1995.
APS Manuscript Number C603-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 10 April 1995.