Sustained muscle contraction induced by agonists,
growth factors, and Ca2+ mediated by
distinct PKC isozymes
K. S.
Murthy,
J. R.
Grider,
J. F.
Kuemmerle, and
G. M.
Makhlouf
Departments of Medicine and Physiology, Medical College of
Virginia, Virginia Commonwealth University, Richmond, Virginia 23298
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ABSTRACT |
The role of
protein kinase C (PKC) in sustained contraction was examined in
intestinal circular and longitudinal muscle cells. Initial contraction
induced by agonists (CCK-8 and neuromedin C) was abolished by
1) inhibitors of Ca2+ mobilization (neomycin and
dimethyleicosadienoic acid), 2) calmidazolium, and
3) myosin light chain (MLC) kinase (MLCK) inhibitor KT-5926. In contrast, sustained contraction was not affected by these inhibitors but was abolished by 1) the PKC inhibitors chelerythrine and
calphostin C, 2) PKC-
antibody, and 3) a
pseudosubstrate PKC-
inhibitor. GDP
S abolished both initial and
sustained contraction, whereas a G
q/11 antibody
inhibited only initial contraction, implying that sustained contraction
was dependent on activation of a distinct G protein. Sustained
contraction induced by epidermal growth factor was inhibited by
calphostin C, PKC-
,
,
antibody, and a pseudosubstrate PKC-
inhibitor. Ca2+ (0.4 µM) induced an initial contraction
in permeabilized muscle cells that was blocked by calmodulin and MLCK
inhibitors and a sustained contraction that was blocked by calphostin C
and a PKC-
,
,
antibody. Thus initial contraction induced by
Ca2+, agonists, and growth factors is mediated by MLCK,
whereas sustained contraction is mediated by specific
Ca2+-dependent and -independent PKC isozymes. G
protein-coupled receptors are linked to PKC activation via distinct G proteins.
calcium sensitization; intestinal smooth muscle; protein kinase C; myosin light chain kinase
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INTRODUCTION |
THE INITIAL
Ca2+ transient induced by contractile agonists in smooth
muscle is accompanied by Ca2+/calmodulin (CaM)-dependent
activation of myosin light chain (MLC) kinase (MLCK) and
phosphorylation of Ser 19 on the 20-kDa regulatory light chain of
myosin II (MLC20), leading to activation of the actin-activated myosin ATPase, interaction of actin and myosin, and
muscle contraction (33, 34, 38).
The initial Ca2+ transient is rapidly dissipated by
extrusion of Ca2+ from the cell and uptake into
intracellular Ca2+ stores, causing a rapid decline in MLCK
activity that is accentuated by phosphorylation of MLCK via
Ca2+/CaM-dependent protein kinase II (35) and
p21-activated kinase (31). Despite the rapid decline of
intracellular Ca2+ concentration
([Ca2+]i) and MLCK activity to near-resting
levels, MLC20 phosphorylation and muscle contraction are
sustained, albeit at lower levels (9, 33,
34, 38).
Various mechanisms that can operate under reduced or resting
Ca2+ levels have been proposed; all involve a regulated
decrease in MLC phosphatase (MLCP) activity and assume that the
increase in MLC20 phosphorylation reflects basal activity
of MLCK or the activation of a Ca2+-independent MLCK
(13, 33, 36, 39).
The contribution of other regulatory proteins, such as caldesmon and
calponin, appears to be small (9, 22,
30, 38). A decrease in MLCP activity could
result from 1) Rho kinase-mediated phosphorylation of the 130-kDa myosin-binding, regulatory subunit of MLCP (7,
12, 16, 36); 2)
arachidonic acid-induced inactivation of the holoenzyme mediated by the
atypical protein kinase C (PKC) isozyme PKC-
(5,
37); and 3) PKC-dependent activation of an
endogenous, 17-kDa inhibitor of MLCP, CPI-17 (3,
14, 19, 21). Inhibition of MLCP
by Rho kinase or via a PKC-dependent mechanism may not be mutually
exclusive. Recent studies (29) in intestinal smooth muscle
have shown that contractile agonists initiate a G protein-dependent cascade involving sequential activation of RhoA, RhoA kinase, phospholipase D (PLD), and PKC, implying that Rho kinase could inhibit
MLCP via a downstream, PKC-dependent mechanism.
Studies of Ca2+ sensitization have contributed greatly to
discovery of the role of MLCP in maintaining MLC20
phosphorylation but are based on the assumption that sustained
MLC20 phosphorylation and contraction reflect G
protein-mediated enhancement or sensitization of
Ca2+-dependent mechanisms. It is equally plausible, as
noted above, that sustained contraction may be mediated by G
protein-dependent pathways that maintain MLC20
phosphorylation and attenuate dephosphorylation, independent of
Ca2+/CaM-dependent activation of MLCK.
The experimental design for measurement of Ca2+
sensitization has yielded conflicting results regarding the role of
PKC. Jensen et al. (10) have reported that in the presence
of a fixed Ca2+ concentration (0.3 µM), the increase in
contraction of rabbit vascular smooth muscle induced by agonists was
either not affected or only partly inhibited by complete or partial
(~70%) downregulation of various PKC isozymes. Lee et al.
(18), however, have reported that the increase in
contraction in ferret aorta and portal vein smooth muscle was blocked
by specific inhibitors of distinct PKC isozymes: the pattern of
inhibition reflected the selective translocation of PKC-
in aortic
muscle and PKC-
and PKC-
in portal vein muscle.
In the present study, we adopted an experimental design that maintained
the sequence of signaling events mediating the initial and sustained
phases of contraction to examine the role of PKC. The results indicate
that sustained contraction of intestinal circular and longitudinal
muscle induced by agonists was independent of Ca2+
mobilization and reflected activation of PKC-
via a distinct G
protein-dependent pathway. In contrast, sustained contraction induced
by epidermal growth factor (EGF) and exogenous Ca2+ was
mediated by Ca2+-dependent PKC isozymes.
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MATERIALS AND METHODS |
Preparation of dispersed intestinal smooth muscle cells.
Muscle cells were isolated separately from the circular and
longitudinal muscle layers of guinea pig intestine by sequential enzymatic digestion, filtration, and centrifugation, as described previously (23, 28). Briefly, muscle strips
were incubated at 31°C for 30 min in HEPES medium with type II
collagenase (0.1%) and soybean trypsin inhibitor (0.1%). The partly
digested strips were washed, and muscle cells were allowed to disperse
spontaneously for 30 min. The cells were harvested by filtration
through 500-µm Nitex and centrifuged twice at 350 g for 10 min.
In experiments with blocking antibodies, the cells were permeabilized
as described previously (23, 28) by
incubation for 10 min with saponin (35 µg/ml) in a medium containing
1% BSA and (in mM) 20 NaCl, 100 KCl, 5 MgSO4, 1 NaH2PO4, 25 NaHCO3, 0.34 CaCl2, and 1 EGTA. The cells were centrifuged at 350 g for 5 min and resuspended in the same medium with 1.5 mM
ATP-regenerating system (5 mM creatine phosphate and 10 U/ml creatine phosphokinase).
Measurement of muscle cell contraction by scanning micrometry.
Contraction was measured in intact and permeabilized muscle cells by
scanning micrometry as described previously (23,
24, 28). An aliquot containing
104 cells/ml was added to 0.1 ml of medium containing CCK-8
(1 nM), neuromedin C (NMC; 100 nM), or EGF (10 nM), and the reaction
was terminated at various intervals with 1% acrolein. The effect of PKC antibodies was determined in permeabilized muscle cells after preincubation for 1 h with different concentrations (1-1,000
ng/ml) of each antibody separately. The mean length of muscle cells
treated with agonist was compared with the mean length of untreated
cells, and contraction was expressed as percent decrease in mean cell length.
Radioreceptor assay for inositol 1,4,5-trisphosphate in dispersed
smooth muscle cells.
Inositol 1,4,5-trisphosphate (IP3) mass was measured in
intact cells as described previously (23, 28)
using an assay system from Amersham. One milliliter of muscle cell
suspension (106 cells/ml) was incubated with
Li+ for 10 min at 31°C, after which EGF (10 nM) or NMC
(100 nM) was added for 30 s and the reaction was terminated with
ice-cold 10% perchloric acid. After centrifugation for 10 min at 750 g, the supernatant was extracted and IP3 content
in the aqueous phase was measured. Results were expressed as picomoles
per 106 cells.
Measurement of [Ca2+]i in dispersed
smooth muscle cells.
[Ca2+]i was measured in suspensions of muscle
cells using the Ca2+ fluorescent dye fura 2 as described
previously (24, 26). Muscle cells were
suspended in a medium containing (in mM) 10 HEPES, 125 NaCl, 5 KCl, 1 CaCl2, 0.5 MgSO4, 5 glucose, 20 taurine, 45 Na
pyruvate, and 5 creatine and incubated with fura 2-AM (2 µM) for 20 min at 31°C. After centrifugation at 350 g for 20 min, the
cells were incubated in fura 2-free medium for immediate measurement of
Ca2+. Fluorescence was monitored at 510 nm, with excitation
wavelengths alternating between 340 and 380 nm, and the measurements
were corrected for autofluorescence of unloaded cells. An estimate of
[Ca2+]i was obtained from observed, maximal,
and minimal fluorescence ratios as described previously
(24, 26).
Measurement of MLCK activity.
MLCK activity was measured by phosphorylation of a smooth muscle MLC
substrate as described by Gilbert et al. (6). After treatment with agonist, the cells were homogenized in a medium containing (in mM) 50 KH2PO4, 4 EDTA, 15 dithiothreitol, 10 NaF, 1 phenylmethylsulfonyl fluoride (pH 6.8), 0.5%
Triton X-100, and 10 µg/ml aprotinin and then centrifuged at 8,000 g for 10 min. The supernatant was added to a mixture
containing (in mM) 0.1 Ca2+, 50 MOPS, 15 dithiothreitol,
and 10 Mg acetate, 0.3 µM CaM, and 18 µM smooth muscle MLC. The
reaction was initiated with 1 mM [
-32P]ATP. Aliquots
were spotted on Whatman filter paper, rinsed successively with 10%
TCA, 4% pyrophosphate, 95% ethanol, and ethyl ether and then dried
for measurement of radioactivity.
Identification of PKC isozymes in intestinal smooth muscle by
Western blot.
Cell homogenates were prepared from dispersed intestinal circular and
longitudinal muscle cells separately and homogenized in a solution
containing (in mM) 10 Tris · HCl (pH 7.5), 5 MgCl2, 2 EDTA, 250 sucrose, 1 dithiothreitol, and 1 phenylmethylsulfonyl fluoride and 20 µg/ml leupeptin and 20 µg/ml aprotinin. The
suspension was centrifuged at 100,000 g for 30 min at 4°C,
and the supernatant was collected as the cytosolic fraction. Pellets
were resuspended, and proteins were extracted by incubation for 30 min
in the homogenization buffer containing 1% Triton X-100 and 1% sodium
cholate. The extract was centrifuged at 1,000 g for 10 min,
and the supernatant was collected as the particulate fraction.
Solubilized membrane proteins (80-100 µg) were resolved by 10%
SDS-PAGE and electrophoretically transferred to nitrocellulose
membranes. After incubation in 5% nonfat dry milk to block nonspecific
antibody binding, the blots were incubated with anti-rabbit IgG
conjugated with horseradish peroxidase. The bands were identified by
enhanced chemiluminescence.
Materials.
[
-32P]ATP was obtained from NEN Life Sciences
Products, HEPES from Research Organics (Cleveland, OH), and soybean
trypsin inhibitor and collagenase (type II) from Worthington. Fura 2-AM
was obtained from Molecular Probes, and calmidazolium, calphostin C,
and chelerythrine chloride were from Calbiochem. Neomycin, KT-5926, and
dimethyleicosadienoic acid (DEDA) were obtained from Biomol (Plymouth
Meeting, PA). G
q/11 and PKC antibodies were from Santa
Cruz Biotechnology (Santa Cruz, CA). All other chemicals were from
Sigma Chemical. Selective N-myristoylated peptide inhibitors
derived from pseudosubstrate sequences of PKC-
, PKC-
,
,
,
PKC-
, and PKC-
were gifts from Drs. D. A. Dartt and D. Zoukhri
(Harvard Medical School).
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RESULTS |
Pathway mediating initial smooth muscle contraction induced by
agonists.
We (24, 26) have previously shown that
Ca2+ mobilization in circular muscle is mediated by
IP3-dependent Ca2+ release, whereas
Ca2+ mobilization in longitudinal muscle is initiated by
phospholipase A2 (PLA2)-dependent
Ca2+ influx, which triggers Ca2+-induced
Ca2+ release via ryanodine receptors/Ca2+
channels (15, 20, 26).
Accordingly, the initial contraction induced by CCK-8 was blocked by
the phosphoinositide inhibitor neomycin in circular muscle cells and by
the PLA2 inhibitor DEDA in longitudinal muscle cells (Fig.
1). The initial Ca2+
transient in both muscle cell types was accompanied by an increase in
MLCK activity that reached a peak within 30 s and declined rapidly
to low levels within 2 min and to basal levels within 5-10 min
(Fig. 2). Results similar to those shown
in Fig. 2 for longitudinal muscle cells were obtained with circular
muscle cells (20). The initial contraction in both cell
types was inhibited by the CaM antagonist calmidazolium and
by Ca2+/CaM-dependent MLCK inhibitor KT-5926 (Fig.
3). The effect of KT-5926 was
concentration dependent with an IC50 of 0.6 nM (Fig. 4). Treatment of permeabilized
circular muscle cells with guanosine 5'-O-(2-thiodiphosphate) (GDP
S) abolished initial and
sustained contraction, implying that both were G protein dependent
(Fig. 5). In contrast,
preincubation of permeabilized circular muscle cells with
G
q/11 antibody (10 µg/ml) for 60 min inhibited the initial contraction but had no effect on sustained contraction (Fig.
5).

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Fig. 1.
Inhibition of CCK-stimulated initial contraction by
neomycin and dimethyleicosadienoic acid (DEDA). Guinea pig intestinal
circular (A) and longitudinal (B) smooth
muscle cells were treated for 10 min with neomycin (50 µM) or DEDA
(10 µM). Contraction was measured at intervals for 20 min in the
presence and absence of each agent. Neomycin and DEDA inhibited initial
contraction in circular and longitudinal muscle cells, respectively,
but had no effect on sustained contraction. Contraction was expressed
as %decrease ( %) in mean cell length from control. Values are
means ± SE of 4 experiments.
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Fig. 2.
Time course of myosin light chain kinase (MLCK) activity
in response to CCK-8. MLCK activity was measured in dispersed
intestinal longitudinal muscle cells as described in MATERIALS
AND METHODS. CCK-8 (1 nM) was added, and samples were obtained at
intervals for 10 min. MLCK activity decreased rapidly, reverting to
basal levels within 5-10 min. Identical results were obtained in
intestinal circular muscle cells. Values are means ± SE of 4 experiments.
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Fig. 3.
Inhibition of CCK-stimulated initial contraction by
calmodulin (CaM) and MLCK inhibitors. Intestinal circular
(A) and longitudinal (B) smooth muscle cells were
treated for 10 min with the CaM inhibitor calmidazolium (1 µM) or the
MLCK inhibitor KT-5926 (1 µM). Contraction was measured at intervals
for 20 min in the presence and absence of each agent. Both inhibitors
blocked initial contraction in circular and longitudinal muscle cells
but had no effect on sustained contraction. Contraction was expressed
as %decrease in mean cell length from control. Values are means ± SE of 4 experiments.
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Fig. 4.
Concentration-dependent inhibition of CCK-stimulated
contraction by MLCK and protein kinase C (PKC) inhibitors. Intestinal
circular smooth muscle cells were treated for 10 min with various
concentrations of the MLCK inhibitor KT-5926, and initial contraction
in response to 1 nM CCK-8 was measured at 30 s (A). In
separate experiments, the muscle cells were treated with various
concentrations of the PKC inhibitor calphostin C, and sustained
contraction was measured at 300 s (B). Contraction was
expressed as %decrease in mean cell length from control.
IC50 of KT-5926 and calphostin C was 0.6 and 12 nM,
respectively. Values are means ± SE of 3 experiments.
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Fig. 5.
Effects of guanosine 5'-O-(2-thiodiphosphate)
(GDP S) and G q/11 antibody (Ab) on CCK-stimulated
contraction. Permeabilized intestinal circular muscle cells were
incubated for 10 min with GDP S or for 60 min with
G q/11 before addition of 1 nM CCK-8. Initial contraction
was measured at 30 s and sustained contraction at 300 s after
addition of CCK-8. GDP S inhibited initial (30 s) and sustained (300 s) contraction, whereas G q/11 antibody inhibited initial
contraction only, suggesting involvement of a distinct G protein in
sustained contraction. Contraction was expressed as %decrease in mean
cell length from control. Values are means ± SE of 4 experiments.
** P < 0.01 vs. control.
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Pathway mediating sustained contraction induced by agonists.
A biphasic increase in DAG and PKC activity occurs in both muscle cell
types consisting of an initial peak that is entirely dependent on
phosphoinositide hydrolysis, followed by a sustained increase resulting
from phosphatidylcholine hydrolysis by PLC and PLD (27).
Nonselective PKC inhibitors as well as isozyme-selective inhibitors and
antibodies were used to determine the involvement of PKC in sustained
contraction. Chelerythrine (10 µM), which blocks the substrate
binding site, abolished sustained contraction in both circular and
longitudinal muscle cells but had no effect on initial contraction
(Fig. 6). Identical results were obtained with calphostin C (1 µM), which blocks the DAG binding site; the effect of calphostin C was concentration dependent with an
IC50 of 12 nM (Fig. 4).

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Fig. 6.
Time course of inhibition of CCK-stimulated contraction
by the PKC inhibitor chelerythrine. Intestinal circular (A)
and longitudinal (B) smooth muscle cells were treated for 10 min with chelerythrine (10 µM), and contraction was measured at
intervals for 20 min in the presence and absence of the inhibitor.
Chelerythrine inhibited sustained contraction but had no effect on
initial contraction. Contraction was expressed as %decrease in mean
cell length from control. Values are means ± SE of 3 experiments.
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Preincubation of permeabilized circular muscle with a specific PKC-
antibody (1 µg/ml) for 1 h abolished sustained contraction but
had no effect on initial contraction, whereas preincubation with a
common PKC-
,
,
antibody (1 µg/ml) had no effect on initial or
sustained contraction (Fig. 7). The
IC50 of PKC-
antibody in inhibiting sustained
contraction was 20 ng/ml (Fig. 7). Similar results were obtained with
selective myristoylated pseudosubstrate PKC inhibitors in intact
circular and longitudinal muscle cells. A selective PKC-
inhibitor
blocked sustained contraction (measured after 5 min of agonist
stimulation) but had no effect on initial contraction (measured at
30 s), whereas selective PKC-
, PKC-
, and PKC-
,
,
inhibitors had no effect on sustained or initial contraction (Fig.
8).

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Fig. 7.
Time course of inhibition of CCK-stimulated contraction
by a specific PKC- antibody. A: permeabilized intestinal
circular smooth muscle cells were incubated for 60 min with a specific
PKC- antibody (1 µg/ml) or a common PKC- , , antibody (1 µg/ml) before addition of 1 nM CCK-8. Contraction was measured at
intervals for 20 min in the presence and absence of antibodies. PKC-
antibody inhibited sustained contraction but had no effect on initial
contraction. PKC- , , antibody had no effect on initial or
sustained contraction. B: effect of various concentrations
of PKC- antibody on sustained contraction (IC50, 20 ng/ml). Contraction was expressed as %decrease in mean cell length
from control. Values are means ± SE of 3 experiments.
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Fig. 8.
Inhibition of CCK-stimulated sustained contraction by a
selective PKC- inhibitor. Intestinal circular (A) and
longitudinal (B) smooth muscle cells were treated for 10 min
with myristoylated pseudosubstrate inhibitors of various PKC isozymes.
Initial (30 s) and sustained (300 s) contraction in response to CCK-8
(1 nM) was measured. Sustained but not initial contraction was
inhibited by the selective PKC- inhibitor; other inhibitors had no
effect. Contraction was expressed as %decrease in mean cell length
from control. Values are means ± SE of 3 experiments.
** P < 0.01 vs. control.
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Western blot analysis using a specific PKC-
antibody and a common
PKC-
,
,
antibody showed that CCK-8 induced delayed
translocation of PKC-
and rapid translocation to the membrane of one
or more of the following: PKC-
, PKC-
, or PKC-
. Translocation
of PKC isozymes attained a plateau within 5 min (Fig.
9). The pattern of translocation was
similar in circular and longitudinal muscle cells.

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Fig. 9.
Translocation of PKC isozymes in response to CCK-8. PKC
isozymes were identified in the particulate fraction after stimulation
of guinea pig intestinal circular muscle cells (A) and
longitudinal muscle cells (B) with CCK-8 (1 nM) using a
specific PKC- antibody and a common PKC- , , antibody.
Densitometric analysis showed delayed (after 30 s) translocation
of PKC- and prompt translocation of one or more of the following
Ca2+-dependent PKC isozymes: PKC- , PKC- , and PKC- .
Values are means ± SE of 3 experiments.
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Pathways mediating initial and sustained contraction induced by EGF
and NMC.
The effects of various inhibitors and PKC antibodies on contraction
induced by EGF and NMC, the active COOH-terminal decapeptide of
gastrin-releasing peptide, were examined in rabbit intestinal circular
muscle cells. The response to CCK-8 in rabbit muscle cells was
previously shown to be closely similar to that in guinea pig muscle
cells (15, 29). EGF (10 nM) and NMC (100 nM)
stimulated IP3 formation (16.0 ± 3.2 and 10.0 ± 2.3 pmol/106 cells, respectively), increased
[Ca2+]i levels (388 ± 53 and 390 ± 20 nM, respectively), and induced contraction. Neomycin abolished
the increase in IP3 and [Ca2+]i
induced by both peptides and inhibited contraction during the first 2 min (Fig. 10). Sustained contraction
was not affected by neomycin but was inhibited by calphostin C (Fig.
10).

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Fig. 10.
Effect of neomycin and calphostin C on epidermal growth
factor (EGF)- and neuromedin C (NMC)-stimulated contraction. Intestinal
circular smooth muscle cells were treated with 10 nM EGF (A)
or 100 nM NMC (B) in the presence and absence of neomycin
(50 µM) or calphostin C (1 µM). Contraction was measured at 30 s (initial contraction) and 300 s (sustained contraction).
Neomycin partly inhibited initial contraction but had no effect on
sustained contraction. Sustained contraction was abolished by
calphostin C. Similar results were obtained with longitudinal muscle
cells (data not shown). Contraction was expressed as %decrease in mean
cell length from control. Values are means ± SE of 3 experiments.
** P < 0.01 vs. control.
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Sustained contraction induced by NMC was abolished by preincubation of
permeabilized circular muscle cells for 1 h with PKC-
antibody
(1 µg/ml) but not with a common PKC-
,
,
antibody (1 µg/ml).
In contrast, sustained contraction induced by EGF was abolished by
preincubation of permeabilized circular muscle cells for 1 h with
a common PKC-
,
,
antibody (1 µg/ml) but not with a PKC-
antibody (Fig. 11). A specific
pseudosubstrate PKC-
inhibitor (1 µM) inhibited EGF-induced
sustained contraction by 63.4 ± 3.7% (P < 0.001); the residual response reflected involvement of PKC-
and/or
PKC-
.

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Fig. 11.
Differential inhibition of EGF- and NMC-stimulated
sustained contraction by PKC isozyme antibodies. Permeabilized
intestinal circular smooth muscle cells were incubated for 60 min with
a specific PKC- antibody (1 µg/ml) or a common PKC- , ,
antibody (1 µg/ml) before addition of EGF (10 nM) or NMC (100 nM);
contraction was measured at 30 s (initial contraction) and
300 s (sustained contraction). PKC- , , antibody inhibited
sustained contraction induced by EGF (A), and PKC-
antibody inhibited sustained contraction mediated by NMC
(B). Contraction was expressed as %decrease in mean cell
length from control. Values are means ± SE of 3 experiments.
**P < 0.01 vs. control.
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Pathways mediating initial and sustained contraction induced by
Ca2+.
Contraction induced by a near-maximal concentration of Ca2+
(0.4 µM) in permeabilized circular and longitudinal muscle cells rose
to a peak within 30 s before declining to a lower sustained level
(Fig. 12). The initial contraction was
abolished by calmidazolium and KT-5926, implying that it was mediated
by activation of Ca2+/CaM-dependent MLCK (Fig.
13). Unexpectedly, sustained
contraction was not affected by calmidazolium or KT-5926 but was
abolished by calphostin C (Fig. 13). Inhibition by
calphostin C, which blocks the DAG binding site of PKC, implied that a
high concentration of Ca2+ activated one or more
phospholipases capable of generating DAG (17).
Preincubation of permeabilized circular and longitudinal muscle cells
with a common PKC-
,
,
antibody abolished sustained contraction
but had no effect on initial contraction; preincubation with a specific
PKC-
antibody had no effect on initial or sustained contraction
(Fig. 12).

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Fig. 12.
Inhibition of Ca2+-stimulated sustained
contraction by PKC- , , antibody. Permeabilized intestinal
circular (A) and longitudinal (B) smooth muscle
cells were exposed to 400 nM Ca2+ alone or after a 60-min
incubation with a specific PKC- antibody (1 µg/ml) or a common
PKC- , , antibody (1 µg/ml). Contraction was measured at
intervals for 20 min in the presence or absence of antibodies.
PKC- , , antibody inhibited sustained contraction but had no
effect on initial contraction. PKC- antibody had no effect on
initial or sustained contraction. Contraction was expressed as
%decrease in mean cell length from control. Values are means ± SE of 3 experiments.
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Fig. 13.
Effect of various inhibitors on
Ca2+-stimulated initial and sustained contraction.
Permeabilized intestinal circular (A) and longitudinal
(B) muscle cells were treated for 10 min with each agent
before raising the Ca2+ concentration to 400 nM. Initial
contraction was measured at 30 s, and sustained contraction was
measured at 300 s in the presence or absence of each inhibitor.
The CaM and MLCK inhibitors blocked initial contraction, whereas
calphostin C blocked sustained contraction in both muscle cell types.
Contraction was expressed as %decrease in mean cell length from
control. Values are means ± SE of 3 experiments. Calmdz,
calmidazolium. ** P < 0.01 vs. control.
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DISCUSSION |
The experimental design adopted in the present study attempted to
maintain the normal sequence of signaling events that mediate the
initial and sustained phases of contraction in smooth muscle. Selective
inhibitors were used in intact muscle cells and specific PKC and G
protein antibodies in permeabilized muscle cells to demonstrate that
agonist-stimulated, sustained contraction was independent of
Ca2+ mobilization and reflected G protein-dependent
activation of PKC-
. Sustained contraction induced by EGF, however,
reflected activation of one or more Ca2+-dependent PKC
isozymes (PKC-
, PKC-
, and/or PKC-
). Unexpectedly, sustained
contraction induced by Ca2+ in permeabilized muscle cells
was also mediated by PKC-
, PKC-
, and/or PKC-
. The evidence on
which these conclusions are based is summarized below.
Inhibition of agonist-stimulated Ca2+ mobilization by
neomycin in intestinal circular muscle (26) and by DEDA in
longitudinal muscle (26) abolished initial contraction but
had no effect on sustained contraction. Because neither
Ca2+ release nor capacitative Ca2+ influx
occurs under these conditions, the mechanism(s) responsible for
sustained contraction did not require an increase in
[Ca2+]i above resting levels. The lack of
effect of DEDA on sustained contraction suggests that PLA2
products, such as arachidonic acid, are not involved in maintaining
contraction (5, 33).
Suppression of Ca2+/CaM-dependent MLCK activity while
maintaining Ca2+ mobilization also abolished initial
contraction but had no effect on sustained contraction. This implied
that MLC20 phosphorylation by
Ca2+/CaM-dependent MLCK is not a prerequisite for sustained
contraction. Sustained MLC20 phosphorylation by other
kinases such as Ca2+-independent MLCK (39) and
RhoA kinase (1) could occur, amplified by inhibition of
MLCP (12, 36).
Suppression of G protein activity by GDP
S abolished both initial and
sustained contraction, whereas blockade of Gq/11, which initiates the cascade that leads to Ca2+ mobilization
(28) abolished initial contraction only, suggesting that
distinct heterotrimeric and/or monomeric G proteins are involved in
sustained contraction.
Sustained contraction induced by CCK-8 was abolished by 1)
chelerythrine and calphostin C, which block the substrate- and DAG-binding sites of PKC, respectively, 2) a specific
PKC-
antibody, and 3) a selective
N-myristoylated pseudosubstrate peptide inhibitor of
PKC-
. The specific involvement of PKC-
in sustained contraction was corroborated by results obtained with NMC, which also interacts with a G protein-coupled receptor (11).
Sustained contraction induced by the growth factor EGF, however, was
abolished by a common PKC-
,
,
antibody and partially inhibited
by a selective myristoylated pseudosubstrate peptide inhibitor of
PKC-
, implying that it was mediated by PKC-
as well as by PKC-
and/or PKC-
. Sustained contraction induced by phorbol 12-myristate
13-acetate was also abolished by a PKC-
,
,
antibody but was not
affected by a PKC-
antibody (K. S. Murthy, unpublished
observations). Thus depending on the agonist,
Ca2+-dependent and -independent PKC isozymes can mediate
sustained contraction.
Unexpectedly, a near-maximal concentration of Ca2+ (0.4 µM) induced an initial contraction mediated by
Ca2+/CaM-dependent MLCK, followed by a sustained
contraction mediated by PKC. Sustained contraction was abolished by
calphostin C and by a common PKC-
,
,
antibody but was not
affected by a CaM antagonist or a MLCK inhibitor. This suggests that
Ca2+/CaM-dependent MLCK was inactive at high
Ca2+ concentrations, possibly inactivated by other kinases,
e.g., Ca2+/CaM-dependent protein kinase II and/or
p21-activated kinase. Inhibition by calphostin C implied that high
Ca2+ concentrations activated one or more phospholipases
capable of generating DAG and activating PKC. Previous studies
(25) had shown that increasing
[Ca2+]i in the absence of receptor activation
stimulates PKC activity.
Thus sustained contraction induced by activation of G protein-coupled
receptors (CCK and NMC) and receptor tyrosine kinases (EGF) or by high
levels of Ca2+ is mediated variously by
Ca2+-dependent or -independent PKC isozymes. Studies
designed to elicit evidence of Ca2+ sensitization, however,
have yielded contradictory results regarding the involvement of PKC in
sustained muscle contraction (8, 10,
18, 37). In these studies,
[Ca2+] is clamped below (0.01 µM) or above (0.3-30
µM) resting [Ca2+]i levels
(0.1 µM) to probe the effects of various agents (agonists, GTP
S,
active phorbol esters, DAG analogs, and MLCP inhibitors). Morgan and
co-workers (8, 9, 18,
22) have studied extensively the response of arterial and
portal vein single smooth muscle cells in the ferret. Their studies
(8, 9, 18, 22)
disclosed the existence of a slowly developing, agonist-stimulated,
PKC-dependent contraction that was mediated by PKC-
in arterial
muscle cells at resting Ca2+ levels and by PKC-
and/or
PKC-
in portal vein muscle cells at higher Ca2+ levels.
The involvement of distinct PKC isozymes reflected the predominant
expression and translocation of these isozymes in the two types of
smooth muscle. A similar tissue-specific translocation of PKC isozymes
was also reported by Sohn et al. (32) in smooth muscle
cells of the cat esophageal body and the lower esophageal sphincter.
Contraction induced by a DAG analog in sphincteric circular muscle,
which resembles intestinal circular muscle in its signaling properties,
was mediated by a splice variant of PKC-
(PKC-
II), whereas
contraction in esophageal muscle was mediated by PKC-
; in the latter
tissue, agonist-stimulated contraction is thought to be
Ca2+ and CaM independent (2, 32).
The involvement of distinct PKC isozymes reflected the expression of
these isozymes in different regions of the esophagus.
Other investigators (10, 37) have used PKC
downregulation to test the involvement of PKC in Ca2+
sensitization. Prolonged exposure of rabbit arterial or portal vein
muscle strips to phorbol dibutyrate completely downregulated some PKC
isozymes (e.g., PKC-
), and drastically downregulated (~70%)
without abolishing other isozymes (e.g., PKC-
, PKC-
1, and
PKC-
). The procedure abolished the response (in the presence of 0.3 µM Ca2+) to phorbol dibutyrate and partly reduced the
response to agonists and GTP
S in arterial muscle without affecting
the response in portal vein muscle. It should be noted that in studies
of Ca2+ sensitization where Ca2+ is clamped at
physiological (0.3 µM) or supraphysiological (30 µM) levels, the
distinction between an initial response to exogenous Ca2+
or agonist that involves activation of MLCK and a sustained response that involves activation of PKC is blurred. This issue is underscored by the results obtained in the present study with exogenous
Ca2+ where the effect of Ca2+ was reflected in
an initial MLCK-dependent response and a subsequent sustained response
mediated by Ca2+-dependent PKC isozymes.
Recent studies (5, 29) have provided a link
between G protein activation and PKC activation that could lead to
inhibition of MLC20 phosphatase and enhancement of
MLC20 phosphorylation. GTP
S-induced translocation of the
monomeric G protein RhoA (4, 7) and
activation of RhoA kinase can lead to direct phosphorylation of
MLC20 at Ser 19 (1), as well as inhibition of
MLC20 phosphatase (12, 16,
36). Our (29) recent studies in intestinal
muscle cells indicate that agonist (CCK)-induced activation of
G13 leads to sequential activation of RhoA, RhoA kinase,
and PLD. Conversion of phosphatidic acid, the primary product of PLD,
to DAG activates PKC, resulting in sustained contraction and
MLC20 phosphorylation. G
13 and RhoA
antibodies inhibited PLD activity; and both antibodies and the PLD
inhibitor PCCG 16 inhibited sustained muscle cell contraction. Eto and
co-workers (3) have recently cloned a 17-kDa PKC-activated
endogenous protein that inhibits MLC20 phosphatase and
could serve as a link between G protein (G13)-dependent
activation of PKC and sustained MLC20 phosphorylation and
contraction. This pathway is consistent with the ability of Rho kinase
to inhibit MLC phosphatase activity. A recently identified
Ca2+-independent MLCK could contribute to the maintenance
of MLC20 phosphorylation at low or resting Ca2+
levels (39).
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-15564.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
G. M. Makhlouf, PO Box 980711, Medical College of Virginia,
Virginia Commonwealth Univ., Richmond, VA 23298-0711 (E-mail: makhlouf{at}hsc.vcu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 19 November 1999; accepted in final form 9 February 2000.
 |
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