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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-epsilon antibody, and 3) a pseudosubstrate PKC-epsilon inhibitor. GDPbeta S abolished both initial and sustained contraction, whereas a Galpha 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-alpha ,beta ,gamma antibody, and a pseudosubstrate PKC-alpha 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-alpha ,beta ,gamma 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-zeta (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-epsilon in aortic muscle and PKC-alpha and PKC-beta 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-epsilon 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 [gamma -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. [gamma -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). Galpha 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-alpha , PKC-alpha ,beta ,gamma , PKC-delta , and PKC-epsilon were gifts from Drs. D. A. Dartt and D. Zoukhri (Harvard Medical School).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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) (GDPbeta S) abolished initial and sustained contraction, implying that both were G protein dependent (Fig. 5). In contrast, preincubation of permeabilized circular muscle cells with Galpha 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 (Delta %) 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) (GDPbeta S) and Galpha q/11 antibody (Ab) on CCK-stimulated contraction. Permeabilized intestinal circular muscle cells were incubated for 10 min with GDPbeta S or for 60 min with Galpha 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. GDPbeta S inhibited initial (30 s) and sustained (300 s) contraction, whereas Galpha 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.

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.

Preincubation of permeabilized circular muscle with a specific PKC-epsilon antibody (1 µg/ml) for 1 h abolished sustained contraction but had no effect on initial contraction, whereas preincubation with a common PKC-alpha ,beta ,gamma antibody (1 µg/ml) had no effect on initial or sustained contraction (Fig. 7). The IC50 of PKC-epsilon 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-epsilon 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-alpha , PKC-delta , and PKC-alpha ,beta ,gamma 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-epsilon antibody. A: permeabilized intestinal circular smooth muscle cells were incubated for 60 min with a specific PKC-epsilon antibody (1 µg/ml) or a common PKC-alpha ,beta ,gamma 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-epsilon antibody inhibited sustained contraction but had no effect on initial contraction. PKC-alpha ,beta ,gamma antibody had no effect on initial or sustained contraction. B: effect of various concentrations of PKC-epsilon 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-epsilon 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-epsilon 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.

Western blot analysis using a specific PKC-epsilon antibody and a common PKC-alpha ,beta ,gamma antibody showed that CCK-8 induced delayed translocation of PKC-epsilon and rapid translocation to the membrane of one or more of the following: PKC-alpha , PKC-beta , or PKC-gamma . 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-epsilon antibody and a common PKC-alpha ,beta ,gamma antibody. Densitometric analysis showed delayed (after 30 s) translocation of PKC-epsilon and prompt translocation of one or more of the following Ca2+-dependent PKC isozymes: PKC-alpha , PKC-beta , and PKC-gamma . Values are means ± SE of 3 experiments.

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.

Sustained contraction induced by NMC was abolished by preincubation of permeabilized circular muscle cells for 1 h with PKC-epsilon antibody (1 µg/ml) but not with a common PKC-alpha ,beta ,gamma 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-alpha ,beta ,gamma antibody (1 µg/ml) but not with a PKC-epsilon antibody (Fig. 11). A specific pseudosubstrate PKC-alpha inhibitor (1 µM) inhibited EGF-induced sustained contraction by 63.4 ± 3.7% (P < 0.001); the residual response reflected involvement of PKC-beta and/or PKC-gamma .


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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-epsilon antibody (1 µg/ml) or a common PKC-alpha ,beta ,gamma 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-alpha ,beta ,gamma antibody inhibited sustained contraction induced by EGF (A), and PKC-epsilon 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.

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-alpha ,beta ,gamma antibody abolished sustained contraction but had no effect on initial contraction; preincubation with a specific PKC-epsilon 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-alpha ,beta ,gamma 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-epsilon antibody (1 µg/ml) or a common PKC-alpha ,beta ,gamma antibody (1 µg/ml). Contraction was measured at intervals for 20 min in the presence or absence of antibodies. PKC-alpha ,beta ,gamma antibody inhibited sustained contraction but had no effect on initial contraction. PKC-epsilon 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-epsilon . Sustained contraction induced by EGF, however, reflected activation of one or more Ca2+-dependent PKC isozymes (PKC-alpha , PKC-beta , and/or PKC-gamma ). Unexpectedly, sustained contraction induced by Ca2+ in permeabilized muscle cells was also mediated by PKC-alpha , PKC-beta , and/or PKC-gamma . 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 GDPbeta 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-epsilon antibody, and 3) a selective N-myristoylated pseudosubstrate peptide inhibitor of PKC-epsilon . The specific involvement of PKC-epsilon 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-alpha ,beta ,gamma antibody and partially inhibited by a selective myristoylated pseudosubstrate peptide inhibitor of PKC-alpha , implying that it was mediated by PKC-alpha as well as by PKC-beta and/or PKC-gamma . Sustained contraction induced by phorbol 12-myristate 13-acetate was also abolished by a PKC-alpha ,beta ,gamma antibody but was not affected by a PKC-epsilon 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-alpha ,beta ,gamma 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, GTPgamma 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-epsilon in arterial muscle cells at resting Ca2+ levels and by PKC-alpha and/or PKC-beta 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-beta (PKC-beta II), whereas contraction in esophageal muscle was mediated by PKC-epsilon ; 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-theta ), and drastically downregulated (~70%) without abolishing other isozymes (e.g., PKC-alpha , PKC-beta 1, and PKC-epsilon ). The procedure abolished the response (in the presence of 0.3 µM Ca2+) to phorbol dibutyrate and partly reduced the response to agonists and GTPgamma 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. GTPgamma 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. Galpha 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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