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Am J Physiol Gastrointest Liver Physiol 293: G699-G710, 2007. First published July 26, 2007; doi:10.1152/ajpgi.00214.2007
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HORMONES AND SIGNALING

Characterization of protein kinase pathways responsible for Ca2+ sensitization in rat ileal longitudinal smooth muscle

Eikichi Ihara, Lori Moffat, Janina Ostrander, Michael P. Walsh, and Justin A. MacDonald

Smooth Muscle Research Group and Department of Biochemistry and Molecular Biology, University of Calgary, Faculty of Medicine, Calgary, Alberta, Canada

Submitted 10 May 2007 ; accepted in final form 24 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We investigated the protein kinases responsible for myosin regulatory light chain (LC20) phosphorylation and regulation of myosin light chain phosphatase (MLCP) activity during microcystin (phosphatase inhibitor)-induced contraction at low Ca2+ concentrations of rat ileal smooth muscle stretched in the longitudinal axis. Application of 1 µM microcystin induced LC20 diphosphorylation and contraction of beta-escin-permeabilized rat ileal smooth muscle at pCa 9. The PKC inhibitor GF-109203x, the MEK inhibitor PD-98059, and the p38 MAPK inhibitor SB-203580 significantly reduced this contraction. These inhibitory effects were abolished when the microcystin concentration was increased to 10 µM, indicating that application of these kinase inhibitors generated an increase in MLCP activity. GF-109203x and PD-98059, but not SB-203580, significantly decreased the phosphorylation level of the myosin-targeting subunit of MLCP, MYPT1, at Thr-697 (rat sequence) during microcystin-induced contraction at pCa 9. On the other hand, SB-203580, but not GF-109203x or PD-98059, significantly reduced the phosphorylation level of the PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17). A zipper-interacting protein kinase (ZIPK) inhibitor (SM1 peptide) and a Rho-associated kinase inhibitor (Y-27632) had little effect on microcystin-induced contraction at pCa 9. In conclusion, PKC, ERK1/2, and p38 MAPK pathways facilitate microcystin-induced contraction at low Ca2+ concentrations by contributing to the inhibition of MLCP activity either through phosphorylation of MYPT1 or CPI-17 [probably mediated by integrin-linked kinase (ILK)]. ILK and not ZIPK is likely to be the protein kinase responsible for LC20 diphosphorylation during microcystin-induced contraction of rat ileal smooth muscle at pCa 9, similar to its recently described role in vascular smooth muscle. The negative regulation of MLCP by PKC and MAPKs during microcystin-induced contraction at pCa 9, which is not observed in vascular smooth muscle, may be unique to phasic smooth muscle.

ileum; protein kinase C; myosin phosphatase; mitogen-activated protein kinase; protein kinase C-potentiated phosphatase inhibitor protein of 17 kDa; myosin-targeting subunit of myosin light chain phosphatase

SMOOTH MUSCLE CONTRACTION is a dynamic and highly regulated process. The contractile state of smooth muscle is mainly regulated by phosphorylation of the 20-kDa myosin regulatory light chain (LC20) (45), which is controlled by the opposing activities of myosin light chain kinase (MLCK) (48) and myosin light chain phosphatase (MLCP) (20). MLCK activity is dependent on Ca2+-calmodulin; hence, the intracellular Ca2+ signal {increased intracellular Ca2+ concentration ([Ca2+]i)} is the primary determinant of smooth muscle contraction. Force can be further increased through signaling pathways that modulate MLCK and/or MLCP activities. The Ca2+ sensitivity of contraction can be affected by any change in the ratio of MLCK:MLCP activity. A decrease in MLCP activity will shift the balance in favor of MLCK, resulting in a greater degree of LC20 phosphorylation and contraction. The phenomenon of Ca2+ sensitization (45), which is associated with inhibition of MLCP activity, is mediated by an agonist-induced, G protein-coupled process. Inhibition of MLCP can occur either directly by phosphorylation of the myosin-targeting subunit of MLCP (MYPT1) (19, 50) or indirectly via phosphorylation of a protein kinase C (PKC)-potentiated phosphatase inhibitor protein of 17 kDa (CPI-17) (33). The Thr-697 site of MYPT1 (rat sequence) has been identified as a key inhibitory phosphorylation site for the regulation of MLCP activity. Rho-associated kinase (ROK) (25), zipper-interacting protein kinase (ZIPK) (3, 24, 34, 40), and integrin-linked kinase (ILK) (28, 39) have been demonstrated to phosphorylate MYPT1 at this inhibitory site. In addition, ROK (32), ZIPK (35), and ILK (9) contribute indirectly to MLCP inhibition by playing a role in CPI-17 phosphorylation. CPI-17, when phosphorylated at the regulatory Thr-38 site, becomes a potent inhibitor of MLCP (13).

When microcystin, a type 1 and 2A protein phosphatase (PP) inhibitor, is applied to permeabilized smooth muscle in low-Ca2+ medium, a sustained contraction is observed that cannot be attributed to MLCK (16, 40, 53). Microcystin unmasks Ca2+-independent protein kinase activity and induces diphosphorylation of LC20 at Ser-19 and Thr-18 to activate cross-bridge cycling and force development (53). In vascular smooth muscle, the microcystin-induced contraction at pCa 9 was inhibited by staurosporine (a general protein kinase inhibitor) but was unaffected by several other protein kinase inhibitors, including the ROK inhibitor Y-27632, the PKC inhibitor GF-109203x, the MLCK inhibitor ML-7, and the MLCK and phosphatidylinositol 3-kinase inhibitor wortmannin. To date, only ILK and/or ZIPK have emerged as bona fide candidates for the Ca2+-independent diphosphorylation of LC20 at Ser-19 and Thr-18 in smooth muscle (55). A synthetic peptide inhibitor, derived from the autoinhibitory domain of MLCK, was used to distinguish between ZIPK and ILK effects in smooth muscle (23). It was concluded that ILK, and not ZIPK, is responsible for Ca2+-independent diphosphorylation of LC20 and contraction of vascular smooth muscle (55).

Gastrointestinal motility is regulated by activation and coupling of muscarinic receptors in numerous cell types, including enteric neurons, interstitial cells of Cajal, and smooth muscle cells. Gastrointestinal smooth muscle expresses both M2 (major isoform) and M3 (minor isoform) muscarinic receptors (58), which are known to be indispensable for gastrointestinal smooth muscle contraction (5). The M3 receptor contributes to the contraction of intestinal smooth muscle in a similar manner to that observed in vascular tissue (15). The stimulation of M3 receptors, which are coupled to Gq/11, activates phospholipase C and produces inositol 1,4,5-trisphosphate and diacylglycerol. These second messengers elicit the activation of PKC and trigger an increase in [Ca2+]i (15). On the other hand, M2 receptors act through Gi/o to regulate adenylyl cyclase. Although the inhibition of adenylyl cyclase is a classical and established effect of M2 receptor activation in smooth muscle, other possible downstream signaling pathways that may be coupled to M2 receptors have been proposed, including Src-family tyrosine kinases (44), ERK1/2 (7), and p38 MAPK (7). Recently, both ERK1/2 and p38 MAPK have been implicated in the Ca2+ sensitization of contraction of esophageal smooth muscle (18). Acetylcholine-induced contraction was reported to be dependent on PKC in the cat esophageal smooth muscle, and this PKC-dependent contraction was mediated by ERK1/2 and p38 MAPK (6). In spite of recent evidence that ERK1/2 and/or p38 MAPK are involved in gastrointestinal smooth muscle contraction, the precise mechanisms by which these protein kinases contribute to smooth muscle contractility have yet to be determined. The phosphorylation of caldesmon and/or calponin is thought to be one mechanism by which ERK1/2 contributes to smooth muscle contraction (26, 31). Alternatively, p38 MAPK has been shown to phosphorylate and activate MAPK-activated protein kinase 2 (MAPKAPK-2), which in turn can phosphorylate heat shock protein (HSP) 27 (8, 42) and contribute to smooth muscle contraction (18).

Although numerous studies have investigated Ca2+ sensitization of smooth muscle contractility, the majority of this research has used vascular smooth muscle as a model system. Thus our understanding of the fundamental mechanisms that regulate Ca2+ sensitization in intestinal smooth muscle is limited. The objective of the present study was to explore the signaling pathway(s) responsible for regulation of MLCP activity and LC20 phosphorylation during contraction of intestinal smooth muscle at low [Ca2+].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. All chemicals were reagent grade unless otherwise indicated. Triton X-100, beta-escin, PD-98059, SB-203580, and GF-109203x were obtained from Sigma (St. Louis, MO). Gö-6976 and A-23187 were from Calbiochem (San Diego, CA). Microcystin LR was obtained from Alexis Biochemical (San Diego, CA). Anti-MAP kinase 1/2 (which recognizes both ERK1 and ERK2) and anti-phospho-MAP kinase (which recognizes the dual-phosphorylated pTEpY motif of ERK1 and ERK2) were from Upstate (Charlottesville, VA). Anti-p38 MAPK (which recognizes various SAPK2 isoforms) and anti-phospho p38 MAPK (which recognizes dual phosphorylation of SAPK2 isozymes at Thr-180 and Tyr-182) were from Stressgen Bioreagents (Ann Arbor, MI). Monoclonal antibody specific for MYPT1 phosphorylated at Thr-697 (anti-[phospho-Thr-697]-MYPT1; rat numbering) was purified as previously described (54). Polyclonal MYPT1 antibody generated against the NH2-terminal fragment of rat MYPT1 was a gift from Dr. Timothy Haystead (Duke University, Durham, NC) and was used to quantify total MYPT1 levels. Polyclonal antibodies specific for total CPI-17 and phosphorylated CPI-17 (anti-[phospho-Thr-38]-CPI-17) were purchased from Upstate. SM1 peptide (sequence: AKKLSKDRMKKYMARRKWQKTG) was produced by the University of Calgary Peptide Synthesis Facility (Calgary, AB), confirmed by amino acid analysis, and shown to be >95% pure by analytical HPLC. The SM1 peptide corresponds to the autoinhibitory domain of smooth muscle MLCK (residues 783–804 of chicken gizzard MLCK) and has recently been shown to be a novel inhibitor of ZIPK (22). This peptide, which has no effect on ILK, can be used to distinguish between ZIPK and ILK effects in smooth muscle tissues.

Tissue preparation and force measurement of longitudinal smooth muscle strips. Ileum was removed from rats anesthetized and euthanized according to protocols approved by the University of Calgary Animal Care and Use Committee. Ileal smooth muscle sheets were dissected and cut into longitudinal smooth muscle strips (250 µm x 2 mm). For force measurement, muscle strips were tied with silk monofilaments to the tips of two fine wires. One wire was fixed, and the other was connected to a force transducer (SensoNor, AE801). The strip was mounted in a well on a stir plate to allow rapid solution exchange. Strips were stretched in the longitudinal axis to 1.3x resting length and were equilibrated for 30 min in normal extracellular solution (NES) containing (in mM) 150 NaCl, 4 KCl, 2 CaMS2, 1 MgMS2, 5.5 glucose, and 5 HEPES, pH 7.3. After obtaining a good contractile response with high-K+ extracellular solution [KES; replacement of NaCl in NES solution with equimolar KMS], muscle strips were permeabilized by incubation with 50 µM beta-escin for 40 min in an intracellular solution (G1), with 10 µM A-23187 added for the final 10 min to deplete intracellular Ca2+ stores. The composition of G1 solution was (in mM) 30 piperazine-1,4-bis(2-ethanesulfonic acid) (K2PIPES), 10 creatine phosphate (Na2CP), 5.16 Na2ATP, 7.31 MgMS2, 74.1 KMS, and 1 ethylenebis(oxyethylenenitrilo)tetraacetic acid (K2EGTA). Different free Ca2+ concentrations (expressed as pCa) were obtained by mixing the CaG and G10 solutions to achieve the desired Ca-EGTA/EGTA ratio. The composition of CaG solution was (in mM) 30 K2PIPES, 10 Na2CP, 5.14 Na2ATP, 7.25 MgMS2, 47.1 KMS, and 10 K2CaEGTA; G10 solution was (in mM) 30 K2PIPES, 10 Na2CP, 5.14 Na2ATP, 7.92 MgMS2, 46.6 KMS, and 10 K2EGTA; pCa 9 solution was (in mM) 30 K2PIPES, 10 Na2CP, 5.14 Na2ATP, 7.91 MgMS2, 46.6 KMS, and 9.96 K2EGTA, with 36.1 µM K2CaEGTA; and pCa 4.5 solution was (in mM) 30 K2PIPES, 10 Na2CP, 5.14 Na2ATP, 7.25 MgMS2, 47.1 KMS, and 9.99 K2CaEGTA, with 6.22 µM K2EGTA. The force levels obtained with relaxing solution (pCa 9 or G10) and pCa 4.5 were designated as 0 and 100%, respectively. Maximal contraction was obtained with maximal [Ca2+] (pCa 4.5) before and after the protocol in all experiments. All contractile measurements were carried out at room temperature (23°C).

Western blot analysis of smooth muscle tissue. Rat ileum was collected, and squares (5 x 5 mm) were mounted in a silicone-bottom dish. The muscles were permeabilized and washed as described in the same section. To test the phosphorylation of ERK1/2, p38 MAPK, and MYPT1, ileum was treated with vehicle or the protein kinase inhibitors GF-109203x, PD-98059, or SB-203580 (Table 1) before the application of microcystin. Tissue was flash frozen in liquid N2 and was homogenized in 10 volumes of sample buffer containing 1% SDS, 30 mM Tris, pH 6.8, 12.5% (vol/vol) glycerol, and (p-amidino-phenyl)methanesulfonyl fluoride (APMSF) by using a glass-glass, hand-operated homogenizer. Homogenates were resolved on 12% SDS-polyacrylamide gels and were transferred to polyvinylidene difluoride (PVDF) membranes in 25 mM Tris, 192 mM glycine, and 20% (vol/vol) methanol at 110 V for 60 min at 4°C. Nonspecific binding sites were blocked with 5% (wt/vol) nonfat dry milk in TBST [25 mM Tris, pH 7.4, 150 mM NaCl, 0.05% (vol/vol) Tween-20]. The blots were washed and incubated for 1 h with primary antibody (1:1,000 dilution of ERK1/2, phospho-ERK1/2, p38 MAPK, phospho-p38 MAPK, and MYPT1 antibodies and 1:500 dilution of phospho-MYPT1 antibody) in 1% (wt/vol) nonfat dry milk in TBST. The blots were washed and then incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution) in 1% (wt/vol) nonfat dry milk in TBST. Blots were developed with enhanced chemiluminescence (GE Healthcare). The bands were quantified by densitometry, and the relative phosphorylation levels of ERK1/2, p38 MAPK, and MYPT1 were expressed as a function of the density of the total protein species.


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  		Table 1. IC50/Ki values for inhibitory compounds of selected protein kinases

 
Western blot analysis of CPI-17. To examine the phosphorylation of CPI-17, ileal smooth muscle strips were frozen by immersion in a dry-ice/acetone slurry with 10% (wt/vol) trichloroacetic acid (TCA) and 10 mM dithiothreitol (DTT). Strips were washed in 10 mM DTT in acetone and were lyophilized overnight. Muscle tissues were homogenized in sample buffer in a glass-glass, hand-operated homogenizer. Aliquots of the homogenate were resolved on 18% SDS-polyacrylamide gels and were transferred to PVDF membrane in 10 mM sodium cyclohexylaminopropane sulfonic acid (pH 11) and 10% (vol/vol) methanol at 50 mA for 4 h at 4°C. Nonspecific binding sites were blocked with I-Block solution (Applied Biosystems, Foster City, CA) in TBST overnight at 4°C. Blots were incubated with primary antibody for total or phospho-CPI-17 (1:2,500 dilution) in 1% (wt/vol) nonfat dry milk in TBST for 1 h. The blots were then washed and incubated with horseradish peroxidase-conjugated secondary antibody (1:50,000 dilution) in 1% (wt/vol) nonfat dry milk in TBST for 1 h at 20°C. The membrane was developed with Supersignal West Femto enhanced chemiluminescence (Pierce Chemical, Rockford, IL). Bands were quantified by densitometry, and the relative phosphorylation levels of CPI-17 were calculated as a function of the density of total CPI-17.

Measurement of LC20 phosphorylation. Urea/glycerol gel electrophoresis and Western blotting to separate and quantify un-, mono-, and diphosphorylated forms of LC20 were performed as previously described (23, 53). Contractile responses were halted by immersion of ileal muscle strips in a dry-ice/acetone solution containing 10% (wt/vol) trichloroacetic acid. The muscle strips were washed with a 10 mM DTT-acetone solution and were lyophilized overnight. Muscle proteins were extracted in a buffer containing 8 M urea, 1 M thiourea, and 10 mM DTT before separation by urea/glycerol gel electrophoresis. Western blotting was carried out with a polyclonal antibody that detects both phosphorylated and unphosphorylated LC20. The stoichiometry of LC20 phosphorylation was calculated from the following equation: mol of Pi/mol LC20 = (y + 2z)/(x + y + z), where x, y, and z are the signal intensities of un-, mono-, and diphosphorylated LC20 bands, respectively.

Data analysis. All smooth muscle contractile data were collected by using a computerized data-acquisition system (PowerLab/8SP data-recording unit and Chart software; ADInstruments, Colorado Springs, CO). All data are expressed as means ± SE. Student's t-test was used to determine statistical significance, with P < 0.05 considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Microcystin-induced contraction of ileal smooth muscle at pCa 9. Because the activity of MLCP (a type 1 protein serine/threonine phosphatase, PP1) is known to be much greater in intestinal smooth muscle than in vascular smooth muscle (16), we hypothesized that the protein kinase networks activated in intestinal smooth muscle, which contribute to contraction and regulation of MLCP, would be different from those in vascular smooth muscle. We set out, therefore, to examine the ability of the phosphatase inhibitor microcystin to induce contractile responses in intestinal smooth muscle. The administration of microcystin (1 µM) to beta-escin-permeabilized smooth muscle strips in relaxing solution (pCa 9) elicited a gradual increase in force, reaching a plateau 45 min after application (Fig. 1A). Because microcystin inhibits both PP1 and PP2A phosphatases with similar potency, control experiments with more selective inhibitors of PP2A (i.e., 1 nM okadaic acid and 0.1 µM fostriecin) were conducted to ascertain whether PP2A contributes to Ca2+ sensitization. There was no development of contractile force when either okadaic acid or fostriecin was applied to permeabilized ileal muscle strips at concentrations that are selective for PP2A (data not shown). Furthermore, pretreatment with these PP2A inhibitors did not affect the rate of contraction or steady-state force elicited by microcystin at pCa 9. We conclude, therefore, that the contractile response to microcystin is due to inhibition of PP1, not PP2A. The fact that microcystin induces LC20 phosphorylation supports the conclusion that the targeted PP1 in this context is indeed MLCP.


Figure 1
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  		Fig. 1. A–C: microcystin (MC)-induced diphosphorylation of 20-kDa myosin regulatory light chain (LC20) and contraction of rat ileal smooth muscle at pCa 9. MC [1 µM (A), 10 µM (B), and 30 µM (C)] elicited contraction of beta-escin-permeabilized rat ileal smooth muscle strips at pCa 9. Reference responses to pCa 4.5 were obtained before and after MC treatment to define maximal contraction (Fmax). D: concentration-dependent contractile responses of rat ileal smooth muscle to 1 µM (open bars), 10 µM (hatched bars), and 30 µM (closed bars) MC were measured 25 and 45 min after addition of MC. Forces observed at pCa 9 and pCa 4.5 were designated as 0 and 100%, respectively. Error bars indicate standard error (n = 5). *, Significantly different (Student's t-test, P < 0.05); n.s., not significantly different. E: following treatment of ileal strips with MC (1 µM) for 45 min, phosphorylated and unphosphorylated LC20 were separated by urea/glycerol gel electrophoresis, detected by Western blotting with anti-LC20, and quantified by scanning densitometry. Different exposure times were used for quantification of these data to ensure that signals lay within linear range of relationship between protein amount and signal intensity in each case. Data are expressed as percentages of total LC20 for unphosphorylated (P0-LC20; open bar), monophosphorylated (P1-LC20; hatched bar), and diphosphorylated (P2-LC20; dotted bar) bands. Error bars indicate standard error (n = 5).

 
We further examined the concentration-dependent contractile response of rat ileal smooth muscle to microcystin. Typical contractile responses to microcystin (10 and 30 µM) are depicted in Fig. 1, B and C, respectively. The force obtained with 1 µM microcystin at 25 and 45 min after application was 31.5 ± 2.7 and 59.2 ± 3.3% of pCa 4.5-induced contraction, respectively. A maximal contractile response was obtained at 10 µM microcystin; the force levels at 25 min (42.5 ± 2.4%; n = 5) and at 45 min (69.3 ± 1.4%; n = 5) were significantly higher than those induced by 1 µM microcystin. However, an increase in the microcystin concentration to 30 µM did not elicit further increases in muscle force. The force induced by 30 µM microcystin after 25 min (37.5 ± 2.2%; n = 5) and 45 min (64.4 ± 0.79%; n = 5) was not significantly different from the corresponding force induced by 1 µM microcystin. However, the time required to reach a plateau of muscle tension at pCa 9 was shortened with the application of higher microcystin concentrations. Interestingly, the steady-state tension developed at any concentration of microcystin did not reach 100% of Fmax (i.e., maximal force obtained at pCa 4.5; Fig. 1D), whereas the steady-state tension induced by 1 µM microcystin in the rat caudal artery reached nearly 100% of Fmax (55).

The contraction induced by 1 µM microcystin at pCa 9 was accompanied by diphosphorylation of LC20 (Fig. 1E). The extent of LC20 mono- and diphosphorylation was determined to be 20.8 ± 1.3 and 1.5 ± 0.4% of total LC20, respectively. This corresponds to an overall phosphorylation stoichiometry of 0.24 mol Pi/mol LC20. In accordance with our force measurements, the amount of LC20 phosphorylation was lower than observed in the caudal artery. For comparison, the extent of LC20 mono- and diphosphorylation induced by microcystin in the rat caudal artery was ~30 and 50% of total LC20, respectively (55).

Effects of Y-27632 on microcystin-induced contraction of ileal smooth muscle at pCa 9. Because ROK is known to be an important mediator of smooth muscle contraction through its regulation of MLCP activity and its potential for LC20 phosphorylation, we first examined whether ROK was involved in the microcystin-induced contraction of rat ileal smooth muscle at pCa 9. The specific inhibitor of ROK, Y-27632, was applied to beta-escin-permeabilized ileal smooth muscle strips 20 min before the application of microcystin. Figure 2 shows representative recordings of the force induced by either 1 µM microcystin (Fig. 2A) or 10 µM microcystin (Fig. 2B) in the presence of 10 µM Y-27632. The initial phase of contraction induced by 1 µM microcystin was inhibited by Y-27632 (Fig. 2C). The force attained 25 min after microcystin application in the presence of Y-27632 (20.6 ± 2.4%; n = 5) was significantly lower than in the absence of the inhibitor (31.5 ± 2.7%; n = 5). However, the effects of Y-27632 on the initial phase of microcystin-induced contraction were abolished when the microcystin concentration was increased to 10 µM (Fig. 2, B and C). Steady-state force elicited by 1 or 10 µM microcystin, on the other hand, was unaffected by Y-27632 (Fig. 2C). These results suggest that ROK does not directly phosphorylate LC20 but does act as a minor contributor to the regulation of MLCP activity during microcystin-induced contraction of rat ileal smooth muscle at pCa 9.


Figure 2
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  		Fig. 2. Effect of the Rho-associated kinase (ROK) inhibitor Y-27632 on MC-induced contraction of rat ileal smooth muscle at pCa 9. Contractile responses to 1 (A) and 10 µM (B) MC were recorded in the presence of 10 µM Y-27632 applied 20 min before administration of MC. Cumulative results (C) are representative of 5 independent experiments. Force was measured 25 and 45 min after application of MC, and data are expressed as %maximal contraction in presence (closed bars) or absence (open bars) of Y-27632. Forces observed at pCa 9 and pCa 4.5 were designated as 0 and 100%, respectively. Error bars indicate standard error. *, Significantly different (Student's t-test, P < 0.05); n.s., not significantly different.

 
PKC involvement in microcystin-induced contraction of ileal smooth muscle at pCa 9. PKC has been linked to microcystin-induced contraction of vascular smooth muscle (40, 55). Therefore, we examined whether PKC was involved in microcystin-induced contraction of ileal smooth muscle. Figure 3, A and B shows representative recordings of force induced by 1 µM microcystin in the presence of either 100 nM GF-109203x or 10 µM chelerythrine. GF-109203x, a broad-specificity PKC inhibitor, reduced contraction induced by 1 µM microcystin. The force levels at 25 min (6.0 ± 1.7%; n = 5) and 45 min (18.1 ± 3.8%; n = 5) following microcystin administration in the presence of GF-109203x were significantly lower than those at 25 min (31.5 ± 2.7%; n = 5) and 45 min (59.2 ± 3.3%; n = 5) in the absence of the inhibitor. Similar results were obtained with chelerythrine, another broad-specificity PKC inhibitor. The forces observed 25 and 45 min after the application of microcystin in the presence of chelerythrine were reduced to 15.2 ± 3.3 and 39.1 ± 4.7% of Fmax, respectively. To elucidate whether conventional PKCs were involved in microcystin-induced contraction at pCa 9, we examined the effects of Gö-6976, a conventional PKC inhibitor, on ileal smooth muscle contraction. Interestingly, Gö-6976 significantly inhibited muscle tension induced by 1 µM microcystin in a similar manner to that observed with GF-109203x and chelerythrine (Fig. 3C). The force levels at 25 and 45 min in the presence of Gö-6976 were 8.4 ± 2.4 and 32.6 ± 3.6% (n = 5), respectively. These data clearly show that conventional PKCs contribute to microcystin-induced contraction of rat ileal smooth muscle at pCa 9.


Figure 3
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  		Fig. 3. Involvement of PKC in MC-induced contraction of rat ileal smooth muscle at pCa 9. Force induced by 1 µM MC was recorded in presence of 100 nM GF-109203x (A), 10 µM chelerythrine (B), or 10 µM Gö-6976 (C). PKC inhibitors were applied 20 min before administration of MC. Cumulative results (D) are presented for force measured 25 and 45 min after application of MC in presence of 100 nM GF-109303x (hatched bars), 10 µM chelerythrine (closed bars), or 10 µM Gö-6976 (dotted bars). Control response (1 µM MC without PKC inhibitors) is included as a reference (open bars). Error bars indicate standard error (n = 5). E: force induced by 10 µM MC in presence of 100 nM GF-109203x. F: effect of GF-109203x on contraction induced by MC at pCa 9. Cumulative results are presented for force measured 25 and 45 min after application of MC in presence (closed bars) or absence (open bars) of 100 nM GF-109303x. Forces observed at pCa 9 and pCa 4.5 were designated as 0 and 100%, respectively. Error bars indicate standard error (n = 5). G: representative recording of force induced by 1 µM MC in G10 solution. *, Significantly different (Student's t-test, P < 0.05); n.s., not significantly different.

 
To further assess the role of PKC in ileal smooth muscle, we investigated the effects of GF-109203x on contraction induced by higher concentrations of microcystin. If PKC lies upstream of the kinase that is responsible for the direct phosphorylation of LC20, rather than inhibiting MLCP activity, then application of GF-109203x should continue to inhibit the microcystin-induced contraction at pCa 9 even in the presence of saturating microcystin concentrations. Interestingly, the inhibitory effect of GF-109203x was reduced, but not abolished, when higher concentrations of microcystin (i.e., 10 and 30 µM) were used (Fig. 3, D and E). These data suggest that PKC-activated signaling pathways contribute to the direct phosphorylation of LC20 during microcystin-induced contraction at pCa 9.

Our pharmacological experiments using PKC inhibitors indicated that conventional, Ca2+-dependent PKCs are likely involved in the microcystin-induced contraction under resting conditions (pCa 9). We conducted further experiments to confirm that pCa 9 was sufficient for the activation of Ca2+-dependent PKCs in ileal smooth muscle. The contractile response to 1 µM microcystin in G10 solution (Ca2+-free solution) was examined. As expected, the force induced by 1 µM microcystin was much lower in G10 solution than in pCa 9 (Fig. 3F). Together, these data suggest that conventional PKC isozymes contribute to microcystin-induced contraction of ileal smooth muscle even at nanomolar levels of Ca2+.

Involvement of MAPKs in microcystin-induced contraction of ileal smooth muscle at pCa 9. MAPKs have been reported to play a role in the regulation of smooth muscle contractility in esophageal phasic smooth muscle (18), so an examination of MAPK involvement in microcystin-induced contraction of ileal smooth muscle at pCa 9 was undertaken. Contraction induced by 1 µM microcystin was recorded in the presence of the MEK antagonist PD-98059 (Fig. 4A) or the p38 MAPK inhibitor SB-203580 (Fig. 4B). PD-98059 reduced 1 µM microcystin-induced force (Fig. 4C). The force developed at 25 min (3.4 ± 2.1%; n = 5) and 45 min (19.4 ± 7.0%; n = 5) in the presence of PD-98059 was significantly lower than at 25 min (31.5 ± 2.7%; n = 5) and 45 min (59.2 ± 3.3%; n = 5) in the absence of the inhibitor. However, the inhibition of microcystin-induced contraction by PD-98059 was completely abolished when the microcystin concentration was increased to 10 µM (Fig. 4C). Similar results were obtained with SB-203580 when contraction was stimulated with 1 µM microcystin. The force generated at 25 and 45 min in the presence of SB-203580 was 4.5 ± 1.9 and 21.4 ± 5.3% (n = 5) of Fmax. This inhibition by SB-203580 was completely abolished when the microcystin concentration was increased to 10 µM (Fig. 4C). From these data, we conclude that both ERK1/2 and p38 MAPK play a role in the negative regulation of MLCP during microcystin-induced contraction of ileal smooth muscle at pCa 9.


Figure 4
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  		Fig. 4. Involvement of ERK1/2 and p38 MAPK in MC-induced contraction of rat ileal smooth muscle at pCa 9. Contractile response to 1 µM MC in presence of 10 µM PD-98059 (MEK inhibitor; A) or 10 µM SB-203580 (p38 MAPK inhibitor; B) are shown. Inhibitors were applied 20 min before administration of MC. Cumulative results (C) are presented for force measurements 25 and 45 min after application of MC (1 and 10 µM) in presence of 10 µM PD-98059 (hatched bars) or 10 µM SB-203580 (closed bars). Control responses (without inhibitors) are included as a reference (open bars). Forces observed at pCa 9 and pCa 4.5 were designated as 0 and 100%, respectively. Error bars indicate standard error (n = 5). *, Significantly different (Student's t-test, P < 0.05); n.s., not significantly different.

 
MYPT1 phosphorylation at Thr-697 by PKC and MAPK pathways in ileal smooth muscle. The best-characterized mechanism of MLCP inhibition involves the phosphorylation of MYPT1 that is accompanied by a decrease in phosphatase activity. PKC may play a role not only in the direct phosphorylation of LC20 but also in the regulation of MLCP activity during microcystin-induced contraction at pCa 9. Thus we examined whether PKC contributes to the phosphorylation of MYPT1. In addition, we investigated whether the negative regulation of MLCP by ERK1/2 and p38 MAPK is mediated by MYPT1 phosphorylation. We examined the effects of GF-109203x, PD-98059, and SB-203580 on the phosphorylation level of MYPT1 at Thr-697. This is the major site of negative regulation of MLCP activity, although phosphorylation at other sites may also have inhibitory effects (38, 54). Ileal smooth muscle tissue was flash frozen 45 min after application of microcystin in the absence and presence of protein kinase inhibitors. Thr-697 phosphorylation of MYPT1 was significantly reduced by administration of either GF-109203x or PD-98059 (Fig. 5). MYPT1 phosphorylation was reduced in the presence of GF-109203x and PD-98059 to 53.3 ± 2.1% (n = 3) and 61.4 ± 10.7% (n = 3) of the control level (absence of protein kinase inhibitors), respectively. The addition of SB-203580 did not have a significant effect on MYPT1 phosphorylation. These data suggest that PKC and ERK1/2 pathways, but not the p38 MAPK pathway, contribute to microcystin-induced contraction at pCa 9 via the inhibition of MLCP activity mediated through MYPT1 phosphorylation at Thr-697.


Figure 5
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  		Fig. 5. Effects of PKC and MAPK inhibitors on the myosin-targeting subunit of myosin light chain phosphatase (MYPT1) phosphorylation during MC-induced contraction of rat ileal smooth muscle at pCa 9. Ileal smooth muscle tissues were flash frozen 45 min after application of MC (1 µM) in presence of PKC (GF-109203x) or MAPK (PD-98059 and SB-203580) inhibitors. Proteins were separated by SDS-PAGE, and MYPT1 phosphorylation at Thr-697 was assessed by Western blotting with anti-[phospho-Thr-697]-MYPT1 and pan-MYPT1 antibodies (A). To account for variations in loading levels, data are expressed as signal-intensity ratios for phosphorylated (P-):total (T-) MYPT1 (B). Ratio for control (MC alone) was set to 1.0, and relative ratios were determined for MYPT1 phosphorylation in tissues treated with protein kinase inhibitors. Error bars indicate standard error (n = 3). *, Significantly different from control (Student's t-test, P < 0.05); n.s., not significantly different from control.

 
Contribution of CPI-17 to MLCP inhibition by PKC and MAPK pathways during microcystin-induced contraction of ileal smooth muscle at pCa 9. Another important mechanism of MLCP inhibition involves the phosphorylation of CPI-17 (14). We examined the possibility that microcystin-induced contraction of the ileum could be a reflection of CPI-17 phosphorylation status. Lower amounts of CPI-17 were observed in intestinal smooth muscle (e.g., colon and ileum) than in vascular smooth muscle beds (Fig. 6A). However, the small amount of CPI-17 that was contained in the ileal smooth muscle cell was tightly associated with the contractile apparatus. Permeabilization with beta-escin or Triton X-100 had no effect on the amount of CPI-17 detected in ileal smooth muscle (Fig. 6B). This is in contrast to our data for caudal artery and previous reports (29) that show the majority of CPI-17 protein diffuses from vascular smooth muscle on treatment with these permeabilization agents. As shown in Fig. 6, C and D, significant amounts of CPI-17 phosphorylation were observed under resting conditions (pCa 9) in beta-escin-permeabilized ileal smooth muscle. Furthermore, CPI-17 phosphorylation was not affected by microcystin treatment alone. To assess the importance of PKC and MAPK signaling on CPI-17 phosphorylation status, ileal muscle strips were incubated with protein kinase inhibitors (GF-109203x, PD-98059, or SB-203580) before microcystin administration and were then subjected to Western blot analysis. CPI-17 phosphorylation was reduced following pretreatment with SB-203580 but was unaffected by GF-109203x or PD-98059 (Fig. 6, C and D). The amount of phosphorylated CPI-17 detected in the presence of SB-203580 was significantly lower than that detected in the control. We conclude that the p38 MAPK pathway, but not the PKC and ERK1/2 pathways, participates in the phosphorylation of CPI-17 in ileal smooth muscle.


Figure 6
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  		Fig. 6. Analysis of PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17) contribution to MC-induced contraction of rat ileal smooth muscle at pCa 9. A: Western blot analysis of CPI-17 expression in vascular (femoral artery, caudal artery, and aorta) and intestinal (colon and ileum) smooth muscle tissues. Representative blots are shown above quantitative data and are normalized to tissue actin content (n = 3). CPI-17 content in ileum was set to 1.0. B: Western blot analysis of CPI-17 in untreated, intact, beta-escin-permeabilized, and Triton X-100-demembranated vascular (caudal artery) and intestinal (ileum) smooth muscle tissues. Representative blots are shown above quantitative data and are normalized to tissue actin content. CPI-17 content of intact tissues (open bars) was set to 1.0, and relative CPI-17 content following Triton X-100 (closed bars) or beta-escin (hatched bars) treatment is quantified (n = 3). C: ileal strips were flash frozen 45 min after application of MC (1 µM) in presence of PKC (GF-109203x) or MAPK (PD-98059 and SB-203580) inhibitors. Proteins were separated by SDS-PAGE, and CPI-17 phosphorylation at Thr-38 was assessed by Western blotting with anti-[phospho-Thr-38]-CPI-17 and pan-CPI-17 antibodies. D: to account for variations in loading levels, data are expressed as signal-intensity ratios for phosphorylated:total CPI-17. Ratio for control (pCa 9, absence of MC) was set to 1.0, and relative ratios were determined for CPI-17 phosphorylation in tissues treated with MC in absence or presence of protein kinase inhibitors. Error bars indicate standard error (n = 5). *, Significantly different from CPI-17 phosphorylation in absence of protein kinase inhibitors (Student's t-test, P < 0.05).

 
Complementation of PKC, ERK1/2, and p38 MAPK pathways in the regulation of MLCP activity during microcystin-induced contraction of ileal smooth muscle at pCa 9. The blockade of either PKC or MAPK (ERK1/2 and p38 MAPK) pathways in ileal muscle with pharmacological agents had similar effects on microcystin-induced contraction at pCa 9. A previous study with esophageal smooth muscle suggested that ERK1/2 and p38 MAPK are likely to participate in PKC-{varepsilon}-dependent contractile pathways (18). Next, we examined whether microcystin-induced force generation in ileal smooth muscle depends on activation of ERK1/2 or p38 MAPK by PKC pathways. Western blot analyses of activated ERK1/2 and p38 MAPK were performed on ileal tissues following the application of microcystin in the presence of GF-109203x, PD-98059, or SB-203580 (Fig. 7, A and C). The phosphorylation level of ERK1 was reduced by PD-98059 but not by GF-109203x (Fig. 7B). ERK1 phosphorylation in the presence of PD-98059 or GF-109203x was 60.2 ± 5.9% (n = 3) and 92.1 ± 17.8% (n = 3), respectively, of the phosphorylation detected in the absence of protein kinase inhibitor. Also, the phosphorylation level of p38 MAPK was not reduced by GF-109203x (Fig. 7D). p38 MAPK phosphorylation in the presence of GF-109203x was 107.1 ± 15.8% (n = 3). These results are consistent with ERK1 and p38 MAPK activation occurring independently of PKC.


Figure 7
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  		Fig. 7. Complementation of PKC, ERK1/2, and p38 MAPK pathways in regulation of myosin light chain phosphatase (MLCP) activity during MC-induced contraction of rat ileal smooth muscle at pCa 9. Analysis of p38 MAPK and ERK1 phosphorylation in response to treatment with inhibitors during MC-induced contraction of rat ileum is shown. Tissues were flash frozen 45 min after application of MC (1 µM) under resting conditions (pCa 9) in presence of PKC (GF-109203x) or MAPK (PD-98059 and SB-203580) inhibitors. Proteins were separated by SDS-PAGE. ERK1 and p38 MAPK phosphorylation were assessed by Western blotting with anti-[phospho-Thr-185/phospho-Tyr-187]-ERK1/2 and pan-ERK1/2 (A and B) or anti-[phospho-Thr-180/phospho-Tyr-182]-p38 MAPK and pan-p38 MAPK antibodies (C and D), respectively. To account for variations in loading levels, data are expressed as signal-intensity ratios for phosphorylated:total p38 MAPK and ERK1 (B and D). Ratio for control (MC alone) was set to 1.0, and relative ratios were determined for p38 MAPK and ERK1 phosphorylation in tissues treated with protein kinase inhibitors. Error bars indicate standard error (n = 3). *, Significantly different from control (Student's t-test, P < 0.05); n.s., not significantly different from control. Total ERK2 was not detected with pan-ERK1/2 antibody; thus only data for ERK1 are presented. E: ileal muscle strips were treated with a combination of 100 nM GF-109203x, 10 µM PD-98059, and 10 µM SB-203580 at pCa 9 before addition of MC (1 µM). F: cumulative data of MC (1 µM) application in presence of various concentrations of PKC and/or MAPK inhibitors. *, Significantly different from values in presence of 100 nM GF-109203x (Student's t-test, P < 0.05); n.s., not significantly different from values in presence of 100 nM GF-109203x.

 
To further assess the integration of PKC and MAPK signaling, we applied the PKC, ERK1/2, and p38 MAPK inhibitors in combination to rat ileal smooth muscle (Fig. 7, E and F). First, we confirmed that 100 nM GF-109203x was sufficient to completely inhibit PKC-dependent contractile pathways. Increasing the GF-109203x concentration to 1 and 10 µM had no further inhibitory effect on the microcystin-induced contraction, demonstrating that 100 nM GF-109203x was sufficient to inhibit PKC contractile signaling in ileal smooth muscle. Addition of either 10 µM PD-98059 or 10 µM SB-203580 in combination with 100 nM GF-109203x did not augment the inhibitory effect of GF-109203x. However, application of all three kinase inhibitors (GF-109203x, PD-98059, and SB-203580) abolished the contractile response to 1 µM microcystin. These data suggest that microcystin-induced contraction of ileal smooth muscle is equally dependent on PKC, ERK1/2, and p38 MAPK pathways. Furthermore, the ERK1/2 and p38 MAPK pathways appear to signal in parallel to PKC and not downstream of this kinase during microcystin-induced contraction at pCa 9.

ZIPK contribution to microcystin-induced contraction of ileal smooth muscle at pCa 9. To determine the contribution of ZIPK to microcystin-induced contraction of rat ileal smooth muscle at pCa 9, we used a recently described inhibitor of ZIPK, the SM1 peptide. This peptide, which is derived from the autoinhibitory domain of MLCK, exhibits inhibitory potency toward ZIPK in vitro and ex vivo (23) but does not have any effect on ILK or ROK (55). SM-1 peptide significantly inhibited the muscle-tension development induced by the addition of exogenous ZIPK to Triton-skinned rat ileal smooth muscle strips (23). We determined that ZIPK was not responsible for LC20 diphosphorylation and microcystin-induced contraction of rat ileal smooth muscle at pCa 9, consistent with results obtained with vascular smooth muscle (55). As shown in Fig. 8, the application of SM1 peptide (50 µM) had no effect on microcystin-induced force generation, implying that this contraction is mediated by ILK.


Figure 8
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  		Fig. 8. Zipper-interacting protein kinase (ZIPK) contribution to MC-induced contraction of rat ileal smooth muscle at pCa 9. Contraction induced by 1 µM MC in presence of 50 µM SM1 peptide, an inhibitor of ZIPK, is shown. SM 1 was applied to muscle in pCa 9 solution 20 min before administration of MC. Cumulative results (B) are presented for force measurement 25 and 45 min after application of MC. Forces observed at pCa 9 and pCa 4.5 were designated as 0 and 100%, respectively. Error bars indicate standard error (n = 5). n.s., Not significantly different from force generated by MC in absence of SM1 (Student's t-test, P > 0.05).

 

   DISCUSSION
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It is widely accepted that pathological alterations in the sensitivity of the smooth muscle contractile response to [Ca2+]i (i.e., Ca2+ sensitization) underlie various disorders associated with smooth muscle dysfunction such as hypertension, vasospasm, bronchial asthma, and inflammatory bowel disease. Although ILK was recently suggested to be involved in microcystin-induced contraction of vascular smooth muscle at low [Ca2+] (55), to our knowledge this is the first study undertaken to define the contribution of protein kinases to Ca2+ sensitization of ileal smooth muscle contraction.

Contraction at low [Ca2+] has been reported in a variety of vascular smooth muscle tissues following agonist stimulation (11, 17, 52) or application of microcystin (53). Ca2+ sensitization not only occurs in vascular smooth muscle but also in intestinal smooth muscle. As shown in the present study, the contractile force elicited by microcystin reached 60% of Fmax in rat ileal smooth muscle even when the microcystin concentration was increased to 30 µM, whereas application of 1 µM microcystin was shown to generate essentially 100% of Fmax in a vascular smooth muscle tissue (rat caudal artery) (55). Consistent with this finding, the extent of LC20 phosphorylation during microcystin-induced contraction at pCa 9 was much smaller in the ileum than in the caudal artery. Furthermore, MLCP activity is much greater in intestinal smooth muscle than in vascular smooth muscle (16, 56). Although it is thought that microcystin elicits a contractile response by inhibiting MLCP activity, and this is supported by the observed increase in LC20 phosphorylation, it should be noted that microcystin also inhibits other type 1 as well as type 2A protein serine/threonine phosphatases. Using selective inhibitors of PP2A, we confirmed that this class of phosphatase is not involved in microcystin-induced contraction of ileal smooth muscle at pCa 9, lending further support to the conclusion that it is inhibition of the type 1 phosphatase MLCP that is responsible for this contractile response. The relative expression levels of MYPT1 (the myosin-targeting subunit of MLCP) correlate with the rate of LC20 dephosphorylation, with higher MLCP content and potential for LC20 dephosphorylation in phasic compared with tonic smooth muscle. The expression levels of MLCP and the phosphatase inhibitory protein, CPI-17, both of which are regulated by phosphorylation, dictate the contractile phenotype of the particular smooth muscle bed. We hypothesized, therefore, that the protein kinase networks contributing to the regulation of MLCP and contraction of intestinal smooth muscle at low [Ca2+] would differ from those in vascular smooth muscle.

The PKC, ERK1/2, and p38 MAPK pathways were shown, through the use of pharmacological agents, to be involved in microcystin-induced contraction of rat ileal smooth muscle at pCa 9. ERK1/2 and p38 MAPK have been previously reported to play a role in contraction of both vascular (37, 41) and intestinal smooth muscle (6). The phosphorylation of caldesmon and/or calponin is thought to be one mechanism whereby ERK1/2 contributes to smooth muscle contraction (26, 31). Alternatively, p38 MAPK has been shown to phosphorylate and activate MAPKAPK-2, which in turn can phosphorylate HSP 27 (8, 42) and contribute to smooth muscle contraction (18). However, the precise mechanism by which these MAPKs contribute to ileal smooth muscle contractility remains to be determined. This study demonstrates that both ERK1/2 and p38 MAPK participate in the regulation of MLCP activity but not in the direct phosphorylation of LC20 at pCa 9. These findings are also consistent with a previous report showing that stimulation of {alpha}1-adrenoceptors with phenylephrine induced ERK1/2 activation and resulted in MLCP inhibition via the phosphorylation of MYPT1 in uterine artery. This MYPT1 phosphorylation and MLCP inhibition were significantly reduced by PD-98059 (57).

Although it appears that both PKC and ERK1/2 modulate contraction of the ileum at low [Ca2+] by contributing to the phosphorylation of MYPT1 at Thr-697 and hence the activity of MLCP, neither kinase has been shown to phosphorylate MYPT1 directly. Several kinases are known to regulate MLCP activity by phosphorylation of the Thr-697 inhibitory site, including ROK (27), ZIPK (34, 40), myotonic dystrophy protein kinase (39), ILK (28, 39), Raf-1 (4), and p21-activated protein kinase (47). It is possible that one or more of these protein kinases are located downstream of PKC and ERK1/2 and are directly responsible for the observed MYPT1 phosphorylation. Harnett and colleagues (18) have previously suggested that ILK is located downstream of the ERK1/2 pathway and that ZIPK is a downstream target of the p38 MAPK pathway. Because the SM1 peptide (a ZIPK inhibitor) did not affect microcystin-induced contraction at pCa 9 (Fig. 8), it is unlikely that ZIPK was a downstream target of PKC and ERK1/2 in the ileum. We suggest that ILK is the most likely downstream target of PKC and ERK1/2, but further investigations will be required to determine the validity of this hypothesis.

Because CPI-17 is an important regulator of MLCP activity, we examined whether PKC and MAPKs were signaling through CPI-17 to regulate MLCP in ileal smooth muscle. CPI-17 has been proposed to be a point of mutual convergence, where various Ca2+-sensitizing pathways meet for inhibition of MLCP (56). CPI-17 was originally identified as a substrate of PKC (13), and both PKC-{alpha} and PKC-{delta} were reported to activate CPI-17 by phosphorylation of Thr-38 (12). However, it was unclear if CPI-17 would play a significant role in regulating MLCP in the ileum. The expression level of CPI-17 in this phasic visceral muscle is low, and the involvement of CPI-17 in PKC-induced Ca2+ sensitization of contraction and LC20 phosphorylation was thought to be minor because phorbol ester-induced LC20 phosphorylation and contraction are much lower than the corresponding GTP{gamma}S-evoked responses, indicating that other mechanisms of G protein-mediated inhibition of MLCP predominate in phasic muscle beds such as ileum (56). In the present study, we investigated the possibility that CPI-17 is an important contributor to Ca2+-sensitized contraction of ileal smooth muscle. In spite of its low expression levels, nearly the entire complement of CPI-17 appears to be tightly associated with the myofilament architecture. More interestingly, we have shown that CPI-17 maintains a high degree of constitutive phosphorylation, and furthermore, p38 MAPK but not PKC appears to contribute to its phosphorylation. Because there is no evidence that p38 MAPK can phosphorylate CPI-17 directly, it is likely that some other kinase operates downstream of p38 MAPK to phosphorylate and activate CPI-17. A recent study by Hersch and colleagues (22) has reported CPI-17 phosphorylation by PKC in isolated rabbit intestinal smooth muscle cells. This phosphorylation was potentiated by low concentrations of okadaic acid and the p38 MAPK inhibitor SB-203580, leading the investigators to conclude that PP2A-mediated CPI-17 dephosphorylation in these cells was activated by p38 MAPK. Our findings differ in two key ways: 1) CPI-17 phosphorylation in rat ileum was not induced by inhibitors of PKC, and 2) basal CPI-17 phosphorylation was high following permeabilization with beta-escin, and PP inhibitors were unable to elicit further CPI-17 activation. It is possible that the participation of PKC and PP2A in the activation of CPI-17 is restricted to rabbit intestinal tissue. Alternatively, isolated tissue may exhibit differences in active signaling pathways from dispersed smooth muscle cells.

Following stimulation of M2 muscarinic receptors with acetylcholine, force is generated in cat esophageal smooth muscle by PKC-{varepsilon} and the subsequent downstream activation of ERK1/2 and p38 MAPK pathways (18). Therefore, we investigated whether PKC could be an upstream modulator of MAPK pathways as previously shown. Treatment of ileum with GF-109203x, however, did not reduce the activating phosphorylation of ERK1 or p38 MAPK. Furthermore, simultaneous inhibition of all three kinase pathways by application of GF-109203x, PD-98059, and SB-203580 reduced microcystin-induced contraction to a greater extent than application of GF-109203x alone. These findings indicate that the ERK1/2 and p38 MAPK pathways operate in parallel with and not upstream of PKC in the regulation of MLCP in rat ileal smooth muscle. We propose that the three protein kinase pathways have different roles in microcystin-induced contraction at pCa 9 (Fig. 9). PKC is associated not only with the direct diphosphorylation of LC20 but also with MLCP inhibition through MYPT1 phosphorylation at the inhibitory Thr-697 site. ERK1/2 is involved in MLCP inhibition through MYPT1 phosphorylation but does not contribute to the direct diphosphorylation of LC20. Finally, p38 MAPK is involved only in MLCP inhibition; however, its actions on MLCP are mediated via CPI-17 phosphorylation and not MYPT1 phosphorylation.


Figure 9
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  		Fig. 9. Schematic representation of signaling pathways involved in MC-induced Ca2+ sensitization of rat ileal smooth muscle contraction. ILK, integrin-linked kinase; MLCK, myosin light chain kinase; PP, protein phosphatase.

 
Ca2+-independent protein kinases can directly contribute to LC20 mono- and diphosphorylation and are thought to play a role in Ca2+ sensitization. Previous reports have implicated several Ca2+-independent MLCKs, including ROK (2), PKC (25), MAPKAPK-2 (30), MAPKAPK-1b (RSK-2) (46), ILK (10), and ZIPK (40). ROK, MAPKAPK-2, and RSK-2 phosphorylation of LC20 is restricted to Ser-19, however, and PKC does not phosphorylate either Ser-19 or Thr-18. Furthermore, among these kinases, only ROK (2), ILK (10), and ZIPK (40) have been shown to induce LC20 phosphorylation and contraction of smooth muscle. Because microcystin induced diphosphorylation of LC20 in low-Ca2+ medium and both Y-27632 and SM1 had no effect on contraction induced by 10 µM microcystin at pCa 9, ILK emerges as the most likely candidate for the Ca2+-independent MLCK in ileal smooth muscle. This conclusion is supported by a recent study (43) that suggested increased [Ca2+]i is required for activation of RhoA and ROK in both KCl- and agonist-induced contractions of vascular smooth muscle. It is possible, therefore, that ILK acts downstream of PKC to contribute to diphosphorylation of LC20 and downstream of MAPKs to regulate MLCP activity.

In conclusion, we describe the mechanism of Ca2+ sensitization in ileal smooth muscle as consisting of several parallel pathways. It appears that PKC, ERK1/2, and p38 MAPK (along with a minor role for ROK) are involved in microcystin-induced contraction through the negative regulation of MLCP activity. The contribution of PKC, ERK1/2, and p38 MAPK to the regulation of LC20 dephosphorylation is thought to be unique to phasic smooth muscle. PKC and ERK1/2 pathways inhibited MLCP activity by modulation of MYPT1 phosphorylation at Thr-697, whereas the p38 MAPK pathway inhibited MLCP activity by modulation of CPI-17 phosphorylation. It is possible that ILK lies downstream of these pathways and PKC, not MAPKs, and may modulate ILK-mediated LC20 diphosphorylation. There are significant differences between the protein kinase networks that contribute to Ca2+ sensitization of vascular (tonic) and intestinal (phasic) smooth muscle contraction. Because these smooth muscle beds have highly specialized functions, it is not unexpected that unique and intricate signaling pathways have developed in each.


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 ABSTRACT
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This work was supported by grants in aid of research from the Crohn's and Colitis Foundation of Canada (J. A. MacDonald) and the Canadian Institutes of Health Research (MGP-13101 to M. P. Walsh). E. Ihara is a recipient of a Uehara Memorial Foundation Fellowship (Japan) and Canadian Association of Gastroenterology/Canadian Institutes of Health Research/AstraZeneca Fellowship. L. Moffat is a recipient of a Studentship from the Canadian Institutes of Health Research. J. A. MacDonald is a recipient of a Canada Research Chair (Tier II) in Smooth Muscle Pathophysiology and a Scholarship from the Heart and Stroke Foundation of Canada. M. P. Walsh is an Alberta Heritage Foundation for Medical Research Scientist and recipient of a Canada Research Chair (Tier I) in Vascular Smooth Muscle Research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. MacDonald, Dept. of Biochemistry and Molecular Biology, Univ. of Calgary, Faculty of Medicine, 3330 Hospital Drive N.W., Calgary, AB, T2N 4N1, Canada (e-mail: jmacdo{at}ucalgary.ca)

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. Section 1734 solely to indicate this fact.


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 REFERENCES
 

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