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J. Biol. Chem., Vol. 277, Issue 6, 4422-4427, February 8, 2002

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Agonist-induced Force Enhancement

THE ROLE OF ISOFORMS AND PHOSPHORYLATION OF THE MYOSIN-TARGETING SUBUNIT OF MYOSIN LIGHT CHAIN PHOSPHATASE^*

Christopher T. Richards, Ozgur Ogut, and Frank V. Brozovich^§¶

From the Departments of ^§ Medicine and  Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication, November 19, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The magnitude of agonist-induced Ca²⁺ sensitization of force is tissue-dependent, but an explanation for this diversity is unknown. Ca²⁺ sensitization is thought to involve a G-protein-mediated inhibition of myosin light chain phosphatase activity by phosphorylation of the myosin-targeting subunit (MYPT). The MYPT has two isoforms that differ by a central insert, which lies near this phosphorylation site. Expression of MYPT isoforms is both developmentally regulated and tissue-specific. We hypothesized that the presence or absence of the central insert determines the magnitude of agonist-induced Ca²⁺ sensitization. Throughout development, the chicken aorta exclusively expresses the splice-in MYPT isoform, and guanosine 5'-O-(thiotriphosphate) (GTPS) produces a significant force enhancement. Early during development, the chicken gizzard expresses the splice-in MYPT isoform, and GTPS produced a Ca²⁺ sensitization. In the gizzard coincident with the shift in expression from the splice-in to splice-out MYPT isoform, GTPS no longer produced force enhancement. In addition, adenosine 5'-O-(thiotriphosphate) (ATPS) phosphorylated only adult gizzard tissue, the only tissue that did not demonstrate a Ca²⁺ sensitization. These results suggest that the relative expression of splice-in/splice-out MYPT isoforms determines the magnitude of agonist-induced force enhancement and that MYPT phosphorylation is not required for Ca²⁺ sensitization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The importance of Ca²⁺-calmodulin-dependent myosin light chain kinase (MLCK)¹ for smooth muscle activation has been well described (1). However, recent evidence suggests that regulation of MLC phosphatase activity is physiologically important for force regulation in smooth muscle (reviewed in Refs. 2-6). This regulation of MLC phosphatase activity can produce a Ca²⁺ sensitization, or an increase in force at a constant [Ca²⁺], and a Ca²⁺ desensitization, or a reduction in force at a constant [Ca²⁺].

Agonist stimulation produces a Ca²⁺ sensitization of the contractile filaments (7). The mechanism for the agonist-induced Ca²⁺ sensitization, or force enhancement, is thought to depend on a G-protein-mediated inhibition of MLC phosphatase activity resulting in an increase in MLC₂₀ phosphorylation (8). The MLC phosphatase is a trimeric protein consisting of a 38-kDa catalytic subunit, a 20-kDa subunit of unknown function, and a large (110-133 kDa) myosin-targeting subunit (MYPT) (reviewed in Ref. 3)). G-protein-induced inhibition of MLC phosphatase activity has been suggested to be regulated by phosphorylation of the MYPT (9), possibly by either Rho-kinase (10, 11) or Zip-like kinase (12). However, direct activation of the G-proteins (8), as well as agonist stimulation (7), does not produce the same magnitude of force enhancement in all smooth muscle tissues, and the mechanism to explain this diversity has yet to be elucidated.

The MYPT has two isoforms that are produced by alternative splicing of a central exon (13). The alternatively spliced segment lies near a phosphorylation site that is thought to be important for regulation of phosphatase activity (14). In the chicken, the presence of the insert shifts this phosphorylation site from Thr-654 to Thr-695 (14). The expression of MYPT isoforms has been shown to be developmentally regulated (15, 16) and tissue-specific (2, 3, 15, 16), raising the possibility that MYPT isoforms could modulate the magnitude of agonist-induced force enhancement. In this study, we tested the hypothesis that the presence or absence of the central insert determines the magnitude of agonist-induced Ca²⁺ sensitization of the contractile filaments.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Force Measurement in Embryonic Chicken Gizzard and Aorta-- Gizzard and aortic smooth muscle strips (~1500 × 500 × 400 µm) were dissected from chicken at embryonic day (ED) 16 and ED 20 and from adult birds in Ca²⁺-free physiological saline solution as described previously (16). Briefly, strips were clamped between two aluminum foil T-clips and skinned in relaxing solution (pCa 9) for 15 min (gizzard) or 30 min (aorta) at room temperature with 400 µM -escin (16). After skinning, the preparation was transferred to a mechanics work station (17) where they were mounted in a 200-µl well containing relaxing solution (pCa 9) between a length driver (Polytec PI, Auburn, MA) and a piezoresistive force transducer (Sensor One, San Francisco) and then stretched to L₀ (length for maximum force) (16, 17).

The smooth muscle strips were activated by changing the well bathing the preparation so that the relaxing solution was replaced with activating solution. After force reached a steady state, the preparation was transferred to an adjacent well containing another solution. In this manner, the tissue was bathed in increasing [Ca²⁺] to generate a force versus Ca²⁺ relationship. The sequence of the Ca²⁺ activating solutions was varied between preparations. After force reached a steady state at each [Ca²⁺], the effect of 100 µM GTPS was determined by replacing the activating or relaxing solution with one containing 100 µM GTPS. In addition, the effect of inhibition of MLCK with 300 µM ML-9 (9), of PKC with 2 µM staurosporine (18), and of Rho-kinase with 2-10 µM Y-27632 (11) on the magnitude of the force enhancement produced by 100 µM GTPS was determined.

For each preparation, force was normalized to the force reached at pCa 4. The force versus Ca²⁺ relationship was fit to a 4-parameter Hill equation: Relative force = F_min + F_max(pCa)ⁿ/[pKⁿ + (pCa)ⁿ], where F_min is a constant, F_max is the maximum relative force, pK is the pCa at half-maximum force level of the Hill fit, and n is the Hill coefficient.

Western Blotting and Determination of MYPT Phosphorylation-- We determined MLC₂₀ phosphorylation using a previously published method (16, 19, 20). Following activation in pCa 9, 5.7, or 5.7 + 100 µM GTPS, the tissues were denatured with 15% trichloroacetic acid in acetone for 30 min on dry ice. Following denaturation, tissues were washed four times in 100 volumes of acetone and allowed to air-dry. MLC₂₀ was solubilized from the tissues by vortexing in 8 M urea, 20 mM Tris, 22 mM glycine, pH 8.6, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. Solubilized MLC₂₀ was resolved in the absence of SDS by 19:1 acrylamide:bisacrylamide 5% gels containing 20% (v/v) glycerol. The running buffer contained 20 mM Tris, 22 mM glycine, pH 8.6, and 1 mM thioglycolic acid. The resolved proteins were transferred to nitrocellulose membrane, and MLC₂₀ was detected using a monoclonal antibody to MLC₂₀ (Sigma). The ratio of phosphorylated/unphosphorylated MLC₂₀ was determined from densitometric analysis of the phosphorylated and unphosphorylated bands on the Western blots.

To determine the level of phosphorylation of MYPT, we used the protocol originally described by Trinkle-Mulcahy et al. (9). Briefly, smooth muscle strips were dissected and skinned as described above and then activated for 10 min in a pCa 9 relaxing solution containing 10 mCi/ml [³⁵S]ATPS, 100 µM unlabeled ATPS, and the MLCK inhibitor ML-9 at 300 µM. Strips were then placed in cold 10% trichloroacetic acid/10 mM dithiothreitol over dry ice for 5 min. The tissue was then brought to room temperature for 1 h and washed several times in acetone/10 mM dithiothreitol. The air-dried tissue was then ground to powder in liquid N₂, and 40-80 µl of SDS sample buffer was added. Samples were resolved by 6% SDS-PAGE. Protein was visualized by Coomassie staining (0.1% Coomassie Blue, 5% methanol, 10% acetic acid). Autoradiography of labeled proteins was done using x-ray film with an image-intensifying screen. Duplicate gels were blotted to nitrocellulose membranes. MYPT protein was visualized by ECL detection of the goat anti-mouse IgG/IgM-labeled MYPT 1⁰ antibody (Berkeley Antibody Co.).

Solutions-- The physiological saline solution contained (mM): 140 NaCl, 4.7 KCl, 1.2 Na₂HPO₄, 0.02 EDTA, 1.2 MgC1₂, 5.6 glucose, 2.0 MOPS, and 0.5 EGTA. Calcium bathing solutions were prepared according to a computer program to result in a desired set of free ion concentrations adjusted for both temperature and ionic strength (17, 21). All solutions had an ionic strength of 200 mM, and experiments were performed at 22 °C. The relaxing solution (pCa 9) contained (mM): 5.0 EGTA, 25.0 creatine phosphate, 9.3 MgCl₂, 5.2 Na₂ATP, 25.0 BES, 68.5 potassium methanesulfonate, 0.02 CaCl₂, pH to 7.1 with 1 N KOH. The activating solution (pCa 4) contained (mM): 5.0 EGTA, 25.0 creatine phosphate, 8.9 MgCl₂, 5.3 Na₂ATP, 25.0 BES, 58.3 potassium methanesulfonate, 5.2 CaCl₂, pH to 7.1, with 1 N KOH. Submaximal Ca²⁺ activating solutions were produced by proportional mixing of relaxing and activating solution. In addition, 1 µM calmodulin was added to all solutions prior to the experiments.

All values given in the text are the mean ± S.E. with n equal to the number of experiments. Means were compared with a Student's t test, and differences were significant at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Force Versus Ca²⁺ in Gizzard and the Effect of GTPS-- Skinned smooth muscle strips were used to determine the Ca²⁺ sensitivity of force. For ED 15-17 gizzard strips, which exclusively express the splice-in isoform of MYPT, the addition of GTPS produces force enhancement at intermediate levels of Ca²⁺ activation but not in relaxing solution or maximal Ca²⁺ activation. The Ca²⁺ sensitivity of force was fit with a pK = 5.30 ± 0.08, (n = 4), and GTPS produced a significant (p < 0.05) Ca²⁺ sensitization with a leftward shift in the Ca²⁺ sensitivity of force (pK = 5.52 ± 0.08, n = 6; Fig. 1a).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Steady-state Ca2+ versus force relationship in -escin skinned gizzard smooth muscle with () and without () GTPS. a, ED 15-17 gizzard: For Ca2+ activation (), Fmin = 2 ± 6, Fmax = 100 ± 7, pK = 5.30 ± 0.08, n = 1.2 ± 1.0 (n = 4); For GTPS (), Fmin = 1 ± 6, Fmax = 100 ± 8, pK = 5.52 ± 0.08, n = 1.5 ± 1.3 (n = 6). b, ED 20 gizzard: For Ca2+ activation (), Fmin = 2 ± 4, Fmax = 100 ± 4, pK = 5.40 ± 0.04, n = 1.8 ± 0.2 (n = 4); For GTPS (), Fmin = 1 ± 5, Fmax = 100 ± 6, pK = 5.50 ± 0.06, n = 1.5 ± 0.3 (n = 4). c, adult gizzard: For Ca2+ activation (), Fmin = 3 ± 4, Fmax = 100 ± 9, pK = 5.60 ± 0.20, n = 0.7 ± 1.5 (n = 4); For GTPS (), Fmin = 7 ± 4, Fmax = 7 ± 7, pK = 5.40 ± 0.20, n = 0.7 ± 0.7 (n = 4).

At ED 20, when gizzard tissue begins to express the splice-out MYPT isoform, data of force versus Ca²⁺ demonstrate that GTPS induces a small force enhancement, but neither force enhancement nor GTPS-induced Ca²⁺ sensitization were significant (pK = 5.40 ± 0.04, n = 4 versus 5.50 ± 0.06, n = 4, p < 0.05; Fig. 1b). In adult chicken, when gizzard tissue exclusively expresses the splice-out MYPT isoform (15, 16), GTPS had no effect on force at any Ca²⁺ (Fig. 1c).

Force Versus Ca²⁺ in -Escin Skinned Aorta Tissue and the Effect of GTPS-- For ED 15-17 aortic strips, the Ca²⁺ sensitivity of force had a pK of 5.40 ± 0.06 (n = 6). GTPS resulted in a force enhancement (p < 0.05), most prominent at lower levels of Ca²⁺ activation and a significant Ca²⁺ sensitization (p < 0.05) with a leftward shift in the force versus Ca²⁺ relationship (pK = 5.60 ± 0.04, n = 4; Fig. 2a). For the aorta at ED 20, GTPS resulted in a significant force enhancement (~40%) at lower levels of Ca²⁺ activation but did not produce a significant shift in the Ca²⁺ sensitivity of force (pK = 5.12 ± 0.07, n = 4 versus 5.07 ± 0.14, n = 4 for Ca²⁺ versus GTPS + Ca²⁺, respectively; Fig. 2b). The differences in the absolute magnitude of the force enhancement or Ca²⁺ sensitization produced by GTPS at ED 15-17 versus ED 20 aortic strips could be explained by changes in the MLCK/MLC phosphatase ratio (22) that occur during development (16).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Steady-state Ca2+ versus force relationship in -escin skinned aortic smooth muscle with () and without () GTPS. a, ED 15-17 aorta: For Ca2+ activation (), Fmin = 2 ± 6, Fmax = 100 ± 7, pK = 5.40 ± 0.06, n = 2.1 ± 0.4 (n = 6); For GTPS (), Fmin = 10 ± 3, Fmax = 91 ± 3, pK = 5.60 ± 0.04, n = 1.6 ± 0.9 (n = 4). b, ED 20 aorta: For Ca2+ activation (), Fmin = 2 ± 5, Fmax = 100 ± 6, pK = 5.12 ± 0.07, n = 1.3 ± 0.2 (n = 4). For GTPS (), Fmin = 40 ± 6, Fmax = 100 ± 7, pK = 5.07 ± 0.14, n = 1.2 ± 0.3 (n = 4).

To determine in smooth muscle from the embryonic chick whether the force enhancement produced by GTPS is mediated by a pathway involving MLCK, PKC, or Rho-kinase, the effect of inhibiting these enzymes on the magnitude of GTPS-induced force enhancement was studied. Neither inhibition of MLCK with ML-9 nor PKC with staurosporine influenced the magnitude of GTPS-induced force enhancement. However, similar to the results of others (11), there is a dose-dependent inhibition of the magnitude of GTPS-induced force enhancement by the Rho-kinase inhibitor Y-27632. Force enhancement was not affected by 2 µM Y-27632, whereas 10 µM Y-27632 significantly blunted the magnitude of GTPS-induced force enhancement (Fig. 3).

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Effect of Rho-kinase inhibition on GTPS-induced force enhancement. Force recording of skinned embryonic aortic smooth muscle activated with GTPS in the presence or absence of Y-27632. In the left panel, the preparation was first activated with Ca2+ (pCa 5.7), then with 100 µM GTPS in the presence of 2 µM Y-27632, and then back to 100 µM GTPS before relaxing the preparation. In the right panel, the experimental protocol was similar except 10 µM Y-27632 was used. As demonstrated, Y-27832 causes a dose-dependent inhibition of GTPS-induced force enhancement.

To demonstrate that GTPS-induced Ca²⁺ sensitization is mediated by an inhibition of MLC phosphatase activity, as has been shown in other tissues (8, 9), we determined MLC₂₀ phosphorylation levels in permeabilized embryonic smooth muscle strips (n = 4) at rest (pCa 9), at pCa 5.7, and at pCa 5.7 with 100 µM GTPS. Similar to the results of others (8, 9), the Ca²⁺ sensitization of force was accompanied by an increase in MLC₂₀ phosphorylation levels; i.e. MLC₂₀ phosphorylation increased by an additional 7 ± 1.3% in pCa 5.7 with 100 µM GTPS compared with pCa 5.7 alone (Fig. 4).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   GTPS induced a significant increase in MLC20 phosphorylation at a constant Ca2+. Western blot demonstrating MLC20 phosphorylation at rest (pCa 9) and with Ca2+ activation (pCa 5.7) and the effect of 100 µM GTPS at pCa 5.7 (pCa 5.7 + GTPS). In this blot, MLC20 phosphorylation was not detected at pCa 9 but increased to 40% at pCa 5.7; and at pCa 5.7 with 100 µM GTPS, MLC20 phosphorylation was 49%.

Thiophosphorylation of MYPT-- To determine the level of phosphorylation of MYPT during force enhancement in embryonic and adult smooth muscle strips, -escin skinned gizzard and aortic strips were activated in pCa 9 with [³⁵S]ATPS, homogenized, and resolved by SDS-PAGE (9). Radiolabeled proteins were detected by autoradiography. Duplicate protein samples were transferred to nitrocellulose for Western blotting. Similar to the results of Trinkle-Mulcahy et al. (9), these autoradiograms demonstrate thiophosphorylation of several proteins between 60 and 110 kDa and the incorporation of radiolabeled ATP into the MYPT. However, the autoradiograms show MYPT phosphorylation occurring only in the adult gizzard preparation but not in adult aortic smooth muscle (Fig. 5).

View larger version (121K): [in this window] [in a new window]   Fig. 5.   Thiophosphorylation of MYPT in aortic and gizzard tissue. Phosphorimaged autoradiograms of whole muscle homogenate of adult aortic and gizzard tissue after treatment with [35S]ATPS in pCa 9 (see "Experimental Procedures"). The location of MYPT (arrow) was confirmed by Western blotting.

To determine whether ATPS treatment results in a significant force enhancement, muscle tissue was incubated in 1 mM ATPS relaxing solution (pCa 9) containing 300 µM ML-9 for 10 min and then activated at submaximal [Ca²⁺]. Incubation of skinned smooth muscle strips in ATPS with ML-9 at pCa 9 does not result in thiophosphorylation of MLC₂₀ (9). In aortic tissue, this protocol produced a force enhancement at a submaximal Ca²⁺ compared with prior to the ATPS treatment. As demonstrated in Fig. 6a, the preparation was first stimulated to contract with pCa 4 and then relaxed. Submaximal activation (pCa 5.7) produced only a small increase in force (12% F_max) prior to the treatment with ATPS, but a substantial increase in force (52% F_max, 40% force enhancement) was produced at the same Ca²⁺ after the treatment with ATPS. Similar results were observed in embryonic gizzard strips. In Fig. 6b, the force in pCa 5.7 was 13% of that in pCa 4 prior to ATPS treatment and 23% (10% force enhancement) of the maximum in pCa 4 after ATPS treatment. The magnitude of the force enhancement produced by ATPS was smaller (p < 0.05) in embryonic gizzard (12 ± 1%, n = 4) than in the aortic tissues (37 ± 2%, n = 4). However, ATPS did not produce force enhancement in adult gizzard tissue (Fig. 6c), the only smooth muscle in which we were able to demonstrate phosphorylation of the MYPT (Fig. 5).

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Force enhancement produced by ATPS in various smooth muscle tissues. a, typical isometric force recording for aortic smooth muscle before and after treatment with 1 mM ATPS at pCa 9 with 300 µM ML-9 for 10 min. b, typical isometric force recording for embryonic gizzard smooth muscle before and after treatment with 1 mM ATPS at pCa 9 with 300 µM ML-9 for 10 min. c, typical isometric force recording for adult gizzard smooth muscle before and after treatment with 1 mM ATPS at pCa 9 with 300 µM ML-9 for 10 min. In all cases, the skinned preparations were first activated at pCa 4 and 5.7 and then incubated in 1 mM ATPS relaxing solution (pCa 9) containing 300 µM ML-9 for 10 min. Then, the preparations were rinsed in relaxing solution and activated again at pCa 5.7 and 4. The open bars indicate relaxing solution (pCa 9).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We used the chicken developmental model to follow the magnitude of agonist-induced force enhancement during natural changes in protein isoform expression. We have previously shown that the default smooth muscle phenotype is tonic with the expression of MLC_17b, noninserted MHC and the splice-in isoform of the MYPT predominating early in development in both aortic and gizzard tissue (15, 16, 23). Expression of these proteins remains relatively constant throughout aortic development (15, 16, 23). In the gizzard, the expression of MHC and MLC₁₇ switch to a phasic phenotype between ED 14 and 16 (23). The MYPT begins to express the splice-out isoform at ED 18 (16) and up-regulates the expression of the splice-out isoform with development (16) until it becomes exclusively splice-out shortly after hatching (15).

Our data shows that G-protein stimulation by GTPS produced a significant force enhancement, or Ca²⁺ sensitization of the contractile filaments, in aortic tissue throughout development (Fig. 2). Early during development in the gizzard, when the tissue expresses only the splice-in MYPT isoform (M133), a significant Ca²⁺ sensitization of the contractile filaments was observed. With the up-regulation of the splice-out isoform (M130), the ability of GTPS to produce a force enhancement decreased and produced no effect when the tissue exclusively expresses M130 (Fig. 1). Others (24) have shown that muscarinic stimulation did not produce Ca²⁺ sensitization in adult gizzard tissue. However in this study, GTPS produced a small force enhancement in -toxin-permeabilized adult chicken gizzard, whereas a robust force enhancement was produced in the cranial tibial artery. It is unclear why the results with GTPS in the adult gizzard are different in our study and that of Anabuki et al. (24), but it could be because of the skinning techniques (25). In addition, Anabuki et al. (24) showed a modest GTPS-induced force enhancement at only one [Ca²⁺] level, and it may be that GTPS at other [Ca²⁺] levels would not have produced a significant increase in force. Our data suggest that the diversity in the magnitude of agonist-induced force enhancement in smooth muscle tissues (8, 24) may be partially because of the relative expression of M133/M130, or splice-in/out MYPT isoforms.

Several pathways for agonist-induced force enhancement have been proposed, all of which converge on an inhibition of MLC phosphatase activity (reviewed in Refs. 2 and 3)). The MYPT has been demonstrated to be one of the few proteins thiophosphorylated under conditions that produced a large decrease in MLC phosphatase activity (9). These data lead to the hypothesis that MYPT phosphorylation has an in vivo role for the physiologic regulation of MLC phosphatase activity (9). Agonist stimulation has been shown to activate the monomeric G-protein, Rho A, which activates Rho-kinase. In embryonic smooth muscle, our results demonstrating a dose dependence of the inhibition of GTPS-induced force enhancement by the Rho-kinase inhibitor Y-27632 (Fig. 3) are similar to those of others (8, 24, 26). The magnitude of force enhancement was not affected by inhibition of MLCK or by inhibition of PKC with a concentration of staurosporine known to inhibit contractions of smooth muscle produced by phorbol esters (18). The GTPS-induced force enhancement was accompanied by an increase in MLC₂₀ phosphorylation (Fig. 4). These results suggest that in embryonic chicken smooth muscle, force enhancement produced by GTPS is mediated by activation of Rho-kinase, which presumably leads to a downstream inhibition of MLC phosphatase activity (10).

Rho-kinase has been shown to phosphorylate the MYPT to inhibit MLC phosphatase activity (10, 11). This pathway has been suggested as a potential physiologically important mechanism for regulation of phosphatase activity and smooth muscle tone. However, the Rho-kinase phosphorylation site has been shown to lie in a COOH-terminal fragment of MYPT (amino acids 753-1004) (3) distinct from the "regulatory site(s)" (residue Thr-654/Thr-695) thought to be important for inhibition of MLC phosphatase activity (14). However more recently, Rho-kinase has been demonstrated to phosphorylate MYPT at Thr-695 (27), which suggests that Rho-kinase-induced phosphorylation of MYPT regulates MLC phosphatase activity. In addition, a Zip-like kinase has recently been shown to phosphorylate mammalian MYPT at its regulatory site (12). However, others have shown that MYPT phosphorylation by Zip-like kinase may not be physiologically important because MYPT is a poor substrate for Zip-like kinase (28).

We have shown that incubation of skinned smooth muscle strips in ATPS produced a significant force enhancement in the aorta throughout development and in embryonic gizzard tissue but not in smooth muscle strips from the adult gizzard (Fig. 6). This is similar to the effect produced by GTPS (Figs. 1 and 2). However, we were unable to demonstrate thiophosphorylation of the MYPT (Fig. 5) in any tissue that displayed a GTPS (Figs. 1 and 2)- and/or an ATPS (Fig. 6)-induced Ca²⁺ sensitization. In fact, GTPS or ATPS stimulation did not produce force enhancement in the only tissue (adult gizzard) that we were able to demonstrate phosphorylation of the MYPT.

It could be that the force enhancement produced by GTPS and ATPS are mediated by different pathways (26). If this is the case, the lack of thiophosphorylation of the MYPT in the aorta and embryonic gizzard would not preclude phosphorylation of the MYPT mediating G-protein stimulated Ca²⁺ sensitization. Nevertheless, the results of the present study do not show a correlation between MYPT phosphorylation and force enhancement. These results could suggest that although MYPT phosphorylation has been shown both to occur in and inhibit MLC phosphatase activity (9, 11, 12), in vivo as suggested by others (28), this event does not participate in force regulation.

It is unclear why others have demonstrated a correlation between Rho-kinase (11)-, ATPS (9)-, and agonist (12)-stimulated force enhancement and MYPT phosphorylation. One explanation is that the guinea pig ileum (11), rabbit portal vein (9), and rabbit bladder (12) could express multiple isoforms of the MYPT, as is the case for these tissues in the chicken and rat (15). Phosphorylation of the splice-out MYPT isoform could occur, but the force enhancement could be the result of an inhibition of the splice-in MYPT isoform, in which "regulatory sites" are not phosphorylated, or via a pathway leading to an increase in MLC₂₀ phosphorylation without an inhibition of MLC phosphatase activity (29, 30). This could explain a correlation between MYPT phosphorylation and force enhancement in other studies (9, 11, 12), which we did not encounter using tissues that exclusively express a single MYPT isoform.

Agonist-induced Ca²⁺ sensitization could also be mediated by PKC (25, 30-32). Agonist activation has been demonstrated to activate PKC, which phosphorylates CPI-17 leading to an inhibition of MLC phosphatase activity without a phosphorylation of the MYPT (25). In smooth muscle, CPI-17 is not detectable after Triton skinning, is reduced by 65% after -escin permeabilization, and is not effected by -toxin (25). In addition, the expression level of CPI-17 is tissue-specific (33). Thus, the contribution of protein kinase C and CPI-17 for Ca²⁺ sensitization will be preparation-dependent, and this could explain some of the variability in the magnitude of force enhancement produced by PKC in different studies. However, CPI-17 has also been demonstrated to be phosphorylated by both Rho-kinase (34, 35) and Zip-like kinase (36). Thus, our results are consistent with a Rho-kinase-mediated (directly or indirectly by Zip-like kinase) phosphorylation of CPI-17 inhibiting MLC phosphatase activity.

Alternatively, others have suggested that the Zip-like kinase participates in the physiologic regulation of force in smooth muscle (28); these investigators showed that Zip-like kinase, rather than phosphorylating the MYPT, phosphorylated MLC₂₀ at both Ser-19 and Thr-18 to increase the actin-activated ATPase activity of myosin at a constant Ca²⁺ and produce a Ca²⁺-independent contraction. However, the antibody used in this study (28) was specific for the mammalian MYPT phosphorylated at Thr-641 (splice-out isoform), and it is possible that Zip-like kinase only phosphorylates the splice-in MYPT isoform (Thr-697). Similarly, integrin-linked kinase has been shown to phosphorylate MLC₂₀ and suggested to participate in the regulation of smooth muscle contraction via a Ca²⁺ independent mechanism (29). However, it is difficult to envision how changes in the relative expression of M130/M133 isoforms of MYPT during chicken development could alter the magnitude of a force enhancement produced by direct MLC₂₀ phosphorylation by Zip-like kinase or integrin-linked kinase. It is possible that Rho-kinase or Zip-like kinase could preferentially phosphorylate the splice-in isoform, compared with the splice-out isoform, of MYPT. Alternatively, Rho-kinase (34, 35) or Zip-like kinase (36) could phosphorylate CPI-17, and the phosphorylated CPI-17 could either preferentially bind to and/or inhibit M133 compared with M130.

Nonetheless, the mechanism to explain how splice variant isoforms of MYPT regulate the magnitude of force enhancement and the physiologically relevant pathway mediating agonist-induced force enhancement will require further investigation.

	ACKNOWLEDGEMENTS

We thank Peter Karagiannis for comments on the text and Marcus Bell, Joe Pili, and Steve DeFeo for the bioinstrumentation.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HL64137.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.

^¶ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, 10900 Euclid Ave., Cleveland, OH 44106-4970. Tel.: 216-844-8955; Fax: 216-368-5586; E-mail: fxb9@po.cwru.edu.

Published, JBC Papers in Press, November 28, 2001, DOI 10.1074/jbc.M111047200

	ABBREVIATIONS

The abbreviations used are: MLCK, myosin light chain kinase; MLC, myosin light chain; MYPT, myosin-targeting subunit; ED, embryonic day; ATPS, adenosine 5'-O-(thiotriphosphate); GTPS, guanosine 5'-O-(thiotriphosphate); PKC, protein kinase C; MOPS, 3-(N-morpholino)propanesulfonic acid; BES, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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