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Am J Physiol Heart Circ Physiol 282: H602-H610, 2002. First published October 18, 2001; doi:10.1152/ajpheart.00221.2001
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Vol. 282, Issue 2, H602-H610, February 2002

Inhibition of ERK attenuates force development by lowering myosin light chain phosphorylation

Gerard D'Angelo1,2 and Leonard P. Adam1,2

1 Boston Biomedical Research Institute, Watertown, Massachusetts 02472; and 2 Bristol-Myers Squibb Pharmaceutical Research Institute, Cardiovascular Drug Discovery, Pennington, New Jersey 08534


    ABSTRACT
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ABSTRACT
INTRODUCTION
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Phosphorylation of the actin-associated protein caldesmon (CaD) by extracellular signal-regulated kinases (ERK1/2) is purported to participate in force maintenance by vascular smooth muscle. We examined the interrelationship among ERK1/2 activity, phosphorylation of the high molecular weight isoform of CaD (h-CaD) and the 20-kDa myosin light chain (LC20), and isometric force in strips of porcine carotid artery stimulated with endothelin-1 (ET-1; 50 nM). After an initial delay, ERK1/2 activity increased in parallel with ET-1-mediated force; h-CaD phosphorylation increased modestly. 2-(2'-Amino-3'-methoxyphenyl)-ox-anaphthalen-4-one (PD-098059; 50 µM), an ERK1/2 kinase inhibitor, significantly reduced basal ERK1/2 activity within 1 h, but only partially attenuated h-CaD phosphorylation at 3 h. The mechanisms underlying the temporal dissociation of ERK1/2 activity from h-CaD phosphorylation are unknown, but include the possibility that a kinase other than ERK1/2 phosphorylates h-CaD or, more likely, that phosphate turnover in h-CaD is very slow. PD-098059 partially inhibited the development of ET-1-stimulated force only in Ca2+-replete physiological saline solution, primarily by reducing LC20 phosphorylation, yet had no effect on myosin light chain kinase in vitro. These inhibitory effects were most evident during the early phase of force production. The inhibitory effect of PD-098059 on force could not be correlated with a corresponding effect on ERK1/2-mediated h-CaD phosphorylation because force in arterial strips stimulated with ET-1 in the absence or presence of PD-098059 tended to approximate each other over time despite significant differences in the level of h-CaD phosphorylation. Force and LC20 phosphorylation in response to KCl depolarization were unaffected by PD-098059. These results show that ERK1/2 may regulate force in arterial smooth muscle, but suggest that the mechanism for this effect is by inhibiting LC20 phosphorylation.

caldesmon; vascular smooth muscle


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES

IN VASCULAR SMOOTH MUSCLE (VSM), it is well established that isometric force production and/or muscle shortening is regulated by Ca2+-dependent phosphorylation of the 20-kDa myosin light chain (LC20). Because both the intracellular Ca2+ concentration and degree of LC20 phosphorylation decline from peak values toward basal levels during sustained contraction of VSM, a state referred to as the "latch" state (reviewed in Ref. 16), it has been proposed that force is maintained by an additional mechanism(s) independent of LC20 phosphorylation. Attention has focused on the role of actin-associated proteins, in particular, caldesmon (CaD), in the regulation of VSM force. CaD is an elongated, protein-possessing binding site for actin, Ca2+-calmodulin, tropomyosin, and myosin. Alternate splicing of the CaD gene yields two isoforms: a high-molecular weight (93 kDa) protein (h-CaD), which is specific to contractile smooth muscle, and the 60-kDa protein l-CaD, which is expressed ubiquitously (see Ref. 25 for review).

To date, there exists some evidence supporting a role for h-CaD in the regulation of VSM force. Biochemical characterization of h-CaD demonstrated that h-CaD inhibits the actin-activated myosin ATPase activity in reconstituted systems of purified contractile proteins (23). Katsuyama et al. (17) found that a peptide fragment of h-CaD, which has no effect on myosin ATPase, produced a concentration-dependent increase in force at a fixed Ca2+ concentration in permeabilized VSM cells. By using antisense nucleotides to produce porcine carotid smooth muscle tissue deficient in h-CaD, Earley et al. (8) showed that unstimulated h-CaD-deficient arterial strips redeveloped force after a stretch-release protocol, whereas h-CaD-containing strips did not. Separately, these authors concluded that h-CaD exerts an inhibitory effect on VSM in the resting state.

Our laboratory (3) has shown phosphorylation of h-CaD parallels isometric force production, lending credence to the notion that h-CaD phosphorylation may reverse, or at least modulate, the effects of h-CaD in vivo. Although phosphorylation of h-CaD by such kinases as protein kinase C (27) and Ca2+-calmodulin-dependent kinase II (23, 26) can reverse its inhibitory effect on myosin ATPase, we have since identified the 44- and 42-kDa isoforms of the extracellular signal-regulated kinase (ERK1/2) as physiologically relevant "CaD kinases" in intact VSM (1, 2, 4). It is unknown, however, whether h-CaD phosphorylation at ERK1/2-specific sites reverses the ability of h-CaD to inhibit actin-activated myosin ATPase. Watts and colleagues (10, 28, 29) have provided evidence of a functional relationship between ERK1/2 and isometric force in VSM, demonstrating that 2-(2'-amino-3'-methoxyphenyl)-ox-anaphthalen-4-one (PD-098059), a novel inhibitor of ERK activation (7), attenuated not only serotonin-induced contraction of rat aorta, but also ERK1/2 activity. On the other hand, studies aimed at determining the role of ERK-dependent phosphorylation of h-CaD in the regulation of smooth muscle force have yielded conflicting results. Whereas recombinant ERK potentiated the Ca2+-mediated contraction of skinned tracheal smooth muscle (12), it had no effect on the Ca2+ sensitivity of skinned preparations of rabbit portal vein or femoral artery (24).

Because ERK1/2 are physiologically relevant CaD kinases that may be involved in contractile regulation, the aim of this study was to characterize the causal relationship among ERK1/2 activation, h-CaD and LC20 phosphorylation, and force maintenance in intact arterial strips. To this end, we determined the temporal relationship among these parameters in porcine carotid arteries stimulated with ET-1, and the effect of PD-098059, an inhibitor of ERK1/2, LC20, and h-CaD phosphorylation, and isometric force. We report that although phosphorylation of ERK1/2 and h-CaD are temporally related to the sustained level of ET-1-stimulated force, attenuation of force caused by ERK1/2 inhibition appears to be mediated principally by a reduction in LC20 phosphorylation.


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Materials. Unless specified otherwise, reagents were purchased from Sigma (St. Louis, MO). Purified LC20 used to determine ATP specific activity for measurement of absolute ERK1/2 enzymatic activity using an in vitro kinase assay and for myosin light chain (MLC) phosphorylation in vitro, was generously given to us by Dr. Renne Lu (Boston Biomedical Research Institute). Calmodulin was purified from bovine testicles according to Klee (18). MLC kinase (MLCK) was purified from chicken gizzards according to the method of Hathaway et al. (15).

Tissue preparation. Porcine carotid artery segments were cleaned of any adhering fat and connective tissue, and denuded of endothelium by gentle abrasion. Subsequently, arterial strips (3 mm wide × 8 mm long) were attached to an isometric force transducer (model FT.03, Grass) and bathed in 3-(N-morpholino)propanesulfonic acid (MOPS)-buffered physiological saline solution (PSS) warmed to 37°C; resting force was adjusted to 2 g. MOPS-buffered PSS contained (in mM) 140 NaCl, 4.7 KCl, 1.2 Na2HPO4, 1.2 MgSO4, 1.6 CaCl2, 5.6 glucose, and 2.0 MOPS (pH 7.4). Tissues were allowed to equilibrate in PSS for 30 min. The arteries were then placed in a high-KCl (110 mM) buffer prepared by isosmotic substitution of KCl for NaCl; all subsequent responses were normalized to the amount of force elicited by KCl depolarization. Vascular strips were permitted to develop force for 30 min, and subsequently relaxed in PSS. Any strip not exhibiting a rapid, robust response was excluded from further study. In certain experiments, tissues were treated with PD-098059 (Calbiochem-Novabiochem; La Jolla, CA) (50 µM) or vehicle [0.5% dimethyl sulfoxide (DMSO)] for the indicated times. Most tissues were stimulated with ET-1 (50 nM) (Alexis; San Diego, CA); at the indicated times, arterial strips were quick-frozen with liquid N2-cooled clamps. When the contractile responses were tested in the absence of extracellular Ca2+, arterial strips were maintained in Ca2+-free PSS for 1 h before and during stimulation with ET-1; strips were similarly quick-frozen with liquid N2-cooled clamps at the indicated times. Ca2+-free PSS was prepared by replacing CaCl2 with 1.0 mM EGTA. To minimize binding of ET-1 to the muscle chambers, all baths were previously treated with SigmaCote (Sigma).

In vitro peptide kinase assay. Frozen tissues were ground to a fine powder while under liquid N2 with a liquid N2-cooled mortar and pestle. Proteins were then extracted into 250-300 µl of ice-cold extraction buffer (4°C) consisting of (in mM) 20 Tris (pH 7.5), 5 EGTA, 1 Na3VO4, 20 beta -glycerophosphate, 10 NaF, and 1 1,4-dithiothreitol (DTT), and the protease inhibitors 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone, and 0.1 mM N-alpha -p-tosyl-L-lysine chloromethyl ketone for 45-60 min. Samples were clarified by centrifugation at 436,000 g for 10 min at 4°C, and the supernatant was withdrawn. ERK specific activity was measured by assaying for phosphotransferase activity using the peptide substrate APRTPGGRR, the sequence based on the ERK1/2 phosphorylation site of myelin basic protein (8). Ten microliters of tissue extract were added to 40 µl of mitogen-activated protein (MAP) kinase buffer to achieve a final concentration of (in mM) 12.5 MOPS (pH 7.2), 0.5 EGTA, 0.5 Na3VO4, 12.5 beta -glycerophosphate, 0.05 NaF, 2.0 DTT, 7.5 MgCl2, and 0.25 [gamma -32P]ATP (NEN Life Sciences; Boston, MA), as well as 500 µM peptide. Kinase reaction was allowed to proceed for 30 min at 25°C, and was terminated by the addition of trichloroacetic acid (final concentration 10% wt/vol). Samples were then centrifuged at 14,000 g for 15 min. Supernatant was spotted onto phosphocellulose paper (P81, Whatman), and filters were washed with 5 × 400 ml of 50 mM H3PO4 for 10 min each. Filters were rinsed briefly (~1 min) with 100 ml of 95% ethanol, and the amount of radioactivity was determined by liquid scintillation counting. Protein content of the 436,000 g supernatant was quantified by the method of Lowry et al. (21). ERK1/2 substrate peptide was synthesized by Dr. Renne Lu of Boston Biomedical Research Institute.

Immunoblot analysis of phosphoCaD and phosphoERK1/2. The frozen tissues were ground to a fine powder. Proteins were extracted into 3% sodium dodecyl sulfate, and heated for 10-15 min, clarified by centrifugation at 14,000 g for 15 min, separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. Relative phosphoCaD and phosphoERK1/2 levels were determined by radioimmunoblotting on duplicate blots with the following: 1) an anti-phosphoCaD antibody raised against a phosphopeptide analogous to the phosphorylation site at serine-789 (Ser789) of human h-CaD (6), 2) an anti-CaD polyclonal antibody generated against full-length porcine stomach h-CaD (3), 3) an anti-phosphoERK1/2 polyclonal antibody specific for the pTEpY sequence of ERK1/2 (New England Biolabs; Beverly, MA), and 4) an anti-ERK1/2-specific polyclonal antibody (Oncogene Research Products; Cambridge, MA). Detection was performed with 125I-labeled protein A as the secondary antibody. Nitrocellulose strips were subjected to autoradiography, and radioactive bands were cut out and counted with the use of a Beckman 5500 gamma counter.

MLC phosphorylation determination of intact tissue. One milliliter of dry-ice-cooled acetone, 10% trichloroacetic acid, and 10 mM DTT was added to liquid N2 frozen arteries in dry-ice-cooled Eppendorf tubes, and allowed to come to room temperature. Tissue strips were then washed at least three times with acetone/10 mM DTT at room temperature and lyophilized. MLCs were extracted into ~50 µl of urea gel extraction buffer for 1 h at room temperature (25°C). Extraction buffer contained 20 mM Tris, 22 mM glycine, 10 mM DTT, 7.25 M urea, and 0.1% bromophenol blue. Stoichiometry of the LC20 phosphorylation was determined by glycerol/urea gel electrophoresis, followed by radioimmunoblotting with an anti-serum developed to the chicken gizzard LC20 (primary antibody), and 125I-labeled protein A (secondary antibody). As previously described, radioactive bands were cut out and counted using a gamma counter (Beckman 5500).

In vitro MLC phosphorylation. Purified LC20 was phosphorylated at room temperature (25°C) by a reaction mixture (10 µl total volume) containing 10 µg LC20, 0.05 µg MLCK, 1 µg calmodulin, 0.25 mM CaCl2, 5.0 mM MgCl2, and 0.25 mM ATP in the absence [vehicle; 1% DMSO (vol/vol)] or presence of 50 (n = 2) or 100 (n = 2) µM PD-098059. Because no difference in either the rate or extent of LC20 phosphorylation was detected for reactions carried out in 50 or 100 µM PD-098059, these data were pooled. Reaction was terminated at the indicated times by the addition of 30-µl urea gel extraction buffer containing 6 M urea, and phosphorylated and unphosphorylated LC20 were separated by glycerol-urea gel electrophoresis. Gels were dried and LC20 phosphorylation was determined by densitometric analysis using NIH Image Software version 1.62.

Statistical analysis. Data are expressed as means ± SE, and n refers to one arterial segment from one animal. All force measurements in response to ET-1 were normalized to the steady-state value (at 30 min) yielded by depolarization with 110 mM KCl in PSS. For in vitro kinase assays, data are expressed as picomoles PO4 per minute per milligram protein, where ATP specific activity was determined by measuring 32P-incorporation into purified, mutant LC20 possessing only the Ser19 phosphorylation site. For Western blot analyses of phosphoCaD and of phosphoERK1/2, data are expressed as the ratio counts of the phosphoprotein to that of the total protein, and are normalized relative to control tissue (which assumes a value of 1). Statistical analysis was made by one- or two-way analysis of variance, followed by Tukey's test for multiple comparisons. Data are considered significant at P < 0.05.


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DISCUSSION
REFERENCES

Time course of ERK1/2 activity and CaD phosphorylation. Initial experiments were aimed at examining the time courses of ERK1/2 activation and of h-CaD phosphorylation in response to ET-1 (50 nM). Because basal ERK1/2 activity progressively declines after application of a preload to arterial strips mounted for measuring isometric force (11), the duration of each experiment was held constant at 4 h. ET-1 (50 nM)- stimulated force rose to 82 ± 6% of the KCl response by 15 min, and decreased only slightly to 72 ± 3% over the next 45 min (Fig. 1). ET-1 also produced a time-dependent increase in ERK1/2 activity, the onset of which was delayed relative to the rise in force. ET-1-mediated ERK1/2 activity peaked at 15 min, reaching nearly twice that of control (46 ± 6 vs. 89 ± 7 pmol · min-1 · mg protein-1, P < 0.001), and remained significantly elevated over the course of the contraction (77 ± 7 pmol · min-1 · mg protein-1 at 60 min, P < 0.05) (Fig. 1). Qualitatively similar results were obtained with Western blot analysis using antibodies that recognize the pTEpY sequence of ERK1/2, and thus are specific for the activated enzyme (data not shown). Aside from the noted difference during the initiation of isometric force, these data show isometric force and ERK1/2 activity exhibited a close temporal relationship.


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  		Fig. 1.   Time course of force production, extracellular signal-regulated kinase (ERK1/2) activity, and caldesmon (CaD) phosphorylation after stimulation with 50 nM endothelin-1 (ET-1) in Ca2+-replete physiological saline solution (PSS). ERK1/2 activity was assayed using an in vitro peptide kinase assay by measuring phosphate incorporation into the peptide substrate APRTPGGRR. Extent of CaD phosphorylation was assayed by Western blot analysis, where increases were assessed by changes in ratio of phosphoCaD/total CaD relative to control (which assumes a value of 1). *P < 0.05 at t = 0.

Accompanying changes in the level of h-CaD phosphorylation were measured separately by Western blot analysis using a polyclonal antibody generated against the phosphopeptide, CQSVDKTS (PO4) PTKV, a sequence that is analogous to Ser789 (pSer789) of the 93-kDa isoform of mammalian CaD. This antibody is specific for CaD phosphorylated at Ser789, one of two sites phosphorylated by ERK in vitro. Phosphorylation at Ser789 in unstimulated tissue was previously found to be 0.24 ± 0.03 mol PO4/mol CaD, whereas the level of phosphorylation at the other ERK-dependent site, Ser759, was <0.005 mol PO4/mol CaD (6). Significant increases in the relative amount of h-CaD phosphorylation were observed after elevation of ET-1-stimulated ERK1/2 activity, i.e., after 15 min. h-CaD phosphorylation levels remained elevated throughout the remainder of the ET-1-stimulated contraction, reaching 1.25 ± 0.05 times that of control.

Effects of PD-098059. We first examined the effect of 50 µM PD-098059 on basal ERK1/2 activity and CaD phosphorylation in arterial segments incubated with PD-098059 for 1, 2, or 3 h. To facilitate the correlation of ERK1/2 activity and CaD phosphorylation, these experiments were performed using an anti-phosphoERK1/2 antibody to assess relative changes in ERK1/2 activity, and accompanying changes in the relative levels of h-CaD phosphorylation were determined using the anti-phosphoCaD antibody described above. To explore the possible regulation of ERK1/2 activity by Ca2+, experiments were performed in Ca2+-free and Ca2+-replete PSS. Arterial strips were maintained in Ca2+-free PSS 1 h before and during administration of PD-098059. Because of the constraints imposed by the time-dependent decrease in basal ERK1/2 activity after the mounting and application of preload to the arterial strips (11), exhaustive depletion of intracellular Ca2+ was not performed. Nevertheless, in control strips, caffeine (25 mM) did not elicit a contraction (data not shown), indicating that 1-h incubation in Ca2+-free PSS is sufficient to completely deplete the caffeine-sensitive Ca2+ stores; moreover, KCl elicited no response indicating Ca2+ influx was completely eliminated. The maximal inhibitory effect of PD-098059 on ERK1/2 activation was evident within 1 h in either Ca2+-free (24 ± 3% of control) or Ca2+-replete conditions (27 ± 4% of control); no further decreases were detected after 3 h (Fig. 2A). By comparison, dephosphorylation of h-CaD proceeded on a much slower time course (Fig. 2B). Whereas the maximal effect of PD-098059 on the basal ERK1/2 phosphorylation level had occurred within 1 h, treatment of arterial strips with 50 µM PD-098059 for 1 h only slightly reduced basal h-CaD phosphorylation in Ca2+-free (88 ± 3% of control) and in Ca2+-replete PSS (79 ± 4% of control). PD-098059 did produce a time-dependent decrease in h-CaD phosphorylation, such that after 3 h, the level of basal h-CaD phosphorylation was lowered to 65 ± 10% (P < 0.001) and 60 ± 8% (P < 0.001) of control values in Ca2+-free and Ca2+-replete PSS, respectively.


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  		Fig. 2.   Time-dependent effect of 2-(2'-amino-3'-methoxyphenyl)-ox-anaphthalen-4-one (PD-098059) on basal ERK1/2 (A) and CaD (B) phosphorylation. Vessels were pretreated with 50 µM PD-098059 or vehicle [0.5% dimethyl sulfoxide (DMSO)] for 1, 2, or 3 h, and were then frozen. For those vessels tested in Ca2+-free PSS, arterial strips were maintained in Ca2+-free medium for 1 h before and during treatment with PD-098059. Extent of ERK1/2 and CaD phosphorylation was assayed by Western blot analysis using an anti-phosphoERK1/2-specific polyclonal antibody and with an anti-phosphoCaD antibody raised against the phosphorylation site at serine 789 (Ser789) of human CaD, respectively. Data are expressed as the ratio of phosphoprotein to total protein, and are normalized relative to control tissue (which assumes a value of 1). *P < 0.05 vs. -PD-098059.

We then tested whether preincubation (1 h) with PD-098059 could impact on ET-1-stimulated isometric force in either Ca2+-free or Ca2+-replete PSS, given the observed inhibitory effect of PD-098059 on basal ERK1/2 activity. Treatment with 50 µM PD-098059 had no effect on either the rate of force development (data not shown) or the maximum force (Fmax) achieved in Ca2+-free PSS (Fig. 3); prolonged incubation up to 3 h similarly had no effect on ET-1-stimulated responses (data not shown). On the other hand, Fmax was significantly reduced in Ca2+-replete PSS (Fig. 3). Moreover, the temporal characteristics of ET-1-stimulated force were dramatically altered by PD-098059. Effects of preincubation with PD-098059 are summarized in Fig. 4. In control strips stimulated with ET-1, isometric force characteristically rose at a consistent rate over the first 15 min, after which time it began to level off. Isometric force was maintained over the next 15 min, where Fmax was equal to 85 ± 4% of the KCl response (at t = 25 min). As previously observed (see Fig. 1), isometric force only partially declined over the ensuing 30 min to 77 ± 3% of the KCl response. Conversely, in arterial strips pretreated with PD-098059, the rate of force development paralleled that for control tissues only over the first 2.5 min, after which time there was a divergence in the rate of force production. Specifically, force increased more slowly than in control tissues such that peak levels were significantly less (65 ± 4% of KCl response; P < 0.01), and were attained at a later time point (i.e., at 45 min or later); force remained invariant up to 60 min (Fig. 4A). By 1 h, however, isometric force tended to converge toward, but did not yet reach, a common level due to the slow decrease in force in control tissues.


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  		Fig. 3.   Summary of effect of PD-098059 on ET-1-mediated maximum isometric force; values shown represent the average of those derived from the peak of each individual force tracing. For those vessels tested in Ca2+-free PSS, arterial strips were maintained in Ca2+-free medium for 1 h before and during stimulation with 50 nM ET-1. Vessels were pretreated with 50 µM PD-098059 or vehicle (0.5% DMSO) for 1 h. *P < 0.001 vs. control.



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  		Fig. 4.   Summary of effect of PD-098059 on ET-1-stimulated isometric force (A) and phosphorylation of the 20-kDa myosin light chain (LC20) (B). Arterial segments were pretreated with 50 µM PD-098059 or vehicle (0.5% DMSO) for 1 h in Ca2+-replete PSS, and frozen at the times indicated in B after the addition of 50 nM ET-1. Data in A reflect averages at discrete time points over the course of the stimulus; all values at time >= 5 min are significant at P < 0.001. Level of LC20 phosphorylation was assayed by Western blot analysis using an antiserum developed to the chicken gizzard LC20. *P < 0.05 vs. +PD-098059 at corresponding time point.

Our finding that PD-098059 inhibited force in Ca2+-replete, but not in Ca2+-free, PSS suggests PD-098059 may affect Ca2+ handling. We therefore wished to determine whether these observed effects of PD098059 could be accounted for, at least in part, by an effect on the extent of LC20 phosphorylation. In Ca2+-free PSS, force was independent of LC20 phosphorylation (control, 0.023 vs. ET-1, 0.025 mol PO4/mol LC20); the level did not rise to that of basal LC20 phosphorylation measured in Ca2+-replete PSS (see Fig. 4B). In Ca2+-replete PSS, there were no differences in basal LC20 phosphorylation between control (vehicle; 0.5% DMSO) and PD-098059-treated strips; however, LC20 phosphorylation levels measured 15 and 30 min after the addition of ET-1 were significantly lower in arterial strips that had been pretreated for 1 h with PD-098059 (Fig. 4B). To determine the relative specificity of PD-098059, we examined its effect on responses to KCl depolarization (Fig. 5, A and B). Whereas PD-098059 did not affect the development of force after the addition of KCl (110 mM), it did cause a slight, though not statistically significant decrease in steady-state force (Fig. 5A). As well, KCl-mediated LC20 phosphorylation was unaffected by PD-098059 (Fig. 5B). Finally, we tested whether the effect on ET-1-stimulated LC20 phosphorylation caused by PD-098059 was due to a direct effect on MLCK by measuring the activity of MLCK in vitro in the absence or presence of PD-098059. As shown in Fig. 6, 50 or 100 µM PD-098059 had no direct effect on either the rate or extent of LC20 phosphorylation by MLCK.


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  		Fig. 5.   Summary of effect of PD-098059 on KCl-stimulated isometric force (A) and phosphorylation of the 20-kDa LC20 (B). Arterial segments were pretreated with 50 µM PD-098059 or vehicle (0.5% DMSO) for 1 h in Ca2+-replete PSS, and frozen at the times indicated in B after adding 50 nM ET-1. A: data reflect averages at discrete time points over the course of the stimulus. Level of LC20 phosphorylation was assayed by Western blot analysis using an anti-serum developed to the chicken gizzard LC20.



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  		Fig. 6.   Summary of effect of PD-098059 on the phosphorylation of the 20-kDa LC20 by myosin LC kinase (MLCK) in vitro. Purified LC20 was phosphorylated by MLCK in either the absence [vehicle; 1% DMSO (vol/vol)] or presence of 50 (n = 2) or 100 (n = 2) µM PD-098059. Reaction was terminated at the indicated times by the addition of 30-µl urea gel extraction buffer containing 6 M urea, and phosphorylated and unphosphorylated LC20 were separated by glycerol-urea gel electrophoresis.

In a separate set of experiments, we also measured the effect of pretreatment with PD-098059 on ET-1-stimulated CaD phosphorylation by immunoblot analysis using the anti-pSer789 antibody. The effect of PD-09859 on ERK1/2 activity was verified by monitoring immunoreactivity with an anti-phosphoERK antibody (Fig. 7B). Because no effect of PD-098059 on isometric force had previously been detected in Ca2+-free PSS, even after prolonged incubation for 3 h (data not shown), experiments were only performed in Ca2+-replete PSS. Arterial segments were stimulated with 50 nM ET-1 after pretreatment with either vehicle (0.5% DMSO) or 50 µM PD-098059, and frozen either after 15 or 60 min, the time points corresponding to the maximal increases in ERK1/2 activity and CaD phosphorylation, respectively. In vehicle control muscle strips, the level of h-CaD phosphorylation increased slightly 15 min after the addition of ET-1 (27 ± 11%) but had more than doubled by 60 min (123 ± 33%) (Fig. 7A). As before, pretreatment with PD-098059 for 1 h had no effect on basal h-CaD phosphorylation. Relative to the vehicle-treated controls, ET-1-stimulated h-CaD phosphorylation rose slightly by 15 min (28 ± 16%) in the presence of PD-098059, and did not increase further at 60 min (34 ± 26%) (Fig. 7A). Because of the large variability in these responses, these values did not reach statistical significance. Relative to the baseline level for PD-098059-treated tissues, ET-1 stimulation produced increases in h-CaD phosphorylation of 35% and 41% at 15 and 60 min, respectively.


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  		Fig. 7.   Summary of effect of PD-098059 on CaD phosphorylation and ERK1/2 activity. Arterial segments were pretreated with 50 µM PD-098059 or vehicle (0.5% DMSO) for 1 h in Ca2+-replete PSS, and frozen at the indicated times after adding 50 nM ET-1. Extent of CaD phosphorylation (A), and of ERK1/2 activity (B) were assayed by Western blot analysis using an anti-phosphoCaD antibody raised against the phosphorylation site at Ser789 of human CaD, and with an anti-phosphoERK1/2-specific polyclonal antibody, respectively. Data are expressed as the ratio of phospho-protein to total protein, and are normalized relative to vehicle control tissue (which assumes a value of 1). *P <0.005 vs. -PD-098059 at corresponding time point; #P < 0.001 vs. vehicle control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dissociation of force and CaD phosphorylation. To address the role of h-CaD phosphorylation by ERK in smooth muscle function, we examined the temporal relationships among isometric force, h-CaD phosphorylation at ERK-modulated sites, and ERK activity in carotid arteries. Specifically, we found that whereas there was a consistent near doubling of ET-1-stimulated ERK1/2 activity, the increase in ERK-dependent h-CaD phosphorylation was much less. In a previous study (6), we found that the stoichiometry of phosphorylation at Ser789, the major site of phosphorylation by ERK in intact tissue, was 0.24 ± 0.03 PO4/mol CaD. On the basis of our previous work, the 30-35% rise observed in the present study would represent an increase to ~0.32 PO4/mol CaD. Comparable results were obtained previously (6), in which stimulation with phorbol dibutyrate only elicited an increase to 0.28 ± 0.04 PO4/mol CaD. Moreover, significant differences in the level of h-CaD phosphorylation were observed at 60 min after ET-1 stimulation in the presence and absence of PD-098059. Despite these differences, the sustained level of force elicited by ET-1 in the presence of PD-098059 approximated the sustained level of force produced in the absence of PD-098059. Collectively, these data argue against a role for ERK1/2-mediated h-CaD phosphorylation.

We also found that the MAP or ERK (MEK) inhibitor PD-098059 exerted a pronounced effect on basal and ET-1-stimulated ERK1/2 activity within 1 h, yet caused only a slight reduction in h-CaD phosphorylation. On the other hand, prolonged incubation (3 h) with PD-098059 was required to produce significant reduction in h-CaD phosphorylation; this effect occurred without any further impact on ERK1/2 activity. This dissociation between ERK1/2 activity and h-CaD phosphorylation suggests either that the phosphate turnover for h-CaD is slow or that a kinase other than ERK phosphorylates h-CaD at Ser789. Our previous findings that phosphorylation at Ser789 of purified CaD was not elevated by treatment with okadaic acid, an inhibitor of protein phosphatases types 1 and 2A, and that the catalytic subunit of protein phosphatase type 1 did not catalyze its dephosphorylation (6) are consistent with the notion that phosphorylation at this site is poorly subject to regulation by a phosphatase. Thus it follows that prolonged incubation with PD-098059 would be necessary to effect a significant decrease in h-CaD phosphorylation at Ser789.

Studies concerning the interrelationship among ERK1/2 activation, h-CaD phosphorylation, and regulation of smooth muscle contraction have yielded conflicting results. Specifically, phosphorylation of gizzard CaD by ERK1 partially reversed the inhibitory effect of CaD on actin sliding velocity in an in vitro sliding filament assay, thereby implicating the involvement of CaD phosphorylation by ERK1/2 in the regulation of smooth muscle contraction (13). In a separate study, Gerthoffer et al. (12) demonstrated that activated recombinant ERK2 could potentiate Ca2+-mediated contraction of Triton X-100-permeabilized tracheal smooth muscle strips. To the contrary, Nixon et al. (24) had shown phosphorylation of CaD by recombinant, activated ERK2 had no effect on the Ca2+ sensitivity of Triton-permeabilized VSM preparations, leading these authors to conclude the phosphorylation of CaD by ERK is temporally associated with, but not involved in the regulation of force by VSM. Stoichiometry of phosphorylation was not determined in either study, and so it is difficult to derive any quantitative correlation between the level of CaD phosphorylation and potentiation of the Ca2+-mediated contraction. From our previously reported stoichiometries (6), it is reasonable to suggest that the small increase in ERK-dependent h-CaD phosphorylation obtained in the present study would not significantly contribute to force maintenance.

While inhibition of MAP kinase results in the attenuation of isometric force, it appears this inhibitory effect occurs independently from its effect on h-CaD phosphorylation. This interpretation is supported by the observed temporal patterns of the ET-1-stimulated responses in the absence and presence of PD-098059. The most pronounced inhibitory effect of PD-098059 occurred during the early, or rising, phase of force development, and not in the later stages of force elicited by ET-1. Specifically, it required nearly twice as long to reach Fmax in PD-098059-treated tissues, and as such, was significantly less than that noted for controls. Whereas force in vehicle-treated tissues declined slightly from a peak level over the time investigated, force in PD-098059-treated strips held constant after attaining a maximal level. Moreover, although force in arterial strips from the two groups tended to approximate each other by 60 min, it was still significantly elevated in control tissues.

Conversely, the relative increases in ET-1-stimulated h-CaD phosphorylation after 15 min were similar in non- and PD-098059-treated arterial strips when normalized to their respective controls, whereas h-CaD phosphorylation was significantly reduced in PD-098059-treated strips at 60 min. Thus, at a time point where force tended to converge, we detected significant differences in the level of h-CaD phosphorylation. Because the early phase of force development in regulated primarily by LC20 phosphorylation, our data are more consistent with a model in which early signaling events, e.g., MLCK activity and LC20 phosphorylation, are modulated directly by ERK1/2 activity, or indirectly by the MEK inhibitor PD-098059. Gorenne et al. (14) reported that PD-098059 had no effect on either basal or stimulated LC20 phosphorylation after the addition of either phorbol 12,13-dibutyrate or histamine. The time points at which LC20 phosphorylation measurements were made, however, was not specified. In our study, there was a significant attenuation of LC20 phosphorylation at the time of peak ET-1-mediated force, but no difference 60 min after the addition of ET-1. Thus, depending on when LC20 phosphorylation is measured, an effect of PD-098059 may or may not be detected.

Interaction between ERK1/2 and MLCK. Similar to the findings of Watts and colleagues (10, 28, 29), we found that pretreating carotid arterial strips for 1 h with the MEK inhibitor PD-098059 attenuated agonist-stimulated isometric force. In the present study, the decrease in the rate of force development caused by PD-098059 was associated with a reduction in LC20 phosphorylation, an outcome that was not predicted by our original hypothesis. This inhibitory effect was detected in Ca2+-replete PSS, but not in Ca2+-free PSS, where the rise in force was not accompanied by an elevation in LC20 phosphorylation. Not even with prolonged incubation (3 h) was there a significant effect on ET-1-mediated force in Ca2+-free PSS. It is unlikely that 1-h incubation in Ca2+-free PSS is sufficient to deplete all the intracellular Ca2+ stores; however, ET-1 did produce a contraction in extracellular Ca2+-depleted muscles that occurred in the absence of measurable changes in LC20 phosphorylation. Thus it follows that ET-1 may not evoke a significant change in intracellular Ca2+ under these conditions, although local Ca2+ responses in response to receptor-activated pools or through other mechanisms (insufficient to yield measurable increases in LC20 phosphorylation) cannot be ruled out. In the latter scenario, where there may be local increases in Ca2+, the lack of effect of PD-098059 on ET-1-stimulated force in Ca2+-free PSS argues against a role for ERK1/2 in modulating the release of Ca2+ from intracellular pools. Nevertheless, PD-098059 attenuated the rise in force only during the situation where force was associated with an increase in LC20 phosphorylation.

We also addressed the possibility that the effect of PD-098059 on force may be nonspecific. In so doing, we found that PD-098059 had no effect on force development and LC20 phosphorylation elicited by KCl depolarization, ruling out the possibility that MLCK was nonspecifically downregulated; there was, however, a small, yet insignificant, attenuation of force at steady state. These data also argue against an effect of ERK inhibition on Ca2+ entry, although it is entirely possible that any effect is simply overcome by the degree of depolarization and ensuing Ca2+ influx. Moreover, neither 50 nor 100 µM PD-098059 had a direct inhibitory effect on MLCK activity in vitro. These data suggest that LC20 serves as a substrate within the signaling cascade downstream of ERK1/2, either directly or through the involvement of an intermediate kinase(s).

Direct evidence of a causal relationship between ERK and MLCK activity was provided by Klemke et al. (19). These authors showed that purified, constitutively active ERK1/2 phosphorylated chicken gizzard MLCK and enhanced its phosphotransferase activity in vitro. Similar results were reported by Morrison et al. (22) using purified preparations of p34cdc2 kinase and of the 44-kDa meiosis-activated myelin basic protein kinase from sea star oocytes. Additional evidence linking LC20 phosphorylation to the MAP kinase pathway was obtained when transient transfection of a mutationally activated MEK1 elicited increases in both MLCK and LC20 phosphorylation in COS-7 cells; increases in both phosphorylation events were reversed by pretreatment with PD-098059 (19). That ERK does not directly phosphorylate LC20 but requires a functional MLCK is suggested by the finding that co-transfection of a mutationally activated MEK1 with a mutationally inactivated MLCK abolished LC20, but not ERK phosphorylation (19). Alternatively, another MAP kinase may participate in the phosphorylation of LC20 via a pathway distinct from MLCK. One of the physiological targets identified for members of the MAP kinase superfamily of proteins is the MAP kinase-activated protein (MAPKAP) kinase-2. Using purified chicken gizzard MLCK and myosin, Komatsu and Hosoya (20) showed MAPKAP kinase-2 phosphorylates LC20 in vitro at the site identical to that catalyzed by MLCK, i.e., Ser19. The data presented herein do not distinguish between these mechanisms for MAP kinase-induced LC20 phosphorylation; nevertheless, it is clear that the predominant mechanism for contractile inhibition by PD-098059 is by reducing the level of LC20 phosphorylation.

In summary, ET-1-stimulated ERK1/2 activity subsequent to the initiation of force, and produced a time-dependent increase in h-CaD phosphorylation. Elevations in h-CaD phosphorylation were observed after a detectable increase in ERK1/2 activity, suggestive of a unique nonlinear temporal relationship between ERK1/2 activation and h-CaD phosphorylation. The increase in ERK-dependent CaD phosphorylation was highly variable, suggesting that it does not play a significant role during force maintenance. Prolonged incubation with PD-098059 had no effect on ET-1-mediated force in Ca2+-free PSS, in which force was independent of LC20 phosphorylation. Conversely, MEK inhibition significantly reduced the rate of ET-1-stimulated force development in Ca2+-replete PSS; this effect can be largely explained by a decrease in LC20 phosphorylation. Together, these results suggest the primary involvement of ERK1/2 activation in VSM function may be in the regulation of MLCK activity, and consequently, of LC20 phosphorylation. Thus the phosphorylation of CaD by ERK1/2 may only be temporally associated with, but not required for ET-1-stimulated force maintenance by VSM.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-09457 (to G. D'Angelo) and HL-56035 (to L. P. Adam).


    FOOTNOTES

Address for reprint requests and other correspondence: G. D'Angelo, Bristol-Myers Squibb Pharmaceutical Research Institute, Neuroscience Drug Discovery, PO Box 5100, Dept. 405, 5 Research Pkwy., Wallingford, CT 06492-7660 (E-mail: gerard.dangelo{at}bms.com).

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.

10.1152/ajpheart.00221.2001

Received 21 March 2001; accepted in final form 16 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 282(2):H602-H610
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society


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