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Am J Physiol Heart Circ Physiol 275: H131-H138, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 1, H131-H138, July 1998

Inhibition of p42 and p44 MAP kinase does not alter smooth muscle contraction in swine carotid artery

Isabelle Gorenne, Xiaoling Su, and Robert S. Moreland

Department of Physiology, MCP/Hahnemann School of Medicine, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19146

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon reverses the inhibition. The caldesmon kinase is believed to be mitogen-activated protein (MAP) kinase. MAP kinases are activated during vascular stimulation, but a cause-and-effect relationship between kinase activity and contraction has not been established. We examined the role of MAP kinase in contraction using PD-098059, an inhibitor of MAP kinase kinase (MEK). MAP kinase activity was assessed using an anti-active MAP kinase antibody and direct measurement of MAP kinase catalyzed phosphorylation of myelin basic protein, MBP-(95---98). MAP kinase phosphorylation, stimulated by histamine (50 µM) or phorbol 12,13-dibutyrate (PDBu, 0.1 µM), was inhibited by PD-098059 (100 µM). PD-098059 did not alter the sensitivity or the maximal level of force in smooth muscle stimulated by histamine or PDBu, nor did PD-098059 affect contraction of beta -escin-permeabilized tissue. Our data suggest that p44 and p42 MAP kinases are not involved in regulation of vascular smooth muscle contraction. These results do not, however, preclude a role for other isoforms of the MAP kinase family.

mitogen-activated protein kinase kinase; PD-098059; vascular smooth muscle; phorbol ester; histamine; phosphorylation; myosin light chain

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CALCIUM- AND CALMODULIN-dependent phosphorylation of the 20-kDa myosin light chain (MLC) has clearly been shown to be a major regulatory step in the activation of smooth muscle contraction (for review, see Ref. 31). However, because stimulation-induced MLC phosphorylation levels are only transiently increased while force is maintained, additional regulatory pathways have been proposed to account for maintained force (for review, see Refs. 18, 33, 36). Most hypotheses that propose an alternate regulatory system to explain the maintenance of force during states of disproportionately low levels of MLC phosphorylation involve the thin filament and target h-caldesmon as the primary regulatory protein (for review, see Refs. 7, 30, 33).

h-Caldesmon is a smooth muscle-specific protein that inhibits actin-activated myosin ATPase activity in its unphosphorylated state; phosphorylation relieves the inhibition (10, 25). h-Caldesmon, in vitro, can be phosphorylated by many kinases including protein kinase C (PKC) (19, 32), calcium/calmodulin-dependent protein kinase II (CaM kinase II) (29), casein kinase (35), cdc2 kinase (22), and mitogen-activated protein kinase (MAP kinase) (2, 4, 9). Phosphorylation of h-caldesmon catalyzed by any of these kinases reverses its inhibition of myosin ATPase activity. Stimulation-induced increases in caldesmon phosphorylation in intact smooth muscle have also been clearly demonstrated (2-4, 16, 26). Significant is the fact that caldesmon phosphorylation has been demonstrated in response to receptor and membrane depolarization-induced contractions (1, 20). However, until recently, suggestions for the identity of the endogenous caldesmon kinase have not been proposed.

Adam et al. (2, 4) proposed MAP kinase as the endogenous caldesmon kinase. This was based on the finding that phosphopeptide maps of endogenous caldesmon phosphorylated in response to cellular stimulation were similar to those obtained from purified caldesmon phosphorylated by MAP kinase. Moreover, microsequencing of the caldesmon phosphopeptides demonstrated that the phosphorylated serines were proline directed, indicative of a MAP kinase. Several laboratories have now shown that all agents tested which produce contraction of smooth muscle also activate MAP kinase (1, 9, 12, 15, 16, 20, 34).

The demonstration that cellular stimulation increases both force and MAP kinase activity is, however, only a correlation and does not strictly reflect cause and effect. The question of a direct role for MAP kinase in contractile regulation remains controversial. Gerthoffer et al. (15) have shown that the addition of activated p44 MAP kinase to a permeabilized airway smooth muscle strip results in a contraction, whereas Nixon et al. (26) performed a similar experiment using permeabilized vascular smooth muscle and obtained negative results. Whether this difference is due to source of tissue or species is not known.

Therefore the goal of this study was to determine whether alterations in p44 MAP kinase activity directly altered contractile activity. If either p42 or p44 MAP kinases are important in smooth muscle contraction, then inhibition of its activity should depress either the sensitivity or the magnitude of a contraction. Activation of MAP kinases is dependent on the activity of a dual-specificity serine/threonine and tyrosine kinase called mitogen-activated/extracellular-regulated protein kinase kinase or MEK (8, 28). MEK phosphorylates MAP kinase on Tyr185 and Thr187, resulting in activation of the enzyme (5, 8, 27). Recently, an inhibitor of MEK, PD-098059, has been described (6, 13). We used this compound to inhibit MEK activity in intact and permeabilized strips of the swine carotid media. Direct measurements of MAP kinase activity as well as quantitation of the magnitude and sensitivity of arterial contraction were performed during inhibition of MEK. Our results are not consistent with a role for p42 and/or p44 MAP kinase in the regulation of contraction and therefore suggest that although stimuli that induce contraction may also activate p42 and p44 MAP kinase, these two end points are not related.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Contraction studies. Swine carotid arteries were obtained from a local slaughterhouse and transported to the laboratory in an ice-cold physiological salt solution (PSS) of the following composition (in mM): 140 NaCl, 4.7 KCl, 1.2 MgSO4, 1.6 CaCl2, 1.2 Na2HPO4, 2 MOPS (pH 7.4), 5 D-glucose, and 0.02 Na2EDTA.

For measurement of isometric force, arteries were cleaned of excess connective tissue, and the endothelium was removed by gently scraping the intima with a cotton swab. Medial strips of swine carotid artery (0.5 × 7 mm) were mounted on a Güth Muscle Research Station (Heidelberg, Germany) at room temperature and allowed to equilibrate in PSS for 90 min. A passive force of 100 mg was applied to all tissues. After equilibration, tissues were maximally contracted with histamine (50 µM) and then washed in PSS until basal force was recovered. The tissues were then incubated for 2 h in either PSS or PSS containing 100 µM PD-098059. After this incubation period, cumulative concentration-response curves to histamine or single stimulations with phorbol 12,13-dibutyrate (PDBu, 0.1 µM) were performed.

In another series of experiments, tissues were permeabilized with beta -escin and then mounted for isometric force recording. The permeabilization was performed in a solution containing 100 µM beta -escin, 2 mM EGTA, 20 mM imidazole (pH 6.8), 1 mM Mg2+, 1 mM dithiothreitol (DTT), 4 mM ATP, and sufficient potassium acetate to maintain ionic strength constant at 120 mM. The tissues were exposed to this solution for 60 min at 4°C and then in fresh solution for 30 min at 30°C. After permeabilization, the tissues were incubated 30 min in a similar relaxing solution without added beta -escin and then contracted with 1 µM Ca2+. The tissues were relaxed in a relaxing solution containing 10 mM EGTA and 10 mM K2HPO4 to increase the rate and magnitude of relaxation. The tissues were then exposed to vehicle or 100 µM PD-098059 for 1 h and contracted a second time with 1 µM Ca2+. To determine the effect of PD-098059 on G protein-dependent pathways in the permeabilized tissues, they were incubated with PD-098059 or vehicle and exposed consecutively to 0.3 µM Ca2+ and 0.3 µM Ca2+ plus 50 µM guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S). After steady-state force was attained, the permeabilized tissues were maximally contracted with 10 µM Ca2+. All studies were performed in the presence of 1 µM ionomycin. The compositions of the contracting solutions were calculated by a computer program that solves the appropriate multiequilibrium association equations and has previously been described in detail (23).

Biochemical studies. Arteries cleaned of excess connective tissue and endothelium were cut into thin strips ~0.5 mm in width and placed in room-temperature PSS. The tissues were incubated for 2 h in the presence of PD-098059 or vehicle alone. After incubation, they were stimulated with either histamine or PDBu. For tissues in which MAP kinase content or phosphotyrosine levels were to be assessed, the reaction was stopped by freezing the tissues in liquid nitrogen followed by homogenization in a solution containing 1% SDS, 10% glycerol, and 40 mM DTT. For tissues in which MAP kinase activity was to be determined, the frozen tissues were homogenized at 4°C in a solution containing 20 mM HEPES (pH 7), 5 mM EGTA, 80 mM beta -glycerophosphate, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM DTT, 10 µM okadaic acid, 0.01% trypsin inhibitor, 0.01% aprotinin, and 0.01% leupeptin. The homogenates from either procedure were clarified by centrifugation at 4°C for 5 min at 14,000 g. Samples for measurement of MAP kinase activity were assayed immediately. Protein concentrations in the samples were determined by the method of Lowry et al. (21).

Phosphorylation of MAP kinase was evaluated using Western blot and immunodetection as previously described (20). The samples were subjected to a 10% SDS-PAGE and transferred to nitrocellulose membranes. Blots were saturated with 3% milk proteins in Tris-buffered saline solution (TBS) and incubated overnight in TBS-1% milk containing a polyclonal antibody directed against the dual threonine/tyrosine phosphorylated form of p42 and p44 MAP kinases (anti-active MAP kinase, Promega; dilution 1:5,000). The membranes were washed three times in TBS-0.05% Tween 20 and incubated with a secondary antibody, anti-IgG conjugated to horseradish peroxidase. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL). Quantitation of the bands on the X-ray film was performed using a Molecular Dynamics personal laser densitometer. The linearity of the ECL signal with respect to MAP kinase concentration was verified using the anti-dual phosphorylated MAP kinase antibody against standard concentrations of a p42 recombinant activated MAP kinase (Upstate Biotechnology) and serial dilutions of arterial tissue homogenates. The membranes were then stripped of all antibodies by incubation in a solution containing 2% SDS, 100 mM beta -mercaptoethanol, and 62.5 mM Tris · HCl (pH 6.8) for 30 min at 50°C. The blots were reprobed with a polyclonal antibody directed against either p44 or p42 MAP kinase (anti-ERK-1 or anti ERK-2, Santa Cruz Biotechnology; dilution 1:4,000) as primary antibody, then processed as described above. Data were quantified by measuring the spot intensity obtained with the anti-dual phosphorylated MAP kinase and normalized with the spot intensity obtained with the anti-p44 MAP kinase antibody for the same spot to provide an index of activation. Additional experiments were performed verifying that linear, quantitative binding to p42 MAP kinase could be measured using the anti-p44 MAP kinase antibody. In all studies presented, total MAP kinase content was quantified using the anti-p44 MAP kinase antibody and measuring densitometrically both p42 and p44 MAP kinases.

MAP kinase phosphotransferase activity was determined in the tissue homogenates by measuring the incorporation of radioactive phosphate into the synthetic peptide derived from myelin basic protein, MBP-(95---98), as previously described (20). The assays were performed at room temperature and contained 0.5 mM MBP-(95---98), 20 mM HEPES (pH 7.2), 20 mM MgCl2, 5 mM EGTA, 1 mM DTT, 100 µM ATP, 10 µM okadaic acid, and 2.5 µCi [gamma -32P]ATP. The reaction was terminated by the addition of TCA (10% final dilution). The solution was allowed to sit for 30 min and centrifuged at 14,000 g for 5 min. The supernatants containing the substrate MBP-(95---98) were spotted onto P-81 Whatman phosphocellulose papers. The papers were washed three times for 20 min each in 75 mM H3PO4 and then rinsed in 95% ethanol. Radioactivity of the filters was counted by liquid scintillation spectrophotometry. MAP kinase activity was expressed as nanomoles of Pi incorporated into MBP-(95---98) per milligram of total cellular protein.

MLC phosphorylation levels were measured in tissues stimulated with 50 µM histamine or 0.1 µM PDBu in the presence and absence of PD-098059. Tissues were frozen by immersion in a dry ice-acetone slurry containing 6% TCA. MLC phosphorylation levels were assayed by two-dimensional gel electrophoresis as previously described (24).

Drugs and statistics. PD-098059 was obtained from New England Biolabs (Beverly, MA); histamine, PDBu, HEPES, EGTA, beta -glycerophosphate, trypsin inhibitor, aprotinin, and leupeptin were obtained from Sigma Chemical (St. Louis, MO); MBP-(95---98) was obtained from GIBCO BRL Life Sciences (Gaithersburg, MD); okadaic acid was obtained from LC Laboratories (Woburn, MA); ECL reagents were obtained from Pierce (Rockford, IL); all electrophoretic and immunoblot reagents were obtained from Bio-Rad Laboratories (Richmond, CA); and all other chemicals were analytical grade or better and obtained from Thomas Scientific (Swedesboro, NJ).

Results are expressed as means ± SE obtained from the number (n) of experiments given. Differences between means were evaluated by use of the Student's t-test for paired or unpaired variables as appropriate. A value of P < 0.05 was taken as significant.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously demonstrated that stimulation of swine carotid medial strips with either histamine or KCl increases MAP kinase activity concomitant with force (20). The following data are the results of experiments designed to determine whether a cause-and-effect relationship or simply a correlation exists between MAP kinase activity and force. One of the primary indexes of MAP kinase activity that we used in these experiments was quantitative analysis of antibody binding to Western blots of homogenates of unstimulated and stimulated intact tissue. The commercial antibodies used in this study included (as listed by provider) anti-phosphotyrosine, anti-p42 MAP kinase, anti-p44 MAP kinase, and anti-active (dual phosphorylated) p44 MAP kinase. To perform quantitative analysis of antibody binding, it must be first verified that we were working in the linear range of detection. This is especially important, since we were using autoradiographs exposed to ECL to visualize and quantify the antibodies, a technique with high potential for saturation of response. Therefore all results used in this study were verified to be in the linear range of antibody binding with respect to protein concentration.

MAP kinase is activated by the dual serine/threonine and tyrosine kinase, MEK. To the best of our knowledge, no specific inhibitors of MAP kinase are available. Recently, however, an inhibitor of MEK, PD-098059, has been developed and made commercially available (6, 13). We used PD-098059 to inhibit MEK, and therefore MAP kinase activity, in the intact swine carotid artery. Intact arterial strips were stimulated with either 50 µM histamine or 0.1 µM PDBu in the presence or absence of PD-098059. The stimulated strips were then homogenized, and immunoblots were developed against total MAP kinase and against the active form of MAP kinase. Figure 1 shows representative ECL-exposed autoradiograms from these experiments. Both histamine and PDBu stimulation produced a significant increase in the active MAP kinase immunoblot signal compared with unstimulated basal strips. PD-098059 (100 µM) significantly depressed the stimulation-induced increase in anti-active MAP kinase signal. Total MAP kinase content was similar in all experiments as shown by the total MAP kinase immunoblot in Fig. 1A.


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  		Fig. 1.   A: immunoblot of swine carotid arterial homogenate for detection of active p44 and p42 mitogen-activated protein kinase (MAPK). Intact strips of swine carotid artery were stimulated by exposure to 50 µM histamine or 0.1 µM phorbol 12,13-dibutyrate (PDBu) in absence and presence of 100 µM PD-098059. Homogenates of stimulated strips were subjected to immunoblotting procedures. A, top: immunoblot is representative of that obtained with carotid strips using a polyclonal anti-p44 MAPK antibody. This antibody also quantitatively binds to p42 MAPK. A, bottom: immunoblot is a representative example showing that anti-active MAPK antibody binding was low in unstimulated tissues (basal) and increased with either histamine or PDBu stimulation. Addition of PD-098059 reduced levels of anti-active MAPK antibody binding below those in basal condition. B: several experiments of type shown in A were performed, and results were combined for analysis. PDBu (20 and 60 min of stimulation) and histamine (10 min of stimulation) significantly increased MAPK phosphorylation levels, indicative of an increase in activity (filled bars). Addition of 100 µM PD-098059 (open bars) significantly decreased MAPK phosphorylation levels during both basal (control) and stimulated conditions. Results shown are means ± SE for 5-8 determinations.

Several experiments of the type shown in Fig. 1A were performed, and the combined results are presented in Fig. 1B. Consistent with previous studies (1, 20), histamine (50 µM) and PDBu (0.1 µM) increased MAP kinase phosphorylation (indicative of MAP kinase activity) in the intact swine carotid arterial strip. The addition of PD-098059 (100 µM) significantly inhibited stimulation-induced increases in MAP kinase phosphorylation. Interestingly, PD-098059 also significantly depressed basal levels of MAP kinase phosphorylation by threefold.

The effect of several concentrations of PD-098059 on histamine-induced MAP kinase phosphorylation was also determined. Figure 2A (top and bottom) shows a representative immunoblot of total MAP kinase and active MAP kinase from swine carotid arterial strips stimulated with 50 µM histamine in the presence of 0-100 µM PD-098059. No change was observed in total MAP kinase, whereas active MAP kinase levels decreased with increasing concentrations of PD-098059. Data from several immunoblots were combined, and the results are shown in Fig. 2B. The percent inhibition of doubly phosphorylated MAP kinase determined from immunoblots of active-MAP kinase is presented as a function of [PD-098059]. PD-098059 produces a concentration-dependent inhibition of MAP kinase phosphorylation in histamine-stimulated swine carotid arteries.


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  		Fig. 2.   A: concentration dependence of PD-098059-induced inhibition of MAPK phosphorylation. Strips of swine carotid artery were incubated in several concentrations of PD-098059, stimulated with 50 µM histamine for 10 min, and then analyzed for total MAPK and active MAPK content. A, top: immunoblot is representative of response of carotid strips to anti-p44 MAPK antibody. A, bottom: immunoblot shows that increasing concentration of PD-098059 from 0-100 µM resulted in decreased binding of anti-active MAPK antibody. B: several experiments of the type shown in A were performed, and results were combined. Percent decrease in anti-active MAPK antibody binding is plotted as a function of PD-098059 concentration. Increasing PD-098059 concentration produces a concentration-dependent inhibition of MAPK phosphorylation levels as measured by anti-active MAPK antibody. Values shown are means ± SE for 6 determinations.

Figure 3 shows the results of experiments to verify that the magnitude of doubly phosphorylated MAP kinase determined using the anti-active MAP kinase antibody directly correlated to the level of MAP kinase phosphotransferase activity. Strips of swine carotid artery were stimulated with histamine in the presence of various concentrations of PD-098059. The strips were frozen in liquid nitrogen and assayed for both doubly phosphorylated MAP kinase levels and phosphotransferase activity. Phosphotransferase activity was measured by the incorporation of 32P from [gamma -32P]ATP into a MAP kinase-specific peptide substrate, MBP-(95---98) (1, 14, 20). Basal MAP kinase phosphotransferase activity was 12 ± 6 pmol · mg-1 · min-1 and increased to 31 ± 8 pmol · mg-1 · min-1 after stimulation by histamine (P < 0.05). The activity measured in tissues treated with a maximal [PD-098059] (100 µM) was 7 ± 1 pmol · mg-1 · min-1, a level significantly different from that measured from stimulated tissues (P < 0.001). Direct addition of 100 µM PD-098059 to the phosphotransferase activity assay in samples from untreated tissues did not alter MAP kinase activity levels. This confirms that the reaction mixture does not contain active MEK and that any activation or inhibition of MAP kinase activity by MEK or PD-098059, respectively, occurred in the intact vascular strip (not shown). The plot of active MAP kinase content as a function of phosphotransferase activity produced a linear relationship (r = 0.97, P < 0.01), demonstrating the utility of using the active MAP kinase antibody as a quantitative measurement of activity.


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  		Fig. 3.   Percent MAPK phosphorylation levels as measured by anti-active MAPK antibody is plotted vs. percent MAPK phosphotransferase activity using myelin basic protein, MBP-(95---98), as substrate. Aliquots of tissue homogenate used for immunoblot analysis shown in Fig. 2 were also used to directly quantify MAPK phosphotransferase activity using MAPK-specific substrate MBP-(95---98). A linear relationship exists between magnitude of anti-active MAPK antibody binding and phosphotransferase activity (r = 0.97, P < 0.01, ANOVA). MAPK activity was calculated as pmol Pi · min-1 · mg protein-1. Values shown are means ± SE for 6 determinations.

To evaluate the effects of MAP kinase inhibition on smooth muscle contraction, cumulative concentration-response curves to histamine were performed in the presence and the absence of 100 µM PD-098059 (Fig. 4). PD-098059 had no effect on tissue basal tone. More importantly, the results shown in Fig. 4 clearly demonstrate that inhibition of MEK and MAP kinase activities did not affect the sensitivity of the intact vascular strip to histamine. Time controls were performed to verify that any difference or lack of difference in histamine concentration-response curves was not due to time-dependent changes in sensitivity. Multiple concentration-response curves to histamine in the absence of PD-098059 showed similar agonist sensitivities (data not shown). The maximal responses to histamine in the intact carotid strips were 109% (control) and 105% (+PD-098059) of an initial maximal contraction to histamine. The pD2 (-log EC50) values were calculated to be 6.64 ± 0.19 and 6.58 ± 0.08 in the absence or presence of PD-098059, respectively.


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  		Fig. 4.   Effects of inhibition of MAPK activity on contraction of swine carotid artery. A: concentration-response curves to histamine stimulation of intact swine carotid arteries were constructed in absence (bullet ) and then presence (black-triangle) of 100 µM PD-098059. B: time course of contraction of swine carotid artery in response to 0.1 µM PDBu in presence of PD-098059 (100 µM; black-triangle) or vehicle (bullet ). Values are means ± SE for 4 determinations.

MAP kinase is activated during PDBu stimulation of vascular smooth muscle. To determine whether PDBu-induced increases in MAP kinase activity may affect smooth muscle contraction, we determined the time course and maximal level of force attained in response to a submaximal [PDBu]. The intact swine carotid arteries were contracted in response to 0.1 µM PDBu, a concentration that produced 86% of the maximal histamine-induced force. Neither the rate of force development nor the magnitude of developed force was altered after inhibition of MAP kinase activity. Contraction in response to PDBu in the presence of PD-098059 was 83% of an initial histamine contraction (Fig. 4).

Compounds that are believed to be reasonably specific inhibitors of distinct kinases may also be weak inhibitors of other kinases or kinase-related reactions. Because MLC phosphorylation catalyzed by the MLC kinase is a critical step in the regulation of smooth muscle contraction, we tested whether PD-098059 may have nonspecific effects on MLC phosphorylation levels. It was possible that any inhibitory affect which PD-098059 may have on histamine or PDBu-induced contraction may be offset or reversed by affects on the reactions responsible for a net increase in MLC phosphorylation. MLC phosphorylation levels were measured at rest and during histamine or PDBu stimulation of intact swine carotid arterial strips in the presence and absence of 100 µM PD-098059. PD-098059 had no effect on MLC phosphorylation levels at rest (vehicle, 0.14 mol Pi/mol MLC; PD-098059, 0.22 mol Pi/MLC), during 0.1 µM PDBu stimulation (vehicle, 0.47 mol Pi/mol MLC; PD-098059, 0.43 mol Pi/MLC), or during 50 µM histamine stimulation (vehicle, 0.56 mol Pi/mol MLC; PD-098059, 0.58 mol Pi/MLC).

The last set of experiments performed examined the possible role of MAP kinase in either the direct Ca2+ activation of a smooth muscle contraction or the G protein-dependent enhancement of myofilament Ca2+ sensitivity. beta -Escin-permeabilized tissues were contracted with a half-maximal [Ca2+] of 1 µM, relaxed, and after a 1-h incubation in 100 µM PD-098059 were then contracted a second time by the addition of 1 µM Ca2+. The direct activation of the contractile apparatus by Ca2+ was unaffected by PD-098059 (Fig. 5, A and B). beta -Escin-permeabilized tissues were also subjected to a subthreshold [Ca2+], followed by the addition of 50 µM GTPgamma S, which augments the contractile response. Maximal force was determined by the final addition of 10 µM Ca2+. The protocol was repeated after a 1-h incubation with 100 µM PD-098059 (Fig. 5C). Inhibition of MAP kinase activity had no effect on either the magnitude of GTPgamma S-dependent enhancement of a contraction or maximal force development in the beta -escin-permeabilized arterial tissue.


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  		Fig. 5.   Effect of inhibition of MAPK activity on contraction of beta -escin-permeabilized carotid artery. Representative tracing of an experiment to determine effect of inhibition of MAPK activity on a contraction in response to Ca2+ alone (A and B) and on enhancement of a Ca2+-dependent contraction by guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S; C and D). Addition of 100 µM PD-098059 had no effect on rate or magnitude of a contraction in response to Ca2+ or on rate or magnitude of enhancement of a contraction by GTPgamma S. Tracings representative of 3 experiments.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, we have confirmed previous results from our laboratory and others (1, 20) that stimulation of swine carotid artery concomitantly increases p42 and p44 MAP kinase activity and force. We have now extended these findings to demonstrate that although p42 and p44 MAP kinase activity and force correlate temporally, there is no cause-and-effect relationship between activation of the kinase and initiation of a contraction. From the results presented, we could find no causal relationship between the activation of the p42 and p44 isoforms of MAP kinase and the sensitivity of intact vascular smooth muscle to an agonist, magnitude of a contraction in response to phorbol ester stimulation, Ca2+-dependent activation of a permeabilized vascular preparation, or enhancement of myofilament Ca2+ sensitivity by receptor and G protein-dependent stimulation.

In addition to the physiologically relevant information summarized above, we also presented new technical information. The commercial availability of an antibody against the active form of MAP kinase enhances the ability to study potential roles of MAP kinase in cellular regulation. Quantitative immunoblot analysis is significantly faster, simpler, and less expensive than the more standard technique of measuring phosphotransferase activity using the MAP kinase-specific substrate, MBP-(95---98) (14, 20). Moreover, the use of an antibody against the dual phosphorylated and therefore active form of the kinase rather than a general anti-phosphotyrosine antibody should provide better specificity. What has been missing, at least in the literature investigating a role for MAP kinase in muscle regulation, is the demonstration of a linear relationship between anti-active MAP kinase antibody binding and actual phosphotransferase activity. We now provide this information. Under the conditions used in this study, a linear relationship was demonstrated between anti-active MAP kinase antibody binding and MBP-(95---98) phosphorylation. We believe that, in addition to providing information about the phosphorylation state of MAP kinase, anti-active MAP kinase immunoblots represent a quantitative indicator of MAP kinase activity.

Adam et al. (1) presented evidence demonstrating that, in resting unstimulated vascular smooth muscle, MAP kinase is partially activated. Our results support this work, since the addition of PD-098059 to unstimulated strips of intact swine carotid artery produced a significant decrease in the level of activated MAP kinase. However, basal levels of stress did not decrease, consistent with our hypothesis that p42 and p44 isoforms of MAP kinase are not causally related to contraction. A more likely role for this enzyme is that a tonic level of MAP kinase activity is required by the smooth muscle cell to maintain the synthetic needs of the tissue or possibly other parameters of contractility such as the rate or magnitude of relaxation.

Previous studies addressing the question of whether MAP kinase is involved in smooth muscle contraction have provided disparate results. Using Triton X-100 detergent-skinned preparations of two different rabbit vascular smooth muscles, Nixon et al. (26) clearly demonstrated that the addition of activated p42 MAP kinase had no effect on resting levels of force. The addition of the active enzyme neither initiated a contraction nor produced a relaxation. Moreover, these investigators demonstrated that the addition of activated p42 MAP kinase had no effect on the Ca2+ sensitivity of contraction of the two permeabilized vascular tissues. Of particular importance was their evidence of activated MAP kinase entering the permeabilized cells and autoradiographic data suggesting that caldesmon was phosphorylated stoichiometrically (26). These results are consistent with our current study and together provide reasonably strong evidence to conclude that at least the p42 and p44 isoforms of MAP kinase are not important in the initiation or sensitivity of smooth muscle contraction.

In contrast to the studies discussed above, Gerthoffer et al. (15, 16) presented a completely different set of results. These investigators used Triton X-100 detergent-skinned preparations of canine tracheal smooth muscle (15) to show that the addition of activated p42 MAP kinase significantly potentiated a Ca2+-induced contraction. Whether this difference in results is due to the source of activated enzyme or the specific smooth muscle used is not known. Because smooth muscle is widely known to exhibit organ- and species-specific differences in regulation, either of these are potential answers. This is especially compelling given the fact that different smooth muscles have significantly different caldesmon contents (17), the putative substrate for active MAP kinase. In intact colonic smooth muscle, which has a high caldesmon content (17), contractile stimulation induces a significant increase in endogenous MAP kinase activity (16). However, MAP kinase activity was increased tonically, whereas the contractions were phasic. This information suggests that the level of MAP kinase activity does not correlate to the level of force and as such is consistent with our present study.

Studies using PD-098059 to inhibit MAP kinase activity in intact smooth muscle tissues have also provided mixed conclusions. Our results presented in this study are not consistent with a regulatory role for MAP kinase in contraction. In contrast, PD-098059 decreased both the sensitivity and magnitude of serotonin-induced contractions in several rat vascular tissues (34). Interestingly, PD-098059 did not affect either PDBu or KCl-induced contractions (34), although we (20, present study) and others (1, 2) have shown that both PDBu and KCl increase MAP kinase activity, at least in the swine carotid artery. PD-098059 has also been shown to inhibit ferret vascular smooth muscle. Dessy et al. (12) presented preliminary evidence demonstrating that the Ca2+-independent fraction of a phenylephrine-induced contraction (11) of ferret aorta was inhibited by PD-098059. The swine carotid artery does not contract in response to agonist activation in the absence of calcium. Therefore our studies utilizing the swine carotid cannot address the potential role for MAP kinase-dependent steps in an agonist-induced, Ca2+-independent contraction.

In summary, we have presented evidence demonstrating that the magnitude of p42 and p44 MAP kinase activity does not correlate to any index of contraction measured. In fact, almost complete abolition of p42 and p44 MAP kinase activity has no demonstrable effect on the sensitivity, rate, or magnitude of a contraction of the swine carotid artery. If caldesmon phosphorylation is to play a role in the contraction-relaxation cycle of vascular smooth muscle, it apparently does not involve either the p42 or p44 isoforms on MAP kinase. Our results do not, however, preclude a role for other members of the MAP kinase family that catalyze proline-directed serine phosphorylation or other classes of kinases.

    ACKNOWLEDGEMENTS

All swine carotid arteries used in this study were obtained from Hatfield Meat Packing Plant, Hatfield, PA. The authors thank William Jack for the reliable and professional delivery of the arteries.

    FOOTNOTES

This study was funded in part by National Heart, Lung, and Blood Institute Grants HL-37956 and HL-46704 (R. S. Moreland) and a Fellowship from the Southeastern Pennsylvania Affiliate of the American Heart Association (I. Gorenne).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: R. S. Moreland, Dept. of Physiology, Allegheny University of the Health Sciences, Graduate Hospital Research Bldg., 415 S. 19th St., Philadelphia, PA 19146.

Received 8 January 1998; accepted in final form 24 March 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Adam, L. P., M. T. Franklin, G. J. Raff, and D. R. Hathaway. Activation of mitogen-activated protein kinase in porcine carotid arteries. Circ. Res. 76: 183-190, 1995[Abstract/Free Full Text].

2.   Adam, L. P., C. J. Gapinski, and D. R. Hathaway. Phosphorylation sequences in h-caldesmon from phorbol ester-stimulated canine aortas. FEBS Lett. 302: 223-226, 1992[Medline].

3.   Adam, L. P., J. R. Haeberle, and D. R. Hathaway. Phosphorylation of caldesmon in arterial smooth muscle. J. Biol. Chem. 264: 7698-7703, 1989[Abstract/Free Full Text].

4.   Adam, L. P., and D. R. Hathaway. Identification of mitogen-activated protein kinase phosphorylation sequences in mammalian h-caldesmon. FEBS Lett. 322: 56-60, 1993[Medline].

5.   Alessandrini, A., C. M. Crews, and R. L. Erikson. Phorbol ester stimulates a protein-tyrosine/threonine kinase that phosphorylates and activates the ERK-1 gene product. Proc. Natl. Acad. Sci. USA 89: 8200-8204, 1992[Abstract/Free Full Text].

6.   Alessi, D. R., A. Cuenda, P. Cohen, D. T. Dudley, and A. R. Saltiel. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270: 27489-27494, 1995[Abstract/Free Full Text].

7.   Allen, B. G., and M. P. Walsh. The biochemical basis of the regulation of smooth-muscle contraction. Trends Biochem. Sci. 19: 362-368, 1994[Medline].

8.   Anderson, N. G., J. L. Maller, N. K. Tonks, and T. W. Sturgill. Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature 343: 651-653, 1990[Medline].

9.   Childs, T. J., M. H. Watson, J. S. Sanghera, D. L. Campbell, S. L. Pelech, and A. S. Mak. Phosphorylation of smooth muscle caldesmon by mitogen-activated protein (MAP) kinase and expression of MAP kinases in differentiated smooth muscle. J. Biol. Chem. 267: 22853-22859, 1992[Abstract/Free Full Text].

10.   Clark, T., P. K. Ngai, C. Sutherland, U. Grüschel-Stewart, and M. P. Walsh. Vascular smooth muscle caldesmon. J. Biol. Chem. 261: 8028-8035, 1986[Abstract/Free Full Text].

11.   Collins, E. M., M. P. Walsh, and K. G. Morgan. Contraction of single vascular smooth muscle cells by phenylephrine at constant [Ca2+]i. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H754-H762, 1992[Abstract/Free Full Text].

12.  Dessy, C., G. D'Angelo, R Laporte, L. P. Adam, and K. G. Morgan. A role for MAPK in contraction of differentiated smooth muscle evoked by alpha -adrenoceptor stimulation or phorbol esters (Abstract). Circulation 96, Suppl. I: I-248, 1997.

13.   Dudley, D. T., L. Pang, S. J. Decker, A. J. Bridges, and A. R. Saltiel. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA 92: 7686-7689, 1995[Abstract/Free Full Text].

14.   Erikson, A. K., D. M. Payne, P. A. Matino, A. J. Rossomando, J. Shabanowitz, M. J. Weber, D. F. Hunt, and T. W. Sturgill. Identification by mass spectrometry of threonine-97 in bovine myelin basic protein as a specific phosphorylation site for mitogen-activated protein kinase. J. Biol. Chem. 265: 19728-19835, 1990[Abstract/Free Full Text].

15.   Gerthoffer, W. T., I. A. Yamboliev, J. Pohl, R. Haynes, S. Dang, and J. McHugh. Activation of MAP kinase in airway smooth muscle. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L244-L252, 1997[Abstract/Free Full Text].

16.   Gerthoffer, W. T., I. A. Yamboliev, M. Shearer, J. Pohl, R. Haynes, S. Dang, K. Sato, and J. R. Sellers. Activation of MAP kinases and phosphorylation of caldesmon in canine colonic smooth muscle. J. Physiol. (Lond.) 495: 597-609, 1996[Abstract/Free Full Text].

17.   Haeberle, J. R., D. R. Hathaway, and C. L. Smith. Caldesmon content of mammalian smooth muscle. J. Muscle Res. Cell Motil. 13: 81-89, 1992[Medline].

18.   Horowitz, A., C. B. Menice, R. Laporte, and K. G. Morgan. Mechanisms of smooth muscle contraction. Physiol. Rev. 76: 967-1003, 1996[Abstract/Free Full Text].

19.   Ikebe, M., and T. Hornick. Determination of the phosphorylation sites of smooth muscle caldesmon by protein kinase C. Arch. Biochem. Biophys. 288: 538-542, 1991[Medline].

20.   Katoch, S. S., and R. S. Moreland. Agonist and membrane depolarization induced activation of MAP kinase in the swine carotid artery. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H222-H229, 1995[Abstract/Free Full Text].

21.   Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. L. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951[Free Full Text].

22.   Mak, A. S., M. H. Watson, C. M. E. Litman, and J. H. Wang. Phosphorylation of caldesmon by cdc2 kinase. J. Biol. Chem. 266: 6678-6681, 1991[Abstract/Free Full Text].

23.   Moreland, R. S., and R. A. Murphy. Determinants of Ca2+-dependent stress maintenance in skinned swine carotid media. Am. J. Physiol. 251 (Cell Physiol. 20): C892-C903, 1986[Abstract/Free Full Text].

24.   Moreland, S., and R. S. Moreland. Effects of dihydropyridines on stress, myosin phosphorylation, and Vo in smooth muscle. Am. J. Physiol. 252 (Heart Circ. Physiol. 21): H1040-H1058, 1987.

25.   Ngai, P. K., and M. P. Walsh. Inhibition of smooth muscle actin-activated myosin Mg2+-ATPase activity by caldesmon. J. Biol. Chem. 259: 13656-13659, 1984[Abstract/Free Full Text].

26.   Nixon, G. F., K. Iizuka, C. M. M. Haystead, T. A. Haystead, A. P. Somlyo, and A. V. Somlyo. Phosphorylation of caldesmon by mitogen-activated protein kinase with no effect on Ca2+ sensitivity in rabbit smooth muscle. J. Physiol. (Lond.) 487: 283-289, 1995[Abstract/Free Full Text].

27.   Pelech, S. L., and J. S. Sanghera. Mitogen-activated protein kinases: versatile transducers for cell signalling. Trends Biochem. Sci. 17: 233-238, 1992[Medline].

28.   Ray, L. B., and T. W. Sturgill. Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo. Proc. Natl. Acad. Sci. USA 84: 1502-1506, 1987[Abstract/Free Full Text].

29.   Scott-Woo, G. C., C. Sutherland, and M. P. Walsh. Kinase activity associated with caldesmon is Ca2+/calmodulin-dependent kinase II. Biochem. J. 268: 367-370, 1990[Medline].

30.   Sobue, K., and J. R. Sellers. Caldesmon, a novel regulatory protein in smooth muscle and nonmuscle actomyosin systems. J. Biol. Chem. 266: 12115-12118, 1991[Free Full Text].

31.   Somlyo, A. P., and A. V. Somlyo. Signal transduction and regulation in smooth muscle. Nature 372: 231-236, 1994[Medline].

32.   Tanaka, T., H. Ohta, K. Kanda, T. Tanaka, H. Hidaka, and K. Sobue. Phosphorylation of high-Mr caldesmon by protein kinase C modulates the regulatory function of this protein on the interaction between actin and myosin. Eur. J. Biochem. 188: 495-500, 1990[Medline].

33.   Walsh, M. P. Smooth muscle caldesmon. Prog. Clin. Biol. Res. 327: 127-140, 1990[Medline].

34.   Watts, S. W. Serotonin activates the mitogen-activated protein kinase pathway in vascular smooth muscle: use of the mitogen-activated protein kinase kinase inhibitor PD-098059. J. Pharmacol. Exp. Ther. 279: 1541-1550, 1996[Abstract/Free Full Text].

35.   Wawrzynow, A., J. H. Collins, N. V. Bogatcheva, A. V. Vorotnikov, and N. B. Gusev. Identification of the site phosphorylated by casein kinase II in smooth muscle caldesmon. FEBS Lett. 289: 213-216, 1991[Medline].

36.   Zhang, Y., S. Moreland, and R. S. Moreland. Regulation of vascular smooth muscle contraction: myosin light chain phosphorylation dependent and independent pathways. Can. J. Physiol. Pharmacol. 72: 1386-1391, 1994[Medline].

Am J Physiol Heart Circ Physiol 275(1):H131-H138
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society


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