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(Circulation Research. 1995;76:183-190.)
© 1995 American Heart Association, Inc.

Articles

Activation of Mitogen-Activated Protein Kinase in Porcine Carotid Arteries

Leonard P. Adam, Michael T. Franklin, Gregory J. Raff, David R. Hathaway

From the Departments of Medicine, Physiology and Biophysics, and the Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis.

Correspondence to Dr Leonard P. Adam, Krannert Institute of Cardiology, 1111 W 10th St, Indianapolis, IN 46202-4800.

	Abstract

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Abstract The thin-filament protein h-caldesmon (the high molecular weight isoform of caldesmon) is phosphorylated in resting and contracted porcine carotid arteries. Phosphorylation of h-caldesmon in intact tissue occurs at sites that are covalently modified by mitogen-activated protein kinase (MAPK) in vitro. In this study, we have evaluated MAPK activation in arteries in response to mechanical load and pharmacological stimulation. MAPK was extracted from resting and stimulated porcine carotid arteries and then partially purified by anion-exchange fast-performance liquid chromatography. MAPK activity was separated into two peaks corresponding to the tyrosine-phosphorylated 42- and 44-kD isoforms of MAPK (p42^MAPK and p44^MAPK, respectively). Of the total MAPK activity, 42% was associated with p42^MAPK, and 58% was associated with p44^MAPK; this percentage was not altered by stimulation of the muscles with either KCl (110 mmol/L) or phorbol 12,13-dibutyrate (PDBu, 1 µmol/L). Both p42^MAPK and p44^MAPK, purified from porcine carotid arteries, phosphorylated h-caldesmon at the same sites and to levels approaching or >1 mol phosphate per mole protein. In unloaded muscle strips, MAPK activity was 39 pmol · min^-1 · mg protein^-1 when assayed with the peptide substrate APRTPGGRR. MAPK activity increased in response to incremental mechanical loading to a maximum of 99 pmol · min^-1 · mg protein^-1 at 16x10³ N/m². MAPK activity could be further increased in loaded muscles by pharmacological stimulation. With KCl stimulation, MAPK activities rose to a peak of 205 pmol · min^-1 · mg protein^-1 at 10 minutes and then declined to basal values at 30 and 60 minutes. Stimulation with PDBu induced a gradual increase in MAPK activity that reached a value of 201 pmol · min^-1 · mg protein^-1 at 60 minutes. These results demonstrate that the level of MAPK activity in vascular smooth muscle is regulated in response to both mechanical load and pharmacological stimulation. Activation of MAPK and the subsequent phosphorylation of h-caldesmon may be important processes that modulate vascular smooth muscle contractility.

Key Words: smooth muscle • mitogen-activated protein kinase • contraction • signal transduction • caldesmon

	Introduction

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The high molecular weight isoform of caldesmon (h-caldesmon; molecular weight, 93 000)¹ is an abundant, smooth muscle–specific, actin-binding protein.^{2 3 4} h-Caldesmon is also a potent inhibitor of actomyosin ATPase activity in vitro^{5 6 7 8} and can tether myosin molecules to actin filaments in various model systems.^{9 10 11} h-Caldesmon is phosphorylated in resting arterial smooth muscle, and the level of phosphorylation increases with pharmacological stimulation.^{12 13} Sequencing of the phosphorylation sites in h-caldesmon purified from aortas identified two major sites of modification: VTS*PTKV and S*PAPK.¹⁴ These sites are consensus phosphorylation sequences for the class of kinases termed proline-directed protein kinases (PDPKs). There are two major types of PDPKs, p34^cdc2 and mitogen-activated protein kinase (MAPK). Both kinase types are present in proliferating smooth muscle cells; however, only MAPK is present in the contractile phenotype of vascular smooth muscle.¹⁵ Importantly, MAPK phosphorylates mammalian h-caldesmon on the same two sites in vitro that are phosphorylated in the intact tissue.¹⁵

The mechanisms for the activation of MAPK and its role in cellular proliferation have been described in various cultured-cell models.^{16 17 18 19} MAPK is a serine/threonine protein kinase that is activated by phosphorylation on both threonine and tyrosine residues. The kinase responsible for this activation is termed MEK, for MAPK and ERK kinase. MEK is also activated by phosphorylation, in this case by the protein kinase raf.²⁰ In proliferating cells, MAPK activity is required for passage through certain checkpoints in the cell cycle (eg, G1/S and G2/M)^{21 22 23} and acts, in part, by phosphorylating specific cytoplasmic and nuclear proteins, including S6 kinase, cPLA₂, and c-fos.^{24 25 26} In highly differentiated and nonproliferating tissues, such as muscle, the function of MAPK is poorly understood. MAPK activity has been detected in smooth and skeletal muscle.^{15 27} However, in neither of these muscle types have quantitative assessments of activity been made. In skeletal muscle, the role of MAPK is unknown but is likely to be involved in signal transduction via the insulin receptor. In smooth muscle, the role of MAPK may be to phosphorylate h-caldesmon, thereby altering a mechanical property of the muscle.

MAPK and other members of the MAPK signaling pathway have been identified only recently in contractile smooth muscle. A series of investigations by Childs and colleagues^{28 29} has confirmed the presence of active MAPK in chicken gizzard and rat aortic smooth muscle. Several sites in gizzard h-caldesmon were phosphorylated by MAPK in vitro; however, only minimal effects of phosphorylation on the activity of h-caldesmon were observed. In particular, caldesmon phosphorylation by MAPK only slightly inhibited its binding to actin.²⁸ Studies by Khalil and Morgan³⁰ in single ferret aortic smooth muscle cells have shown that MAPK is translocated from the cytosol to the membrane on pharmacological stimulation. Thus, there is cytochemical evidence for MAPK activation in fully differentiated smooth muscle. However, there has been no report of direct quantitative measurements of MAPK activity in the contractile phenotype of vascular smooth muscle.

In the present study, we have partially purified MAPK from porcine carotid arteries and quantified MAPK-specific activities after stretch and agonist stimulation. These data not only demonstrate that MAPK is activated by contractile agonists but also provide supporting evidence that MAPK most likely phosphorylates h-caldesmon in vascular smooth muscle.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Most chemicals and reagents were purchased from Sigma Chemical Co. [³²P]Orthophosphate, [³²P]ATP, and [¹²⁵I]protein A were from Dupont–New England Nuclear. Nitrocellulose sheets were from either Hoefer or Schleicher & Schuell. Antibodies specific for MAPK and phosphotyrosine (PY20) were purchased from Biodesign and ICN, respectively. Biotinylated goat anti-mouse antibody and streptavidin–horseradish peroxidase were from Amersham.

Methods
Physiological Preparation
Porcine carotid arteries were transported from the slaughterhouse in an ice-cold physiological saline solution (PSS) that consisted of (mmol/L) NaCl 140, KCl 4.7, Na₂HPO₄ 1.2, MgSO₄ 1.2, CaCl₂ 1.6, EDTA 0.02, glucose 5.6, and MOPS 2, pH 7.4. Arteries were dissected free of fat and connective tissue and either used immediately or stored overnight at 4°C in fresh PSS before use. Strips of artery 5 mm in width were dissected and attached to a force transducer (Grass Instrument Co) for the measurement of tension at 37°C in PSS. Muscles were allowed to relax in this solution for at least 60 minutes before the addition of agonists. Experiments were timed so that all muscles were freeze-clamped at precisely 2 hours after attachment to the transducers. Experimental solutions consisted of either (1) PSS with the addition of pharmacological agents or (2) PSS with the replacement of KCl for NaCl to give a final KCl concentration of 110 mmol/L (KPSS). Loads initially applied to muscles were calculated by dividing the amount of force applied to the muscle by the cross-sectional area of the muscle. Cross-sectional areas were determined by dividing the weight of the muscle by the length of the muscle; a tissue density value of 1.05x10³ kg/m³ was used.

In some experiments, the measurement of MAPK activity was made in porcine carotid arteries that were frozen immediately after dissection from the animals. In these experiments, arteries were dissected, rinsed briefly with PSS, and frozen within 1 minute. Extreme care was used to be certain that the arteries were not stretched on dissection. In one set of experiments, carotid arteries were clamped with hemostats in an anesthesized pig, and segments of the arteries distal to the clamp were rapidly frozen without dissection. The results from experiments in which the arteries were briefly rinsed with PSS and experiments in which arteries were frozen without dissection were not significantly different from one another and were, therefore, combined.

Measurement of MAPK Activity
Frozen muscle strips were ground to a fine powder under liquid N₂, and MAPK was extracted into 500 µL of extraction buffer containing (mmol/L) Tris 20, pH 7.5, EGTA 5, Na₃VO₄ 1, ß-glycerophosphate 20, NaF 10, and dithiothreitol (DTT) 1, along with 1 µg/mL aprotinin and 0.1 mmol/L each of phenylmethylsulfonyl fluoride (PMSF), N-tosyl-L-phenylalanine chloromethyl ketone, and N-p-tosyl-L-lysine chloromethyl ketone. After extraction for 30 minutes at 4°C, the samples were clarified by centrifugation at 100 000g for 10 minutes. MAPK activity was assayed in the supernatant fraction immediately after extraction and clarification. In some circumstances, extracts were separated by chromatography on a 1-mL Mono-Q fast-performance liquid chromatography (FPLC) column. For these studies, extracts from three muscles treated in the same manner were combined before chromatography. The column was equilibrated in a buffer containing (mmol/L) Tris 20, pH 7.5, EGTA 2, Na₃VO₄ 1, ß-glycerophosphate 10, DTT 1, and PMSF 0.1, along with 1 µg/mL aprotinin. After sample loading, the column was washed with 10 vol equilibration buffer, and bound proteins were eluted with a 60-mL gradient of 0 to 400 mmol/L NaCl in the same buffer. Fractions of 625 µL were collected in tubes containing 625 µg bovine serum albumin. In these experiments, separations were performed immediately after extraction and clarification, and MAPK activities were measured immediately after chromatography. Although not shown, we found that kinase activities in extracts stored at 4°C were stable for several days.

MAPK-specific activity was measured by assaying for phosphotransferase activity by use of the peptide substrate APRTPGGRR.³¹ Briefly, 10 µL of sample (tissue extract or column fraction) was incubated with peptide (500 µmol/L) for 30 minutes at room temperature in 50 µL of a buffer that consisted of (mmol/L) MOPS 12.5, pH 7.2, ß-glycerophosphate 12.5, MgCl₂ 7.5, EGTA 0.5, NaF 0.05, Na₃VO₄ 0.5, DTT 2, and [³²P]ATP 0.25. Reactions were terminated by the addition of trichloroacetic acid (final concentration, 10% [wt/vol]) and centrifuged for 5 minutes at 14 000g. The supernatant was spotted onto phosphocellulose paper (P81, Whatman) and washed four times in 500 mL of 50 mmol/L H₃PO₄ at 4°C. Filters were washed briefly with 100 mL of 95% ethanol, and the amount of radioactivity was determined by liquid scintillation counting. Protein was quantified by the method of Lowry et al.³²

Phosphorylation of Caldesmon In Vitro and Phosphopeptide Mapping
Porcine stomach h-caldesmon (0.33 µmol/L), purified according to Bretscher,³³ with certain modifications,⁶ was phosphorylated at room temperature in 150 µL of a buffer containing 7.5 mmol/L MgCl₂, 0.25 mmol/L [³²P]ATP, and 75 µL of FPLC column fraction. h-Caldesmon was separated from assay reactants by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), and the amount of radioactivity in the protein was determined by liquid scintillation counting. Phosphopeptide maps of h-caldesmon were generated after transfer of the phosphorylated protein to nitrocellulose and digestion with Staphylococcus aureus protease. In these studies, phosphopeptides were separated by thin layer electrophoresis in a buffer of acetic acid:formic acid:water (15:5:80), according to a method we have described previously.¹²

Gel Electrophoresis and Immunoassays
SDS-PAGE was performed by using the buffer system of Porzio and Pearson.³⁴ Immunostaining of proteins transferred to nitrocellulose was performed by using MAPK or phosphotyrosine antibody at dilutions of 1:5000 and 1:1000, respectively. Antibody binding was detected either by using a secondary biotinylated antibody (anti-mouse) and streptavidin–horseradish peroxidase or by using a secondary antibody (anti-mouse) developed in rabbits and [¹²⁵I]protein A. Color detection was obtained by using hydrogen peroxide and 2,2'-diaminobenzidine.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Because of the difficulty usually encountered in extracting proteins from arterial smooth muscle, we performed two types of experiments to ensure that all MAPK was recovered from the tissue. In the first set of experiments, we assayed MAPK by immunoblot in the supernatant and pellet fractions after extraction. Under conditions described in "Materials and Methods," essentially all kinase was extracted into the supernatant fraction (Fig 1). Because small amounts of MAPK in the pellet, not detectable by immunoblot, could be responsible for significant amounts of MAPK activity, thereby affecting our quantification, we performed a second set of experiments. In these studies, kinase activities were measured under conditions described in "Materials and Methods" or with the following modifications: (1) reduction of the extraction buffer to 300 µL, (2) addition of 0.5% Triton X-100, (3) addition of 400 mmol/L NaCl, or (4) addition of both Triton X-100 and NaCl. As shown in Fig 2, the addition of detergent and/or high salt increased the total amount of protein extracted, thereby decreasing the specific activity of MAPK (expressed as picomoles per minute per milligram). However, these modifications had no effect on the total units of MAPK activity extracted (Fig 2C).

View larger version (32K): [in this window] [in a new window]   Figure 1. Identification of mitogen-activated protein kinase (MAPK) in extracts of porcine carotid arteries. Frozen ground arterial tissue (50 mg) was incubated with 500 µL of extraction buffer as described in "Materials and Methods." After centrifugation, proteins in the supernatant (S) or remaining in the pellet (P) (and subsequently solubilized by 3% sodium dodecyl sulfate [SDS]) were separated by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose. Equal fractions of the supernatant and pellet were loaded on the gel and subsequently probed with MAPK-specific antibodies.

View larger version (25K): [in this window] [in a new window]   Figure 2. Bar graphs showing effects of different buffers on the extraction of mitogen-activated protein kinase (MAPK). MAPK was extracted from identical amounts of frozen ground muscle in the following buffers: (1) 500 µL of extraction buffer exactly as described in "Materials and Methods," (2) 300 µL of the same extraction buffer, (3) 500 µL of extraction buffer with 0.5% Triton X-100, (4) 500 µL of extraction buffer with 400 mmol/L NaCl, and (5) 500 µL of extraction buffer with a combination of 0.5% Triton X-100 and 400 mmol/L NaCl. Extracts were assayed for MAPK activity (A) and protein content (B). Total MAPK activity extracted from the muscles (C) was calculated from the data in panels A and B (1 unit=1 pmol phosphate transferred to the peptide substrate per minute).

When extracts from porcine carotid arteries were fractionated by Mono-Q FPLC, two peaks of kinase activity were detected (Fig 3A). All phosphotransferase activity eluted from the column was contained in these two peaks; no activity was observed in either the flow-through or column wash. To prove that the two peaks corresponded specifically to MAPK, Western blots were generated from the appropriate fractions and probed with MAPK-specific antibodies (Fig 3B). Immunoblots with MAPK antibody detected bands corresponding to the 42- and 44-kD isoforms of MAPK (p42^MAPK and p44^MAPK, respectively). p44^MAPK was separated into two peaks: one peak contained peptide phosphotransferase activity (fraction 54); the other did not (fraction 48). p42^MAPK was not separated into two discrete peaks; however, several fractions containing this isoform did not exhibit kinase activity. For example, fraction 38 contained p42^MAPK that had no phosphotransferase activity, whereas fraction 42 contained active p42^MAPK. Because tyrosine phosphorylation of MAPK is required for its activation, we tested for this modification by using an anti-phosphotyrosine antibody (Fig 3C). Fractions containing phosphotransferase activity also were found to contain MAPK that reacted with the phosphotyrosine antibody. No significant amount of phosphotyrosine antibody labeling was detected in MAPK fractions devoid of phosphotransferase activity. As an additional control, no amount of phosphotyrosine antibody labeling was detected in immunoblots where free phosphotyrosine (5 mmol/L) was added during incubation, thus confirming that the active MAPK was truely phosphorylated on tyrosine residues (data not shown).

View larger version (22K): [in this window] [in a new window]   Figure 3. Line graph (A) and immunoblots (B and C) showing separation of mitogen-activated protein kinase (MAPK) isoforms by Mono-Q fast-performance liquid chromatography. Extracts from three muscles were combined and separated on a 1-mL Mono-Q column as described in "Materials and Methods." Aliquots from each fraction were assayed for MAPK activity (A). Only fractions containing activity above background are presented for clarity. Proteins, from specified fractions, were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and assayed by immunoblot for the presence of either MAPK (B) or phosphotyrosine (PTyr) (C).

Studies with partially purified MAPK showed that both arterial isoforms could phosphorylate h-caldesmon (Fig 4). The slow nature of the phosphorylation time course was due to the low levels of kinase present in the FPLC fractions. Phosphorylation of h-caldesmon by either of the two isoforms was to levels approaching or >1 mol phosphate per mole protein, and the relative activities of p42^MAPK:p44^MAPK for h-caldesmon were similar to the relative activities for the peptide. These stoichiometries for phosphorylation are in good agreement with sequence data showing that purified recombinant p44^MAPK phosphorylates h-caldesmon at two sites. Importantly, phosphopeptide maps generated from h-caldesmon, phosphorylated by either p42^MAPK or p44^MAPK, were indistinguishable (Fig 5), suggesting that the same sites were covalently modified by the two different isoforms.

View larger version (19K): [in this window] [in a new window]   Figure 4. Line graphs showing phosphorylation of the high molecular weight isoform of caldesmon (h-caldesmon) by the 42- and 44-kD isoforms of mitogen-activated protein kinase (MAPK). Proteins were extracted from porcine carotid arteries and separated by Mono-Q fast-performance liquid chromatography, and column fractions were assayed for MAPK activity as described in "Materials and Methods" (A). Fractions enriched in the two MAPK isoforms, along with a background, were used to phosphorylate purified h-caldesmon in vitro (B).

View larger version (44K): [in this window] [in a new window]   Figure 5. Phosphopeptide maps of the high molecular weight isoform of caldesmon (h-caldesmon) phosphorylated by the 42- and 44-kD isoforms of mitogen-activated protein kinase (p42 and p44, respectively). Purified h-caldesmon was phosphorylated in vitro with either porcine carotid p42 or p44 and then digested with Staphylococcus aureus protease. Phosphopeptides were separated by thin-layer electrophoresis and detected by autoradiography as described in "Materials and Methods." Two major spots (labeled 1 and 2) were identified.

To investigate alterations of MAPK activity after muscle stimulation, the measurement of phosphotransferase activity in unfractionated extracts was desirable. However, to make these measurements two potential problems needed to be addressed. First, because stimulation of the muscle might have specifically altered the expression of one isoform activity over the other, measurements were made of the ratio of p42^MAPK activity to total MAPK activity on agonist stimulation. The fraction of total MAPK activity associated with the 42-kD isoform was not altered by stimulation of the muscles with either KCl or phorbol 12,13-dibutyrate (PDBu, 1 µmol/L) (Fig 6). The second potential problem in measuring kinase activities in unfractionated tissue extracts was the specificity of the phosphotransferase assay. To address this potential problem, we measured kinase activities in representative muscle extracts before and after fractionation on a Mono-Q column. Recoveries significantly different from 100% could confound interpretations of results derived when measuring MAPK-specific activity in unfractionated extracts. Under all conditions tested, the amount of kinase activity recovered from the column was similar to the amount of kinase activity loaded on the column (Fig 7). There were no differences in the recovery of kinase activity among all conditions tested (ANOVA, P>.25), and only the control point was significantly elevated above 100% (t test, P<.05) with a mean recovery of 131±12%. Collectively, these data suggest that (1) the peptide can be used in carotid artery extracts to specifically and quantitatively measure MAPK activity, (2) all kinase activity is recovered from the column under our running conditions, and (3) kinase activity measured in extracts is representative of total MAPK activity in the muscle.

View larger version (37K): [in this window] [in a new window]   Figure 6. Bar graph showing effect of muscle stimulation on the proportion of mitogen-activated protein kinase (MAPK) activity associated with the 42-kD isoform (p42). Unstimulated (control) and stimulated muscles were freeze-clamped and ground to a fine powder as described in "Materials and Methods." Proteins were extracted from the tissue and separated by Mono-Q fast-performance liquid chromatography, and column fractions were assayed for MAPK activity. The fraction of total MAPK activity associated with p42 was calculated for each experimental intervention. PDBu indicates phorbol 12,13-dibutyrate. Data are presented as the mean±SEM for each muscle set (n=3).

View larger version (60K): [in this window] [in a new window]   Figure 7. Bar graph showing recovery of mitogen-activated protein kinase (MAPK) in tissue extracts after Mono-Q chromatography. MAPK activity was measured in tissue extracts before and after fractionation by Mono-Q fast-performance liquid chromatography. The recovery of MAPK activity was calculated as the amount of peptide-specific phosphotransferase activity eluted from the column divided by the amount of activity loaded on the column. PDBu indicates phorbol 12,13-dibutyrate. Data are presented as mean±SEM (n=3).

In preliminary experiments, MAPK activity in unstimulated muscles stored overnight at 4°C and then dissected, stretched to optimal length (ie, loaded), and warmed to 37°C was found to be 99 pmol · min^-1 · mg protein^-1. This high level of activity could have resulted from several factors, including overnight storage, dissection, or stretch due to mechanical loading. Overnight storage per se had no effect on basal MAPK activity (data not shown). However, stretch and possibly removal of arteries from the animals did modulate MAPK activity (Fig 8). Arteries freeze-clamped in situ, maintained at 4°C (ie, either freshly isolated or stored overnight), or mechanically loaded at 4°C had virtually identical MAPK activities (Fig 8A). Warming muscles to 37°C doubled basal MAPK activity (from 17 to 43 pmol · min^-1 · mg protein^-1), suggesting that the process of removal and dissection does lead to MAPK activation, possibly as a result of injury and local release of neurotransmitters and/or growth factors. More prominent, however, was the effect of load (Fig 8B). As shown in Fig 8B (inset), application of a load at 37°C resulted in rapid activation of MAPK activity (ie, <1 minute). The level of MAPK activity was dependent on the load applied up to a maximum of 99 pmol · min^-1 · mg protein^-1 at 16x10³ N/m².

View larger version (36K): [in this window] [in a new window]   Figure 8. Effects of load on mitogen-activated protein kinase (MAPK) activity in unstimulated porcine carotid arteries. A, Bar graph showing MAPK activities measured in arteries frozen immediately on dissection from animals (in situ) or after storage and incubation in physiological saline solution (PSS) at the indicated temperature. Muscles were either unloaded (no) or had 12.5 g of tension applied (yes). B, Line graph showing MAPK activities measured in muscles to which various initial loads were applied at 37°C. In the inset to panel B, kinase activities were measured under maximal load after various times of incubation in PSS at 37°C. Each time point represents the results of from three to six experiments.

The activity of MAPK could be further increased by pharmacological stimulation. For example, with KCl stimulation, kinase activity rose to approximately twice background at 10 minutes but was not different from resting levels at 30 and 60 minutes (Fig 9). In addition, PDBu (1 µmol/L) stimulation led to a more slowly developing, yet sustained, increase in kinase activity (Fig 10).

View larger version (12K): [in this window] [in a new window]   Figure 9. Line graph showing effect of KCl stimulation on mitogen-activated protein kinase (MAPK) activity. Porcine carotid arteries were stimulated for the times indicated with KCl and then freeze-clamped as described in "Materials and Methods." MAPK activity was determined in tissue extracts, and data are presented as mean±SEM. Values in parentheses indicate the number of experiments. *P<.005 vs control.

View larger version (11K): [in this window] [in a new window]   Figure 10. Line graph showing effects of phorbol 12,13-dibutyrate (PDBu) stimulation on mitogen-activated protein kinase (MAPK) activity. Arteries were stimulated with PDBu (1 µmol/L) for the times indicated and assayed for MAPK activity as described in "Materials and Methods." Data are presented as mean±SEM for each time point. Values in parentheses indicate the number of experiments. *P<.005 vs control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we have established conditions for measuring MAPK activity in vascular smooth muscle and have used these procedures to determine how MAPK activities are altered during contraction. We show, by immunoblot analysis, that only the tyrosine-phosphorylated form of the kinase is active in contracted smooth muscle. After partial purification by Mono-Q FPLC, both MAPK isoforms are separated into non–tyrosine-phosphorylated and tyrosine-phosphorylated fractions. Consistent with reports in the literature,^{35 36 37 38} phosphotransferase activity was only associated with the tyrosine-phosphorylated isoforms of MAPK.

There are two isoforms of MAPK in smooth muscle: p42^MAPK and p44^MAPK. Although we did observe a significant increase in MAPK activity in response to pharmacological stimulation, the relative amounts of activity associated with each isoform were not altered by either KCl or PDBu treatment. On the other hand, it is possible that other pharmacological agents could preferentially activate one or the other isoform. Interestingly, the levels of MAPK activity in mechanically loaded unstimulated muscles were found to be quite high (100 pmol · min^-1 · mg protein^-1). This finding is consistent with observations we have made previously. For example, h-caldesmon, which is postulated to be a substrate for the kinase in carotid arteries, is phosphorylated to significant levels in unstimulated muscles treated methodologically similar.^{12 13} h-Caldesmon is phosphorylated to a level of 0.4 mol phosphate per mole protein in unstimulated carotid arteries, and after stimulation with various agonists, levels increase by only a factor of 2 or 3. If h-caldesmon is phosphorylated by MAPK, then it would be expected that resting kinase levels would be high. In addition, our studies provide at least a partial explanation for high resting levels of MAPK activity. MAPK is activated by applying a load to the muscle. This finding is in agreement with the results of others,³⁹ who have shown that MAPK can be activated by stretch in cardiac cells, and suggests that MAPK activation in arterial smooth muscle is subject to regulation by a combination of both mechanical load and pharmacological manipulation. Since the MAPK pathway may modulate growth processes, including protein synthesis, the sensitivity of MAPK activity to load raises a potentially important link to pathophysiological processes such as hypertension and vascular hypertrophy. One particularly interesting finding is that MAPK levels in arteries quick-frozen in situ are low, whereas the activities in muscles attached to force transducers and stretched under "physiological" loads are high. It is possible that removal of arteries from the animal and subsequent manipulations inactivate a suppressor of MAPK activity. It is noteworthy in this regard that we did not take special precautions to prevent endothelial denudation.

The time courses for activation of MAPK with PDBu and KCl are similar to the time courses observed for the activation of T cells by phorbol 12-myristate 13-acetate⁴⁰ and HSWP cells by ionomycin,⁴¹ respectively. Thus, the results we observe are consistent with reports in the literature showing both agonist and calcium-dependent activation of the MAPK cascade. The time course for activation of MAPK with PDBu stimulation correlates well with PDBu-dependent phosphorylation of h-caldesmon and contraction.¹² However, it is somewhat surprising that MAPK activation is transient with KCl stimulation, whereas h-caldesmon phosphorylation and force are sustained. There are several possible explanations for this apparent discrepancy. First, h-caldesmon phosphorylation levels may be regulated by phosphatase activity. The inhibition of a phosphatase during KCl stimulation would have the effect of increasing h-caldesmon phosphorylation in the background of maintained kinase activity. A similar dual-control mechanism has been hypothesized to regulate the levels of myosin light chain phosphorylation.^{42 43 44} Second, a small pool of MAPK may be responsible for phosphorylating h-caldesmon. Total cellular MAPK activity measurements may not accurately reflect the activity of this pool of kinase. Third, MAPK may not be h-caldesmon kinase. A possibility to be considered is that another proline-directed protein kinase is responsible for phosphorylating h-caldesmon in arterial smooth muscle. Whereas we suppose that h-caldesmon is the physiological substrate for MAPK, it is likely that other kinases will phosphorylate h-caldesmon and that other proteins are substrates of MAPK.

In cultured cells, the low molecular weight isoform of caldesmon (l-caldesmon) is thought to be phosphorylated by the proline-directed protein kinase, p34^cdc2.^{45 46 47} This kinase is active during mitosis, and phosphorylation of l-caldesmon may be required for the cytoskeletal rearrangements that occur during cell cycle progression. In fully differentiated vascular smooth muscle, there are no data showing that p34^cdc2 is expressed¹⁵ and that the predominant form of caldesmon is the high molecular weight isoform (h-caldesmon).⁴⁸ Although p34^cdc2 does phosphorylate h-caldesmon in vitro, the stoichiometry (>4 mol phosphate per mole protein) is greater than that observed for MAPK, reflecting the larger number of potential proline-directed protein kinase phosphorylation sites in h-caldesmon.^{15 49}

Although MAPK is activated during contraction of fully differentiated vascular smooth muscle, several analogies can be drawn to the activation of MAPK in proliferating cells. For example, in cultured cells the kinase is activated by phorbol esters, and cAMP can inhibit this activation.^{50 51 52} Similar signal transduction systems seem to operate in fully differentiated smooth muscle, except that the end product of MAPK activation may be an alteration of contractility. A general observation can be made that contractile agents for fully differentiated smooth muscle are often stimulatory for cell proliferation and increase MAPK activity, whereas smooth muscle relaxants inhibit proliferation and MAPK activity. The activation of MAPK as well as other proline-directed protein kinases and the subsequent phosphorylation of caldesmon appear to be important regulatory factors modulating growth and contractility of vascular smooth muscle.

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-06308, the American Heart Association, Indiana Affiliate, Inc, and the Herman C. Krannert Fund.

Received July 7, 1994; accepted October 11, 1994.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
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