Stretch-dependent activation and desensitization of mitogen-activated protein kinase in carotid arteries

Michael T. Franklin1, C. L.-Albert Wang2,3, and Leonard P. Adam2,3

1 Krannert Institute of Cardiology, Indiana University, Indianapolis, Indiana 46202; 2 Harvard Medical School, Boston 02215; and 3 Boston Biomedical Research Institute, Boston, Massachusetts 02114

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Arterial smooth muscle stretch is an important physiological modulator of vascular function. To identify intracellular processes altered during muscle stretch, we found previously that extracellular signal-regulated kinase-mitogen-activated protein kinase (MAPK) activity increased in response to the application of mechanical loads. In the present study, stretch-dependent activation of MAPK in porcine carotid arteries was investigated as was the phosphorylation of the thin filament-binding protein caldesmon, which is known to be a substrate for the kinase in fully differentiated smooth muscle. MAPK activity was 67 pmol · min-1 · mg protein-1 in unloaded muscle strips immediately after attachment to force transducers and 139 pmol · min-1 · mg protein-1 within 30 s of muscle stretch. When muscle strips were continually stretched, MAPK activity remained elevated for ~2 h and then decreased over 16 h to 16 pmol · min-1 · mg protein-1. When muscle strips were stretched and then unloaded, MAPK activity decreased within 1 h to the level present in the muscle before the stretch. These effects of muscle stretch on MAPK activity were additive to the effects of KCl or phorbol ester stimulation and were partially inhibited by reducing extracellular Ca2+. Eliminating extracellular Ca2+ had no effect on phorbol 12,13-dibutyrate (PDBu)-dependent contractions or MAPK activity; however, KCl-dependent contractions and MAPK activity were completely abolished by this procedure. An antibody specific for detecting caldesmon phosphorylated by MAPK, vs. protein kinase C (PKC), was developed and used to assess relative caldesmon phosphorylation in unstimulated and PDBu-stimulated muscle strips. In all cases investigated, the level of MAPK activity correlated with phosphocaldesmon immunoreactivity. Because arterial MAPK activity is regulated by PKC- and stretch-dependent mechanisms, these data are consistent with a role for MAPK and the subsequent phosphorylation of caldesmon as mediators in the stretch activation of vascular smooth muscle.

smooth muscle; phorbol esters; caldesmon; phosphorylation; antibody

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

WHEN SMOOTH MUSCLE is stretched by the application of a mechanical load, a number of changes occur in the muscle cell. Stretch activates potassium channels in the gut (8, 27), opens stretch-activated cationic selective channels in the urinary bladder (35) and coronary vascular smooth muscle cells (18), increases the intracellular free Ca2+ concentration in a number of smooth muscle types (9, 15, 21), and closes certain stretch-inactivated channels in stomach muscle (13). The functions altered in response to stretch include myosin phosphorylation (presumably due to alterations in intracellular free Ca2+ concentration) and contractility (6). In addition, stretch is also thought to be an important activator of cardiac muscle growth or hypertrophy (19, 29-31, 36). Perhaps the best known response of smooth muscle to mechanical stretch is the generation of myogenic tone (7, 14, 24), i.e., the intrinsic increase in muscle force that results from stretch of the muscle or increased intraluminal pressure in arteries.

Although an increase in sarcolemmal Ca2+ currents occurs in many types of smooth muscle in response to mechanical perturbations, not all changes due to stretch are accounted for by an increase in intracellular Ca2+. Intracellular mediators other than Ca2+ that are activated upon muscle stretch, and the pathways controlled by these mediators, are incompletely known. However, much work has focused on the potential involvement of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in cellular stretch responses. In smooth muscle, PKC is implicated in the generation of myogenic tone (7, 12, 20, 24). In endothelial cells, shear stress activates PKC and subsequently MAPK (33). Finally, in cultured cardiac myocytes, cellular stretching activates PKC, MAPK, p90RSK, p21RAS, and phospholipase C (29). Although stretch is a potent modulator of MAPK in endothelial and cardiac cells, it is not understood how the kinase contributes to the ultimate functional effects that occur in response to mechanical manipulation. In smooth muscle, the only described functional result of MAPK activation is the phosphorylation of the thin filament-associated protein caldesmon (1, 2).

The early signaling events that result in the activation of smooth muscle extracellular signal-regulated kinase (ERK)-MAPK (herein referred to simply as MAPK) as well as the temporal relationships among stretch, MAPK activity, and caldesmon phosphorylation are unknown. We therefore investigated the time course and extracellular Ca2+ requirements for MAPK activation in porcine carotid arteries. MAPK is activated rapidly in response to stretch in vascular smooth muscle when compared with stretch activation of the kinase in cardiac myocytes; however, during continuous muscle stretching, MAPK activity desensitizes along a very much longer time scale (hours vs. minutes). With the use of antibodies specific for MAPK-phosphorylated caldesmon, we find that caldesmon phosphorylation levels parallel this effect on MAPK activity. Finally, the activation of MAPK in response to muscle stretch is only partially dependent on extracellular Ca2+ but can be enhanced by increased sarcolemmal Ca2+ influx (i.e., KCl stimulation) or by phorbol ester stimulation and the activation of PKC.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials

Most chemicals and reagents were purchased from Sigma. [gamma -32P]ATP and 125I-labeled protein A were purchased from NEN. Phosphocellulose (P81) paper was from Whatman, and nitrocellulose was from Hoefer. Antibodies directed against MAPK were purchased from Oncogene Science (MAPK antibody 2), and antibodies specific for phosphorylated MAPK were from New England Biolabs.

Methods

Physiological preparation. Porcine carotid arteries were transported from the slaughterhouse in an ice-cold physiological saline solution (PSS) that consisted of (in mM) 140 NaCl, 4.7 KCl, 1.2 Na2HPO4, 1.2 MgSO4, 1.6 CaCl2, 0.02 EDTA, 5.6 glucose, and 2 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.4. Arteries were dissected free of fat and connective tissue and either were used immediately or stored overnight at 4°C in fresh PSS before use. Strips of artery 5 mm in width were dissected, the endothelium was gently rubbed off, and the muscle strip was attached to a force transducer (Grass Instrument) for the measurement of tension at 37°C in PSS. 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 mM. In experiments in which muscle strips were stretched, a mechanical load of 12.5 g was applied initially. The application of this load is required to place the muscles at their physiologically optimal length for maximal contraction by agonists. Although this is the load initially applied to the muscles, the final load on any given muscle strip varied but was typically <5 g due to the subsequent isometric relaxation of the muscle.

Measurement of MAPK peptide phosphotransferase activity. Freeze-clamped muscle strips were ground to a fine powder under liquid N2, and MAPK was extracted into 500 µl of extraction buffer containing 20 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.5, 5 mM ethylene glycol-bis (beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM Na3VO4, 20 mM beta -glycerophosphate, 10 mM NaF, 1 mM dithiothreitol (DTT), 1 mg/ml aprotinin, and 0.1 mM each of phenylmethylsulfonyl fluoride (PMSF), N-tosyl-L-phenylalanine chloromethyl ketone, and Nalpha -p-tosyl-L-lysine chloromethyl ketone. After extraction for 30 min at 4°C, the samples were clarified by centrifugation at 100,000 g for 10 min. MAPK activity was assayed in the supernatant fraction immediately after extraction and clarification.

MAPK-specific activity was measured by assaying for phosphotransferase activity using the peptide substrate APRTPGGRR. Briefly, 10 µl of tissue extract were incubated with peptide (500 µM) for 30 min at room temperature in 50 µl of a buffer that consisted of (in mM) 12.5 MOPS, pH 7.2, 12.5 beta -glycerophosphate, 7.5 MgCl2, 0.5 EGTA, 0.05 NaF, 0.5 Na3VO4, 2 DTT, and 0.25 [gamma -32P]ATP. Reactions were terminated by the addition of trichloroacetic acid (10% final concentration wt/vol) and were centrifuged for 5 min at 14,000 g. The supernatant was spotted onto phosphocellulose paper and was washed four times in 500 ml of 50 mM 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 quantitated by the method of Lowry et al. (22). This method has been shown to detect only MAPK activity because of the specificity of the peptide substrate that is used (and, specifically, ERK1 and ERK2 activities) in porcine carotid arteries and is quantitative when performed as described (1).

Quantitation of MAPK content. The amount of MAPK in tissue was quantitated using immunoblotting techniques. Proteins in tissue extracts (prepared as described in Measurement of MAPK peptide phosphotransferase activity) were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and were transferred to nitrocellulose. The nitrocellulose sheets were then incubated with a buffer containing 5% bovine serum albumin (BSA) followed by incubation with anti-MAPK antibodies and radiolabeled protein A. Autoradiography was performed to identify MAPK, the bands were cut out, and the amount of radioactivity was determined using a gamma counter. MAPK content was normalized to the amount of protein in the extract as determined by the method of Lowry et al. (22).

In-gel kinase assay. Proteins were extracted from frozen ground tissue into a buffer containing 3% SDS and were immediately separated on a polyacrylamide gel polymerized with 0.5 mg/ml myelin basic protein. The gel was washed two times in a buffer containing 20% 2-propanol and 50 mM Tris, pH 8.0, and then two times in a buffer containing 50 mM Tris, pH 8.0, and 5 mM beta -mercaptoethanol. Proteins in the gel were denatured in 50 mM Tris, pH 8.0, 5 mM beta -mercaptoethanol, and 6 M guanidine and subsequently were renatured overnight in 50 mM Tris, pH 8.0, 5 mM beta -mercaptoethanol, and 0.04% Tween 40. The gel was washed free of detergent with 40 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 8.0, 2 mM DTT, 0.1 mM EGTA, and 5 mM MgCl2 and then was incubated for 1 h in the same buffer containing 25 µM [gamma -32P]ATP (3-4 µCi/ml). The gel was washed free of residual [gamma -32P]ATP with 10 washes of 5% trichloroacetic acid and 1% sodium pyrophosphate, dried, and subjected to autoradiography.

To obtain roughly similar protein loadings on the gels, the extracts from equivalent amounts of tissue (determined by wet weight) were separated by SDS-PAGE. Intensities of the phosphomyelin basic protein bands were normalized to MAPK content as determined by quantitative immunoblot analysis. Normalization of the relative band intensity was performed in this manner vs. protein content because it was deemed that this methodology prevented any bias in the data due to inherent, individual, sample variabilities in MAPK content per amount of protein extracted and was more direct in its approach. This method of normalization was also performed with immunoblots using anti-phospho-ERK1 antibodies.

Antibody production and purification. Affinity purified polyclonal rabbit anti-phosphopeptide antibodies were generated as follows. The phosphopeptide PDGNKS(PO4)PAPKPGC was synthesized, coupled to keyhole limpet hemacyanin, injected into rabbits, and, at appropriate times, serum was collected. This sequence is analogous to one of the MAPK phosphorylation sites on caldesmon (serine-702 of chicken caldesmon or serine-759 of human caldesmon). Antibodies in the serum were purified from proteases and other protein components by chromatography using DEAE Affi-Gel Blue (Bio-Rad) according to the manufacturer's instructions. Affinity purified antibodies were prepared by passage over a column of the phosphopeptide covalently bound to SulfoLink Coupling Gel (Pierce) also according to the manufacturer's directions. Polyclonal antiserum against caldesmon was generated in rabbits using full-length caldesmon.

Immunoblotting techniques and caldesmon phosphorylation. Proteins were extracted from frozen, ground, tissue as described previously (2) and were heated to denature most proteins. Proteins in the boiled supernatant fraction, which included essentially all of the caldesmon (2), were separated by SDS-PAGE and were transferred to nitrocellulose. Extracts from 20 mg of tissue (wet weight) were loaded onto two lanes of the gel so that equal amounts of caldesmon from a given muscle strip were present in the duplicate lanes. The Western blots were blocked with a solution of 5% BSA and then incubated overnight in buffer containing BSA and an appropriate dilution of either affinity purified anti-phosphopeptide antibodies or polyclonal anti-caldesmon serum. The blots were washed with a solution containing 0.1% Nonidet P-40 and then were incubated for 2 h with 125I-protein A (NEN). After further washing with Nonidet P-40, the nitrocellulose strips were subjected to autoradiography, and the amount of radioactivity was determined by gamma counting.

Protein kinase purification and caldesmon phosphorylation. The 44-kDa MAPK, ERK1, was produced by recombinant means using the baculovirus expression system and was purified, briefly, as follows. Cells from 1 liter of culture were centrifuged and homogenized in buffer A, which contained (in mM) 50 beta -glycerophosphate, pH 7.4, 5 EGTA, 1 DTT, 0.5 Na3VO4, 0.25 PMSF, and 1 benzamidine. The homogenate was centrifuged for 45 min at 100,000 g, and the supernatant was passed over a 5-ml column of phenyl-Sepharose (Sigma) equilibrated in buffer A. The column was washed and proteins eluted with fractions of 10, 30, and 60% ethylene glycol in buffer A. Fractions enriched in ERK1 were passed over a mono-Q column equilibrated in buffer that contained 20 mM Tris, pH 7.5, 10 mM NaF, and 1 mM each of EGTA, EDTA, DTT, and benzamidine. Proteins were eluted with a gradient of 0-500 mM NaCl in the same buffer. Fractions enriched in ERK1 were combined and dialyzed against buffer C that contained 20 mM Tris, pH 6.5, and 1 mM each of EGTA, EDTA, DTT, and benzamidine. The dialysate was passed over a 5-ml column of SP-Sepharose Fast Flow (Pharmacia) equilibrated in buffer C, and proteins were eluted with buffer C containing 100, 200, 300, 400, and 500 mM NaCl. Purified ERK1 was concentrated using a Centricon-10 followed by a Microcon-10 and was stored in aliquots at -80°C. Purified ERK1 was activated by ERK kinase (MEK) that was partially purified in its active form from rabbit skeletal muscle. MEK was partially purified by a combination of chromatographic techniques using columns of DEAE-Sephacel, Q-Sepharose FF, mono-Q, SP-Sepharose, and ammonium sulfate precipitation.

Porcine stomach caldesmon (3.58 µM) was phosphorylated using ERK1 and MEK in a buffer described previously (1) and by PKC that was partially purified from guinea pig brains, as described in Adam et al. (3). Phosphorylation reactions were performed in duplicate under identical conditions except that, in one reaction, [32P]ATP (0.25 mM, 311 counts · min-1 · pmol-1) was used, whereas the other reaction contained nonradioactive ATP. Caldesmon that was phosphorylated using radiolabeled ATP was used to estimate the level of phosphorylation in the duplicate reaction containing nonradiolabeled ATP. Only nonradiolabeled caldesmon was used for antibody binding to prevent any interference in the immunoblot from 32P.

Data analysis. Statistical analysis for all of the data was performed using a Student's t-test. Data are expressed as means ± SE. An error bar not appearing in a graph results from the error being within the symbol.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

When porcine carotid arterial muscle strips are stretched by the application of mechanical loads, MAPK activity increases (Fig. 1). In agreement with previously published data, the activity in unstretched arteries is elevated above that in arteries stored at 4°C (1) or in arteries frozen in situ (data not shown). MAPK activity is maximal at 30 s of stretch, the first time for which measurements are made, and remains elevated during 30 min of stretching. If carotid arteries are continually stretched for a time span of several hours, this initial increase in MAPK activity accommodates (Fig. 2) and the levels decrease to values less than those of unloaded muscles immediately after attachment to the transducers (compare the activity at 16 or 24 h in Fig. 2 with time 0 for Fig. 1). This accommodation begins approximately 4 h after stretching and is complete within 16 h. The accommodation of MAPK activity during prolonged stretch is more properly termed desensitization since this activity does not increase when the muscles are stretched further. Although not shown, quantitative immunoblot analysis demonstrated that the amount of MAPK in carotid arteries, expressed per milligram of protein extracted from the tissue, is not different after 16 h of stretch from the amount in arteries stretched for only 1 h. Therefore, the decrease in MAPK activity that is measured after 16 h of muscle stretch is not due to a decrease in MAPK protein content. These data compare with the results of Fig. 3 that show a more rapid decrease in MAPK activity when the mechanical load is removed from the muscle by making the muscle slack. In this set of experiments, stretch-dependent increases in MAPK activity are observed when a mechanical load is applied. When the load is removed, MAPK activity reverts to the level before the application of the load within 1 h.


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Fig. 1.   Mitogen-activated protein kinase (MAPK) activation in response to porcine carotid arterial muscle stretch. Muscle strips were dissected in ice-cold buffer and were frozen immediately (4°C) or attached to force transducers at 37°C for a total time of 90 min. Three muscle strips were unloaded for the entire 90 min (0), whereas other muscle strips were stretched for either 30 s or 1, 2, 5, or 30 min before the end of the 90-min time course. An additional set of muscles was stretched for the entire 90 min. All time points are elevated above the time 0 point (P < 0.005, n = 3 experiments for each bar), and all data are presented as means + SE.


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Fig. 2.   Time course for desensitization of MAPK activity during continual stretch. Carotid arterial muscle strips were attached to force transducers at 37°C for the times indicated, and then MAPK activity was determined as described under EXPERIMENTAL PROCEDURES. Data are expressed as means ± SE; n is at least 3 for each data point.


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Fig. 3.   Time for reduction of MAPK activity after removal of mechanical load. All muscle strips were incubated at 37°C for 90 min. Muscles were unloaded for the entire time (0 Load), stretched for 90 min (Control), or stretched for 5 min to maximally activate MAPK and unloaded for 0, 30, or 60 min before the end of the experiment. The 30- and 60-min time points are significantly different from control and time 0 points (P < 0.005). All time points, except for 60 min, are elevated above 0 Load (P < 0.005). Data are expressed as means + SE; n = 9 for each data point.

Stimulation of carotid arteries with either KCl or phorbol 12,13-dibutyrate (PDBu) leads to an increase in force along the time course shown in Fig. 4A. Stimulation with these agents also results in an increase in MAPK activity. With KCl stimulation, MAPK activity rises rapidly (peaking before the peak of contraction) and then decreases over the 2-h time course for which measurements are made (Fig. 4B). PDBu stimulation results in a slowly developing rise in MAPK activity that persists in the continued presence of the contractile agonist and that occurs over a time course that parallels that of contraction. As shown in Fig. 5, the increase in activity due to KCl or PDBu stimulation is similar in muscles stretched for 2 h (A) and muscles stretched for 17 h (B). After 2 and 17 h of stretch, incubation with KCl results in an elevation of MAPK activity of 65 and 61 pmol · min-1 · mg protein-1, respectively; incubation with PDBu results in an elevation of 137 and 116 pmol · min-1 · mg protein-1, respectively. Thus the increase in MAPK activity due to KCl or PDBu stimulation is not affected by the factors that result in a decrease in activity during the accommodation of stretch (i.e., during stretch desensitization).


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Fig. 4.   Time course for activation of MAPK in response to KCl and phorbol 12,13-dibutyrate (PDBu). Muscle strips were stimulated with KCl, allowed to relax, and then stimulated with either KCl or PDBu (1 µM) for the times indicated. Tension was monitored at all times and is shown in A as a percentage of the force developed in the initial KCl contraction. In B, MAPK activity in the muscle strips is shown. Closed triangles depict the level of MAPK activity in a control set of muscles that were stimulated to contract with KCl and allowed to relax but that were not stimulated a second time with either KCl or PDBu. Data are expressed as means ± SE.


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Fig. 5.   Effects of KCl or PDBu on MAPK activity after 2 or 17 h of stretch. Muscles were stretched at 37°C for a total time of 2 (A) or 17 (B) h. Muscle strips were stimulated with KCl or PDBu (1 µM) for 10 or 60 min, respectively, before the end of the experiment. Data are expressed as means + SE (n = 3). MAPK activity in KCl- or PDBu-stimulated muscles was greater than the activity in unstimulated muscles (P < 0.005).

To investigate the proximate signals resulting in an increase in MAPK activity upon stimulation of carotid arteries, the effects of changing the extracellular Ca2+ concentration were investigated in a series of experiments. As shown in Fig. 6, the removal of extracellular Ca2+ has no apparent effect on resting MAPK activity in unstretched carotid arteries not stimulated to contract. The effects of stretch on MAPK activity that normally lead to an increase in this activity were only partially inhibited by removing extracellular Ca2+. Verification that extracellular Ca2+ is removed is shown by the lack of effect of KCl in increasing MAPK activity; although not shown, KCl-dependent contractions were completely inhibited under these conditions. These data suggest that the influx of Ca2+ accompanying KCl stimulation, and not depolarization, per se, is the signal resulting in MAPK activation in these arteries. To more clearly demonstrate the Ca2+ dependence of MAPK activation, muscles were stimulated, or not, after 16 h of stretch (Fig. 7). At this time, unstimulated arteries had very low levels of MAPK activity that were not altered by changing extracellular Ca2+ concentration. The increase in MAPK activity associated with KCl stimulation was completely inhibited by removing extracellular Ca2+ concentration; however, the increase in kinase activity due to phorbol ester stimulation was independent of extracellular Ca2+ concentration.


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Fig. 6.   Effects of extracellular Ca2+ on stretch-induced increases in MAPK activity in carotid arteries. Muscle strips were unstretched (unloaded or "-" load) or stretched in the presence or absence of extracellular Ca2+ for 5 min. In an additional set of experiments, stretched muscles were stimulated with KCl (110 mM) in the absence of extracellular Ca2+. Buffers without Ca2+ also contained 1 mM EGTA. Mechanically loaded muscles in the absence of extracellular Ca2+ had MAPK activities different from the activity in either unloaded muscles or muscles loaded in the presence of Ca2+ (P < 0.005, n = 6).


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Fig. 7.   Effects of removing extracellular Ca2+ on the activation of MAPK by KCl and PDBu after 17 h of stretch. Carotid arterial muscle strips were stretched for a total of 17 h at 37°C in normal physiological saline solution (PSS) or PSS containing 1 mM EGTA in place of Ca2+. Muscle strips were stimulated with KCl or PDBu (1 µM) for the last 10 or 60 min, respectively, before termination of the experiment. Data are expressed as means + SE, n = 3.

The effects of prolonged stretch and phorbol ester stimulation of the arteries on MAPK activity were documented further by the use of in-gel kinase assays and by monitoring phospho-MAPK levels (Fig. 8). As shown in Fig. 8A, phorbol ester stimulation at either 2 or 16 h of muscle stretch resulted in an increase in anti-phospho-MAPK levels using antibodies specific for the tyrosine-phosphorylated and active forms of ERK1 and ERK2. These antibodies are not known to cross-react with other members of the MAPK family of protein kinases. Similar results were observed using an "in-gel" kinase assay with increases in the intensities of two bands having mobilities corresponding to ERK1 and ERK2 (Fig. 8B). To confirm that the increase in phospho-MAPK reactivity or in-gel kinase activity was not accounted for by an alteration in the amount of MAPK in the samples, MAPK content was assessed using anti-ERK antibodies (Fig. 8C). When the data in Fig. 8, A and B, were normalized to the amount of MAPK in Fig. 8C, we calculated a reduction in control phospho-ERK immunoreactivity and in-gel kinase activity of 73 and 79%, respectively, at 16 vs. 2 h. These data compare to an 88% reduction in peptide phosphotransferase activity over the same time period (Fig. 2). The variability in immunoreactive MAPK observed in Fig. 8C was due to alterations in the total amount of protein (tissue extract) loaded per lane and not due to differences in the amount of kinase present in the tissue. Thus, although the amount of immunoreactive MAPK in the various samples was different, when we normalized the data as described in EXPERIMENTAL PROCEDURES, similar results were obtained using the in-gel kinase assay and by quantitating phospho-ERK immunoreactivity as were obtained with the peptide phosphotransferase assay.


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Fig. 8.   Phosphoextracellular signal-regulated kinase (ERK) and in-gel kinase assays of porcine carotid arteries. Carotid arterial muscle strips were stretched for a total of either 2 or 16 h and either stimulated or not (UnStim) with PDBu (1 µM) for 1 h before termination of the experiment. A is an immunoblot of tissue extracts probed with anti-phospho-ERK antibodies to assess phosphorylation and therefore activation level of the ERK-MAPK. B is an autoradiogram from an in-gel kinase assay performed using the same tissue extracts as in A. C is an immunoblot using nonselective ERK antibodies to normalize for MAPK content in the tissue extracts.

To compare the activity of MAPK with the only known functional effect of MAPK activation in contractile smooth muscle, caldesmon phosphorylation was assessed under several conditions using phosphopeptide antibodies. As shown in Fig. 9, affinity purified antibodies are specific for detecting ERK1-phosphorylated vs. PKC-phosphorylated caldesmon. Caldesmon was phosphorylated for 15, 30, or 60 min with either ERK1 (MAPK) or PKC, using either nonradiolabeled ATP for subsequent immunoblot analyses (Fig. 9A) or radiolabeled ATP for quantitation of the level of phosphate incorporated into caldesmon (Fig. 9B). A phosphorylation level of ~1 mol phosphate/mol caldesmon was achieved with either ERK1 or PKC. As depicted in Fig. 9A, immunoreactivity increased in concordance with the level of MAPK phosphorylation but not PKC phosphorylation even though the phosphorylation sites are near one another. Although not shown, these antibodies did not detect ERK1-phosphorylated myelin basic protein; therefore, they are also specific for the sequence in caldesmon, vs. myelin basic protein, phosphorylated by MAPK.


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Fig. 9.   Anti-phospho-caldesmon antibody binding to purified and phosphorylated porcine stomach caldesmon (CaD). Anti-phospho-caldesmon-specific antibodies were produced and affinity purified as described in EXPERIMENTAL PROCEDURES. These antibodies were used to bind to caldesmon that was phosphorylated for the indicated times with either ERK1 or protein kinase C (PKC), subjected to SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose (A). Antibody binding was detected by autoradiography after incubation with 125I-labeled protein A. In a parallel reaction, the level of phosphate incorporated into caldesmon was quantitated using [32P]ATP to verify that caldesmon was phosphorylated to significant levels by either ERK1 or PKC (B).

Anti-phospho-caldesmon antibodies were used in immunoblots to assess the increase in caldesmon phosphorylation under various conditions (Fig. 10). However, antibody reactivity to the amount of caldesmon that was able to be loaded on a single gel lane was very low. Also, bands other than, but near in mobility to, caldesmon were detected with greater intensity than was caldesmon in unpurified tissue Western blots with the use of these antibodies. For these two reasons, a partial purification of caldesmon was necessary as described in Methods. Significant anti-phosphopeptide binding to caldesmon was detected in unstimulated muscles attached to force transducers for 2 h; this binding was significantly attenuated in muscles allowed to remain attached overnight (16 h). Stimulation of muscles with phorbol esters (PDBu), but not restretching of muscles, resulted in a significant increase in anti-phosphopeptide antibody binding at either 2 or 16 h of stretch.


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Fig. 10.   Caldesmon phosphorylation in porcine carotid arteries. Porcine carotid arterial muscle strips were stretched in PSS at 37°C in all experiments for a total of either 2 or 16 h. As in Fig. 8, muscle strips were stimulated or not with 1 µM PDBu (PDBu) for 1 h before the end of the experiment. Proteins were extracted from the muscles and heated. Equal amounts of the heat-stable proteins were separated in two lanes of an SDS-polyacrylamide gel and transferred to nitrocellulose. Western blots were probed with polyclonal anti-phosphopeptide antibodies (A) or polyclonal anti-caldesmon antibodies (B) as described in EXPERIMENTAL PROCEDURES and visualized by autoradiography after incubation with 125I-protein A.

The absolute level of phosphate incorporated into caldesmon could not be determined using the phosphocaldesmon antibodies; however, phosphorylation levels under various experimental conditions could be determined relative to control values. In three experiments, the level of MAPK activity was compared with the level of caldesmon phosphorylation in the same muscle strips stretched overnight (as a control) or in strips that were stretched overnight and then restretched. There was no significant increase in either MAPK activity or caldesmon immunoreactivity under these circumstances. The MAPK activity in restretched muscle strips divided by the activity in control arteries was 1.1 ± 0.1, and the phosphocaldesmon immunoreactivity in re-stretched muscle strips divided by the immunoreactivity in control arteries was 1.2 ± 0.1. In six additional experiments, PDBu stimulation resulted in a 3.45 ± 0.30-fold increase in MAPK activity and a 4.43 ± 0.61-fold increase in anti-phosphopeptide antibody reactivity. Thus caldesmon phosphorylation on MAPK phosphorylation sites paralleled MAPK activity in carotid arterial smooth muscle.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Stretch of smooth muscle is well known to activate or inhibit a number of different types of surface membrane ion channels (8, 13, 18, 27, 35) and to alter muscle contractile function; in particular, stretch induces myogenic tone (7, 14, 24). Although many of the cellular responses to stretch are known, the understanding of the intracellular signaling processes altered by stretch is an area of active investigation. We describe here experiments investigating some of the events, including stretch, KCl, and phorbol ester stimulation, that lead to MAPK activation in porcine carotid arteries. Smooth muscle stretch activates MAPK rapidly compared with cardiac muscle or endothelial cells, and this activity desensitizes over a period of several hours. Although it is possible that stretch activation of the kinase is due to the release of some factor from the muscle cells or associated neurons, this seems unlikely, since the carotid artery is not known to be heavily innervated and any such factor should be washed away in the muscle superfusate. It is most likely, therefore, that the activation of the kinase is due directly to the mechanical stretch applied to the muscle strip. Furthermore, the prolonged inactivation of the kinase is not due to a decrease in MAPK content; rather, it is due to the inactivation of existing kinase resulting from a desensitization in the pathway connecting muscle stretch to kinase activation, since restretching of the muscle does not lead to a further increase in MAPK activity. Such a prolonged stimulation of kinase activity is unusual for MAPK. For example, during cell proliferation, MAPK activity peaks only during certain transition checkpoints of the cell cycle (32, 34); during cultured cardiac myocyte stretch, MAPK activity peaks at 10 min and then decreases to basal levels within 30 min (29, 36).

Although stretch-dependent desensitization of MAPK occurs over a several hour time period, not all mechanisms or pathways leading to MAPK activation are desensitized. KCl or PDBu stimulation of muscles immediately after attachment to the force transducers effects an increase in kinase activity comparable to that observed after stretch desensitization. Because the response to PDBu is not altered by continual stretch (Fig. 5), these data suggest that PKC is not specifically desensitized during stretch. Likewise, none of the elements in the pathway connecting PKC to MAPK activation are desensitized during prolonged muscle stretch. Furthermore, because the response to KCl is not altered, the pathway leading from an increase in Ca2+ to MAPK activation is also not affected by stretch desensitization.

Our finding that the increase in intracellular Ca2+ resulting from depolarization (KCl stimulation) is sufficient to effect an increase in MAPK activity is in agreement with previously published results in porcine carotid (1, 16) and rat vascular smooth muscle cells (23). However, the increase in MAPK activity due to muscle stretch most likely does not result primarily from an increase in trans-sarcolemmal Ca2+ influx, since this response is only slightly inhibited by removing extracellular Ca2+ (Fig. 6), a procedure known to inhibit stretch-dependent increases in intracellular free Ca2+ concentration (28). Alternatively, it is possible that intracellular free Ca2+ concentration may rise due to the release of Ca2+ from intracellular stores (9) in response to muscle stretch, and this may contribute to the activation of MAPK in the absence of extracellular Ca2+. The activation of MAPK by Ca2+ in smooth muscle is consistent with the observation that Ca2+/calmodulin-dependent kinase II activates MAPK in cultured cells (25), further suggesting the possibility that Ca2+/calmodulin-dependent kinase II may lead to the activation of MAPK in this contractile smooth muscle. Despite this possibility, however, the intracellular free Ca2+ concentration response to stretch is transient in the absence of extracellular Ca2+ (9); therefore, it is unlikely that an increase in Ca2+ alone is sufficient to support the prolonged activation of MAPK.

As depicted in Fig. 2, MAPK activity desensitizes over a time period of ~4-24 h. This raises the question if it is more appropriate to understanding the physiological role of MAPK in smooth muscle by characterizing the modulation of its activity in muscles stretched overnight or in muscles immediately after incubation at 37°C. In another study, we found that the low level of MAPK activity in muscles stretched overnight was similar to the level in unstimulated arteries (carotid and coronary) in anesthetized pigs in vivo. These data might suggest that an analysis of MAPK in muscles stretched overnight is more appropriate. However, a difference between these two arterial models (in vivo vs. stretched overnight, in vitro) can be found in that muscles stretched overnight have MAPK activities that are desensitized to further stretching, whereas MAPK activity increases dramatically in arteries stretched using a balloon angioplasty catheter in anesthetized pigs in vivo (data not shown). Thus the muscle stretched for prolonged periods of time may not be a good model for assessing how muscle length modulates basal kinase levels but may help to provide insight for what mechanisms are involved in the stretch-dependent activation of the kinase.

Given the multitude of proteins that are substrates for MAPK, in vitro and in vivo, a definitive function for the kinase in stretch activation can not be determined at present. However, in fully differentiated smooth muscle, an abundant substrate for MAPK is the thin filament protein caldesmon. To assess the functionality of the activated MAPK and to determine if its activation parallels caldesmon phosphorylation, we developed an antibody specific for caldesmon phosphorylated on one of the two MAPK-specific phosphorylation sites (serine-702 of gizzard caldesmon and serine-759 of human caldesmon). Using this antibody, the increase in MAPK activity resulting from agonist-dependent stimulation or stretch of the muscle paralleled that of phosphocaldesmon immunoreactivity. These data provide further support that caldesmon is an endogenous substrate for MAPK in intact porcine carotid arteries. The data also help to solidify the link between PKC and MAPK activation. Previous work showed that caldesmon was phosphorylated on similar sites in unstimulated or phorbol ester-stimulated carotid arteries and that phosphorylation was not on sites directly modified by PKC (2, 3). These earlier observations, and the work of Khalil and Morgan (17) who showed PKC-dependent translocation of MAPK to the surface membrane (subsequently followed by a further translocation to the cytoplasm) in smooth muscle cells, are consistent with the data of Fig. 10 in confirming that PKC stimulation leads to the activation of MAPK that, in turn, phosphorylates caldesmon. Because caldesmon phosphorylation occurs concomitant with MAPK activation during muscle stretch, caldesmon phosphorylation is part of the stretch response of arterial smooth muscle. A role for this process in the stretch-dependent functional changes of smooth muscle will require further investigation. Caldesmon is also phosphorylated during muscle contraction (3-5, 11); however, the identification of a specific functional effect due to this phosphorylation has remained elusive, and, although it is speculated that caldesmon phosphorylation plays a role in contraction, this hypothesis remains controversial (10, 26). Clearly, however, these results show that MAPK is activated downstream of PKC so that effects on smooth muscle function resulting from PKC stimulation may in fact result from the subsequent activation of MAPK.

Collectively, these data show that MAPK is activated in porcine carotid arteries by a number of stimuli acting independently of one another. The influx of Ca2+ alone is sufficient to support the activation of MAPK; the activation of PKC by phorbol esters is also sufficient to activate MAPK. Finally, stretch of the muscle leads to a rapid activation of the kinase that desensitizes after several hours. The effects of stretch are additive to the effects of Ca2+ or PKC stimulation, and desensitization due to stretch does not alter the responsiveness of the muscle to stimulation by either KCl or PDBu. Based on these results, we conclude that MAPK and caldesmon are intracellular mediators for muscle stretch and may therefore play a role in stretch-dependent functions of smooth muscle.

    ACKNOWLEDGEMENTS

We thank Drs. Yanhua Li and Katsuhide Mabuchi for help in antibody production, Shaobin Zhuang for baculovirus expression of recombinant ERK1, and Samantha Matson for technical expertise in helping to perform these experiments.

    FOOTNOTES

This work was supported by National Institutes of Health Grants HL-56035 (to L. P. Adam) and AR-41637 (to C. L.-A. Wang).

Address for reprint requests: L. P. Adam, Boston Biomedical Research Institute, 20 Staniford St., Boston, MA 02114.

Received 7 April 1997; accepted in final form 31 July 1997.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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AJP Cell Physiol 273(6):C1819-C1827
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