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Protein Kinase C Activation during Ca2+-Independent Vascular Smooth Muscle Contraction
JOURNAL OF SURGICAL RESEARCH ARTICLE NO.
78, 48 –53 (1998)
JR985368
Protein Kinase C Activation during Ca21-Independent Vascular Smooth Muscle Contraction Douglas C. Throckmorton, M.D.,*,†,‡ C. Subah Packer, Ph.D.,§ and Colleen M. Brophy, M.D.*,†,‡,¶ ¶
Department of Surgery, *Department of Cell Biology and Anatomy, and †Institute for Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912; ‡Augusta VA Medical Center, Augusta, Georgia 30901; and §Department of Physiology and Biophysics, Indiana University, Indianapolis, Indiana 46202 Submitted for publication December 4, 1997
vasospasm. While the initiation of vascular smooth muscle contraction has been attributed to an increase in Ca21, leading to activation of myosin light chain kinase (MLCK) and phosphorylation of the 20-kDa myosin light chain (MLC20) [1, 2]; less is known about the signalling events that lead to the maintainance of force as occurs in vasospasm. For example, sustained increases in intracellular Ca21 are not required to maintain contraction [3], and the temporal relationship between MLC20 phosphorylation and tension development is variable [1, 2, 4]. We and others have hypothesized that protein kinase C (PKC) activation plays a major role in mediating the sustained phase of vascular smooth muscle contraction [5– 8]. In support of this hypothesis, many agonists of smooth muscle contraction, including angiotensin II, bradykinin, endothelin, and histamine, activate PKC [9 –11]. Phorbol 12,13-dibutyrate (PDBu) and other phorbol esters, which are pharmacologic activators of PKC, cause contraction of smooth muscle from several different species and types of vessels [8, 12–14]. PKC activation may contribute to the maintenance of force through a kinase cascade involving mitogen-activated protein (MAP) kinase and increases in the phosphorylation of the actin regulatory protein, caldesmon [15, 16]. To examine the role of PKC activation in sustained contraction of bovine carotid artery smooth muscle, we measured phorbol ester-induced phosphorylation changes in bovine carotid artery smooth muscles under conditions where the Ca21 concentrations were physiologic (1.5 mM) compared to conditions where the intracellular Ca21 concentrations are low and fixed (‘‘Ca21-free’’ conditions) [8]. We hypothesized that under Ca21-free conditions, there would be no activation of the Ca21dependent MLC20 pathway, yet Ca21-independent isoforms of PKC would be activated leading to increases in the phosphorylation of the specific PKC substrate MARCKS (myristolated, alanine-rich, C-kinase substrate). Activation of PKC would then lead to activa-
The cellular signaling mechanisms that modulate the sustained vascular smooth muscle contractions that occur in vasospasm are not known. We and others have hypothesized that a kinase cascade involving protein kinase C (PKC) modulates sustained vascular smooth muscle contraction. The purpose of this investigation was to develop a model in which the traditional contractile pathways involving myosin light chain phosphorylation are not activated and determine if the PKC pathway is activated under these conditions. The phosphorylation of caldesmon, myosin light chain (MLC20), and the specific PKC substrate, MARCKS (myristoylated, alanine-rich C-kinase substrate) was measured in bovine carotid arterial smooth muscle (BCASM) stimulated with phorbol 12,13dibutyrate (PDBu) under Ca21-containing and Ca21free conditions. PDBu stimulation led to increases in caldesmon and MARCKS phosphorylation to the same degree in the presence or absence of Ca21. PDBu stimulation but did not lead to increases in MLC20 phosphorylation over basal levels in Ca21-free conditions. Immunoblot analysis of BCASM using PKC isoformspecific antibodies demonstrated the presence of one ‘‘Ca21- dependent’’ PKC isoform: alpha, and two of the ‘‘Ca21-independent’’ isoforms: epsilon and zeta. These data suggest that Ca21-independent isoforms of PKC may play a role in the sustained phase of BCASM contractions through a kinase cascade that involves caldesmon and MARCKS phosphorylation but not MLC20 phosphorylation. © 1998 Academic Press Key Words: smooth muscle; vascular; protein kinase C; caldesmon; myosin light chain; MARCKS; protein phosphorylation.
INTRODUCTION
The dynamic caliber of blood vessels is determined by the contractile state of the smooth muscle cells in the wall of the vessel. Pathologic increases in the contractile state of the smooth muscle constitutes
0022-4804/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
48
THROCKMORTON ET AL.: PKC ACTIVATION DURING Ca21-INDEPENDENT CONTRACTION
tion of a kinase cascade that would ultimately phosphorylate the actin binding protein caldesmon. MATERIALS AND METHODS Materials. The 12-deoxyphorbol 13-isobutyrate (PDBu) was purchased from LC services (Woburn, MA), the [32P]orthophosphate was obtained from DuPont-New England Nuclear (Belerica, MA) and BAPTA-AM from Calbiochem (La Jolla, CA). Characterization of antibodies to the PKC isoforms alpha, beta I, beta II, and gamma has been previously described [17]. Antibodies to the PKC isoforms delta, epsilon, and zeta were obtained commercially (Gibco BRL, Grand Island, NY). The reagents used for electrophoretic analysis were purchased from Bio-Rad (Hercules, CA). All other chemicals were of analytical grade and were obtained from Sigma (St. Louis, MO) Preparation of bovine carotid arterial smooth muscle (BCASM) strips. Fresh bovine calf carotid arteries were obtained from a local abattoir and dissected free of adventitial tissues. The arteries were opened longitudinally and the endothelium was denuded with gentle rubbing of the intimal surface. The strips were equilibrated in Krebs bicarbonate solution (KRB: 120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4, 1.0 mM NaHPO4, 10 mM glucose, 1.5 mM CaCl2, and 25 mM Na2HCO3) at 37°C and gassed with 95% O2, 5% CO2 for at least 90 min. For experiments in calcium-free conditions, the muscles were equilibrated in KRB without CaCl2, containing 4 mM EGTA (an extracellular Ca21 chelator) and 0.1 mM BAPTA/AM (an intracellular Ca21 chelator). Depletion of extracellular calcium under these conditions has been previously confirmed by repeating the high extracellular KCl contraction and demonstrating that the muscle does not contract when exposed to KCl [8]. Smooth muscle phosphorylation. Following equilibration in KRB, the strips were placed in a low-phosphate Hepes buffer containing: 140 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4, 10 mM glucose, 1 mM CaCl2, and 10 mM Hepes (pH 7.4). For experiments performed in the absence of Ca21, strips were incubated in low-phosphate Hepes buffer containing 0 CaCl2, 4 mM EGTA, and 0.1 mM BAPTA/AM. The strips were incubated for 90 min at 37°C, with intermittent oxygenation every 20 min. Muscle strips were then incubated in the same Hepes buffer containing 250 mCi/ml [32P]orthophosphate for an additional 90 min to label cellular ATP pools. Concurrent contraction measurements were performed on strips from the same vessel to confirm muscle viability and contractile response. The strips were stimulated using the appropriate protocol and then immersed in acetone/dry ice. The tissue was then crushed using mortar and pestle under liquid N2. Immunoprecipitation of caldesmon was performed as described by Adam et al. utilizing polyclonal antiserum against chicken gizzard caldesmon [15]. After protein weighting, the immunoprecipitates were analyzed by SDS–PAGE using 8% gels, followed by autoradiography. Changes in phosphorylation were quantified by digitalizing the images with a CCD camera interfaced with an IS 1000 Digital Imaging System (Alpha Innotech Corp., San Leandro, CA). Whole cell phosphorylation changes were assessed using 2-dimensional gel electrophoresis as previously described [18]. Acetone-precipitated proteins were solubilized in 9.0 M urea/2% Chaps. Protein weighted aliquots of these samples were resolved with isoelectric focusing (IEF) using 2% v/v ampholines (LKB Pharmacia, Upsala Sweden). After loading, the samples were overlayed with 20 ml of 8 M urea containing 1% ampholines and run for 18 h at 500 V and 1 h at 950 V. The tube gels were then layered on SDS– PAGE slab gels of 8% acrylamide with 4% stacking gels, and run at 50 V for 18 h. Phosphorylation changes were quantitated using densitometric analysis with a CCD camera interfaced with a 2D analyzer software package (Innovision Software, Bioimage Corp., Ann Arbor, MI). MLC20 phosphorylation was measured on arterial strips that were freeze-clamped with tongs cooled to the temperature of liquid N2 at specific time points. The frozen samples were placed in a frozen slurry of the denaturation solution [90% acetone, 10% trichloroacetic acid (TCA), and 10 mM dithiothreoitol (DTT)], then thawed over a 30-min
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period. The denatured tissues were homogenized in ice-cold homogenization solution (10% TCA and 10 mM DTT), then centrifuged at 1000 rpm for 1 min. The supernatant was discarded and the remaining pellet was washed three times with ether then resuspended in urea sample buffer (6.7 M urea, 18 mM Tris, 20 mM glycine, 9 mM DTT, 4.6% saturated sucrose, and 0.004% bromphenol blue). The samples were subjected to electrophoresis urea glycerol minigels (40% glycerol, 10% acrylamide, 0.5% bisacrylamide, 20 mM Tris, and 22 mM glycine) [19]. Electrophoretic transfer of proteins from the gels onto nitrocellulose membranes was carried out in a buffer containing 10 mM Na2HPO4 (pH 7.6) at 1.5 A for 1.5 h at 20°C. The blot was blocked in 0.3% milk solution (150 mM NaCl and 10 mM Tris, pH 7.4) for 1 h, then incubated overnight with antiserum raised to bovine tracheal smooth muscle MLC20. The blot was then placed in goat anti-rabbit IgG-peroxidase and developed in a 4-chloro-1napthol/H2O2 solution. The MLC20 bands were measured densitometrically (Bio-Rad densitometer), and the relative amounts of the phosphorylated forms of MLC20 over the total amount of MLC20 were determined [20]. PKC isoform determination. BCASM strips were boiled 10 mM EGTA, 2 mM EDTA, 10 mM b-mercaptoethanol, 1% Glycerol, and 4% SDS in 60 mM Tris, pH 7.0, followed by centrifugation (14,000g) to remove insoluble material. Protein normalized aliquots were resolved using SDS–PAGE using 8% gels. The proteins were then electroblotted to Immobilon (Millipore, Bedford, MA) and the blots blocked with 1% bovine serum albumin (BSA) and 1% Tween-20 in 25 mM Tris-buffered saline (pH 7.4). Separate blots were incubated in buffer containing antibodies to the following isoforms of PKC for 3 h at room temperature: alpha, beta I, beta II, gamma, delta, epsilon, and zeta. The blots were washed, and specific binding was detected using 125I-protein A, or 125I-anti-IgG antibody (Amersham, Arlington Heights, IL) for 30 min. After washing, the blots were dried for exposure to film (Kodak XAR-5) for autoradiography. To identify the PKC band, a lane containing rat brain cytosol was included on all Western blots [17]. PKC was identified as a band of the appropriate molecular weight using molecular weight standards (Rainbow Markers, Amersham). Statistical analysis. Data are presented as the mean 6 standard error of the mean. Statistical analysis was performed by unpaired Student’s t test or one way analysis of variance (ANOVA) followed by Newman–Keul’s test for comparing two mean values and multiple means, respectively, with a P value of , 0.05 considered indicative of significant differences.
RESULTS
MLC20 Phosphorylation Treatment of BCASM with high extracellular KCl leads to increases in intracellular Ca21 and increases in the ratio of phosphorylated MLC20 to total MLC20 (Fig. 1). KCl stimulation resulted in an increase in MLC20 phosphorylation level with an early peak (30 s) and subsequent decline (15 min) (Fig. 1). However, stimulation with PDBu (1027 M for 45 min) had no effect on MLC20 phosphorylation in BCASM in the presence or absence of Ca21 (Fig. 2). Densitometric analysis of whole cell phosphorylation and 2D gels also revealed no increase in MLC20 phosphorylation in response to PDBu stimulation under Ca21-free conditions (data not shown). The lack of phosphorylation of MLC20 suggests that the MLCK pathway is not activated under these Ca21-free conditions. MARCKS Phosphorylation We next sought to determine if the PKC pathway was activated under Ca21-free conditions. MARCKS
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FIG. 1. KCl-induced myosin light chain phosphorylation. Comparison of the ratios of phosphorylated to total smooth muscle 20-kDa myosin light chain (MLC20-P) from bovine carotid arterial smooth muscle under resting conditions (0 time) or stimulated with high potassium (110 mM) in physiologic Ca21 (1.5 mM, 1) or Ca21-free conditions (2). KCl stimulation resulted in a MLC20 phosphorylation transient with an early peak (0.5 min, *P , 0.01 compared with resting BCASM) and subsequent decline (15 min., **P , 0.05 compared with BCASM at 0.5 min).
is an 80-kDa phosphoprotein with a pI of 4.3– 4.5 that is a specific PKC substrate [21]. Stimulation of bovine carotid artery smooth muscle strips with PDBu (1027 M) for 45 min resulted in an increase in the phosphorylation of a band with a similar Mr and pI as MARCKS (Fig. 3). Densitometric analysis of this band revealed a three- to fourfold increase in phosphorylation in BCASM stimulated with PDBu (1027 M) for 45 min in the presence or absence of Ca21 (Fig. 5).
FIG. 3. Phosphorylation of MARCKS-like protein. Representative autoradiograms of 2-dimensional gels of whole cell phosphorylation of BCASM demonstrated MLC20 phosphorylation (closed arrows). PDBu stimulation resulted in an increase in the phosphorylation of an 80-kDa phosphoprotein with a pI of 4.3– 4.5 which corresponds to MARCKS (open arrows) in the presence and absence of Ca21.
Caldesmon is an actin binding phosphoprotein that has been implicated in the maintenance of force. In order to determine if caldesmon was phosphorylated under Ca21-free conditions, BCASM was whole
cell phosphorylated, stimulated with PDBu (1027 M) for 45 min, and caldesmon was immunoprecipitated. Stimulation with PDBu resulted in an increase in phosphorylation of a band at Mr 140 kDa, in autoradiographs of immunoprecipitates from BCASM homogenates (Fig. 4). Analysis of Western blots of immunoprecipitates confirmed that the Mr 140 band is caldesmon (data not shown). Less than 15% of the caldesmon remained in the supernatant as determined by Western blot analysis on supernatants following immunoprecipitation. Stimulation of bo-
FIG. 2. PDBu-induced myosin light chain phosphorylation. Comparison of the ratios of phosphorylated to total smooth muscle 20-kDa myosin light chain (MLC20-P) from bovine carotid arterial smooth muscle under resting conditions (0 time) or stimulated with phorbol dibutyrate (PDBu, 15 and 45 min) in physiologic Ca21 (1.5 mM, 1) or Ca21-free conditions (2). PDBu stimulation had no effect on the level of phosphorylated MLC20 in the presence or absence of Ca21 (P . 0.05).
FIG. 4. Caldesmon immunoprecipitation from bovine carotid artery smooth muscle. This is a representative autoradiogram after caldesmon immunoprecipitation from phosphorylated BCASM. Strips were exposed to control (CNT) conditions or PDBU (1027 M for 45 min) in the presence (1Ca21) or absence (2Ca21) of calcium. The band at 145 kDa represents caldesmon. Molecular mass standards of 200 and 97 kDa are indicated by small arrows.
Caldesmon Phosphorylation
THROCKMORTON ET AL.: PKC ACTIVATION DURING Ca21-INDEPENDENT CONTRACTION
FIG. 5. Quantitation of Caldesmon and MARCKS-like protein phosphorylation. Densitometric analysis demonstrated a three-fold increase in caldesmon and MARCKS phosphorylation in the presence (1Ca21, open bars) or absence (2Ca21, filled bars) of calcium (*P , 0.05, n 5 4 separate experiments) with PDBU stimulation (1027 M, 45 min).
vine carotid artery smooth muscle strips with PDBu (1027M) for 45 min stimulated a threefold increase in the amount of caldesmon phosphorylation, in the presence or absence of Ca21 (Fig. 5). Protein Kinase C (PKC) Isoforms in BCASM The isoforms of PKC present in fresh BCASM were determined with immunoblotting using isoform specific antibodies (Fig. 6). The ‘‘classical’’ (calciumdependent) isoform, alpha, the ‘‘calcium-independent’’ isoform, epsilon, and the ‘‘atypical’’ isoform, zeta, were detected by immunoblotting in BCASM (Fig. 6). The beta I, beta II, gamma, and delta isoforms were not detected in BCASM (data not shown). DISCUSSION
Sustained smooth muscle contraction occurs in response to the phorbol esters, phorbol dibutyrate (PDBu) in the the presence of the extracellular Ca21
FIG. 6. PKC isoforms in BCASM. Western blots of BCASM arterial homogenates using isoform-specific antibodies to protein kinase C detected the classical alpha (calcium-dependent) isoform (Mr 80 kDa), and the ‘‘Ca21-independent’’ epsilon (Mr 90 kDa) and zeta (Mr 80 kDa) isoforms.
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FIG. 7. Cellular signalling pathways in vascular smooth muscle contraction. Agonist stimulation of vascular smooth muscle leads to receptor-mediated increases in inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 causes the sarcoplasmic reticulum to release Ca21 and DAG activates protein kinase C (PKC). Increases in intracellular Ca21 activate myosin light chain kinase (MLCK) which leads to increases in the phosphorylation of the 20-kDa myosin light chains (MLC20). RHO kinase also inactivates MLC20 phosphatase (PPase) and is an alternate pathway for increasing MLC20 phosphorylation. PKC activates a protein kinase pathway leading to increases in the phosphorylation of the extracellular regulated kinase, ERK1 (otherwise known as MAP kinase), which has been shown to phosphorylate caldesmon. Caldesmon is a thin filament regulatory protein which when phosphorylated, dissociates from actin, thus allowing actin–myosin interactions and contraction. Finally, contractile agonists such as thrombin have been shown to activate another extracellular regulated kinase, ERK3 (also known as P38 MAP kinase), which phosphorylates MAPKAP kinase-2, leading to increases in the phosphorylation of a small heat shock protein, HSP27. HSP27 is an actin binding protein that has been implicated in modulating actin filament dynamics.
chelator (EGTA), and the intracellular Ca21 chelator BAPTA-AM [8]. The Ca21-free PDBu-induced contractions are slowly developing and maximal tension is reached at 45 min. [8]. The magnitude of PDBuinduced contractions are similar under physiologic Ca21 conditions (1.5 mM) and Ca21-free conditions. In the present study we confirm that the Ca21-dependent myosin light chain kinase (MLCK) pathway is not activated under Ca21-free conditions in that there were no increases in the phosphorylation of MLC20 (Fig. 2). Under physiologic Ca21 conditions depolarization of the membrane with high extracellular KCl (110 mM) led to significant increases in MLC20 (Fig. 1). However, high extracellular KCl did not lead to increases in MLC20 phosphorylation under Ca21-free conditions (Fig. 1). Thus, the use of Ca21-chelators provides a model in which signalling pathways independent of the Ca21-dependent MLCK pathway can be isolated and examined. Vascular smooth muscle contractions in physiologic Ca21 conditions, and Ca21-free conditions, were associated with increases in the phosphorylation of the specific PKC substrate phosphoprotein, MARCKS and the thin filament actin binding protein, caldesmon (Figs. 3–5). We and others have suggested that activation of PKC initiates a kinase cascade in which MAP kinase is activated leading to increases in caldesmon phosphorylation [8, 22, 23]. Caldesmon is a thin filament regulatory protein
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that when phosphorylated dissociates from actin thus allowing actin–myosin interaction and force development. More recently, two additional regulatory pathways have been implicated in vascular smooth muscle contraction. Investigators have recently focused on the RHO kinase pathway. RHO is a small GTP binding protein that activates RHO kinase. Activation of RHO kinase leads to increases in the phosphorylation of MLC20 and inhibition of MLC20 phosphatase activity [24, 25]. However, while these data support another pathway for the regulation of MLC20-phosphorylation, the data from this system and others, in which contractile force is dissociated from MLC 20-phosphorylation suggests that there are alternate pathways leading to sustained contraction (Fig. 7). Finally, vascular smooth muscles contain an abundant amount of the small heat shock protein, HSP27, and this protein has been implicated in modulating bombesin-induced contraction in rectal smooth muscle [26]. We have recently demonstrated that thrombin stimulation leads to vascular smooth muscle contraction and these contractions are associated with activation of the MLCK/MLC20 pathway, the traditional MAP kinase pathway, and activation of the stress kinase pathway which leads to increases in the phosphorylation of HSP27 [28, 29]. HSP27 is another actin binding protein that may regulate actin filament dynamics [27]. Thus, it will be of interest to determine if increases in the phosphorylation of HSP27 occur under Ca21free conditions. These data provide a model for sustained smooth muscle contraction under conditions where the Ca21dependent MLCK/MLC20 pathway does not appear to be activated. Under these Ca21-free conditions, phorbol ester stimulation may activate Ca21-independent isoforms of PKC, which are present in bovine carotid artery smooth muscles. This may result in the activation of a kinase cascade which leads to increases in the phosphorylation of caldesmon and possibly other contractile regulatory proteins. ACKNOWLEDGMENTS The authors thank Shapiro’s Meat Packing Plant for generously providing access to bovine tissue. The authors thank Shannon Lamb (Medical College of Georgia) and Lisa Harden (Indiana University) for technical assistance. The authors are grateful to Dr. James T. Stull for supplying the antigen (bovine tracheal muscle MLC20) to which the MLC20 antibody used in this study was raised and to Drs. Len Adam and David Hathaway for providing antibodies to caldesmon. C. M. Brophy is a recipient of a Clinician Scientist Award from the American Heart Association and a VA Merit Review Award. C. S. Packer is a recipient of an American Lung Association Career Investigator Award.
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Silver, P., and Stull, J. T. Regulation of myosin light chain and phosphorylase phosphorylation in tracheal smooth muscle. J. Biol. Chem. 288: 925–929, 1982.
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Miller-Jance, W. C., Miller, J., Wells, J. W., Stull, J. T., and Kamm, K. Biochemical events associated with activation of smooth muscle contractions. J. Biol. Chem. 262: 13979 –13982, 1988. Jaing, M., and Morgan, K. G. Intracellular calcium levels in phorbol ester-induced contractions of vascular muscle. Am. J. Physiol. 253: H1365–1371, 1987. Gerthoffer, W. T. Dissociation of myosoin phosphorylation and active tension during muscarinic stimulation of tracheal smooth muscle. J. Pharm. Exp. Ther. 240: 8 –15, 1987. Rasmussen, H., Takuwa, Y., and Park, S. Protein kinase C in the regulation of smooth muscle contraction. FASEB J. 1: 177– 185, 1987. Andrea, J. E., and Walsh, M. P. Protein kinase C of smooth muscle. Hypertension 20: 585–595, 1992. Khalil, R. A., and Morgan, K. G. Protein kinase C: A second E-C coupling pathway in vascular smooth muscle? NIPS 7: 10 –15, 1992. Whitney, E. G., Throckmorton, D., Yeh, J., Isales, C., Rasmussen, H., and Brophy, C. M. Kinase activation and smooth muscle contraction in the presence and absence of calcium. J. Vasc. Surg. 22: 37– 44, 1995. Griendling, K., Rittenhouse, S. E., Brock, T. A., Ekstein, L. S., Gimbrone, M. A., and Alexander, R. W. Sustained diacylglycerol formation from inositol phospholipids in angiotensin IIstimulated vascular smooth muscle cells. J. Biol. Chem. 261: 5901–5906, 1986. Haller, H., Smallwood, J. I., and Rasmussen, H. Protein kinase C. Translocation and contraction in intact vascular smooth muscle strips. Biochem. J. 270: 375–381, 1990. Singer, H. A., Schworer, C. M., Sweeley, C., and Benscoter, H. Activation of protein kinase C isozymes by contractile stimuli in arterial smooth muscle. Arch. Biochem. Biophys. 299: 320 –329, 1992. Rasmussen, H., Forder, J., Kojima, I., and Scriabine, A. TPA-induced contraction of isolated rabbit vascular smooth muscle. Biochem. Biophys. Res. Commun. 2: 776 – 784, 1984. Danthulure, N. R., and Deth, R. C. Phorbol ester induced contraction of arterial smooth muscle and inhibition of alphaadrenergic response. Biochem. Biophys. Res. Commun. 125: 1103–1109, 1984. Chatterjee, M., and Tejada, M. Phorbol ester-induced contraction in chemically skinned vascular smooth muscle. Am. J. Physiol. 251: C356 –C361, 1986. Adam, L. P., Haeberle, J. R., and Hathaway, D. R. Phosphorylation of caldesmon in arterial smooth muscle. J. Biol. Chem. 264: 7698 –7703, 1989. Childs, T. J., Watson, M. H., Sanghera, J. S., Campbell, D. L., Pelech, S. L., and Mak, A. S. Phosphorylation of smooth muscle caldesmon by mitogen activated protein kinase and expression of MAP kinase in differentiated smooth muscle cells. J. Biol. Chem. 267: 22853–22859, 1992. Ganesan, S., Calle, R., Zawalich, K., Smallwood, J. I., Zawalich, W. S., and Rasmussen, H. Glucose-induced translocation of protein kinase C in rat pancreatic islets. Proc. Natl. Acad. Sci. USA 87: 9893–9897, 1990. Parks, S., and Rasmussen, H. Carbachol-induced protein phosphorylation changes in bovine tracheal smooth muscle. J. Biol. Chem. 261: 15734 –15739, 1986. Hathaway, D. R., and Haeberle, J. R. A radioimmunoblotting method for measuring myosin light chain phosphorylation levels in smooth muscle. Am. J. Physiol. 249: C345–C351, 1985. Persechini, A., Kamm, K. E., and Stull, J. T. Different phosphorylated forms of myosin in contracting tracheal smooth muscle. J. Biol. Chem. 261: 6293– 6299, 1986.
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Aderem, A. The MARCKS family: A family of protein kinase C substrates. Cell 71: 713–716, 1992. 22. Epstein, A. M., Throckmorton, D., and Brophy, C. M. MAP kinase activation: An alternative signalling pathway for sustained vascular smooth muscle contraction. J. Vasc. Surg. 26: 327–332, 1997. 23. Katsuyama, H., and Morgan, K. Mechanisms of Ca 21 independent contraction in single permeabilized ferret aorta cells. Circ. Res. 72: 651– 657, 1993. 24. Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Matsuura, Y., and Kaibuchi, K. Phosphorylation and activation of myosin by Rho-associated kinase. J. Biol. Chem. 271: 20246 –20249, 1996. 25. Kureishi, Y., Kobayashi, S., Amano, M., Kimura, K., Kanaide, H., Nakano, T., Kaibuchi, K., and Ito, M. Rho-associated kinase
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directly induces smooth muscle contraction through myosin light chain phosphorylation. 26. Yamada, H., Strahler, J., Welsh, M. J., and Bitar, K. N. Activation of MAP kinase and translocation with HSP27 in bombesin-induced contraction of rectosigmoid smooth muscle. Am. J. Physiol. 269: G683– 691, 1995. 27. Jerius, H., Beall, A., Woodrum, D., Epstein, A., and Brophy, C. Thrombin-induced vasospasm: Cellular signalling mechanisms. Surgery 123: 46 –50, 1998. 28. Brophy, C. M., Woodrum, D., Dickinson, M., and Beall, A. Thrombin activates MAPKAP-2 kinase in vascular smooth muscle. J. Vasc. Surg. 27: 963–969, 1998. 29. Lavoie, J. N., Hickey, E., Weber, L. A., and Landry, J. Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of HSP27. J. Biol. Chem. 268: 24210–24214, 1993.
78, 48 –53 (1998)
JR985368
Protein Kinase C Activation during Ca21-Independent Vascular Smooth Muscle Contraction Douglas C. Throckmorton, M.D.,*,†,‡ C. Subah Packer, Ph.D.,§ and Colleen M. Brophy, M.D.*,†,‡,¶ ¶
Department of Surgery, *Department of Cell Biology and Anatomy, and †Institute for Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912; ‡Augusta VA Medical Center, Augusta, Georgia 30901; and §Department of Physiology and Biophysics, Indiana University, Indianapolis, Indiana 46202 Submitted for publication December 4, 1997
vasospasm. While the initiation of vascular smooth muscle contraction has been attributed to an increase in Ca21, leading to activation of myosin light chain kinase (MLCK) and phosphorylation of the 20-kDa myosin light chain (MLC20) [1, 2]; less is known about the signalling events that lead to the maintainance of force as occurs in vasospasm. For example, sustained increases in intracellular Ca21 are not required to maintain contraction [3], and the temporal relationship between MLC20 phosphorylation and tension development is variable [1, 2, 4]. We and others have hypothesized that protein kinase C (PKC) activation plays a major role in mediating the sustained phase of vascular smooth muscle contraction [5– 8]. In support of this hypothesis, many agonists of smooth muscle contraction, including angiotensin II, bradykinin, endothelin, and histamine, activate PKC [9 –11]. Phorbol 12,13-dibutyrate (PDBu) and other phorbol esters, which are pharmacologic activators of PKC, cause contraction of smooth muscle from several different species and types of vessels [8, 12–14]. PKC activation may contribute to the maintenance of force through a kinase cascade involving mitogen-activated protein (MAP) kinase and increases in the phosphorylation of the actin regulatory protein, caldesmon [15, 16]. To examine the role of PKC activation in sustained contraction of bovine carotid artery smooth muscle, we measured phorbol ester-induced phosphorylation changes in bovine carotid artery smooth muscles under conditions where the Ca21 concentrations were physiologic (1.5 mM) compared to conditions where the intracellular Ca21 concentrations are low and fixed (‘‘Ca21-free’’ conditions) [8]. We hypothesized that under Ca21-free conditions, there would be no activation of the Ca21dependent MLC20 pathway, yet Ca21-independent isoforms of PKC would be activated leading to increases in the phosphorylation of the specific PKC substrate MARCKS (myristolated, alanine-rich, C-kinase substrate). Activation of PKC would then lead to activa-
The cellular signaling mechanisms that modulate the sustained vascular smooth muscle contractions that occur in vasospasm are not known. We and others have hypothesized that a kinase cascade involving protein kinase C (PKC) modulates sustained vascular smooth muscle contraction. The purpose of this investigation was to develop a model in which the traditional contractile pathways involving myosin light chain phosphorylation are not activated and determine if the PKC pathway is activated under these conditions. The phosphorylation of caldesmon, myosin light chain (MLC20), and the specific PKC substrate, MARCKS (myristoylated, alanine-rich C-kinase substrate) was measured in bovine carotid arterial smooth muscle (BCASM) stimulated with phorbol 12,13dibutyrate (PDBu) under Ca21-containing and Ca21free conditions. PDBu stimulation led to increases in caldesmon and MARCKS phosphorylation to the same degree in the presence or absence of Ca21. PDBu stimulation but did not lead to increases in MLC20 phosphorylation over basal levels in Ca21-free conditions. Immunoblot analysis of BCASM using PKC isoformspecific antibodies demonstrated the presence of one ‘‘Ca21- dependent’’ PKC isoform: alpha, and two of the ‘‘Ca21-independent’’ isoforms: epsilon and zeta. These data suggest that Ca21-independent isoforms of PKC may play a role in the sustained phase of BCASM contractions through a kinase cascade that involves caldesmon and MARCKS phosphorylation but not MLC20 phosphorylation. © 1998 Academic Press Key Words: smooth muscle; vascular; protein kinase C; caldesmon; myosin light chain; MARCKS; protein phosphorylation.
INTRODUCTION
The dynamic caliber of blood vessels is determined by the contractile state of the smooth muscle cells in the wall of the vessel. Pathologic increases in the contractile state of the smooth muscle constitutes
0022-4804/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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THROCKMORTON ET AL.: PKC ACTIVATION DURING Ca21-INDEPENDENT CONTRACTION
tion of a kinase cascade that would ultimately phosphorylate the actin binding protein caldesmon. MATERIALS AND METHODS Materials. The 12-deoxyphorbol 13-isobutyrate (PDBu) was purchased from LC services (Woburn, MA), the [32P]orthophosphate was obtained from DuPont-New England Nuclear (Belerica, MA) and BAPTA-AM from Calbiochem (La Jolla, CA). Characterization of antibodies to the PKC isoforms alpha, beta I, beta II, and gamma has been previously described [17]. Antibodies to the PKC isoforms delta, epsilon, and zeta were obtained commercially (Gibco BRL, Grand Island, NY). The reagents used for electrophoretic analysis were purchased from Bio-Rad (Hercules, CA). All other chemicals were of analytical grade and were obtained from Sigma (St. Louis, MO) Preparation of bovine carotid arterial smooth muscle (BCASM) strips. Fresh bovine calf carotid arteries were obtained from a local abattoir and dissected free of adventitial tissues. The arteries were opened longitudinally and the endothelium was denuded with gentle rubbing of the intimal surface. The strips were equilibrated in Krebs bicarbonate solution (KRB: 120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4, 1.0 mM NaHPO4, 10 mM glucose, 1.5 mM CaCl2, and 25 mM Na2HCO3) at 37°C and gassed with 95% O2, 5% CO2 for at least 90 min. For experiments in calcium-free conditions, the muscles were equilibrated in KRB without CaCl2, containing 4 mM EGTA (an extracellular Ca21 chelator) and 0.1 mM BAPTA/AM (an intracellular Ca21 chelator). Depletion of extracellular calcium under these conditions has been previously confirmed by repeating the high extracellular KCl contraction and demonstrating that the muscle does not contract when exposed to KCl [8]. Smooth muscle phosphorylation. Following equilibration in KRB, the strips were placed in a low-phosphate Hepes buffer containing: 140 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4, 10 mM glucose, 1 mM CaCl2, and 10 mM Hepes (pH 7.4). For experiments performed in the absence of Ca21, strips were incubated in low-phosphate Hepes buffer containing 0 CaCl2, 4 mM EGTA, and 0.1 mM BAPTA/AM. The strips were incubated for 90 min at 37°C, with intermittent oxygenation every 20 min. Muscle strips were then incubated in the same Hepes buffer containing 250 mCi/ml [32P]orthophosphate for an additional 90 min to label cellular ATP pools. Concurrent contraction measurements were performed on strips from the same vessel to confirm muscle viability and contractile response. The strips were stimulated using the appropriate protocol and then immersed in acetone/dry ice. The tissue was then crushed using mortar and pestle under liquid N2. Immunoprecipitation of caldesmon was performed as described by Adam et al. utilizing polyclonal antiserum against chicken gizzard caldesmon [15]. After protein weighting, the immunoprecipitates were analyzed by SDS–PAGE using 8% gels, followed by autoradiography. Changes in phosphorylation were quantified by digitalizing the images with a CCD camera interfaced with an IS 1000 Digital Imaging System (Alpha Innotech Corp., San Leandro, CA). Whole cell phosphorylation changes were assessed using 2-dimensional gel electrophoresis as previously described [18]. Acetone-precipitated proteins were solubilized in 9.0 M urea/2% Chaps. Protein weighted aliquots of these samples were resolved with isoelectric focusing (IEF) using 2% v/v ampholines (LKB Pharmacia, Upsala Sweden). After loading, the samples were overlayed with 20 ml of 8 M urea containing 1% ampholines and run for 18 h at 500 V and 1 h at 950 V. The tube gels were then layered on SDS– PAGE slab gels of 8% acrylamide with 4% stacking gels, and run at 50 V for 18 h. Phosphorylation changes were quantitated using densitometric analysis with a CCD camera interfaced with a 2D analyzer software package (Innovision Software, Bioimage Corp., Ann Arbor, MI). MLC20 phosphorylation was measured on arterial strips that were freeze-clamped with tongs cooled to the temperature of liquid N2 at specific time points. The frozen samples were placed in a frozen slurry of the denaturation solution [90% acetone, 10% trichloroacetic acid (TCA), and 10 mM dithiothreoitol (DTT)], then thawed over a 30-min
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period. The denatured tissues were homogenized in ice-cold homogenization solution (10% TCA and 10 mM DTT), then centrifuged at 1000 rpm for 1 min. The supernatant was discarded and the remaining pellet was washed three times with ether then resuspended in urea sample buffer (6.7 M urea, 18 mM Tris, 20 mM glycine, 9 mM DTT, 4.6% saturated sucrose, and 0.004% bromphenol blue). The samples were subjected to electrophoresis urea glycerol minigels (40% glycerol, 10% acrylamide, 0.5% bisacrylamide, 20 mM Tris, and 22 mM glycine) [19]. Electrophoretic transfer of proteins from the gels onto nitrocellulose membranes was carried out in a buffer containing 10 mM Na2HPO4 (pH 7.6) at 1.5 A for 1.5 h at 20°C. The blot was blocked in 0.3% milk solution (150 mM NaCl and 10 mM Tris, pH 7.4) for 1 h, then incubated overnight with antiserum raised to bovine tracheal smooth muscle MLC20. The blot was then placed in goat anti-rabbit IgG-peroxidase and developed in a 4-chloro-1napthol/H2O2 solution. The MLC20 bands were measured densitometrically (Bio-Rad densitometer), and the relative amounts of the phosphorylated forms of MLC20 over the total amount of MLC20 were determined [20]. PKC isoform determination. BCASM strips were boiled 10 mM EGTA, 2 mM EDTA, 10 mM b-mercaptoethanol, 1% Glycerol, and 4% SDS in 60 mM Tris, pH 7.0, followed by centrifugation (14,000g) to remove insoluble material. Protein normalized aliquots were resolved using SDS–PAGE using 8% gels. The proteins were then electroblotted to Immobilon (Millipore, Bedford, MA) and the blots blocked with 1% bovine serum albumin (BSA) and 1% Tween-20 in 25 mM Tris-buffered saline (pH 7.4). Separate blots were incubated in buffer containing antibodies to the following isoforms of PKC for 3 h at room temperature: alpha, beta I, beta II, gamma, delta, epsilon, and zeta. The blots were washed, and specific binding was detected using 125I-protein A, or 125I-anti-IgG antibody (Amersham, Arlington Heights, IL) for 30 min. After washing, the blots were dried for exposure to film (Kodak XAR-5) for autoradiography. To identify the PKC band, a lane containing rat brain cytosol was included on all Western blots [17]. PKC was identified as a band of the appropriate molecular weight using molecular weight standards (Rainbow Markers, Amersham). Statistical analysis. Data are presented as the mean 6 standard error of the mean. Statistical analysis was performed by unpaired Student’s t test or one way analysis of variance (ANOVA) followed by Newman–Keul’s test for comparing two mean values and multiple means, respectively, with a P value of , 0.05 considered indicative of significant differences.
RESULTS
MLC20 Phosphorylation Treatment of BCASM with high extracellular KCl leads to increases in intracellular Ca21 and increases in the ratio of phosphorylated MLC20 to total MLC20 (Fig. 1). KCl stimulation resulted in an increase in MLC20 phosphorylation level with an early peak (30 s) and subsequent decline (15 min) (Fig. 1). However, stimulation with PDBu (1027 M for 45 min) had no effect on MLC20 phosphorylation in BCASM in the presence or absence of Ca21 (Fig. 2). Densitometric analysis of whole cell phosphorylation and 2D gels also revealed no increase in MLC20 phosphorylation in response to PDBu stimulation under Ca21-free conditions (data not shown). The lack of phosphorylation of MLC20 suggests that the MLCK pathway is not activated under these Ca21-free conditions. MARCKS Phosphorylation We next sought to determine if the PKC pathway was activated under Ca21-free conditions. MARCKS
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FIG. 1. KCl-induced myosin light chain phosphorylation. Comparison of the ratios of phosphorylated to total smooth muscle 20-kDa myosin light chain (MLC20-P) from bovine carotid arterial smooth muscle under resting conditions (0 time) or stimulated with high potassium (110 mM) in physiologic Ca21 (1.5 mM, 1) or Ca21-free conditions (2). KCl stimulation resulted in a MLC20 phosphorylation transient with an early peak (0.5 min, *P , 0.01 compared with resting BCASM) and subsequent decline (15 min., **P , 0.05 compared with BCASM at 0.5 min).
is an 80-kDa phosphoprotein with a pI of 4.3– 4.5 that is a specific PKC substrate [21]. Stimulation of bovine carotid artery smooth muscle strips with PDBu (1027 M) for 45 min resulted in an increase in the phosphorylation of a band with a similar Mr and pI as MARCKS (Fig. 3). Densitometric analysis of this band revealed a three- to fourfold increase in phosphorylation in BCASM stimulated with PDBu (1027 M) for 45 min in the presence or absence of Ca21 (Fig. 5).
FIG. 3. Phosphorylation of MARCKS-like protein. Representative autoradiograms of 2-dimensional gels of whole cell phosphorylation of BCASM demonstrated MLC20 phosphorylation (closed arrows). PDBu stimulation resulted in an increase in the phosphorylation of an 80-kDa phosphoprotein with a pI of 4.3– 4.5 which corresponds to MARCKS (open arrows) in the presence and absence of Ca21.
Caldesmon is an actin binding phosphoprotein that has been implicated in the maintenance of force. In order to determine if caldesmon was phosphorylated under Ca21-free conditions, BCASM was whole
cell phosphorylated, stimulated with PDBu (1027 M) for 45 min, and caldesmon was immunoprecipitated. Stimulation with PDBu resulted in an increase in phosphorylation of a band at Mr 140 kDa, in autoradiographs of immunoprecipitates from BCASM homogenates (Fig. 4). Analysis of Western blots of immunoprecipitates confirmed that the Mr 140 band is caldesmon (data not shown). Less than 15% of the caldesmon remained in the supernatant as determined by Western blot analysis on supernatants following immunoprecipitation. Stimulation of bo-
FIG. 2. PDBu-induced myosin light chain phosphorylation. Comparison of the ratios of phosphorylated to total smooth muscle 20-kDa myosin light chain (MLC20-P) from bovine carotid arterial smooth muscle under resting conditions (0 time) or stimulated with phorbol dibutyrate (PDBu, 15 and 45 min) in physiologic Ca21 (1.5 mM, 1) or Ca21-free conditions (2). PDBu stimulation had no effect on the level of phosphorylated MLC20 in the presence or absence of Ca21 (P . 0.05).
FIG. 4. Caldesmon immunoprecipitation from bovine carotid artery smooth muscle. This is a representative autoradiogram after caldesmon immunoprecipitation from phosphorylated BCASM. Strips were exposed to control (CNT) conditions or PDBU (1027 M for 45 min) in the presence (1Ca21) or absence (2Ca21) of calcium. The band at 145 kDa represents caldesmon. Molecular mass standards of 200 and 97 kDa are indicated by small arrows.
Caldesmon Phosphorylation
THROCKMORTON ET AL.: PKC ACTIVATION DURING Ca21-INDEPENDENT CONTRACTION
FIG. 5. Quantitation of Caldesmon and MARCKS-like protein phosphorylation. Densitometric analysis demonstrated a three-fold increase in caldesmon and MARCKS phosphorylation in the presence (1Ca21, open bars) or absence (2Ca21, filled bars) of calcium (*P , 0.05, n 5 4 separate experiments) with PDBU stimulation (1027 M, 45 min).
vine carotid artery smooth muscle strips with PDBu (1027M) for 45 min stimulated a threefold increase in the amount of caldesmon phosphorylation, in the presence or absence of Ca21 (Fig. 5). Protein Kinase C (PKC) Isoforms in BCASM The isoforms of PKC present in fresh BCASM were determined with immunoblotting using isoform specific antibodies (Fig. 6). The ‘‘classical’’ (calciumdependent) isoform, alpha, the ‘‘calcium-independent’’ isoform, epsilon, and the ‘‘atypical’’ isoform, zeta, were detected by immunoblotting in BCASM (Fig. 6). The beta I, beta II, gamma, and delta isoforms were not detected in BCASM (data not shown). DISCUSSION
Sustained smooth muscle contraction occurs in response to the phorbol esters, phorbol dibutyrate (PDBu) in the the presence of the extracellular Ca21
FIG. 6. PKC isoforms in BCASM. Western blots of BCASM arterial homogenates using isoform-specific antibodies to protein kinase C detected the classical alpha (calcium-dependent) isoform (Mr 80 kDa), and the ‘‘Ca21-independent’’ epsilon (Mr 90 kDa) and zeta (Mr 80 kDa) isoforms.
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FIG. 7. Cellular signalling pathways in vascular smooth muscle contraction. Agonist stimulation of vascular smooth muscle leads to receptor-mediated increases in inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 causes the sarcoplasmic reticulum to release Ca21 and DAG activates protein kinase C (PKC). Increases in intracellular Ca21 activate myosin light chain kinase (MLCK) which leads to increases in the phosphorylation of the 20-kDa myosin light chains (MLC20). RHO kinase also inactivates MLC20 phosphatase (PPase) and is an alternate pathway for increasing MLC20 phosphorylation. PKC activates a protein kinase pathway leading to increases in the phosphorylation of the extracellular regulated kinase, ERK1 (otherwise known as MAP kinase), which has been shown to phosphorylate caldesmon. Caldesmon is a thin filament regulatory protein which when phosphorylated, dissociates from actin, thus allowing actin–myosin interactions and contraction. Finally, contractile agonists such as thrombin have been shown to activate another extracellular regulated kinase, ERK3 (also known as P38 MAP kinase), which phosphorylates MAPKAP kinase-2, leading to increases in the phosphorylation of a small heat shock protein, HSP27. HSP27 is an actin binding protein that has been implicated in modulating actin filament dynamics.
chelator (EGTA), and the intracellular Ca21 chelator BAPTA-AM [8]. The Ca21-free PDBu-induced contractions are slowly developing and maximal tension is reached at 45 min. [8]. The magnitude of PDBuinduced contractions are similar under physiologic Ca21 conditions (1.5 mM) and Ca21-free conditions. In the present study we confirm that the Ca21-dependent myosin light chain kinase (MLCK) pathway is not activated under Ca21-free conditions in that there were no increases in the phosphorylation of MLC20 (Fig. 2). Under physiologic Ca21 conditions depolarization of the membrane with high extracellular KCl (110 mM) led to significant increases in MLC20 (Fig. 1). However, high extracellular KCl did not lead to increases in MLC20 phosphorylation under Ca21-free conditions (Fig. 1). Thus, the use of Ca21-chelators provides a model in which signalling pathways independent of the Ca21-dependent MLCK pathway can be isolated and examined. Vascular smooth muscle contractions in physiologic Ca21 conditions, and Ca21-free conditions, were associated with increases in the phosphorylation of the specific PKC substrate phosphoprotein, MARCKS and the thin filament actin binding protein, caldesmon (Figs. 3–5). We and others have suggested that activation of PKC initiates a kinase cascade in which MAP kinase is activated leading to increases in caldesmon phosphorylation [8, 22, 23]. Caldesmon is a thin filament regulatory protein
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that when phosphorylated dissociates from actin thus allowing actin–myosin interaction and force development. More recently, two additional regulatory pathways have been implicated in vascular smooth muscle contraction. Investigators have recently focused on the RHO kinase pathway. RHO is a small GTP binding protein that activates RHO kinase. Activation of RHO kinase leads to increases in the phosphorylation of MLC20 and inhibition of MLC20 phosphatase activity [24, 25]. However, while these data support another pathway for the regulation of MLC20-phosphorylation, the data from this system and others, in which contractile force is dissociated from MLC 20-phosphorylation suggests that there are alternate pathways leading to sustained contraction (Fig. 7). Finally, vascular smooth muscles contain an abundant amount of the small heat shock protein, HSP27, and this protein has been implicated in modulating bombesin-induced contraction in rectal smooth muscle [26]. We have recently demonstrated that thrombin stimulation leads to vascular smooth muscle contraction and these contractions are associated with activation of the MLCK/MLC20 pathway, the traditional MAP kinase pathway, and activation of the stress kinase pathway which leads to increases in the phosphorylation of HSP27 [28, 29]. HSP27 is another actin binding protein that may regulate actin filament dynamics [27]. Thus, it will be of interest to determine if increases in the phosphorylation of HSP27 occur under Ca21free conditions. These data provide a model for sustained smooth muscle contraction under conditions where the Ca21dependent MLCK/MLC20 pathway does not appear to be activated. Under these Ca21-free conditions, phorbol ester stimulation may activate Ca21-independent isoforms of PKC, which are present in bovine carotid artery smooth muscles. This may result in the activation of a kinase cascade which leads to increases in the phosphorylation of caldesmon and possibly other contractile regulatory proteins. ACKNOWLEDGMENTS The authors thank Shapiro’s Meat Packing Plant for generously providing access to bovine tissue. The authors thank Shannon Lamb (Medical College of Georgia) and Lisa Harden (Indiana University) for technical assistance. The authors are grateful to Dr. James T. Stull for supplying the antigen (bovine tracheal muscle MLC20) to which the MLC20 antibody used in this study was raised and to Drs. Len Adam and David Hathaway for providing antibodies to caldesmon. C. M. Brophy is a recipient of a Clinician Scientist Award from the American Heart Association and a VA Merit Review Award. C. S. Packer is a recipient of an American Lung Association Career Investigator Award.
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