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Protein kinase C isoforms and A1 adenosine receptors in porcine coronary smooth muscle cells
Vascular Pharmacology 39 (2002) 47 – 54 www.elsevier.com/locate/vph
Protein kinase C isoforms and A1 adenosine receptors in porcine coronary smooth muscle cells Mohammed A. Nayeem, S. Jamal Mustafa* Department of Pharmacology, Brody School of Medicine, East Carolina University, Greenville, NC 27858-2735, USA Received 1 November 2001; received in revised form 1 April 2002; accepted 1 April 2002
Abstract We have previously reported that prolonged exposure of porcine coronary arteries to adenosine agonists upregulates protein kinase C (PKC) through the activation of adenosine A1 receptor-coupled to pertussis toxin sensitive G-protein(s) [Am. J. Physiol. 264 (1993) H1465; Am. J. Physiol. 269 (1995) H1619]. The mechanism(s) by which A1 adenosine receptor upregulates PKC (isoforms) are not yet clearly understood. In the present study, we identified the a, b1, b2, g, e, and z PKC isoforms that were upregulated by adenosine A1 receptor agonist as a possible mechanism(s) involved for this upregulation. Incubation of porcine coronary smooth muscle cells (PCSMC) with adenosine A1 receptor agonist (2s)-N 6-[2-endo-norbornyl]adenosine (ENBA) caused an upregulation of PKC (isoforms), which were blocked by adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Western blot analysis using specific antibodies to PKC isoforms indicated that all the isoforms tested (a, bI, bII, m, g, d, e, and z) were present in the primary cultured smooth muscle cells from porcine coronary artery. Western blot studies indicated that PKC a, bI, bII, g, e, and z isoforms were upregulated in a dose dependent manner by adenosine agonist (ENBA) and PKC d and m were not altered. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Protein kinase C; Porcine coronary smooth muscle cells; A1 adenosine receptor
1. Introduction Adenosine, a natural nucleoside, is produced from cardiac myocytes and produces a variety of responses including bradycardia, hypotension, and coronary vasodilation (Mustafa, 1980). Adenosine recognizes specific cell surface receptors originally classified as A1 and A2 subtypes. This classification was based on the comparison of the agonist potency profiles and their ability to modulate adenylate cyclase. The high affinity adenosine receptors (A1) inhibit adenylate cyclase, whereas the low affinity receptors (A2) stimulate the cyclase (Londos et al., 1980). Since then, however, at least four adenosine receptor subtypes (A1, A2A, A2B, and A3) have been identified and cloned from various species (Linden et al., 1993). All these receptor subtypes are coupled to guanine nucleotide binding proteins (G-proteins) (Sabouni et al., 1989, 1991; Hussain and Mustafa, 1993; Marala and Mustafa, 1993). Subsequent to the binding to its receptor(s) * Corresponding author. Tel.: +1-252-816-2736; fax: +1-252-8163203. E-mail address: [email protected] (S.J. Mustafa).
adenosine initiates a cascade of events for its signal transduction. The most characterized mechanism is the effect on adenylate cyclase (Hussain and Mustafa, 1993; Sabouni et al., 1991; Marala and Mustafa, 1993). In addition, yet another system involving protein kinase C (PKC) has been postulated to play a key role in adenosine signalling. Pretreatment of porcine coronary artery with adenosine or its analogs (2-chloroadenosine and NECA) attenuated the phorbol ester induced contractions through receptor mechanism (Crushing et al., 1991). Phorbol esters are known to activate PKC directly, independent of any receptor systems (Marala and Mustafa, 1995a,b). Downregulation of PKC attenuated contractions produced by agents like PDBu, KCl, and endothelin in porcine arterial rings (Marala et al., 1993). Bradley et al. (1991) demonstrated that adenosine prevents phorbol ester-induced injury to rabbit lung without altering the levels of leukotrienes and tumor necrotic factor. Earlier studies from this laboratory have demonstrated that adenosine analog (CAD) regulates PKC in porcine coronary artery (Marala et al., 1993). In this study, CAD was shown to protect the activation and depletion of PKC by PDBu (Marala et al., 1993). It also demonstrated that
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prolonged exposure to CAD alone causes the upregulation of PKC in porcine coronary artery (Marala et al., 1993), a phenomenon that could explain the protection against the depletion of PKC during prolonged exposure to PDBu. Using relatively specific A1 and A2A receptor agonists (ENBA and CGS 21680, respectively) and antagonists (N0861 or DPCPX and DMPX, respectively), the receptor subtype involved in such upregulation of PKC was established to be A1 subtype (Marala and Mustafa, 1995a,b). ENBA [(2s)-N6-[2-endo-norbornyl]adenosine], an A1 receptor agonist, has been demonstrated to be of high affinity (Kd=0.3 nM) and selectivity (4700-fold) (Trivedi et al., 1989). CGS 21680 [2-p-(2-carboxyethyl)phenethylamino50-N-ethylcarboxamidoadenosine] was used as A2 receptor agonist, since it was demonstrated to bind to the receptor subtype with high selectivity (>170-fold) and low affinity (Kd=15.5 nM). In subsequent studies, bacterial toxins (cholera and pertussis) were used to covalently modify G-proteins in order to identify the G-protein(s) involved in mediation of the adenosine A1 receptor induced upregulation of PKC in porcine coronary artery (Marala and Mustafa, 1995a,b). These studies demonstrated that the effects of A1 selective agonist ENBA could be blocked by preincubation with pertussis toxin, indicating the involvement of pertussis toxin-sensitive Gi/Go protein(s) (Marala and Mustafa, 1995a,b). The exact mechanism(s) by which adenosine upregulates PKC are not yet clearly understood. This study was, therefore, designed to further elucidate the PKC isoforms involved with the activation A1 adenosine receptor for such signal transduction pathways.
dissected from each heart and cleaned of the adherent fat and connective tissue. 2.2.1. Isolation of PCSMC Smooth muscle cells from porcine coronary artery were isolated and cultured as described by Marala and Mustafa (1998) in this laboratory with minor modifications. Briefly, the arteries were cut open longitudinally and the endothelium was removed by rubbing the internal surface gently with scalpel blade. Tissue was rinsed twice with sterile Hank’s balanced salt solution (HBSS) containing (in mM): KCl 5.0, KH2PO4 0.3, NaCl 138, NaHCO3 4.0, Na2HPO47H2O 0.3, D-glucose 5.6, and HEPES 10.0. Tissue was then soaked for 10 min at ambient temperature in sterile HBSS. Arteries were minced with the scissors and incubated for 3 to 4 h at 37 C with vigorous shaking in sterile HBSS containing 1 mg/ml collagenase type II (300 U/mg), 0.5 mg/ml soybean trypsin inhibitor, and 3% bovine serum albumin. Then, it was filtered through nylon gauze and centrifuged at 100g for 10 min. The pellet was washed twice with Dulbecco’s modified Eagle’s medium (DMEM, GIBCO-BRL) and finally resuspended in DMEM containing 10% FBS and cultured at 37 C in 95% O2+5% CO2 incubator. Media was changed twice a week and, when the cells became confluent (about 1 week), they were split at 1:3 ratio using trypsin (0.25%). 2.2.2. Experimental protocol PCSMC were incubated in DMEM for 24 h with A1 agonist ENBA (109, 106, and 105 M) at 37 C and washed out with HBSS. In another group, ENBA+DPCPX
2. Materials and methods 2.1. Materials DPCPX and ENBA were purchased from Research Biochemicals International (Natick, MA). RPN 77 kit was fetal bovine serum (FBS) and purchased from Amersham (Arlington Heights, IL). DMEM, anti-PKC a, b1, b2, m, g, d, e, and z antibodies (GIBCO BRL), polyclonal antibody cross-reacting to adenosine A1 receptor protein (Alpha Diagnostic, San Antonio, TX) and were purchased. All other reagents were of analytical grade and purchased commercially. 2.2. Tissue preparation Porcine hearts from either sex were obtained from the local slaughterhouse within 30 min of sacrifice and transported to the laboratory in ice-cold Krebs-Henseleit (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 10 mM glucose, pH 7.4) oxygenated with 95% O2+5% CO2. The left anterior descending (LAD) and circumflex coronary arteries were
Fig. 1. (A) represents Western blot analysis, whereas (B) represents the densitometric analysis (mean of two) in arbitrary units for A1AR (36 kDa). Samples were taken from PCSMC after ENBA (105 M) alone, ENBA (105 M)+DPCPX (1 mM) for 24-h treatment.
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(1 mM, A1 antagonist) were incubated together with PCSMC for 24 h. 2.2.3. Gel electrophoresis and Western blotting Cells were rinsed with PBS and lysed with boiling lysis solution, containing 1% SDS, 1 mM sodium vanadate, and 10 mM Tris and pH 7.4. Cells were scraped and transferred to a microcentrifuge tube and boiled for an additional 5 min. The samples were then sonicated briefly and centrifuged (12,000g 15 C) for 5 min. Protein was measured using Bio-Rad protein assay based on the Bradford dye binding procedure with bovine serum albumin as a standard. The protein was divided into aliquots and stored at 80 C. At the time of analysis, experimental samples as well as pig brain (positive controls, data not shown) were thawed and approximately 20 mg of total protein per lane was loaded on a slab gel. Proteins were separated by sodium dodecyl sulfate-
Fig. 3. (A) represents Western blot analysis for bI-PKC (80 kDa), whereas (B) represents the densitometric analysis (mean of two) in arbitrary units. Samples were taken from PCSMC after ENBA (105, 106 and 109 M) treatment for 24 h. (C) represents Western blot analysis after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h, whereas (D) represents the densitometric analysis (mean of two) in arbitrary units.
Fig. 2. (A) represents Western blot analysis for a-PKC (82 kDa), whereas (B) represents the densitometric analysis (mean of two) in arbitrary units. Samples were taken from PCSMC after ENBA (105, 106 and 109 M) treatment. (C) represents Western blot analysis after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h, whereas (D) represents the densitometric analysis (mean of two) in arbitrary units.
polyacrylamide gel electrophoresis (SDS-PAGE) using 10% acrylamide gels (1-mm-thick). After electrophoresis, the proteins on the gel were transferred to polyvinyllidene difluoride (PVDF) membrane (Schleicher and Schuell, Keene, NH) by electroelution. Protein transfer was confirmed by employing prestained molecular weight markers (Bio-Rad Laboratories, Hercules, CA). Complete transfer of the protein was ascertained by staining the gel in Coomassie blue and transfer of prestained molecular weight markers on the PVDF membrane. Following blocking with nonfat dry milk, the PVDF membranes were incubated with monoclonal and polyclonal antibodies cross-reacting to adenosine A1 receptor protein, PKC isoforms (a, bI, bII, m, g, d, e, and z). The second antibody was a horseradish peroxidase conjugated antirabbit IgG. The membranes were developed using enhanced chemiluminescence (Amersham Life Sciences, CA) and exposed to X-ray film for appropriate time. As for as all the experiments regarding A1 adenosine receptor agonist (ENBA) for the receptor activation and antagonist
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(DPCPX) for the blocking of receptor activation related to expression of A1AR, and various PKC isoforms are concerned we chose only one concentration of ENBA (105 M) and one concentration of DPCPX (1 mM). Whereas in other sets of experiments we chose different concentrations of ENBA (109, 106, and 105 M) in relation to various level of expression of different PKC isoforms in PCSMC.
3. Results Incubation of PCSMC (2 –3 passages) with ENBA (105 M) alone for 24 h caused an elevation of A1 adenosine receptor expression (36 kDa) compared to control (Fig. 1A). To block A1 adenosine receptor activation in PCSMC, the cells were treated with ENBA+DPCPX together for 24 h, which caused an attenuation of A1 adenosine receptor expression (Fig. 1A). The densitometric scan analysis of the Western blot revealed 1.45-fold increase in expression of A1 adenosine receptor with ENBA treatment when compared to nontreated controls (Fig. 1B) and this upregulation of A1 adenosine receptor with ENBA was blocked by DPCPX treatment (Fig. 1B). In a separate experiment, the data suggest that the antibody we used in this report is solely recognizing the A1 adenosine receptor. Experiments were performed with A1 adenosine receptor antibody displacement (data not shown.) using corresponding peptide to rat A1AR gene, provided by Alpha Diagnostic International, San Antonio, TX. Fig. 2A shows a dose-dependent (109, 106, and 105 M of ENBA) upregulation of PKC a (82 kDa) in PCSMC compared to control. The densitometric scan analysis of the Western blot revealed a concentration-dependant increase in
Fig. 4. (A) represents Western blot analysis, whereas (B) represents the densitometric analysis (mean of two) in arbitrary units for bII-PKC (80 kDa). Samples were taken from PCSMC after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h.
Fig. 5. (A) represents Western blot analysis for e-PKC (90 kDa), whereas (B) represents the densitometric analysis (mean of two) in arbitrary units. Samples were taken from PCSMC after ENBA (105, 106 and 109 M) treatment for 24 h. (C) represents Western blot analysis after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h, whereas (D) represents the densitometric analysis (mean of two) in arbitrary units.
expression of PKC a (82 kDa) by 1.06 – 1.31- and 1.79-fold with ENBA at 109, 106, and 105 M, respectively, when compared to nontreated control (Fig. 2B). To block adenosine receptor in PCSMC, the cells were incubated with ENBA (105 M) +DPCPX (1 mM) for 24 h, which caused an attenuation of PKC a (Fig. 2C). The densitometric scan analysis of the Western blot revealed 1.45-fold increase in expression of PKC a with ENBA treatment when compared to nontreated controls (Fig. 2D) and this upregulation of PKC a with ENBA was blocked by DPCPX treatment (Fig. 2D). In separate experiments, the data suggest that the antibodies we used in this report are solely recognizing the PKC isoforms (a, bI, bII, m, g, d, e, and z, data not shown). Treatment with ENBA (109, 106, and 105 M) shows a concentration related increase of PKC bI (80 kDa, Fig. 3A) and the densitometric scan analysis of the Western blot revealed a concentration-dependent increase in expression of PKC bI by 1.03– 1.15- and 2.10-fold, respectively, com-
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pared to nontreated PCSMC (Fig. 3B). DPCPX attenuated PKC bI expression in ENBA treated PCSMC (Fig. 3C). The densitometric scan analysis of the Western blot revealed 2.25-fold increase in expression of PKC bI with ENBA treatment when compared to nontreated controls (Fig. 3D) and this upregulation of PKC bI with ENBA was blocked with DPCPX treatment (1.45-fold, Fig. 3D). Incubation of PCSMC with ENBA (105 M) alone for 24 h caused a slight elevation of PKC bII (80 kDa) compared to control (Fig. 4A). The cells were incubated with ENBA+DPCPX for 24 h, which caused a significant downregulation of PKC bII expression (Fig. 4A). The densitometric scan analysis of the Western blot revealed a slight increase in expression of PKC bII with ENBA treatment when compared to nontreated controls (Fig. 4B) and this slight upregulation of PKC bII with ENBA was followed by a significant downregulation of bII with DPCPX treatment (Fig. 4B). Fig. 5A shows concentration-dependent (109, 106, and 5 10 M of ENBA) upregulation of PKC e (90 kDa) in PCSMC compared to control. The densitometric scan analysis of the Western blot revealed a concentration-dependent increase in expression of PKC e (90 kDa) by 1.73 –3.60- and 4.68-fold with ENBA at 109, 106, and 105 M, respectively, when compared to nontreated control (Fig. 5B). To block A1 adenosine receptor (36 kDa), the cells were incubated with ENBA+DPCPX (1 mM, A1 adenosine receptor antagonist) for 24 h, which caused an attenuation of PKC e (Fig. 5C). The ENBA (105 M, 4.68 fold) increased expression of PKC e was blocked with DPCPX treatment (2.12fold, Fig. 5D). Treatment with ENBA (109, 106, and 105 M) shows a concentration-dependent increase of PKC g (80 kDa, Fig. 6A) by 4.19- and 5.91-fold compared to nontreated PCSMC (Fig. 6B), respectively. No further change of PKC g expres-
Fig. 6. (A) represents Western blot analysis, whereas (B) represents the densitometric analysis (mean of two) in arbitrary units for g-PKC (80 kDa). Samples were taken from PCSMC after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h.
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Fig. 7. (A) represents Western blot analysis for z-PKC (72 kDa), whereas (B) represents the densitometric analysis (mean of two) in arbitrary units. Samples were taken from PCSMC after ENBA (105, 106 and 109 M) treatment for 24 h. (C) represents Western blot analysis after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h, whereas (D) represents the densitometric analysis (mean of two) in arbitrary units.
sion was noted between 106 and 109 M ENBA, these doses may be less than a required threshold dose (105 M) of ENBA to activate A1 adenosine receptor for the upregulation of PKC g. Fig. 7A shows dose-dependent (109, 106, and 105 M of ENBA) upregulation of PKC z (72 kDa) in PCSMC compared to control. The densitometric scan analysis of the Western blot revealed a concentration-dependent increase in expression of PKC z (72 kDa) by 6.09 –38.72- and 52.82fold with ENBA at 109, 106, and 105 M, respectively, when compared to nontreated control (Fig. 7B). The cells were incubated with ENBA+DPCPX (1 mM, A1 adenosine receptor antagonist) for 24 h, which caused an attenuation of PKC z (Fig. 7C). The densitometric scan analysis of the Western blot revealed only a three-fold increase in expression of PKC z with ENBA (105 M) treatment when compared to nontreated controls (52.82-fold, Fig. 7D) and blocked by DPCPX (Fig. 7D).
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Fig. 8. (A) represents Western blot analysis, whereas (B) represents the densitometric analysis (mean of two) in arbitrary units for d-PKC (78 kDa). Samples were taken from PCSMC after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h. (C) represents Western blot analysis, whereas (D) represents the densitometric analysis (mean of two) in arbitrary units for m-PKC (115 kDa).
Treatment of PCSMC with ENBA (109, 106, and 105 M) shows no change of PKC d (78 kDa, Fig. 8A and B) compared to nontreated group. Similarly, there was no change in the level of PKC m in ENBA-treated compared to nontreated PCSMC (Fig. 8C and D). These data served as negative control.
4. Discussion This study demonstrated the role of adenosine-receptor subtype in the upregulation and downregulation of several isoforms of PKC in PCSMC. It also provides the evidence that the upregulation of several isoforms of PKC is a receptor-mediated phenomenon and A1 adenosine receptors are responsible for such an effect. Additionally, this study also demonstrates that DPCPX (A1 adenosine receptor antagonist) blocked this upregulation of these PKC isoforms. PKC has been implicated to play an important role in adenosine receptor signal transduction pathways. Earlier studies
from this laboratory demonstrated that porcine coronary arterial rings, when preincubated with adenosine analogues [2-chloroadenosine and 50-(N-ethylcarboxamido) adenosine] caused an attenuation of phorbol ester-induced contractions, thus, indicating a possible role of PKC (Crushing et al., 1991). It has also been previously shown from this laboratory that adenosine analogue 2-chloroadenosine blocked the activation of PKC (translocation from cytosol to membrane) upon short-term incubation (20 min), whereas upon longterm incubation (24 – 48 h), it produced an upregulation of PKC in porcine coronary artery (Marala and Mustafa, 1995a; Marala et al., 1993). Prolong exposure to adenosine analogues also partially blocked the phorbol ester-induced PKC depletion (Marala and Mustafa, 1995b; Marala and Mustafa, 1993). In the present study with the use of relatively specific A1-receptor agonist (ENBA) and antagonist (DPCPX), it is possible to upregulate and block the upregulation of A1AR and different isoforms of PKCs. PKC consists of a family of at least 11 isoenzymes, and it has become increasingly clear that many of the functions of PKC reflect the action of specific isoenzymes. The distribution of these isoenzymes varies among cell types, and even the function of a particular isoenzyme may differ from one cell to another. To establish which isoenzymes are present in PCMSC as well as which isoenzymes are upregulated through A1 adenosine receptor agonist (ENBA) treatment, we evaluated various PKC isoforms using Western blot technique. There are several studies (Lu et al., 1996; Sasaguri et al., 1993; Webb et al., 1997a,b) in which the isotypes of PKC in smooth muscle cells have been reported from a variety of vascular sources other than porcine coronary artery. Further, the present study specifically provides the first evidence for the presence of different isoforms of PKC (a, bI, bII, m, g, d, e, and z) and the regulation of several PKC isoforms (a, bI, bII, g, e, and z) through A1 adenosine receptor activation through ENBA treatment in PCSMC. A1 adenosine receptor agonist ENBA stimulates upregulation of both Ca 2+-dependent and Ca 2+-independent PKC isoforms in PCSMC. Immunoblot analysis demonstrated that PKC-a, bI, bII, g, e, and z isoforms are the isoforms responsible for ENBA-induced upregulation of PKC in PCSMC. Interestingly, upregulation of A1 adenosine receptor in ENBAtreated PCSMC was also observed. It is well known that desensitization of A1AR occurs due to chronic exposure to A1AR agonist. This could be simply due to the expression of A1AR without any functional activity. This may lead to a cascade of signaling events causing an upregulation of specific PKC isoforms. The existence of multiple isotypes provide an explanation for the many and diverse physiological actions that follow activation of PKC. In vascular smooth muscle cells, proliferation and migration are only two of the many such cellular functions stimulated by PKC. Other includes induction of smooth muscle cells contraction, secretion of anticoagulant substances such as PGI2 and tissue plasminogen activator, and production of extracelullar matrix (Pomerantz et al.,
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1997). It is conceivable that each of these functions is mediated by a different isotype of PKC. There is evidence in other cells types as well as smooth muscle cells that the various isotypes of PKC have distinct physiological effects (Horowitz et al., 1996). Bovine aortic smooth muscle cells express at least five (5) PKC isoforms, a, bI, bII, d, e, and z during mechanical strain (Assender et al., 1994; Khalil et al., 1992; Paul et al., 1997; Sasaguri et al., 1993), with the predominant expression of PKC-a and -z isoforms (Assender et al., 1994; Paul et al., 1997). Similarly, in the present study, we found upregulation of at least six (6) PKC isoforms, a, bI, bII, g, e, and z through activation of A1 adenosine receptor out of eight PKC isoforms, a, bI, bII, m, g, d, e, and z identified, with the predominant expression of PKC-e-g and -z in PCSMC. PKC-z is known to be critically important for regulating a number of cellular functions, including proliferation and activation of nuclear factor NF-kB (Dominguez et al., 1993). In the present study, increased expression of PKC-z through activation of A1 adenosine receptor by 6.09-, 38.72-, and 52.82-fold with A1 adenosine receptor agonist (ENBA) at 109, 106, and 105 M, respectively, when compared with nontreated control (Fig. 7B) is highly significant. Previous reports indicated that PKC-z acts downstream of Ras in the kinase cascade leading to mitogenesis (Berra et al., 1995). Stimulation of Ras initiates a kinase cascade that culminates in the activation of MAP kinase, which is required for cell growth and other cell responses. However, the specific cellular function of PKC-z and other PKC isoforms (a, bI, bII, g, and e) upregulation through activation of A1 adenosine receptor activation in PCSMC have yet to be determined. In summary, activation of A1 adenosine receptor leads to an upregulation of A1 adenosine receptor protein and PKC isoforms (a, bI, bII, g, e, and z) shown in this study. Even though the exact mechanism of upregulation of these isoforms for such an action of A1 adenosine receptor remains vague. On the other hand PKC d and m were not affected by A1 adenosine receptor activation. Eight out of 11 currently known isoforms of PKC were identified in PCSMC. Acknowledgements The authors would like to thank Weixi Qin for technical help in this work. This work was supported by HL 27339 from National Institutes of Health to S.J. Mustafa. M.A. Nayeem was supported by beginning-grant-in-aid from American Heart Association (mid-Atlantic) and a starter grant from East Carolina University, Brody School of Medicine. References Assender, J.W., Kontny, E., Fredholm, B.B., 1994. Expression of protein kinase C isoforms in smooth muscle cells in various states of differentiation. FEBS Lett. 342, 76 – 80.
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Protein kinase C isoforms and A1 adenosine receptors in porcine coronary smooth muscle cells Mohammed A. Nayeem, S. Jamal Mustafa* Department of Pharmacology, Brody School of Medicine, East Carolina University, Greenville, NC 27858-2735, USA Received 1 November 2001; received in revised form 1 April 2002; accepted 1 April 2002
Abstract We have previously reported that prolonged exposure of porcine coronary arteries to adenosine agonists upregulates protein kinase C (PKC) through the activation of adenosine A1 receptor-coupled to pertussis toxin sensitive G-protein(s) [Am. J. Physiol. 264 (1993) H1465; Am. J. Physiol. 269 (1995) H1619]. The mechanism(s) by which A1 adenosine receptor upregulates PKC (isoforms) are not yet clearly understood. In the present study, we identified the a, b1, b2, g, e, and z PKC isoforms that were upregulated by adenosine A1 receptor agonist as a possible mechanism(s) involved for this upregulation. Incubation of porcine coronary smooth muscle cells (PCSMC) with adenosine A1 receptor agonist (2s)-N 6-[2-endo-norbornyl]adenosine (ENBA) caused an upregulation of PKC (isoforms), which were blocked by adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Western blot analysis using specific antibodies to PKC isoforms indicated that all the isoforms tested (a, bI, bII, m, g, d, e, and z) were present in the primary cultured smooth muscle cells from porcine coronary artery. Western blot studies indicated that PKC a, bI, bII, g, e, and z isoforms were upregulated in a dose dependent manner by adenosine agonist (ENBA) and PKC d and m were not altered. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Protein kinase C; Porcine coronary smooth muscle cells; A1 adenosine receptor
1. Introduction Adenosine, a natural nucleoside, is produced from cardiac myocytes and produces a variety of responses including bradycardia, hypotension, and coronary vasodilation (Mustafa, 1980). Adenosine recognizes specific cell surface receptors originally classified as A1 and A2 subtypes. This classification was based on the comparison of the agonist potency profiles and their ability to modulate adenylate cyclase. The high affinity adenosine receptors (A1) inhibit adenylate cyclase, whereas the low affinity receptors (A2) stimulate the cyclase (Londos et al., 1980). Since then, however, at least four adenosine receptor subtypes (A1, A2A, A2B, and A3) have been identified and cloned from various species (Linden et al., 1993). All these receptor subtypes are coupled to guanine nucleotide binding proteins (G-proteins) (Sabouni et al., 1989, 1991; Hussain and Mustafa, 1993; Marala and Mustafa, 1993). Subsequent to the binding to its receptor(s) * Corresponding author. Tel.: +1-252-816-2736; fax: +1-252-8163203. E-mail address: [email protected] (S.J. Mustafa).
adenosine initiates a cascade of events for its signal transduction. The most characterized mechanism is the effect on adenylate cyclase (Hussain and Mustafa, 1993; Sabouni et al., 1991; Marala and Mustafa, 1993). In addition, yet another system involving protein kinase C (PKC) has been postulated to play a key role in adenosine signalling. Pretreatment of porcine coronary artery with adenosine or its analogs (2-chloroadenosine and NECA) attenuated the phorbol ester induced contractions through receptor mechanism (Crushing et al., 1991). Phorbol esters are known to activate PKC directly, independent of any receptor systems (Marala and Mustafa, 1995a,b). Downregulation of PKC attenuated contractions produced by agents like PDBu, KCl, and endothelin in porcine arterial rings (Marala et al., 1993). Bradley et al. (1991) demonstrated that adenosine prevents phorbol ester-induced injury to rabbit lung without altering the levels of leukotrienes and tumor necrotic factor. Earlier studies from this laboratory have demonstrated that adenosine analog (CAD) regulates PKC in porcine coronary artery (Marala et al., 1993). In this study, CAD was shown to protect the activation and depletion of PKC by PDBu (Marala et al., 1993). It also demonstrated that
1537-1891/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 7 - 1 8 9 1 ( 0 2 ) 0 0 3 0 7 - 5
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prolonged exposure to CAD alone causes the upregulation of PKC in porcine coronary artery (Marala et al., 1993), a phenomenon that could explain the protection against the depletion of PKC during prolonged exposure to PDBu. Using relatively specific A1 and A2A receptor agonists (ENBA and CGS 21680, respectively) and antagonists (N0861 or DPCPX and DMPX, respectively), the receptor subtype involved in such upregulation of PKC was established to be A1 subtype (Marala and Mustafa, 1995a,b). ENBA [(2s)-N6-[2-endo-norbornyl]adenosine], an A1 receptor agonist, has been demonstrated to be of high affinity (Kd=0.3 nM) and selectivity (4700-fold) (Trivedi et al., 1989). CGS 21680 [2-p-(2-carboxyethyl)phenethylamino50-N-ethylcarboxamidoadenosine] was used as A2 receptor agonist, since it was demonstrated to bind to the receptor subtype with high selectivity (>170-fold) and low affinity (Kd=15.5 nM). In subsequent studies, bacterial toxins (cholera and pertussis) were used to covalently modify G-proteins in order to identify the G-protein(s) involved in mediation of the adenosine A1 receptor induced upregulation of PKC in porcine coronary artery (Marala and Mustafa, 1995a,b). These studies demonstrated that the effects of A1 selective agonist ENBA could be blocked by preincubation with pertussis toxin, indicating the involvement of pertussis toxin-sensitive Gi/Go protein(s) (Marala and Mustafa, 1995a,b). The exact mechanism(s) by which adenosine upregulates PKC are not yet clearly understood. This study was, therefore, designed to further elucidate the PKC isoforms involved with the activation A1 adenosine receptor for such signal transduction pathways.
dissected from each heart and cleaned of the adherent fat and connective tissue. 2.2.1. Isolation of PCSMC Smooth muscle cells from porcine coronary artery were isolated and cultured as described by Marala and Mustafa (1998) in this laboratory with minor modifications. Briefly, the arteries were cut open longitudinally and the endothelium was removed by rubbing the internal surface gently with scalpel blade. Tissue was rinsed twice with sterile Hank’s balanced salt solution (HBSS) containing (in mM): KCl 5.0, KH2PO4 0.3, NaCl 138, NaHCO3 4.0, Na2HPO47H2O 0.3, D-glucose 5.6, and HEPES 10.0. Tissue was then soaked for 10 min at ambient temperature in sterile HBSS. Arteries were minced with the scissors and incubated for 3 to 4 h at 37 C with vigorous shaking in sterile HBSS containing 1 mg/ml collagenase type II (300 U/mg), 0.5 mg/ml soybean trypsin inhibitor, and 3% bovine serum albumin. Then, it was filtered through nylon gauze and centrifuged at 100g for 10 min. The pellet was washed twice with Dulbecco’s modified Eagle’s medium (DMEM, GIBCO-BRL) and finally resuspended in DMEM containing 10% FBS and cultured at 37 C in 95% O2+5% CO2 incubator. Media was changed twice a week and, when the cells became confluent (about 1 week), they were split at 1:3 ratio using trypsin (0.25%). 2.2.2. Experimental protocol PCSMC were incubated in DMEM for 24 h with A1 agonist ENBA (109, 106, and 105 M) at 37 C and washed out with HBSS. In another group, ENBA+DPCPX
2. Materials and methods 2.1. Materials DPCPX and ENBA were purchased from Research Biochemicals International (Natick, MA). RPN 77 kit was fetal bovine serum (FBS) and purchased from Amersham (Arlington Heights, IL). DMEM, anti-PKC a, b1, b2, m, g, d, e, and z antibodies (GIBCO BRL), polyclonal antibody cross-reacting to adenosine A1 receptor protein (Alpha Diagnostic, San Antonio, TX) and were purchased. All other reagents were of analytical grade and purchased commercially. 2.2. Tissue preparation Porcine hearts from either sex were obtained from the local slaughterhouse within 30 min of sacrifice and transported to the laboratory in ice-cold Krebs-Henseleit (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 10 mM glucose, pH 7.4) oxygenated with 95% O2+5% CO2. The left anterior descending (LAD) and circumflex coronary arteries were
Fig. 1. (A) represents Western blot analysis, whereas (B) represents the densitometric analysis (mean of two) in arbitrary units for A1AR (36 kDa). Samples were taken from PCSMC after ENBA (105 M) alone, ENBA (105 M)+DPCPX (1 mM) for 24-h treatment.
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(1 mM, A1 antagonist) were incubated together with PCSMC for 24 h. 2.2.3. Gel electrophoresis and Western blotting Cells were rinsed with PBS and lysed with boiling lysis solution, containing 1% SDS, 1 mM sodium vanadate, and 10 mM Tris and pH 7.4. Cells were scraped and transferred to a microcentrifuge tube and boiled for an additional 5 min. The samples were then sonicated briefly and centrifuged (12,000g 15 C) for 5 min. Protein was measured using Bio-Rad protein assay based on the Bradford dye binding procedure with bovine serum albumin as a standard. The protein was divided into aliquots and stored at 80 C. At the time of analysis, experimental samples as well as pig brain (positive controls, data not shown) were thawed and approximately 20 mg of total protein per lane was loaded on a slab gel. Proteins were separated by sodium dodecyl sulfate-
Fig. 3. (A) represents Western blot analysis for bI-PKC (80 kDa), whereas (B) represents the densitometric analysis (mean of two) in arbitrary units. Samples were taken from PCSMC after ENBA (105, 106 and 109 M) treatment for 24 h. (C) represents Western blot analysis after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h, whereas (D) represents the densitometric analysis (mean of two) in arbitrary units.
Fig. 2. (A) represents Western blot analysis for a-PKC (82 kDa), whereas (B) represents the densitometric analysis (mean of two) in arbitrary units. Samples were taken from PCSMC after ENBA (105, 106 and 109 M) treatment. (C) represents Western blot analysis after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h, whereas (D) represents the densitometric analysis (mean of two) in arbitrary units.
polyacrylamide gel electrophoresis (SDS-PAGE) using 10% acrylamide gels (1-mm-thick). After electrophoresis, the proteins on the gel were transferred to polyvinyllidene difluoride (PVDF) membrane (Schleicher and Schuell, Keene, NH) by electroelution. Protein transfer was confirmed by employing prestained molecular weight markers (Bio-Rad Laboratories, Hercules, CA). Complete transfer of the protein was ascertained by staining the gel in Coomassie blue and transfer of prestained molecular weight markers on the PVDF membrane. Following blocking with nonfat dry milk, the PVDF membranes were incubated with monoclonal and polyclonal antibodies cross-reacting to adenosine A1 receptor protein, PKC isoforms (a, bI, bII, m, g, d, e, and z). The second antibody was a horseradish peroxidase conjugated antirabbit IgG. The membranes were developed using enhanced chemiluminescence (Amersham Life Sciences, CA) and exposed to X-ray film for appropriate time. As for as all the experiments regarding A1 adenosine receptor agonist (ENBA) for the receptor activation and antagonist
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(DPCPX) for the blocking of receptor activation related to expression of A1AR, and various PKC isoforms are concerned we chose only one concentration of ENBA (105 M) and one concentration of DPCPX (1 mM). Whereas in other sets of experiments we chose different concentrations of ENBA (109, 106, and 105 M) in relation to various level of expression of different PKC isoforms in PCSMC.
3. Results Incubation of PCSMC (2 –3 passages) with ENBA (105 M) alone for 24 h caused an elevation of A1 adenosine receptor expression (36 kDa) compared to control (Fig. 1A). To block A1 adenosine receptor activation in PCSMC, the cells were treated with ENBA+DPCPX together for 24 h, which caused an attenuation of A1 adenosine receptor expression (Fig. 1A). The densitometric scan analysis of the Western blot revealed 1.45-fold increase in expression of A1 adenosine receptor with ENBA treatment when compared to nontreated controls (Fig. 1B) and this upregulation of A1 adenosine receptor with ENBA was blocked by DPCPX treatment (Fig. 1B). In a separate experiment, the data suggest that the antibody we used in this report is solely recognizing the A1 adenosine receptor. Experiments were performed with A1 adenosine receptor antibody displacement (data not shown.) using corresponding peptide to rat A1AR gene, provided by Alpha Diagnostic International, San Antonio, TX. Fig. 2A shows a dose-dependent (109, 106, and 105 M of ENBA) upregulation of PKC a (82 kDa) in PCSMC compared to control. The densitometric scan analysis of the Western blot revealed a concentration-dependant increase in
Fig. 4. (A) represents Western blot analysis, whereas (B) represents the densitometric analysis (mean of two) in arbitrary units for bII-PKC (80 kDa). Samples were taken from PCSMC after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h.
Fig. 5. (A) represents Western blot analysis for e-PKC (90 kDa), whereas (B) represents the densitometric analysis (mean of two) in arbitrary units. Samples were taken from PCSMC after ENBA (105, 106 and 109 M) treatment for 24 h. (C) represents Western blot analysis after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h, whereas (D) represents the densitometric analysis (mean of two) in arbitrary units.
expression of PKC a (82 kDa) by 1.06 – 1.31- and 1.79-fold with ENBA at 109, 106, and 105 M, respectively, when compared to nontreated control (Fig. 2B). To block adenosine receptor in PCSMC, the cells were incubated with ENBA (105 M) +DPCPX (1 mM) for 24 h, which caused an attenuation of PKC a (Fig. 2C). The densitometric scan analysis of the Western blot revealed 1.45-fold increase in expression of PKC a with ENBA treatment when compared to nontreated controls (Fig. 2D) and this upregulation of PKC a with ENBA was blocked by DPCPX treatment (Fig. 2D). In separate experiments, the data suggest that the antibodies we used in this report are solely recognizing the PKC isoforms (a, bI, bII, m, g, d, e, and z, data not shown). Treatment with ENBA (109, 106, and 105 M) shows a concentration related increase of PKC bI (80 kDa, Fig. 3A) and the densitometric scan analysis of the Western blot revealed a concentration-dependent increase in expression of PKC bI by 1.03– 1.15- and 2.10-fold, respectively, com-
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pared to nontreated PCSMC (Fig. 3B). DPCPX attenuated PKC bI expression in ENBA treated PCSMC (Fig. 3C). The densitometric scan analysis of the Western blot revealed 2.25-fold increase in expression of PKC bI with ENBA treatment when compared to nontreated controls (Fig. 3D) and this upregulation of PKC bI with ENBA was blocked with DPCPX treatment (1.45-fold, Fig. 3D). Incubation of PCSMC with ENBA (105 M) alone for 24 h caused a slight elevation of PKC bII (80 kDa) compared to control (Fig. 4A). The cells were incubated with ENBA+DPCPX for 24 h, which caused a significant downregulation of PKC bII expression (Fig. 4A). The densitometric scan analysis of the Western blot revealed a slight increase in expression of PKC bII with ENBA treatment when compared to nontreated controls (Fig. 4B) and this slight upregulation of PKC bII with ENBA was followed by a significant downregulation of bII with DPCPX treatment (Fig. 4B). Fig. 5A shows concentration-dependent (109, 106, and 5 10 M of ENBA) upregulation of PKC e (90 kDa) in PCSMC compared to control. The densitometric scan analysis of the Western blot revealed a concentration-dependent increase in expression of PKC e (90 kDa) by 1.73 –3.60- and 4.68-fold with ENBA at 109, 106, and 105 M, respectively, when compared to nontreated control (Fig. 5B). To block A1 adenosine receptor (36 kDa), the cells were incubated with ENBA+DPCPX (1 mM, A1 adenosine receptor antagonist) for 24 h, which caused an attenuation of PKC e (Fig. 5C). The ENBA (105 M, 4.68 fold) increased expression of PKC e was blocked with DPCPX treatment (2.12fold, Fig. 5D). Treatment with ENBA (109, 106, and 105 M) shows a concentration-dependent increase of PKC g (80 kDa, Fig. 6A) by 4.19- and 5.91-fold compared to nontreated PCSMC (Fig. 6B), respectively. No further change of PKC g expres-
Fig. 6. (A) represents Western blot analysis, whereas (B) represents the densitometric analysis (mean of two) in arbitrary units for g-PKC (80 kDa). Samples were taken from PCSMC after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h.
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Fig. 7. (A) represents Western blot analysis for z-PKC (72 kDa), whereas (B) represents the densitometric analysis (mean of two) in arbitrary units. Samples were taken from PCSMC after ENBA (105, 106 and 109 M) treatment for 24 h. (C) represents Western blot analysis after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h, whereas (D) represents the densitometric analysis (mean of two) in arbitrary units.
sion was noted between 106 and 109 M ENBA, these doses may be less than a required threshold dose (105 M) of ENBA to activate A1 adenosine receptor for the upregulation of PKC g. Fig. 7A shows dose-dependent (109, 106, and 105 M of ENBA) upregulation of PKC z (72 kDa) in PCSMC compared to control. The densitometric scan analysis of the Western blot revealed a concentration-dependent increase in expression of PKC z (72 kDa) by 6.09 –38.72- and 52.82fold with ENBA at 109, 106, and 105 M, respectively, when compared to nontreated control (Fig. 7B). The cells were incubated with ENBA+DPCPX (1 mM, A1 adenosine receptor antagonist) for 24 h, which caused an attenuation of PKC z (Fig. 7C). The densitometric scan analysis of the Western blot revealed only a three-fold increase in expression of PKC z with ENBA (105 M) treatment when compared to nontreated controls (52.82-fold, Fig. 7D) and blocked by DPCPX (Fig. 7D).
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Fig. 8. (A) represents Western blot analysis, whereas (B) represents the densitometric analysis (mean of two) in arbitrary units for d-PKC (78 kDa). Samples were taken from PCSMC after ENBA (105 M) alone, ENBA (105 M) +DPCPX (1 mM) treatment for 24 h. (C) represents Western blot analysis, whereas (D) represents the densitometric analysis (mean of two) in arbitrary units for m-PKC (115 kDa).
Treatment of PCSMC with ENBA (109, 106, and 105 M) shows no change of PKC d (78 kDa, Fig. 8A and B) compared to nontreated group. Similarly, there was no change in the level of PKC m in ENBA-treated compared to nontreated PCSMC (Fig. 8C and D). These data served as negative control.
4. Discussion This study demonstrated the role of adenosine-receptor subtype in the upregulation and downregulation of several isoforms of PKC in PCSMC. It also provides the evidence that the upregulation of several isoforms of PKC is a receptor-mediated phenomenon and A1 adenosine receptors are responsible for such an effect. Additionally, this study also demonstrates that DPCPX (A1 adenosine receptor antagonist) blocked this upregulation of these PKC isoforms. PKC has been implicated to play an important role in adenosine receptor signal transduction pathways. Earlier studies
from this laboratory demonstrated that porcine coronary arterial rings, when preincubated with adenosine analogues [2-chloroadenosine and 50-(N-ethylcarboxamido) adenosine] caused an attenuation of phorbol ester-induced contractions, thus, indicating a possible role of PKC (Crushing et al., 1991). It has also been previously shown from this laboratory that adenosine analogue 2-chloroadenosine blocked the activation of PKC (translocation from cytosol to membrane) upon short-term incubation (20 min), whereas upon longterm incubation (24 – 48 h), it produced an upregulation of PKC in porcine coronary artery (Marala and Mustafa, 1995a; Marala et al., 1993). Prolong exposure to adenosine analogues also partially blocked the phorbol ester-induced PKC depletion (Marala and Mustafa, 1995b; Marala and Mustafa, 1993). In the present study with the use of relatively specific A1-receptor agonist (ENBA) and antagonist (DPCPX), it is possible to upregulate and block the upregulation of A1AR and different isoforms of PKCs. PKC consists of a family of at least 11 isoenzymes, and it has become increasingly clear that many of the functions of PKC reflect the action of specific isoenzymes. The distribution of these isoenzymes varies among cell types, and even the function of a particular isoenzyme may differ from one cell to another. To establish which isoenzymes are present in PCMSC as well as which isoenzymes are upregulated through A1 adenosine receptor agonist (ENBA) treatment, we evaluated various PKC isoforms using Western blot technique. There are several studies (Lu et al., 1996; Sasaguri et al., 1993; Webb et al., 1997a,b) in which the isotypes of PKC in smooth muscle cells have been reported from a variety of vascular sources other than porcine coronary artery. Further, the present study specifically provides the first evidence for the presence of different isoforms of PKC (a, bI, bII, m, g, d, e, and z) and the regulation of several PKC isoforms (a, bI, bII, g, e, and z) through A1 adenosine receptor activation through ENBA treatment in PCSMC. A1 adenosine receptor agonist ENBA stimulates upregulation of both Ca 2+-dependent and Ca 2+-independent PKC isoforms in PCSMC. Immunoblot analysis demonstrated that PKC-a, bI, bII, g, e, and z isoforms are the isoforms responsible for ENBA-induced upregulation of PKC in PCSMC. Interestingly, upregulation of A1 adenosine receptor in ENBAtreated PCSMC was also observed. It is well known that desensitization of A1AR occurs due to chronic exposure to A1AR agonist. This could be simply due to the expression of A1AR without any functional activity. This may lead to a cascade of signaling events causing an upregulation of specific PKC isoforms. The existence of multiple isotypes provide an explanation for the many and diverse physiological actions that follow activation of PKC. In vascular smooth muscle cells, proliferation and migration are only two of the many such cellular functions stimulated by PKC. Other includes induction of smooth muscle cells contraction, secretion of anticoagulant substances such as PGI2 and tissue plasminogen activator, and production of extracelullar matrix (Pomerantz et al.,
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1997). It is conceivable that each of these functions is mediated by a different isotype of PKC. There is evidence in other cells types as well as smooth muscle cells that the various isotypes of PKC have distinct physiological effects (Horowitz et al., 1996). Bovine aortic smooth muscle cells express at least five (5) PKC isoforms, a, bI, bII, d, e, and z during mechanical strain (Assender et al., 1994; Khalil et al., 1992; Paul et al., 1997; Sasaguri et al., 1993), with the predominant expression of PKC-a and -z isoforms (Assender et al., 1994; Paul et al., 1997). Similarly, in the present study, we found upregulation of at least six (6) PKC isoforms, a, bI, bII, g, e, and z through activation of A1 adenosine receptor out of eight PKC isoforms, a, bI, bII, m, g, d, e, and z identified, with the predominant expression of PKC-e-g and -z in PCSMC. PKC-z is known to be critically important for regulating a number of cellular functions, including proliferation and activation of nuclear factor NF-kB (Dominguez et al., 1993). In the present study, increased expression of PKC-z through activation of A1 adenosine receptor by 6.09-, 38.72-, and 52.82-fold with A1 adenosine receptor agonist (ENBA) at 109, 106, and 105 M, respectively, when compared with nontreated control (Fig. 7B) is highly significant. Previous reports indicated that PKC-z acts downstream of Ras in the kinase cascade leading to mitogenesis (Berra et al., 1995). Stimulation of Ras initiates a kinase cascade that culminates in the activation of MAP kinase, which is required for cell growth and other cell responses. However, the specific cellular function of PKC-z and other PKC isoforms (a, bI, bII, g, and e) upregulation through activation of A1 adenosine receptor activation in PCSMC have yet to be determined. In summary, activation of A1 adenosine receptor leads to an upregulation of A1 adenosine receptor protein and PKC isoforms (a, bI, bII, g, e, and z) shown in this study. Even though the exact mechanism of upregulation of these isoforms for such an action of A1 adenosine receptor remains vague. On the other hand PKC d and m were not affected by A1 adenosine receptor activation. Eight out of 11 currently known isoforms of PKC were identified in PCSMC. Acknowledgements The authors would like to thank Weixi Qin for technical help in this work. This work was supported by HL 27339 from National Institutes of Health to S.J. Mustafa. M.A. Nayeem was supported by beginning-grant-in-aid from American Heart Association (mid-Atlantic) and a starter grant from East Carolina University, Brody School of Medicine. References Assender, J.W., Kontny, E., Fredholm, B.B., 1994. Expression of protein kinase C isoforms in smooth muscle cells in various states of differentiation. FEBS Lett. 342, 76 – 80.
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