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20-Hydroxyeicosatetraenoic Acid-induced Vasoconstriction and Inhibition of Potassium Current in Cerebral Vascular Smooth Muscle Is Dependent on Activation of Protein Kinase C

1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês

10.1074/jbc.272.43.27345

1083-351X

Andrew R. Lange, Debebe Gebremedhin, Jayashree Narayanan, David R. Harder,

Cancer, Hypoxia, and Metabolism

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 metabolite of arachidonic acid, is a potent vasoconstrictor, and has been implicated in the myogenic activation of renal and cerebral arteries. We examined the role of protein kinase C (PKC) in the signal transduction pathway by which 20-HETE induces vasoconstriction and inhibition of whole-cell K+current in cat cerebral vascular smooth muscle. 20-HETE induced a concentration-dependent constriction in isolated pressurized cat middle cerebral arteries (−29 ± 87 at 1 ॖm). However, in the presence of anN-myristoylated PKC pseudosubstrate inhibitor peptide (MyrΨPKC-I(19–27)), 20-HETE induced a concentration-dependent vasodilation (26 ± 47 at 1 ॖm). In whole-cell voltage clamp studies, application of 20-HETE inhibited whole-cell K+ current recorded in cat cerebral vascular smooth muscle cells, an effect that was attenuated by MyrΨPKC-I(19–27). Further evidence for the role of PKC activation in response to 20-HETE is the finding that 20-HETE increased the phosphorylation of myristoylated, alanine-rich PKC substrate in cultured cat cerebral vascular smooth muscle cells in a concentration- and PKC-dependent manner. These data provide evidence that PKC is an integral part of the signal transduction pathway by which 20-HETE elicits vasoconstriction of cerebral arteries and inhibition of whole-cell K+ current in cat cerebral vascular smooth muscle. 20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 metabolite of arachidonic acid, is a potent vasoconstrictor, and has been implicated in the myogenic activation of renal and cerebral arteries. We examined the role of protein kinase C (PKC) in the signal transduction pathway by which 20-HETE induces vasoconstriction and inhibition of whole-cell K+current in cat cerebral vascular smooth muscle. 20-HETE induced a concentration-dependent constriction in isolated pressurized cat middle cerebral arteries (−29 ± 87 at 1 ॖm). However, in the presence of anN-myristoylated PKC pseudosubstrate inhibitor peptide (MyrΨPKC-I(19–27)), 20-HETE induced a concentration-dependent vasodilation (26 ± 47 at 1 ॖm). In whole-cell voltage clamp studies, application of 20-HETE inhibited whole-cell K+ current recorded in cat cerebral vascular smooth muscle cells, an effect that was attenuated by MyrΨPKC-I(19–27). Further evidence for the role of PKC activation in response to 20-HETE is the finding that 20-HETE increased the phosphorylation of myristoylated, alanine-rich PKC substrate in cultured cat cerebral vascular smooth muscle cells in a concentration- and PKC-dependent manner. These data provide evidence that PKC is an integral part of the signal transduction pathway by which 20-HETE elicits vasoconstriction of cerebral arteries and inhibition of whole-cell K+ current in cat cerebral vascular smooth muscle. Arachidonic acid metabolites of the cytochrome P450 monooxygenase pathway have recently been found to play a major role in modulating vascular tone in the renal and cerebral circulations (1Harder D.R. Narayanan J. Gebremedhin D. Roman R.J. Trends Cardiovasc. Med. 1995; 5: 7-14Crossref PubMed Scopus (45) Google Scholar, 2Roman R.J. Harder D.R. J. Am. Soc. Nephrol. 1993; 4: 986-996PubMed Google Scholar, 3Harder D.R. Campbell W.B. Roman R.J. J. Vasc. Res. 1995; 32: 79-92Crossref PubMed Scopus (280) Google Scholar). The major cytochrome P450 metabolite of arachidonic acid produced in the cerebral and renal vasculature is 20-hydroxyeicosatetraenoic acid (20-HETE) 1The abbreviations used are: 20-HETE, 20-hydroxyeicosatetraenoic acid; KCa, large conductance, calcium-activated potassium channel; PKC, protein kinase C; MARCKS, myristoylated, alanine-rich PKC substrate; VSMC, vascular smooth muscle cell; PSS, physiological salt solution; PMA, phorbol 12-myristate 13-acetate; MyrΨPKC-I(19–27), N-myristoylated PKC pseudosubstrate inhibitor peptide. (4Harder D.R. Gebremedhin D. Narayanan J. Jefcoat C. Falck J.R. Campbell W.B. Roman R. Am. J. Physiol. 1994; 266: H2098-H2107PubMed Google Scholar, 5Imig J.D. Zou A.P. Stec D.E. Harder D.R. Falck J.R. Roman R.J. Am. J. Physiol. 1996; 270: R217-R227Crossref PubMed Google Scholar, 6Ma Y.H. Gebremedhin D. Schwartzman M.L. Falck J.R. Clark J.E. Masters B.S. Harder D.R. Roman R.J. Circ. Res. 1993; 72: 126-136Crossref PubMed Google Scholar). 20-HETE is a potent vasoconstrictor in isolated cat cerebral and rat renal microvessels over the concentration range of 10−11 to 10−9m (4Harder D.R. Gebremedhin D. Narayanan J. Jefcoat C. Falck J.R. Campbell W.B. Roman R. Am. J. Physiol. 1994; 266: H2098-H2107PubMed Google Scholar, 5Imig J.D. Zou A.P. Stec D.E. Harder D.R. Falck J.R. Roman R.J. Am. J. Physiol. 1996; 270: R217-R227Crossref PubMed Google Scholar). The underlying cellular-ionic mechanism of this vasoconstrictor response appears to be depolarization-induced influx of calcium secondary to inhibition of large conductance calcium-activated potassium channels (KCa) (4Harder D.R. Gebremedhin D. Narayanan J. Jefcoat C. Falck J.R. Campbell W.B. Roman R. Am. J. Physiol. 1994; 266: H2098-H2107PubMed Google Scholar, 6Ma Y.H. Gebremedhin D. Schwartzman M.L. Falck J.R. Clark J.E. Masters B.S. Harder D.R. Roman R.J. Circ. Res. 1993; 72: 126-136Crossref PubMed Google Scholar, 7Zou A.P. Fleming J.T. Falck J.R. Jacobs E.R. Gebremedhin D. Harder D.R. Roman R.J. Am. J. Physiol. 1996; 270: R228-R237PubMed Google Scholar). Independent of the depolarization induced by inhibitory effects on KCa, recent data indicate that 20-HETE also activates L-type calcium channels in a concentration-dependent manner, an effect that is antagonized by nifedipine (8Harder D.R. Lange A.R. Gebremedhin D. Birks E.K. Roman R.J. J. Vasc. Res. 1997; 34: 237-243Crossref PubMed Scopus (150) Google Scholar, 9Gebremedhin D. Lange A.R. Narayanan J. Jacobs E.R. Harder D.R. J. Physiol. (Lond.). 1997; (in press)Google Scholar). Several reports identify a role for 20-HETE in the regulation of renal tubular ion transport. In cells of the thick ascending limb of the rat kidney, 20-HETE decreases the open state probability of an apical 70 pS K+ channel (10Wang W. Lu M. J. Gen. Physiol. 1995; 106: 727-743Crossref PubMed Scopus (100) Google Scholar), thus regulating K+ recycling across the membrane and Na+ resorption. In the medullary thick ascending limb of the loop of Henle, Na+-K+-(NH4+)-2Cl−transport activity is reduced by 20-HETE (11Amlal H. Legoff C. Vernimmen C. Paillard M. Bichara M. Am. J. Physiol. 1996; 271: C455-C463Crossref PubMed Google Scholar). In proximal tubular epithelial cells, the activity of the Na+-K+ATPase is reduced by 20-HETE, an effect that is dependent upon activation of protein kinase C (PKC) (12Ominato M. Satoh T. Katz A.I. J. Membr. Biol. 1996; 152: 235-243Crossref PubMed Scopus (120) Google Scholar, 13Ribeiro C.M.P. Dubay G.R. Falck J.R. Mandel L.J. Am. J. Physiol. 1994; 266: F497-F505PubMed Google Scholar, 14Nowicki S. Chen S. Aizman O. Cheng X. Li D. Nowicki C. Nairn A. Greengard P. Aperia A. J. Clin. Invest. 1997; 99: 1224-1230Crossref PubMed Scopus (152) Google Scholar). These observations implicate 20-HETE in a diverse array of effector functions. However, the exact signal transduction pathway by which 20-HETE exerts these effects is unknown. Most of the effects described above could be related to an increased activity of PKC (15Liu J.-P. Mol. Cell. Endocrinol. 1996; 116: 1-29Crossref PubMed Scopus (213) Google Scholar, 16Blobe G.C. Khan W.A. Hannun Y.A. Prostaglandins Leukotrienes Essent. Fatty Acids. 1995; 52: 129-135Abstract Full Text PDF PubMed Scopus (46) Google Scholar, 17Khan W.A. Blobe G.C. Hannun Y.A. Cell. Signal. 1995; 7: 171-184Crossref PubMed Scopus (219) Google Scholar). Because severalcis-unsaturated fatty acids, including arachidonic acid and its metabolites, activate PKC (18Hansson A. Serhan C.N. Haeggstrom J. Ingelman-Sundberg M. Samuelsson B. Morris J. Biochem. Biophys. Res. Commun. 1986; 134: 1215-1222Crossref PubMed Scopus (171) Google Scholar, 19Murakami K. Chan S.Y. Routtenberg A. J. Biol. 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Pharmacol. 1994; 72: 1392-1399Crossref PubMed Scopus (91) Google Scholar), we hypothesize that the effects of 20-HETE on cerebral arterial tone and whole-cell K+ channel current involve activation of PKC. In this report, we provide functional evidence indicating that 20-HETE promotes cerebral vasoconstriction and inhibition of whole-cell K+ current via a pathway that involves PKC. We also provide biochemical evidence that 20-HETE increases the phosphorylation of myristoylated, alanine-rich PKC substrate (MARCKS) in cultured cat cerebral vascular smooth muscle cells (VSMCs) in a concentration-related and PKC-dependent manner. Isolated cat middle cerebral arteries (outside diameter, 200–400 ॖm; length, 10–12 mm) were placed in a perfusion chamber, cannulated with glass micropipettes, and secured in place with 8-O polyethylene suture (Ethicon, Inc., Somerville, NJ), and side branches were tied off with 10-O polyethylene suture using a stereomicroscope (Carl Zeiss, Inc., Berlin, Germany). The arterial segments were bathed in physiological salt solution (PSS) equilibrated with a 957 O2-57 CO2 gas mixture at 37 °C. During the experiment, the outflow cannula was clamped off, and the vessels were pressurized to 80 mm Hg. The inflow cannula was connected in series with a volume reservoir and a pressure transducer (Gould Instruments Division, Cleveland, Ohio) to monitor intraluminal pressure. Internal diameters of the vessels were measured with a video system composed of a CCTV camera (KP-130AU, Hitachi, Tokyo, Japan), a TV monitor (CVM-1271, Sony, Tokyo, Japan), and a videomicrometer system (model 305, Colorado Video, Inc., Boulder, CO). After an equilibration period of 30 min, the arterial segments were preconstricted with 5 ॖmserotonin, and the vasodilator response to acetylcholine (1 ॖm) was determined. Vessels that exhibited no response to either serotonin or acetylcholine were excluded from the study. Cumulative concentration-response curves for 20-HETE (0.1–1000 nm) were obtained, in the absence or presence of the PKC inhibitor MyrΨPKC-I(19–27), by adding it to the bath and allowing a 10-min equilibration period. Internal diameters were measured 2–5 min after application of 20-HETE. Cerebral microvessels were isolated according to a protocol published previously (4Harder D.R. Gebremedhin D. Narayanan J. Jefcoat C. Falck J.R. Campbell W.B. Roman R. Am. J. Physiol. 1994; 266: H2098-H2107PubMed Google Scholar). Briefly, adult mongrel cats were anesthetized as described above, and vessels were isolated by microdissection. Isolated vessels were minced and placed in a low-Ca2+ PSS containing 134 mm NaCl, 5.4 mm KCl, 1.2 mm MgSO4, 0.24 mm KH2PO4, 0.05 mmCaCl2, 11 mm glucose, and 10 mmHEPES, pH adjusted to 7.4 with NaOH. Vessel fragments were transferred to a vial containing 88.5 units/ml collagenase type II, 2 mm dithiothreitol, and 1 mm trypsin inhibitor in low-Ca2+ PSS. The vial was placed in a water-jacketed beaker on a microstirrer, and the tissue was stirred (12 rpm) at 37 °C for a total of 1 h in the enzyme solution. At 5-min intervals, the supernatant fractions were collected and checked for appearance of dispersed cells under a microscope; fresh enzyme solution was added to the vessels for continued digestion and fraction collection. Pieces of vessel were disrupted mechanically by forcing them repeatedly through a Pasteur pipette. Fractions containing the cell suspension were transferred to a test tube and diluted with normal PSS and placed on ice. Aliquots of cells were removed from the suspension for immunofluorescence staining with anti-smooth muscle α-actin antibody (Cy3 conjugate, Sigma) and anti-factor VIII antibody (FITC conjugate, Atlantic) to confirm vascular smooth muscle origin of the cells and to assess possible contamination with endothelial cells. VSMCs thus isolated were found to be free of endothelial cell contamination and were used for seeding of cultures and for electrophysiological experiments. Outward whole-cell K+ current were recorded at room temperature from cerebral arterial muscle cells using pipette or intracellular solution containing 145 KCl mm, 1.8 mmCaCl2, 1 mm MgCl2, 5 mmEGTA, 2 mm magnesium adenosine triphosphate, 0.1 mm GTP, and 10 mm HEPES, with the final pH adjusted to 7.2 with KOH. The external solution bathing the cells was composed of 140 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, and 10 mm glucose, with pH adjusted to 7.4 with NaOH. Outward whole-cell K+ current were elicited every 1 s by depolarizing pulses of 300-ms duration from a holding potential of −70 mV to 80 mV in 10-mV increments. The effect of increasing concentrations of 20-HETE was studied before and after application of the cell-permeable PKC inhibitor, MyrΨPKC-I(19–27) (Calbiochem). The effect of the PKC activator phorbol 12-myristate 13-acetate (PMA; Sigma) on whole-cell K+ current was studied by addition to the bath. The identification of this outward current as Ca2+-activated K+ current was justified by its sensitivity to blockade by low concentration (1 mm) of tetraethylammonium chloride or charybdotoxin (50 nm) and activation by the calcium ionophore A23187 (1 ॖm) (data not shown). Freshly dissociated cat cerebral VSMCs were prepared as detailed above. Cells were washed three times in RPMI 1640 containing 207 fetal bovine serum and 17 penicillin-streptomycin, then plated onto 35-mm tissue culture dishes, and incubated at 37 °C, 57 CO2. Cell culture medium was changed twice daily for the first 3 days of culture. After 6 days of culture, the cells were suspended by treatment with trypsin-EDTA in PBS and transferred to 75-cm2 tissue culture flasks and grown in media as described above for 1 week. After 1 week, cells were split into 12-well plates for MARCKS assay or frozen in liquid nitrogen for future use. Cat cerebral VSMCs were maintained in RPMI 1640 (Life Technologies, Inc.) supplemented with 207 fetal bovine serum (Sigma) and 17 penicillin-streptomycin (Life Technologies, Inc.) and incubated at 37 °C with 957 humidity and 57 CO2. Cells were seeded into 12-well plates at equal density (104cells/well) and allowed to reach 807 confluence (107cells/well). MARCKS extraction was performed based on a modification of a protocol published previously and normalized to cell number prior to lysis (26Robinson P.J. Liu J.P. Chen W. Wenzel T. Anal. Biochem. 1993; 210: 172-178Crossref PubMed Scopus (43) Google Scholar). Prior to [32P]orthophosphate labeling, cells were serum starved for 48 h in DMEM F-12/Ham's medium with 17 penicillin-streptomycin. Cells were washed two times with phosphate-free DMEM (Life Technologies, Inc.), and 100 ॖCi of32Pi (DuPont NEN) were added in 1 ml of phosphate-free DMEM per well and incubated for 6 h at 37 °C, 57 CO2. After 6 h, test compounds 20-HETE, PMA, and PKC inhibitor were added, followed by incubation for 5 min at 37 °C. The medium was removed rapidly, the wells were washed once with PBS, and 175 ॖl of lysis buffer (10 mm Tris-HCl, pH 7.4, 1 mm ZnSO4, 1 mmNa3VO4, 5 mg/ml saponin, 0.27 glycerol, and 0.57 Triton X-100) were immediately added. After 5 min of incubation in lysis buffer at room temperature, cells were scraped into a 1.5-ml microcentrifuge tube, vortexed 20 s, and centrifuged at 20,000 × g for 5 min to pellet debris. The supernatant was removed to a 1.5-ml tube, and 700 ॖl of ice-cold methanol, 175 ॖl of ice-cold chloroform, and 550 ॖl of deionized H20 were added, vortexed for 30 s, and centrifuged at 9000 ×g for 2 min at room temperature. The aqueous phase was removed, and 600 ॖl of ice-cold methanol were added, followed by centrifugation at 20,000 × g for 5 min to pellet precipitated protein. The supernatant was removed, and the samples were dried in a vacuum concentrator for 5 min to remove residual chloroform/methanol. The pellets were resuspended in 130 ॖl of 2 × SDS Laemmli sample buffer by vigorous vortexing. 130 ॖl of 807 acetic acid were added, and the samples were incubated on ice for 30 min. The acid-insoluble material was pelleted by centrifugation at 14,000 × g at 4 °C for 10 min. Acid-soluble proteins (MARCKS) in the supernatant were removed to a fresh tube and dried in a vacuum concentrator for 3 h, washed by resuspension in 500 ॖl of distilled water, and dried for an additional 3 h under high heat. The final sample was resuspended in 50 ॖl of 0.25 × SDS sample buffer by vigorous vortexing, boiled 3 min, and loaded along with molecular weight standards (Bio-Rad) onto 3.57 stacking/107 resolving SDS-polyacrylamide gel electrophoresis gels (Bio-Rad Ready Gels). Following SDS-polyacrylamide gel electrophoresis, the gels were electrophoretically transferred to nitrocellulose membranes. Membranes were washed once with PBS, wrapped in plastic, and autoradiographed using DuPont Reflections film. Western blotting was performed to confirm equal loading and the identity of MARCKS by probing the membranes with a monoclonal antibody directed against human MARCKS (Upstate Biotechnology, Inc.) (data not shown). Quantitation was achieved by scanning densitometry of autoradiograms (Molecular Dynamics, Personal Densitometer) and verified by scintillation counting of excised nitrocellulose squares corresponding to MARCKS identified by Western blotting. Data are presented as the mean percentage of change in autoradiogram density for experimental treatments (PMA, 20-HETE) from the mean vehicle-treated autoradiogram density. Data are presented as mean ± S.E. where appropriate. Significant differences represent ap < 0.05 using a paired Student's ttest. 20-HETE was purchased from BioMol (Plymouth Meeting, PA), MyrΨPKC-I(19–27) was from Calbiochem (San Diego, CA), and [32P]orthophosphate was from DuPont NEN (Boston, MA). DMEM, RPMI 1640, and antibiotics were from Life Technologies, Inc. (Bethesda, MD). All other chemicals were supplied by Sigma unless otherwise noted. The effects of increasing concentrations of 20-HETE on the inner diameter of isolated pressurized (80 mm Hg) cat middle cerebral arteries, in the absence or presence of MyrΨPKC(19–27) (50 ॖm), are depicted in Fig. 1. Inhibition of PKC by this pseudosubstrate peptide has been demonstrated previously to be potent and highly specific (27House C. Kemp B.E. Science. 1987; 238: 1726-1728Crossref PubMed Scopus (837) Google Scholar, 28Eichholtz T. de Bont D.B.A. de Widt J. Liskamp R.M.J. Ploegh H.L. J. Biol. Chem. 1993; 268: 1982-1986Abstract Full Text PDF PubMed Google Scholar). Under control conditions, cumulative addition of increasing concentrations of 20-HETE (1 nm, 100 nm, 300 nm, and 1 ॖm) to the bath resulted in a concentration-dependent reduction in inner diameter. The percentage of change in diameter from baseline averaged −6 ± 37 (mean ± S.E.) at 1 nm, −13 ± 37 at 100 nm, −21 ± 47 at 300 nm, and −29 ± 77 at 1 ॖm (n = 6 for all concentrations studied). The vasoconstrictor effect of 20-HETE reached a maximum within 2–5 min. After washout of 20-HETE from the bath, the cannulated arterial segment was pretreated for 10 min with MyrΨPKC-I(19–27) (50 ॖm) by addition to the bath. No significant changes in the baseline diameter of the arterial segment were observed for a 10-min period after addition of the inhibitor alone. Cumulative addition of 20-HETE (1 nmto 1 ॖm) in the presence of MyrΨPKC-I(19–27) resulted in a concentration-dependent increase in diameter; the percentage of change in diameter averaged 4 ± 37 (mean ± S.E.) at 1 nm, 13 ± 67 at 100 nm, 20 ± 77 at 300 nm, and 27 ± 117 at 1 ॖm (n = 6 for all concentrations studied). The arterial preparations were washed repeatedly with fresh PSS for 30 min, after which time the effect of cumulative addition of 20-HETE (1 nm to 1 ॖm) to the bath was redetermined. The vasoconstrictor response to 20-HETE returned to the pre-inhibitor treatment level; the percentage of change in diameter from baseline averaged −13 ± 67 (mean ± S.E.) at 1 nm, −19 ± 117 at 100 nm, −28 ± 167 at 300 nm, and −34 ± 197 at 1 ॖm(n = 6 for all concentrations studied). These results demonstrate that the vasoconstrictor action of 20-HETE can be reversibly abolished by inhibition of PKC and indicates that this response is dependent on a signal transduction pathway in which PKC plays an integral role. The effects of 20-HETE on whole-cell K+ current in freshly dispersed cat cerebral VSMCs were studied using the whole-cell voltage clamp technique (29Hamill O.P. Marty A. Neher E. Sakmann B. Sigwort F.J. Pflugers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15371) Google Scholar). Addition of increasing concentrations of 20-HETE (100–300 nm) to the bathing solution resulted in significant inhibition of peak whole-cell K+ current by 38 ± 47 (n = 5) at 100 nm and by 58 ± 97 (n = 5) at 300 nm (p < 0.001 compared with control) (Fig.2). 20-HETE did not appear to shift the current-voltage relationship, thus suggesting that 20-HETE decreases whole-cell current amplitude by decreasing either the probability of channel opening or the number of active channels. Addition of the MyrΨPKC-I(19–27) (100 nm) alone did not change the amplitude of the whole-cell K+ current as compared with control (Fig. 3). In the presence of MyrΨPKC-I(19–27) (100 nm), addition of 20-HETE (300 nm) to the bath failed to inhibit whole-cell K+ current (Fig. 3), indicating that inhibition of PKC prevents the inhibitory action of 20-HETE on whole-cell K+ current. In a separate series of experiments, activation of PKC by addition of 100 nm PMA resulted in a diminution of peak whole-cell outward current (Fig.4), indicating that known activators of PKC, such as PMA (30Castagna M. Takai Y. Kaibuchi K. Sano K. Kikkawa U. Nishizuka Y. J. Biol. Chem. 1982; 257: 7847-7851Abstract Full Text PDF PubMed Google Scholar), reduce the amplitude of whole-cell K+ current in cat cerebral VSMCs. The inhibitory effects of PMA on whole-cell K+ current were prevented by prior addition of 100 nm MyrΨPKC-I(19–27). Taken together, these results indicate that 20-HETE inhibits whole-cell K+ current in cat cerebral VSMCs by a mechanism that involves PKC activation.Figure 3Inhibition of PKC attenuates the inhibition of whole-cell K+ currents by 20-HETE. A,averaged whole-cell K+ current tracings demonstrating that the PKC inhibitor, MyrΨPKC-I(19–27) (100 nm), does not effect control whole-cell K+current. Addition of MyrΨPKC-I(19–27) (100 nm) did not alter whole-cell K+ current (middle panel), and 20-HETE (300 nm) failed to reduce whole-cell K+ current in the presence of this PKC inhibitor (bottom panel). B, averaged peak current-voltage relation before (○), after addition of MyrΨPKC-I(19–27) (100 nm) (•), and after addition of 300 nm 20-HETE in the continued presence of 100 nm MyrΨPKC-I(19–27) (▾). 20-HETE did not reduce whole-cell K+ current amplitude following PKC inhibition. n = 5 for each experiment. Bars,S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Activation of PKC by PMA inhibits whole-cell K+ currents in cat cerebral VSMCs. A, averaged whole-cell K+ current tracings showing that application of the known PKC activator; PMA (100 nm) markedly inhibits whole-cell K+ current (middle panel as compared with left panel), and addition of PMA (100 nm) in the presence of 100 nm MyrΨPKC-I(19–27)failed to reduce the amplitude of whole-cell K+ currents (right panel). B, averaged peak current-voltage relation under control conditions (○), after addition of 100 nm PMA (•), and after addition of 100 nm PMA in the presence of 100 nmMyrΨPKC-I(19–27) (▾). n = 5 for each experiment. Asterisks represent significant difference from control at p < 0.05. Bars, S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT) MARCKS is a ubiquitously expressed substrate that has been shown to be the major in vivo target for phosphorylation by PKC (31Albert K.A. Walaas S.I. Wang J.K.T. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2822-2826Crossref PubMed Scopus (153) Google Scholar, 32Albert K.A. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7046-7050Crossref PubMed Scopus (109) Google Scholar, 33Herget T. Oehrlein S.A. Pappin D.J.C. Rozengurt E. Parker P.J. Eur. J. Biochem. 1995; 233: 448-457Crossref PubMed Scopus (80) Google Scholar). MARCKS is a heat-stable, acid-soluble protein that migrates at 80–87 kDa on SDS-polyacrylamide gel electrophoresis gels (26Robinson P.J. Liu J.P. Chen W. Wenzel T. Anal. Biochem. 1993; 210: 172-178Crossref PubMed Scopus (43) Google Scholar). Previous studies in rat aortic smooth muscle cells have demonstrated expression of MARCKS, and the phosphorylation of MARCKS in these cells can be modulated by activators of PKC such as PMA and angiotensin II (26Robinson P.J. Liu J.P. Chen W. Wenzel T. Anal. Biochem. 1993; 210: 172-178Crossref PubMed Scopus (43) Google Scholar). We exploited these properties of MARCKS to determine if 20-HETE induces a PKC-mediated phosphorylation of MARCKS in primary cultures of cat cerebral VSMCs. Acetic acid extraction of proteins from 32Pi-labeled VSMCs treated with PMA (100 nm) in the absence or presence of MyrΨPKC-I(19–27) (100 ॖm) or 20-HETE (100 nm and 1 ॖm) demonstrated a PKC-dependent effect of PMA and a concentration-related effect of 20-HETE on MARCKS phosphorylation (Fig.5). Treatment with PMA (100 nm) increased MARCKS phosphorylation by 73 ± 167, as assessed by scanning densitometry of autoradiograms. This PMA-induced increase in MARCKS phosphorylation was completely abolished by pretreatment of cells for 5 min with 100 ॖmMyrΨPKC-I(19–27). Similarly, 20-HETE also induced an increase in MARCKS phosphorylation by 29 ± 187 at 100 nm and 46 ± 107 at 1 ॖm, as assessed by scanning densitometry of autoradiograms. The relative number of moles of 32P incorporated into MARCKS in response to the different treatments was assessed by scintillation counting of excised nitrocellulose squares corresponding to MARCKS. Baseline incorporation was 2.08 ± 0.14 fmol 32P, whereas treatment with PMA increased incorporation to 9.72 ± 0.53 fmol 32P, and treatment with 20-HETE increased incorporation to 6.23 ± 0.49 fmol and 7.47 ± 0.65 fmol at 100 nm and 1 ॖm, respectively (p < 0.05 for all with respect to baseline, n = 6). To determine if the 20-HETE induced increase in MARCKS phosphorylation was dependent on PKC activation, we treated cells with 1 ॖm 20-HETE and examined the effect of increasing concentrations of MyrΨPKC-I(19–27). These results are depicted in Fig.6. 20-HETE (1 ॖm)-induced MARCKS phosphorylation was inhibited by MyrΨPKC-I(19–27)in a concentration-dependent manner and averaged 15 ± 37 (mean ± S.E.) at 1 ॖm, 26 ± 67 at 10 ॖm, 79 ± 27 at 50 ॖm, and 93 ± 0.47 at 100 ॖm. A fit of this data by a single exponential yielded a value for an IC50 of 30.64 ± 13.11 ॖm MyrΨPKC-I(19–27).Figure 6MyrΨPKC-I(19–27) inhibits 20-HETE induced phosphorylation in a concentration-dependent manner. Increasing concentrations of MyrΨPKC-I(19–27) reduced the increase in 87-kDa MARCKS phosphorylation in cat cerebral VSMCs in response to treatment with 1 ॖm 20-HETE. A, representative autoradiogram depicting the concentration-related inhibitory effects of MyrΨPKC-I(19–27) on 20-HETE-induced phosphorylation MARCKS. B, summary of data from six such experiments run in parallel. Data are represented as the percentage of inhibition of 20-HETE-induced MARCKS phosphorylation by increasing concentrations of MyrΨPKC-I(19–27). Mean data were fitted with a single exponential function. IC50, 30.64 ± 13.11 ॖm. Bars, S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Recent reports have described several potential roles for 20-HETE in mediating an array of cellular functions, ranging from regulation of vascular tone and myogenic reactivity (4Harder D.R. Gebremedhin D. Narayanan J. Jefcoat C. Falck J.R. Campbell W.B. Roman R. Am. J. Physiol. 1994; 266: H2098-H2107PubMed Google Scholar, 5Imig J.D. Zou A.P. Stec D.E. Harder D.R. Falck J.R. Roman R.J. Am. J. Physiol. 1996; 270: R217-R227Crossref PubMed Google Scholar, 6Ma Y.H. Gebremedhin D. Schwartzman M.L. Falck J.R. Clark J.E. Masters B.S. Harder D.R. Roman R.J. Circ. Res. 1993; 72: 126-136Crossref PubMed Google Scholar, 7Zou A.P. Fleming J.T. Falck J.R. Jacobs E.R. Gebremedhin D. Harder D.R. Roman R.J. Am. J. Physiol. 1996; 270: R228-R237PubMed Google Scholar, 8Harder D.R. Lange A.R. Gebremedhin D. Birks E.K. Roman R.J. J. Vasc. Res. 1997; 34: 237-243Crossref PubMed Scopus (150) Google Scholar, 34Zou A. Imig J.D. 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