Thromboxane A2-induced Bi-directional Regulation of Cerebral Arterial Tone
2008; Elsevier BV; Volume: 284; Issue: 10 Linguagem: Inglês
10.1074/jbc.m807040200
ISSN1083-351X
AutoresRonald L. Neppl, Lubomir T. Lubomirov, Ko Momotani, Gabriele Pfitzer, Masumi Eto, Avril V. Somlyo,
Tópico(s)Traumatic Brain Injury and Neurovascular Disturbances
ResumoMyosin light chain phosphatase plays a critical role in modulating smooth muscle contraction in response to a variety of physiologic stimuli. A downstream target of the RhoA/Rho-kinase and nitric oxide (NO)/cGMP/cyclic GMP-dependent kinase (cGKI) pathways, myosin light chain phosphatase activity reflects the sum of both calcium sensitization and desensitization pathways through phosphorylation and dephosphorylation of the myosin phosphatase targeting subunit (MYPT1). As cerebral blood flow is highly spatio-temporally modulated under normal physiologic conditions, severe perturbations in normal cerebral blood flow, such as in cerebral vasospasm, can induce neurological deficits. In nonpermeabilized cerebral vessels stimulated with U-46619, a stable mimetic of endogenous thromboxane A2 implicated in the etiology of cerebral vasospasm, we observed significant increases in contractile force, RhoA activation, regulatory light chain phosphorylation, as well as phosphorylation of MYPT1 at Thr-696, Thr-853, and surprisingly Ser-695. Inhibition of nitric oxide signaling completely abrogated basal MYPT1 Ser-695 phosphorylation and significantly increased and potentiated U-46619-induced MYPT1 Thr-853 phosphorylation and contractile force, indicating that NO/cGMP/cGKI signaling maintains basal vascular tone through active inhibition of calcium sensitization. Surprisingly, a fall in Ser-695 phosphorylation did not result in an increase in phosphorylation of the Thr-696 site. Although activation of cGKI with exogenous cyclic nucleotides inhibited thromboxane A2-induced MYPT1 membrane association, RhoA activation, contractile force, and regulatory light chain phosphorylation, the anticipated decreases in MYPT1 phosphorylation at Thr-696/Thr-853 were not observed, indicating that the vasorelaxant effects of cGKI are not through dephosphorylation of MYPT1. Thus, thromboxane A2 signaling within the intact cerebral vasculature induces "buffered" vasoconstrictions, in which both the RhoA/Rho-kinase calcium-sensitizing and the NO/cGMP/cGKI calcium-desensitizing pathways are activated. Myosin light chain phosphatase plays a critical role in modulating smooth muscle contraction in response to a variety of physiologic stimuli. A downstream target of the RhoA/Rho-kinase and nitric oxide (NO)/cGMP/cyclic GMP-dependent kinase (cGKI) pathways, myosin light chain phosphatase activity reflects the sum of both calcium sensitization and desensitization pathways through phosphorylation and dephosphorylation of the myosin phosphatase targeting subunit (MYPT1). As cerebral blood flow is highly spatio-temporally modulated under normal physiologic conditions, severe perturbations in normal cerebral blood flow, such as in cerebral vasospasm, can induce neurological deficits. In nonpermeabilized cerebral vessels stimulated with U-46619, a stable mimetic of endogenous thromboxane A2 implicated in the etiology of cerebral vasospasm, we observed significant increases in contractile force, RhoA activation, regulatory light chain phosphorylation, as well as phosphorylation of MYPT1 at Thr-696, Thr-853, and surprisingly Ser-695. Inhibition of nitric oxide signaling completely abrogated basal MYPT1 Ser-695 phosphorylation and significantly increased and potentiated U-46619-induced MYPT1 Thr-853 phosphorylation and contractile force, indicating that NO/cGMP/cGKI signaling maintains basal vascular tone through active inhibition of calcium sensitization. Surprisingly, a fall in Ser-695 phosphorylation did not result in an increase in phosphorylation of the Thr-696 site. Although activation of cGKI with exogenous cyclic nucleotides inhibited thromboxane A2-induced MYPT1 membrane association, RhoA activation, contractile force, and regulatory light chain phosphorylation, the anticipated decreases in MYPT1 phosphorylation at Thr-696/Thr-853 were not observed, indicating that the vasorelaxant effects of cGKI are not through dephosphorylation of MYPT1. Thus, thromboxane A2 signaling within the intact cerebral vasculature induces "buffered" vasoconstrictions, in which both the RhoA/Rho-kinase calcium-sensitizing and the NO/cGMP/cGKI calcium-desensitizing pathways are activated. Physiologic control of cerebral circulation is modulated metabolically via PO2 and PCO2, as well as via eicosanoids, endothelin, and nitric oxide (NO). 2The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase; MLCP, myosin light chain phosphatase; RLC, regulatory light chain; cGKI, cyclic GMP-dependent kinase; MCA, middle cerebral artery; TXA2, thromboxane A2; TXA2R, TXA2 receptor; l-NAME, NG-nitro-l-arginine methyl ester; PKA, protein kinase A; LZ, leucine zipper; MLCK, myosin light chain kinase; PDE, phosphodiesterase; SNP, sodium nitroprusside; VASP, vasodilator-stimulated protein; 6-Bnz-cAMP, N6-benzoyl-cAMP; 8-Br-cGMP, 8-bromo-cGMP. Disruptions in the blood-brain barrier either by traumatic head injury or subarachnoid hemorrhage can cause prolonged and severe perturbations to normal cerebral blood flow. Cerebral vasospasm following subarachnoid hemorrhage is characterized by an extensive prolonged narrowing of cerebral arteries, which may result in neurological deficits (1Nishizawa S. Laher I. Trends Cardiovas. Med. 2005; 15: 24-34Crossref PubMed Scopus (79) Google Scholar, 2Hansen-Schwartz J. Vajkoczy P. Macdonald R.L. Pluta R.M. Zhang J.H. Trends Pharmacol. Sci. 2007; 28: 252-256Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Eicosanoids, such as the prostaglandins, leukotrienes, and thromboxanes, have been implicated in the etiology of cerebral vasospasm following subarachnoid hemorrhage (1Nishizawa S. Laher I. Trends Cardiovas. Med. 2005; 15: 24-34Crossref PubMed Scopus (79) Google Scholar). The Rho-kinase inhibitor fasudil is used clinically as an effective treatment for cerebral vasospasm (3Shimokawa H. Takeshita A. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1767-1775Crossref PubMed Scopus (422) Google Scholar), suggesting the importance of RhoA signaling in the cerebrovasculature. Thus a better understanding of the signaling pathways regulating normal and pathological cerebral blood flow is warranted and is the basis of this study.Thromboxane A2 (TXA2), a platelet-secreted, short lived derivative of arachidonic acid, is known to induce vasoconstriction in multiple vascular smooth muscles (4Huang J.S. Ramamurthy S.K. Lin X. Le Breton G.C. Cell. Signal. 2004; 16: 521-533Crossref PubMed Scopus (163) Google Scholar, 5Ellis E.F. Oelz O. Roberts II, L.J. Payne N.A. Sweetman B.J. Nies A.S. Oates J.A. Science. 1976; 193: 1135-1137Crossref PubMed Scopus (391) Google Scholar, 6Wilson D.P. Susnjar M. Kiss E. Sutherland C. Walsh M.P. Biochem. J. 2005; 389: 763-774Crossref PubMed Scopus (168) Google Scholar), including the cerebral microvessels (7Hou X. Gobeil Jr., F. Peri K. Speranza G. Marrache A.M. Lachapelle P. Roberts II, J. Varma D.R. Chemtob S. Ellis E.F. Stroke. 2000; 31: 516-525Crossref PubMed Google Scholar). TXA2 receptors (TXA2R) signal through both Gq, resulting in the activation of phospholipase Cβ catalyzing the generation of inositol 1,4,5-trisphosphate and diacylglycerol, and G12/13, resulting in Ca2+ sensitization through the activation of RhoA (4Huang J.S. Ramamurthy S.K. Lin X. Le Breton G.C. Cell. Signal. 2004; 16: 521-533Crossref PubMed Scopus (163) Google Scholar, 8Somlyo A.P. Somlyo A.V. Physiol. Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1666) Google Scholar). Activation of the TXA2R is generally considered to preferentially activate calcium sensitization pathways (9Bradley A.B. Morgan K.G. J. Physiol. (Lond.). 1987; 385: 437-448Crossref Scopus (132) Google Scholar, 10Himpens B. Kitazawa T. 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Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1666) Google Scholar) results in reduced MLCP activity thus increasing RLC20 phosphorylation and force. The role of CPI-17 in the cerebral vasculature is not presently known.Ca2+ desensitization leads to a decrease in RLC20 phosphorylation and relaxation of vascular smooth muscle at constant [Ca2+]i through inhibition of MLCK activity, activation of MLCP, or inhibition of Ca2+-sensitizing pathways (8Somlyo A.P. Somlyo A.V. Physiol. Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1666) Google Scholar, 13Murthy K.S. Annu. Rev. Physiol. 2006; 68: 345-374Crossref PubMed Scopus (297) Google Scholar). Cyclic nucleotide-induced relaxation of vascular smooth muscle occurs through several potential downstream signaling pathways initiating Ca2+ desensitization, which include phospho-inhibition of RhoA·GTP at Ser-188 by cyclic GMP-dependent kinase (cGKI) (14Sauzeau V. Le Jeune H. Cario-Toumaniantz C. Smolenski A. Lohmann S.M. Bertoglio J. 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Acad. Sci. U. S. A. 2008; 105: 6702-6707Crossref PubMed Scopus (103) Google Scholar). Although the molecular mechanism whereby cGKI alters MLCP activity remains unknown, recent evidence in permeabilized smooth muscles would suggest that phosphorylation of MYPT1 at Ser-695, resulting in the reduction of phosphorylation at the adjacent inhibitory Thr-696 site (23Nakamura K. Koga Y. Sakai H. Homma K. Ikebe M. Circ. Res. 2007; 101: 712-722Crossref PubMed Scopus (86) Google Scholar, 24Wooldridge A.A. MacDonald J.A. Erdodi F. Ma C. Borman M.A. Hartshorne D.J. Haystead T.A. J. Biol. Chem. 2004; 279: 34496-34504Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), may play an important physiologic role.Cyclic nucleotides, generated in response to endothelium-derived NO, can induce relaxation by decreasing [Ca2+]i through the inhibition of calcium influx (25Sausbier M. Schubert R. Voigt V. Hirneiss C. Pfeifer A. Korth M. Kleppisch T. Ruth P. Hofmann F. Circ. 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Chem. 2003; 278: 51190-51202Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Furthermore, the nitric oxide donor SNP was shown to decrease CPI-17 phosphorylation coincident in time with the rise in [cGMP] and MLCP activity in porcine carotid arteries (31Etter E.F. Eto M. Wardle R.L. Brautigan D.L. Murphy R.A. J. Biol. Chem. 2001; 276: 34681-34685Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). It remains uncertain whether activation of cGKI in cerebrovascular smooth muscle similarly inhibits RhoA activation and MYPT1 phosphorylation as in cell culture systems (32Seko T. Ito M. Kureishi Y. Okamoto R. Moriki N. Onishi K. Isaka N. Hartshorne D.J. Nakano T. Circ. Res. 2003; 92: 411-418Crossref PubMed Scopus (276) Google Scholar), or whether cyclic nucleotide-mediated phosphorylation of telokin, such as in phasic smooth muscles or dephosphorylation of CPI-17, contributes to activation of MLCP in the cerebral vasculature, which is a focus of this study.Coincident with the Ca2+ sensitization-induced phosphorylation of MYPT1 at Thr-696, MLCP has been observed to translocate from the cytosol to the cell membrane following stimulation with prostaglandin F2α and sphingosine 1-phosphate in freshly isolated ferret portal vein SMCs (33Shin H.M. Je H.D. Gallant C. Tao T.C. Hartshorne D.J. Ito M. Morgan K.G. Circ. Res. 2002; 90: 546-553Crossref PubMed Scopus (86) Google Scholar) and in hamster resistance arteries (34Bolz S.S. Vogel L. Sollinger D. Derwand R. de Wit C. Loirand G. Pohl U. Circulation. 2003; 107: 3081-3087Crossref PubMed Scopus (118) Google Scholar), suggesting that removal of MLCP from the actomyosin contractile apparatus may be an additional mechanism of Ca2+ sensitization. Though presently an interesting observation associated with Ca2+ sensitization, the underlying function, magnitude, and mechanism of its translocation to the membrane is presently unknown. In this study we examined the downstream signaling events of TXA2 receptor stimulation in concert with cGKI activation in the intact cerebral vasculature. We demonstrate cGKI-dependent inhibition of MYPT1 membrane association and RhoA activation following TXA2R stimulation, without the anticipated reduction of MYPT1 phosphorylation at Thr-696, recently demonstrated in permeabilized ileum (24Wooldridge A.A. MacDonald J.A. Erdodi F. Ma C. Borman M.A. Hartshorne D.J. Haystead T.A. J. Biol. Chem. 2004; 279: 34496-34504Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) and femoral artery (23Nakamura K. Koga Y. Sakai H. Homma K. Ikebe M. Circ. Res. 2007; 101: 712-722Crossref PubMed Scopus (86) Google Scholar). Surprisingly, phosphorylation of the MYPT1 Ser-695 site, generally associated with cyclic nucleotide signaling, is present in the resting state, significantly increased in response to thromboxane A2 signaling, and completely abrogated by l-NAME pretreatment or permeabilization, suggesting a critical role for TXA2 signaling in endothelium-smooth muscle coupling within the cerebral vasculature.MATERIALS AND METHODSTissue Preparation-Adult male Sprague-Dawley rats (150–175 g) and adult male New Zealand White rabbits (4–5 pounds) were euthanized according to protocols approved by the Animal Care and Use Committee at the University of Virginia. The posterior and middle cerebral as well as the superior cerebellar arteries were removed from the brain, placed in ice-cold HEPES-buffered Krebs solution, and cleaned of their adventitia. Histological sections of the middle cerebral artery (supplemental Fig. 1) show that the vessels consists of 2–3 layers of circular smooth muscle cells with an intact endothelial lining. Morphometric measurements show that endothelial cells contribute ∼16.5 ± 2.9% of the total cross-sectional area of the vessel wall.Immunofluorescence Microscopy-Rat cerebral arteries were fixed 10 min post-stimulus in 4% paraformaldehyde in phosphate-buffered saline at room temperature for 15 min. Arteries were permeabilized with 0.03% Triton X-100 for 20 min and blocked with 5% normal donkey serum. Primary antibodies were visualized by a rhodamine Red-X conjugated secondary antibody (Jackson ImmunoResearch, West Grove. PA) at 1:500 dilution. Cell nuclei were visualized with TO-PRO-3 Iodide (Invitrogen). All images were acquired under identical laser power and gain settings using an Olympus Fluoview 300 confocal microscope (Olympus).Protein Phosphorylation-Protein phosphorylation was preserved by freeze substitution in 10% in trichloroacetic acid in acetone at -80 °C. The trichloroacetic acid was removed by successive washes with pure acetone, after which the vessels were allowed to dry, homogenized in SDS buffer, and boiled for 10 min. Following centrifugation at 10,000 × g for 10 min, the total protein content was determined by the RC DC protein assay (Bio-Rad) to ensure equal loading. Following transfer, gels were stained with Coomassie Blue, and the filamin bands were scanned densitometrically as an additional internal loading control.Membrane Fractionation-Membrane-bound proteins were separated from cytosolic proteins according to the methods described in Gong et al. (35Gong M.C. Fujihara H. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1997; 272: 10704-10709Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Briefly, rabbit cerebral vessels were homogenized in ice-cold homogenization buffer (10 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 2 mm EDTA, 250 mm sucrose, 1 mm dithiothreitol) with protease inhibitor mixture (Sigma) at 1:100 dilution and centrifuged at 100,000 × g for 30 min at 4 °C (Optima™ TLX ultracentrifuge, TLA 120.1 rotor, Beckman Instruments). The supernatant was collected as the cytosolic fraction. Pellets were resuspended in ice-cold homogenization buffer supplemented with 1% Triton X-100 and 1% sodium cholate and incubated for 30 min. The detergent-soluble fraction was separated from the detergent-insoluble (pellet) fraction by centrifugation at 1000 × g for 10 min. Fractions were separated by gel electrophoresis and transferred to polyvinylidene difluoride.Rhotekin Pulldown-RhoA activation was determined by precipitation of active GTP-bound RhoA (RhoA·GTP) with a glutathione S-transferase fusion protein of the Rho-binding domain of the Rho effector rhotekin as described previously (36Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1354) Google Scholar).Western Blotting-Proteins were transferred to polyvinylidene difluoride or to nitrocellulose for CPI-17 blotting and visualized using either the Odyssey system (Li-Cor, Lincoln, NE) or ECL. For Odyssey imaging, membranes were blocked with Odyssey Blocking Buffer and probed with primary antibody in Blocking Buffer. Primary antibodies were visualized using either a goat anti-mouse Alexa 680 (Invitrogen) or a goat anti-rabbit IRDye800 (Rockland Immunochemicals, Gilbertsville, PA)-conjugated secondary antibody. Chicken IgY was detected using a rabbit anti-chicken HiLyte 680 (AnaSpec, San Jose CA)-conjugated secondary antibody. Detection and quantification of the infrared signal were performed using the Odyssey system software. ECL blots were processed following previously published methods (37Lubomirov L.T. Reimann K. Metzler D. Hasse V. Stehle R. Ito M. Hartshorne D.J. Gagov H. Pfitzer G. Schubert R. Circ. Res. 2006; 98: 1159-1167Crossref PubMed Scopus (37) Google Scholar).Isometric Force Measurements-Helical strips (100 μm wide × 3 mm long) were cut from the rat middle cerebral artery (inner diameter ≈75–100 μm) and mounted on a 100-μm stainless steel rod for ease of cutting and to remove the endothelium. In some cases to preserve the endothelium, helical strips were cut from an unmounted MCA. Isometric tension was measured at room temperature with a force transducer (AE 801; SensoNor A.S., Horten, Norway), and a length-adjusting device was mounted on a "bubble plate" (38Somlyo A.V. Horiuti K. Trentham D.R. Kitazawa T. Somlyo A.P. J. Biol. Chem. 1992; 267: 22316-22322Abstract Full Text PDF PubMed Google Scholar) or on a newly developed wire myograph system with low bath volumes (900SM, Danish Myo Technology, Aarus, Denmark). Strips were stretched 1.1 × resting length in a bicarbonate Krebs buffer and allowed to equilibrate for 30 min prior to depolarization with 154 mm K+. The bicarbonate Krebs buffer containing (mm) 115.2 NaCl, 22.14 NaHCO3, 7.88 d-glucose, 4.7 KCl, 1.18 KH2PO4, 1.16 MgSO4, 1.80 CaCl2, 0.114 ascorbic acid, and 0.027 Na2EDTA was continuously bubbled with 95% O2, 5% CO2. Strips permeabilized with α-toxin and treated with 10 μm A23187 in G1 solution, as described previously (39Khromov A.S. Wang H. Choudhury N. McDuffie M. Herring B.P. Nakamoto R. Owens G.K. Somlyo A.P. Somlyo A.V. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2440-2445Crossref PubMed Scopus (57) Google Scholar, 40Kitazawa T. Gaylinn B.D. Denney G.H. Somlyo A.P. J. Biol. Chem. 1991; 266: 1708-1715Abstract Full Text PDF PubMed Google Scholar), were used to determine the free calcium EC50 (supplemental Fig. 2) and the effects of 8-Br-cGMP on contractile force in the presence of fixed [Ca2+]i. Maximal forces were normalized to that of pCa 5 solution, and curves were fit to the data using Origin-Pro software (Northampton MA).Cyclic Nucleotide Measurements-Following stimulation, rat cerebral arteries were immediately homogenized in 95% ethanol at pH 3.0, after which the homogenate was left overnight at -20 °C for extraction of cyclic nucleotides (41Gray D.W. Marshall I. Br. J. Pharmacol. 1992; 107: 691-696Crossref PubMed Scopus (133) Google Scholar). Samples were centrifuged at 10,000 × g for 10 min. The supernatant was evaporated to dryness under N2, and cyclic nucleotide content was measured using acetylated radioimmunoassay cAMP and cGMP kits according to the manufacturer's protocol (BTI Inc., Stoughton, MA). The protein pellet was dissolved in 0.5 m NaOH and assayed for protein content as above. Cyclic nucleotide content was normalized to total protein content.Antibodies and Reagents-The following antibodies were used: mouse monoclonal anti-MYPT1 (Covance) for indirect immunofluorescence localization of MYPT1 and a monoclonal anti-MYPT1 (BD Biosciences, San Jose CA) for Western blotting; phospho-specific rabbit polyclonal antibodies anti-phospho-MYPT1 Thr-696 and anti-phospho-MYPT1 Thr-850 (Upstate, Charlottesville VA); mouse monoclonal anti-MLC20 antibody for total MLC20 (Sigma); total and phospho-vasodilator-stimulated protein (VASP; Ser-157 and Ser-239), as well as the rabbit polyclonal anti-phospho-MLC20 antibodies (Cell Signaling, Danvers MA); mouse monoclonal anti-RhoA (Santa Cruz Biotechnology Inc., Santa Cruz CA); rabbit polyclonal total CPI-17 (42Senba S. Eto M. Yazawa M. J. Biochem. (Tokyo). 1999; 125: 354-362Crossref PubMed Scopus (81) Google Scholar), and a chicken IgY anti-phospho-CPI-17 Thr-38 (43Eto M. Kitazawa T. Matsuzawa F. Aikawa S. Kirkbride J.A. Isozumi N. Nishimura Y. Brautigan D.L. Ohki S.Y. Structure (Lond.). 2007; 15: 1591-1602Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Rabbit polyclonal anti-phospho-MYPT1 Ser-695 and mouse monoclonal anti-MYPT1 leucine zipper (LZ) antibodies were generous gifts from Dr. Timothy Haystead (Duke University, Durham, NC) and Dr. Frank Brozovich (Mayo Clinic, Rochester, MN), respectively.Statistical Analysis-Statistical significance with respect to control (*) was determined using the Student's t test (Microsoft Excel). The level of significance was set at p < 0.05.RESULTSEffects of Thromboxane A2 and cGMP Signaling on Middle Cerebral Artery Contractility-To assess the general effects of TXA2R stimulation and cGMP signaling in the intact cerebral vasculature, we examined the effects of the stable thromboxane A2 mimetic U-46619 and 8-Br-cGMP on the contractility of the intact middle cerebral artery (MCA, Fig. 1). The maximal contractile force induced by 300 nm U-46619 (10 min) is reversible upon addition of the Rho-kinase inhibitor Y-27632 (Fig. 1A, inset), although not completely, suggesting a component of TXA2R-mediated contractile force is independent of Rho-kinase. Additionally, small changes in [Ca2+]i above resting values may contribute to the Rho-kinase-independent component of the TXA2-signaled contractile force, as in the rat caudal artery (6Wilson D.P. Susnjar M. Kiss E. Sutherland C. Walsh M.P. Biochem. J. 2005; 389: 763-774Crossref PubMed Scopus (168) Google Scholar). Pretreatment with 8-Br-cGMP (10 min) significantly inhibited the U-46619-induced contraction by ∼40% (Fig. 1A, n = 4, p < 0.05), similar to the nearly 40% reduction in U-46619-induced contractile force upon addition of (post-treatment) 8-Br-cGMP (Fig. 1B). The efficacy of 8-Br-cGMP to inhibit (pretreatment) or reverse (post-treatment) U-46619-induced contractile force was not significantly different (Fig. 1C). MCA preparations were permeabilized with α-toxin and exhibited a normal pCa-tension relationship (supplemental Fig. 2). Addition of 8-Br-cGMP reduced pCa 6.3 force by ∼70% (n = 4, p < 0.05; data not shown) under conditions where Ca2+ was clamped with EGTA buffer. This finding plus the ability of the Rho-kinase inhibitor to relax the U-46619-contracted nonpermeabilized MCA demonstrate that 8-Br-cGMP-induced relaxation is not solely because of decreases in cytoplasmic [Ca2+].8-Br-cGMP Pretreatment Inhibits TXA2 Receptor-coupled RhoA Activation-To elucidate the convergence of signaling pathways activated by both 8-Br-cGMP and U-46619 as it relates to TXA2R-mediated contractile force, we performed rhotekin pulldowns on homogenates of intact cerebral vessels to measure changes in RhoA activation (Fig. 2A). We observe a greater than 2.5-fold significant increase (2.72 ± 0.57, n = 3, p < 0.05) in active RhoA following stimulation with U-46619 as compared with control (Fig. 2B). Pretreatment with 8-Br-cGMP significantly inhibits U-46619-induced RhoA activation by ∼38% (n = 3, p < 0.05), resulting in a net 1.6-fold increase (1.66 ± 0.21, n = 3, p < 0.05) in active RhoA as compared with control.FIGURE 2U-46619-induced RhoA activation is inhibited with 8-Br-cGMP pretreatment. A, representative RhoA Western blots following Rhotekin pulldown of stimulated intact cerebral vasculature. Increased RhoA in pulldown fraction with respect to whole homogenate (WH) are observed following U-46619 treatment. B, quantification of changes in RhoA activation following U-46619 alone, and in the presence of (following pretreatment) 8-Br-cGMP. Rabbit cerebral vessels were used for the rhotekin pulldown assay because of protein requirements. Error bars represent mean ± S.D; *, p < 0.05 with respect to control, n = 3 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)8-Br-cGMP Pretreatment Uncouples MLCP Inhibitory Phosphorylation of MYPT1 from RhoA·GTP in the Intact Cerebral Vasculature-To determine whether the observed decreases in RhoA activation (Fig. 2) following U-46619 stimulation in the presence of 8-Br-cGMP results in decreased MLCP inhibitory phosphorylation of MYPT1, we performed quantitative Western blot analysis on MYPT1 phosphorylation at Thr-696 and Thr-853. Representative Western blots are shown depicting the effects of U-46619 and 8-Br-cGMP treatment on the phosphorylation of MYPT1 at Thr-696 and Thr-853 by both LiCor (Fig. 3A) and ECL (supplemental Fig. 4A) detection methodologies. Treatment with U-46619 results in significant increases in RLC20 phosphorylation, as well as MYPT1 phosphorylation at both Thr-696 and Thr-853 (Fig. 3B), suggesting inhibition of MLCP activity, and consistent with prior studies demonstrating large contractile force with minimal increases in [Ca2+]i (9Bradley A.B. Morgan K.G. J. Physiol. (Lond.). 1987; 385: 437-448Crossref Scopus (132) Google Scholar, 10Himpens B. Kitazawa T. Somlyo A.P. Pfluegers Arch. 1990; 417: 21-28Crossref PubMed Scopus (152) Google Scholar, 11Nobe K. Paul R.J. Circ. Res. 2001; 88: 1283-1290Crossref PubMed Scopus (88) Google Scholar) on other smooth muscles (8Somlyo A.P. Somlyo A.V. Physiol. Rev. 2003; 83: 1325-1358Crossref PubMed Scopus (1666) Google Scholar). Surprisingly, pretreatment with 8-Br-cGMP has no significant effect on U-46619-induced phosphorylation of MYPT1 at either Thr-696 or Thr-853 (Fig. 3B), despite a significant reduction in RhoA·GTP levels (Fig. 2B), suggesting that the activity of the kinase(s) that phosphorylate
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