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KT5823 inhibits cgmp-dependent protein kinase activity in vitro but not in intact human platelets and rat mesangial cells

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第43期275卷 33536-33541页

作者： Burkhardt, M Glazova, M Gambaryan, S Vollkommer, T Butt, E Bader, B Heermeier, K Lincoln, TM Walter, U Palmetshofer, A Med Univ Klin Inst Klin Biochem & Pathobiochem D-97080 Wurzburg Germany Med Univ Klin Div Nephrol D-97080 Wurzburg Germany Russian Acad Sci Sechenov Evolutionary Physiol & Biochem Inst St Petersburg 194223 Russia Univ Alabama Birmingham Div Mol & Cellular Pathol Birmingham AL 35294 USA

Many signal transduction pathways are mediated by the second messengers cgmp and cAMP, cgmp- and cAMP-dependent protein kinases (cGK and PHA), phosphodiesterases, and ion channels. To distinguish among the different cgmp effecters, inhibitors of cGK and PKA have been developed including the K-252 compound KT5823 and the isoquinolinesulfonamide H89, KT5823, an in vitro inhibitor of cGK, has also been used in numerous studies with intact cells to implicate or rule out the involvement of this protein kinase in a given cellular response, However, the efficacy and specificity of KT5823 as cGK inhibitor in intact cells or tissues have never been demonstrated. Here, we analyzed the effects of both KT5823 and H89 on cyclic-nucleotide-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in intact human platelets and rat mesangial cells. These two cell types both express high levels of cGK. KT5823 inhibited purified cGK, However, with both intact human platelets and rat mesangial cells, KT5823 failed to inhibit cGK-mediated serine 157 and serine 239 phosphorylation of VASP induced by nitric oxide, atrial natriuretic peptide, or the membrane-permeant cgmp analog 8-pCPT-cgmp, KT5823 enhanced 8-pCPT-cgmp-stimulated VASP phosphorylation in platelets and did not inhibit forskolin-stimulated VASP phosphorylation in either platelets or mesangial cells, In contrast H89, an inhibitor of both PKA. and cGK, clearly inhibited 8-pCPT-cgmp and forskolin-stimulated VASP phosphorylation in the two cell types. The data indicate that KT5823 inhibits purified cGK but does not affect a cGK-mediated response in the two different cell types expressing cGK I. These observations indicate that data that interpret the effects of KT5823 in intact cells as the major or only criteria supporting the involvement of cGK clearly need to be reconsidered.

关键词：生物碱类/药理学血小板/酶学卡唑类细胞黏附分子/代谢环gmp/类似物和衍生物环gmp/药理学环gmp依赖性蛋白激酶类/拮抗剂和抑制剂酶抑制剂/药理学福司柯林/药理学肾小球膜/酶学吲哚类异喹啉类/药理学微丝蛋白质类一氧化氮/生理学磷蛋白类/代谢磷酰化磺胺类动物牛人类大鼠

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Activation of mitogen-activated protein kinase pathways by cyclic gmp and cyclic gmp-dependent protein kinase in contractile vascular smooth muscle cells

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第48期274卷 34301-34309页

作者： Komalavilas, P Shah, PK Jo, HJ Lincoln, TM Univ Alabama Birmingham Div Mol & Cellular Pathol Dept Pathol Birmingham AL 35294 USA

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic gmp (cgmp) signaling, the cgmp-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cgmp activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cgmp on ERK and JNK activation mere also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cgmp signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cgmp and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC.

关键词： 8-溴环腺苷一磷酸/药理学生物碱类/药理学主动脉卡唑类细胞培养的环gmp/类似物和衍生物环gmp/药理学环gmp依赖性蛋白激酶类/拮抗剂和抑制剂环gmp依赖性蛋白激酶类/遗传学环gmp依赖性蛋白激酶类/代谢酶激活/药物作用酶抑制剂/药理学基因表达吲哚类/药理学 JNK丝裂原活化蛋白激酶类 MAP激酶激酶4 MAP激酶激酶激酶类/代谢丝裂原活化蛋白激酶1/代谢丝裂原活化蛋白激酶3 丝裂原激活蛋白激酶激酶类/代谢丝裂原激活蛋白激酶类/代谢肌收缩/药物作用肌收缩/生理学肌平滑血管/细胞学肌平滑血管/药物作用肌平滑血管/酶学血小板源性生长因子/药理学蛋白质丝氨酸苏氨酸激酶吡咯类/药理学大鼠 Sprague-Dawley 重组融合蛋白质类/药物作用重组融合蛋白质类/遗传学重组融合蛋白质类/代谢硫代核苷酸类/药理学时间因素转染肿瘤坏死因子α/药理学 p38丝裂原活化蛋白激酶类动物大鼠

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NO-induced modulation of calcium-oscillations in pulmonary vascular smooth muscle

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CELL CALCIUM 2000年第6期27卷 329-338页

作者： Pauvert, O Marthan, R Savineau, JP Univ Bordeaux 2 INSERM E9937 Lab Physiol Cellulaire Resp F-33076 Bordeaux France

The effect of the nitric oxide (NO) donor sodium nitroprusside (SNP) on both [Ca2+](i) and mechanical activity was studied in the rat isolated pulmonary artery (RPA). In freshly isolated myocytes loaded with 1 mu M indo-lace-toxymethyl ester for 30 min, short (40-60 s) application of ATP (100 mu M) or ET-1 (0.1 mu M) induced 3-6 cyclic rises in [Ca2+](i) (Ca-oscillations) of decreasing amplitude. Preincubation of cells with SNP (10-250 mu M) for 10 min had no effect on the resting [Ca2+](i) value, but progressively abolished the oscillations. A similar effect was obtained with 8-bromo-cgmp (100-500 mu M). SNP (0.001-100 mu M) concentration-dependently relaxed ATP (10 mM, n = 4) and ET-1 (0.1 mu M, n = 4)-precontracted RPA. 1H-[1,2,4]oxadiazolol [4,3,-a]quinoxalin-1-one (ODQ, 10 mu M), a potent inhibitor of the cytosolic guanylyl cyclase, fully reversed the effect of SNP on ATP-induced [Ca2+](i) oscillations as well as on ATP-precontracted RPA. In contrast, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8, 10 mu M), a potent inhibitor of cgmp-dependent protein kinase (PKG), did not alter the effect of SNP Caffeine (5 mM) induced only one transient [Ca2+](i)-increase (n = 24), the amplitude of which was altered neither by SNP nor by 8-bromo-cgmp. Our results show that the relaxing effect of NO in RPA is related, at least in part, to its action on the Ca-signalling pathway. NO interacts with inositol trisphosphate pathway without interacting with the ryanodine-sensitive receptor. Finally, the effect of NO involves an increase in cgmp but appears independent of activation of PKG. (C) 2000 Harcourt Publishers Ltd.

关键词： 8-溴环腺苷一磷酸/药理学腺苷三磷酸/药理学咖啡因/药理学钙/代谢环gmp依赖性蛋白激酶类/拮抗剂和抑制剂内皮缩血管肽1/药理学吲哚类/诊断应用等长收缩吗多明/类似物和衍生物吗多明/药理学肌平滑血管/代谢一氧化氮供体/药理学硝普钠/药理学青霉胺/类似物和衍生物青霉胺/药理学肺动脉/药物作用肺动脉/代谢大鼠 Wistar S-亚硝基-N-乙酰基青霉胺肌浆网/药物作用肌浆网/代谢血管舒张/药物作用血管舒张药/药理学动物男(雄)性大鼠

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Regulation of pacemaker currents in interstitial cells of Cajal from murine small intestine by cyclic nucleotides

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JOURNAL OF PHYSIOLOGY-LONDON 2000年第1期527卷 149-162页

作者： Koh, XD Kim, TW Jun, JY Glasgow, NJ Ward, SM Sanders, KM Univ Nevada Sch Med Dept Physiol & Cell Biol Reno NV 89557 USA

1. Electrical rhythmicity (slow waves) in gastrointestinal muscles (GI) is generated by interstitial cells of Cajal (ICC). Cultured ICC from the murine small intestine were studied with the patch-clamp technique to characterize regulation of pacemaker currents by cyclic nucleotides. Cyclic nucleotide agonists were also tested on intact strips of murine small intestine. 2. Nitric oxide donors slowed the frequency of pacemaker currents in a concentration-dependent manner. These effects depended on cgmp formation and were reduced by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The effects of nitric oxide donors were mimicked by meabrane-permeable analogues of cgmp. The specific cgmp phosphodiesterase inhibitor zaprinast reduced the frequency of spontaneous pacemaker currents. 3. The cgmp-dependent effects on pacemaker currents were not affected by okadaic acid or KT-5823, an inhibitor of protein kinase G. 4. Forskolin, but not dideoxy forskolin, reduced the frequency of spontaneous pacemaker activity and activated a sustained outward current. The latter was likely to be due to ATP-dependent K+ channels because it was blocked by glibenclamide. 5. The effects of forskolin were not mimicked by membrane-permeable cAMP analogues. A membrane-permeable inhibitor of protein kinase A, myristoylated PKA inhibitor, and the adenylyl cyclase inhibitor SQ-22536, had no effect on responses to forskolin. 6. Responses of intact muscles to cgmp and cAMP agonists were similar to the responses of pacemaker cells. Changes in resting membrane potential and slow wave amplitude, however, were noted in intact jejunal muscles that were not observed in ICC. Differences in responses may have been due to the effects of cyclic nucleotide agonists on smooth muscle cells that would sum with responses of ICC in intact jejunal muscle strips. 7. A cgmp-dependent mechanism regulates slow wave frequency, but this occurs through direct action of cgmp not via protein phosphorylation. Regulation of pac

关键词：细胞培养的环AMP/类似物和衍生物环AMP/代谢环AMP/生理学环AMP依赖性蛋白激酶类/拮抗剂和抑制剂环gmp/类似物和衍生物环gmp/拮抗剂和抑制剂环gmp/生理学环gmp依赖性蛋白激酶类/拮抗剂和抑制剂导电性胃肠活动鸟苷酸环化酶/拮抗剂和抑制剂小肠/细胞学小肠/药物作用小肠/生理学小鼠近交BALBC 一氧化氮/代谢硝普钠/药理学核苷酸类环/激动剂核苷酸类环/拮抗剂和抑制剂核苷酸类环/生理学膜片钳术磷酸二酯酶抑制剂/药理学动物小鼠

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Dual modulation of swelling-activated chloride current by NO and NO donors in rabbit portal vein myocytes

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JOURNAL OF PHYSIOLOGY-LONDON 2000年第1期528卷 15-24页

作者： Ellershaw, DC Greenwood, IA Large, WA St George Hosp Sch Med Dept Pharmacol & Clin Pharmacol Cardiovasc Res Grp London SW17 0RE England

1. The effects of authentic NO and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) on swelling-activated chloride currents (I-swell) were investigated in freshly dispersed rabbit portal vein smooth muscle cells. I-swell was recorded with the perforated patch configuration of the whole-cell patch clamp technique. 2. In approximately 50 % of cells NO and SNAP inhibited the amplitude of I-swell by about 45 % in a, voltage-independent manner. I-swell was also inhibited by an inhibitor of NO-sensitive guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and by KT5823, an inhibitor of cCMP-dependent protein kinase. 3. In other cells both NO and SNAP enhanced I-swell by about 40% in a voltage-independent manner. A similar increase was produced by application of the cell-permeable cgmp analogue 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br-cgmp). However, 8-Br-cgmp had no effect on current amplitude in cells pre-treated with KT5823. In contrast 8-Br-cgmp increased the amplitude of I-swell in cells which had been pre-treated with ODQ. 4. XNAP also modulated I-swell recorded in the conventional whole-cell configuration with internal solutions containing 10 mM EGTA to rule out any contribution from Ca2+-activated Cl- currents. 5. These data suggest that the amplitude of I-swell can be enhanced by NO via a cgmp-dependent phosphorylation and inhibited by NO in a cgmp-independent manner.

关键词：细胞大小/药物作用细胞大小/生理学细胞培养的螯合剂/药理学氯化物通道/药物作用氯化物通道/代谢环gmp/类似物和衍生物环gmp/药理学环gmp依赖性蛋白激酶类/拮抗剂和抑制剂环gmp依赖性蛋白激酶类/代谢鸟苷酸环化酶/拮抗剂和抑制剂葡甲胺/代谢葡甲胺/药理学膜电位/药物作用肌平滑血管/细胞学肌平滑血管/代谢一氧化氮/代谢一氧化氮/药理学一氧化氮供体/代谢一氧化氮供体/药理学膜片钳术青霉胺/类似物和衍生物青霉胺/药理学门静脉/细胞学门静脉/代谢 S-亚硝基-N-乙酰基青霉胺钠/代谢动物兔

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Identification of a conserved residue responsible for the autoinhibition of cgmp-dependent protein kinase Iα and β

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FEBS LETTERS 2000年第1期466卷 175-178页

作者： Yuasa, K Michibata, H Omori, K Yanaka, N Tanabe Seiyaku Co Ltd Discovery Res Lab Yodogawa Ku Osaka 5328505 Japan Tanabe Seiyaku Co Ltd Discovery Res Lab Toda Saitama 3358505 Japan

We isolated a constitutively active form of cgmp-dependent protein kinase I alpha (cGK I alpha) by PCR-driven random mutagenesis. The replacement of Ile-63 by Thr in the autoinhibitory domain results in the enhancement of autophosphorylation and the basal kinase activity in the absence of cgmp. The hydrophobicity at position 63 is essential for the inactive state of cGK I alpha, and Ile-78 of cGK I beta is also required for the autoinhibitory property. Furthermore, cGK I alpha (Ile-63-Thr) is constitutively active in vivo. These findings suggest that a conserved residue in the autoinhibitory domain was involved in the autoinhibition of both cGK Is. (C) 2000 Federation of European Biochemical Societies.

关键词：氨基酸序列碱基序列 COS细胞催化域/遗传学保守序列环gmp依赖性蛋白激酶类/拮抗剂和抑制剂环gmp依赖性蛋白激酶类/遗传学环gmp依赖性蛋白激酶类/代谢 DNA引物/遗传学诱变磷酰化蛋白质结构三级重组蛋白质类/拮抗剂和抑制剂重组蛋白质类/遗传学重组蛋白质类/代谢动物牛人类

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Endothelin-1 as a protective factor against beta-adrenergic agonist-induced apoptosis in cardiac myocytes

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JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY 2000年第4期36卷 1411-1418页

作者： Araki, M Hasegawa, K Iwai-Kanai, E Fujita, M Sawamura, T Kakita, T Wada, H Morimoto, T Sasayama, S Kyoto Univ Grad Sch Med Dept Cardiovasc Med Sakyo Ku Kyoto 6068507 Japan Kyoto Univ Coll Med Technol Kyoto 6068507 Japan Natl Cardiovasc Ctr Osaka Japan

OBJECTIVES The purpose of this study was to investigate the regulation of beta-adrenergic agonist-induced apoptosis by endothelin-1 (ET-1) in cardiac myocytes. BACKGROUND Numerous hormonal factors including norepinephrine and ET-1 are activated in patients with heart failure. These factors may be involved in the positive and negative regulation of myocardial cell apoptosis observed in failing hearts. Recently, it has been shown that norepinephrine can induce myocardial cell apoptosis via a beta-adrenergic receptor-dependent pathway. METHODS Primary cardiac myocytes were prepared from neonatal rats. These cells were stimulated with the beta-adrenergic agonist isoproterenol (ISO) in the presence or absence of ET-1. RESULTS The administration of 10(-7) mol/liter of ET-1 completely blocked Iso-induced apoptosis. An endothelin type A receptor antagonist, FR139317, negated the inhibitory effect of ET-1 on apoptosis, while the endothelin type B receptor antagonist BQ788 did not show such a negation. Endothelin-1 also inhibited apoptosis induced by a membrane-permeable cAMP analogue (8-Br-cAMP), which bypassed Gi. The effect of ET-1 was neutralized by an MEK-1-specific inhibitor (PD098059), a phosphatidylinositol 3' kinase inhibitor (wortmannin) and its downstream pp70 S6-kinase inhibitor, rapamycin. CONCLUSIONS These findings suggest that ET-1 represents a protective factor against myocardial cell apoptosis in heart failure and that this effect is mediated mainly through endothelin type A receptor-dependent pathways involving multiple downstream signalings in cardiac myocytes. (J Am Cell Cardiol 2000;36:1411- 8) (C) 2000 by the American College of Cardiology.

关键词：肾上腺素能β激动剂/毒性动物新生抗菌药/药理学细胞凋亡/药物作用吖庚因类/药理学钙-钙调素依赖性蛋白激酶类/拮抗剂和抑制剂细胞培养的环gmp/类似物和衍生物环gmp/毒性环gmp依赖性蛋白激酶类/拮抗剂和抑制剂内皮缩血管肽1/药理学酶抑制剂/药理学类黄酮物质/药理学心脏/药物作用吲哚类/药理学异丙肾上腺素/毒性心肌/病理学寡肽类/药理学哌啶类/药理学大鼠 Sprague-Dawley 受体内皮素A 西罗莫司/药理学动物大鼠

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