ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

We tested whether opening of mitochondrial ATP-sensitive K+(mitoKATP) channels depolarizes mitochondrial membrane potential (&Dgr;&PSgr;m) and thereby prevents the mitochondrial Ca2+overload. With the use of a Nipkow disk confocal system, the mitochondrial Ca2+concentration ([Ca2+]m) and &Dgr;&PSgr;min rat ventricular myocytes were measured by loading cells with Rhod-2 and JC-1, respectively. Exposure to ouabain (1 mmol/L) for 30 minutes produced mitochondrial Ca2+overload, and the intensity of Rhod-2 fluorescence significantly increased to 173±16% of baseline (P<0.001). Treatment of myocytes with the mitoKATPchannel opener diazoxide (100 &mgr;mol/L) blunted the ouabain-induced mitochondrial Ca2+overload (131±10% of baseline;P<0.001 versus ouabain). Moreover, diazoxide significantly depolarized the &Dgr;&PSgr;mand reduced the intensity of JC-1 fluorescence during application of ouabain to 89±2% of baseline (P<0.05). These effects of diazoxide were blocked by the mitoKATPchannel blocker 5-hydroxydecanoate (500 &mgr;mol/L). These results indicate that opening of mitoKATPchannels prevents a mitochondrial Ca2+overload in association with &Dgr;&PSgr;mdepolarization and thereby protects myocardium against ischemic damage.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

The leukocyte integrin Mac-1 (&agr;M&bgr;2, CD11b/CD18) regulates important cell functions in inflammation, including adhesion, phagocytosis, and oxidative burst. Deficiency of Mac-1 reduces vessel wall inflammation and neointimal thickening after murine carotid artery injury. Although Mac-1 has been implicated in modulating AP-1 and NF-&kgr;B activity, the signal transduction pathways involved are undefined. cDNA array analysis of Mac-1–clustered compared with –nonclustered monocytic THP-1 cells showed increased expression of the signal transducer TRAF6 (TNF receptor–associated factor 6), leading us to consider the possibility that Mac-1 used a Toll/IL-1 receptor family–like signaling pathway. Mac-1–dependent activation of NF-&kgr;B was potentiated by wild-type, and attenuated by dominant negative, TRAF6- and TGF-&bgr;–activated kinase (TAK1) constructs. IRAK1 (IL-1 receptor associated kinase), a kinase immediately upstream of TRAF6, coimmunoprecipitated with Mac-1. Taken together, these observations indicate that Mac-1 recruits a Toll/IL-1 receptor family–like cascade to modulate NF-&kgr;B activity. This represents a new pathway for integrin-dependent modulation of gene expression.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

3-Hydroxy-3-methylglutaryl (HMG)–coenzyme A reductase inhibitors or statins exert direct beneficial effects on the endothelium in part through an increase in nitric oxide (NO) production. Here, we examined whether posttranslational modifications of the endothelial NO synthase (eNOS) could account for the proangiogenic effects of statins. We used endothelial cells (ECs) isolated from cardiac microvasculature, aorta, and umbilical veins, as well as dissected microvessels and aortic rings, that were cultured on reconstituted basement membrane matrix (Matrigel). Tube or precapillary formation was evaluated after statin treatment, in parallel with immunoblotting and immunoprecipitation experiments. Atorvastatin stimulated NO-dependent angiogenesis from both isolated and outgrowing (vessel-derived) ECs, independently of changes in eNOS expression. We found that in macro- but not microvascular ECs, atorvastatin stabilized tube formation through a decrease in caveolin abundance and its inhibitory interaction with eNOS. We also identified the chaperone protein hsp90 as a key target for the proangiogenic effects of statins. Using geldanamycin, an inhibitor of hsp90 function, and overexpression of recombinant hsp90, we documented that the statin-induced phosphorylation of eNOS on Ser1177 was directly dependent on the ability of hsp90 to recruit Akt in the eNOS complex. Finally, we showed that statin promoted the tyrosine phosphorylation of hsp90 and the direct interaction of hsp90 with Akt, which further potentiated the NO-dependent angiogenic processes. Our study provides new mechanistic insights into the NO-mediated angiogenic effects of statins and underscores the potential of these drugs and other modulators of hsp90 and caveolin abundance to promote neovascularization in disease states associated or not with atherosclerosis.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

The giant muscle protein titin contains a unique sequence, the PEVK domain, the elastic properties of which contribute to the mechanical behavior of relaxed cardiomyocytes. Here, human N2-B–cardiac PEVK was expressed inEscherichia coliand tested—along with recombinant cardiac titin constructs containing immunoglobulin-like or fibronectin-like domains—for a possible interaction with actin filaments. In the actomyosin in vitro motility assay, only the PEVK construct inhibited actin filament sliding over myosin. The slowdown occurred in a concentration-dependent manner and was accompanied by an increase in the number of stationary actin filaments. High [Ca2+] reversed the PEVK effect. PEVK concentrations ≥10 &mgr;g/mL caused actin bundling. Actin-PEVK association was found also in actin fluorescence binding assays without myosin at physiological ionic strength. In cosedimentation assays, PEVK-titin interacted weakly with actin at 0°C, but more strongly at 30°C, suggesting involvement of hydrophobic interactions. To probe the interaction in a more physiological environment, nonactivated cardiac myofibrils were stretched quickly, and force was measured during the subsequent hold period. The observed force decline could be fit with a three-order exponential-decay function, which revealed an initial rapid-decay component (time constant, 4 to 5 ms) making up 30% to 50% of the whole decay amplitude. The rapid, viscous decay component, but not the slower decay components, decreased greatly and immediately on actin extraction with Ca2+-independent gelsolin fragment, both at physiological sarcomere lengths and beyond actin-myosin overlap. Steady-state passive force dropped only after longer exposure to gelsolin. We conclude that interaction between PEVK-titin and actin occurs in the sarcomere and may cause viscous drag during diastolic stretch of cardiac myofibrils. The interaction could also oppose shortening during contraction.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Protein kinase C (PKC) &egr; and PKC&dgr; translocation in neonatal rat ventricular myocytes (NRVMs) is accompanied by subsequent activation of the ERK, JNK, and p38MAPKcascades; however, it is not known if either or both novel PKCs are necessary for their downstream activation. Use of PKC inhibitors to answer this question is complicated by a lack of isoenzyme specificity, and the fact that many PKC inhibitors stimulate JNK and p38MAPKactivity. Therefore, replication-defective adenoviruses (Advs) encoding constitutively active (ca) mutants of PKC&egr; and PKC&dgr; were used to test if either or both of these PKCs are sufficient to activate ERKs, JNKs, and/or p38MAPKin NRVMs. Adv-caPKC&egr; infection (1 to 25 multiplicities of viral infection (MOI); 4 to 48 hours) increased total PKC&egr; levels in a time- and dose-dependent manner, with maximal expression observed 8 hours after Adv infection. Adv-caPKC&egr; induced a time- and dose-dependent increase in phosphorylated p42 and p44 ERKs, as compared with a control Adv encoding &bgr;-galactosidase (Adv-ne&bgr;gal). Maximal ERK phosphorylation occurred 8 hours after Adv infection. In contrast, JNK was only minimally activated, and p38MAPKwas relatively unaffected. Adv-caPKC&dgr; infection (1 to 25 MOI, 4 to 48 hours) increased total PKC&dgr; levels in a similar fashion. Adv-caPKC&dgr; (5 MOI) induced a 29-fold increase in phosphorylated p54 JNK, and a 15-fold increase in phosphorylated p38MAPK24 hours after Adv infection. In contrast, p42 and p44 ERK were only minimally activated. Whereas neither Adv induced NRVM hypertrophy, Adv-caPKC&dgr;, but not Adv-caPKC&egr;, induced NRVM apoptosis. We conclude that the novel PKCs differentially regulate MAPK cascades and apoptosis in an isoenzyme-specific and time-dependent manner.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Mitochondrial ATP-sensitive potassium (mitoKATP) channels play a key role in ischemic preconditioning of the heart. However, the mechanism of cardioprotection remains controversial. We measured rhod-2 fluorescence in adult rabbit ventricular cardiomyocytes as an index of mitochondrial matrix Ca2+concentration ([Ca2+]m), using time-lapse confocal microscopy. To simulate ischemia and reperfusion (I/R), cells were exposed to metabolic inhibition (50 minutes) followed by washout with control solution. Rhod-2 fluorescence gradually increased during simulated ischemia and rose even further with reperfusion. The mitoKATPchannel opener diazoxide attenuated the accumulation of [Ca2+]mduring simulated I/R (EC50=18 &mgr;mol/L). These effects of diazoxide were blocked by the mitoKATPchannel antagonist 5-hydroxydecanoate (5HD). In contrast, inhibitors of the mitochondrial permeability transition (MPT), cyclosporin A and bongkrekic acid, did not alter [Ca2+]maccumulation during ischemia, but markedly suppressed the surge in rhod-2 fluorescence during reperfusion. Measurements of mitochondrial membrane potential, &Dgr;&PSgr;m, in permeabilized myocytes revealed that diazoxide depolarized &Dgr;&PSgr;m(by 12% at 10 &mgr;mol/L,P<0.01) in a 5HD-inhibitable manner. Our data support the hypothesis that attenuation of mitochondrial Ca2+overload, as a consequence of partial mitochondrial membrane depolarization by mitoKATPchannels, underlies cardioprotection. Furthermore, mitoKATPchannels and the MPT differentially affect mitochondrial calcium homeostasis: mitoKATPchannels suppress calcium accumulation during I/R, while the MPT comes into play only upon reperfusion.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Nuclear factor-&kgr;B (NF&kgr;B) plays a significant role in the coordinated transactivation of cytokine, inducible NO synthase (iNOS), and adhesion molecule genes. Although inflammation is an essential pathological feature of myocarditis, the role of NF&kgr;B in this process remains obscure. We examined the role of NF&kgr;B in the progression of rat experimental autoimmune myocarditis (EAM) and tested the hypothesis that NF&kgr;B blockade with a decoy against theciselement of NF&kgr;B can prevent the progression of EAM. Lewis rats were immunized with purified porcine cardiac myosin to establish EAM on day 0. NF&kgr;B decoy was infused into the rat coronary artery on day 0 (group NF0), 7 (group NF7), or 14 (group NF14) and harvested on day 21. Scrambled decoy was infused on day 0 (group SD0), 7 (group SD7), or 14 (group SD14) and served for control groups. The ratios of myocarditis-affected areas to the ventricular cross-sectional area of all treatment groups were significantly lower than those of the control groups (group NF0, 33±18% versus SD0, 53±14%; group NF7, 19±15% versus SD7, 50±16%; and group NF14, 34±10% versus SD14, 52±14%). Immunohistochemical and immunoblot analyses showed expression of ICAM-1, iNOS, IL-2, and TNF&agr; in myocardium of scrambled decoy groups, and this expression was effectively suppressed by NF&kgr;B decoy treatment. Thus, we found that NF&kgr;B is a key regulator in the progression of EAM and that in vivo transfection of NF&kgr;B decoy reduces the severity of EAM.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID