摘要:

The identification of a majority of the polypeptides in mitochondria would be invaluable because they play crucial and diverse roles in many cellular processes and diseases. The endogenous production of reactive oxygen species (ROS) is a major limiter of life as illustrated by studies in which the transgenic overexpression in invertebrates of catalytic antioxidant enzymes results in increased lifespans. Mitochondria have received considerable attention as a principal source—and target—of ROS. Mitochondrial oxidative stress has been implicated in heart disease including myocardial preconditioning, ischemia/reperfusion, and other pathologies. In addition, oxidative stress in the mitochondria is associated with the pathogenesis of Alzheimer’s disease, Parkinson’s disease, prion diseases, and amyotrophic lateral sclerosis (ALS) as well as aging itself. The rapidly emerging field of proteomics can provide powerful strategies for the characterization of mitochondrial proteins. Current approaches to mitochondrial proteomics include the creation of detailed catalogues of the protein components in a single sample or the identification of differentially expressed proteins in diseased or physiologically altered samples versus a reference control. It is clear that for any proteomics approach prefractionation of complex protein mixtures is essential to facilitate the identification of low-abundance proteins because the dynamic range of protein abundance within cells has been estimated to be as high as 107. The opportunities for identification of proteins directly involved in diseases associated with or caused by mitochondrial dysfunction are compelling. Future efforts will focus on linking genomic array information to actual protein levels in mitochondria.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Although activation of protein kinase C (PKC) &egr; and mitogen-activated protein kinases (MAPKs) are known to play crucial roles in the manifestation of cardioprotection, the spatial organization of PKC&egr; signaling modules in naïve and protected myocardium remains unknown. Based on evidence that mitochondria are key mediators of the cardioprotective signal, we hypothesized that PKC&egr; and MAPKs interact, and that they form functional signaling modules in mitochondria during cardioprotection. Both immunoblotting and immunofluorescent staining demonstrated that PKC&egr;, ERKs, JNKs, and p38 MAPK co-localized with cardiac mitochondria. Moreover, transgenic activation of PKC&egr; greatly increased mitochondrial PKC&egr; expression and activity, which was concomitant with increased mitochondrial interaction of PKC&egr; with ERKs, JNKs, and p38 as determined by co-immunoprecipitation. These complex formations appeared to be independent of PKC&egr; activity, as the interactions were also observed in mice expressing inactive PKC&egr;. However, although both active and inactive PKC&egr; bound to all three MAPKs, increased phosphorylation of mitochondrial ERKs was only observed in mice expressing active PKC&egr; but not in mice expressing inactive PKC&egr;. Examination of potential downstream targets of mitochondrial PKC&egr;-ERK signaling modules revealed that phosphorylation of the pro-apoptotic protein Bad was elevated in mitochondria. Together, these data show that PKC&egr; forms subcellular-targeted signaling modules with ERKs, leading to the activation of mitochondrial ERKs. Furthermore, formation of mitochondrial PKC&egr;-ERK modules appears to play a role in PKC&egr;-mediated cardioprotection, in part by the phosphorylation and inactivation of Bad.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

The p53 tumor suppressor gene regulates growth arrest and apoptosis after DNA damage. Recent studies suggest that p53 is inactive in vascular smooth muscle cells (VSMCs) in human angioplasty restenosis, promoting VSMC accumulation and vessel stenosis. In contrast, the success of irradiation (brachytherapy) for in-stent restenosis argues that DNA-damage p53 responses are intact. We examined p53 expression and function in human VSMCs from normal vessels (n-VSMCs) and angioplasty/in-stent restenosis sites (r-VSMCs). p53 expression was uniformly low in all VSMCs and was induced by DNA damage. However, p53 induced profoundly different biological effects in r-VSMCs versus n-VSMCs, causing growth arrest and apoptosis in r-VSMCs only. In addition, dominant-negative p53 promoted cell proliferation and apoptosis in r-VSMCs but not n-VSMCs. Cytotoxic drug– or irradiation-induced growth arrest and apoptosis in both cell types was mediated only partly by p53. In contrast, cyclin D degradation in response to DNA damage, a critical early mediator of growth arrest, was impaired in r-VSMCs, an effect that required p53. We conclude that p53 expression and function are normal or increased in r-VSMCs and may underlie the success of brachytherapy. We also identify a restenosis VSMC-specific defect in cyclin D degradation induced by DNA damage.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

cGMP-dependent protein kinase (PKG) expression is highly variable and decreases in cultured vascular smooth muscle cells (VSMCs), exposure of cells to nitric oxide (NO), or in response to balloon catheter injury in vivo. In this study, the mechanisms of human type I PKG-&agr; (PKG-I&agr;) gene expression were examined. Three structurally unrelated NO donors decreased PKG-I&agr; promoter activity after transfection of a promoter/luciferase construct in VSMCs. Promoter deletion analysis demonstrated that (1) a 120-bp promoter containing tandem Sp1 sites was sufficient to drive basal PKG-I&agr; promoter activity, and (2) NO was inhibitory at this site. Cyclic nucleotide analogues also suppressed PKG-I&agr; promoter activity with cAMP being more potent than cGMP. The effects of cyclic nucleotides to suppress PKG-I&agr; promoter activity were attenuated by a specific cAMP-dependent protein kinase (PKA) inhibitor. Single or double mutation of Sp1 binding sites abolished PKG-I&agr; expression. Moreover, Sp1 binding activity on the PKG-I&agr; promoter was detected in A7r5 cells, and this binding was inhibited by NO and cyclic nucleotides. These results indicate that PKG-I&agr; gene expression is driven by an Sp1 transcription mechanism, and that NO and cAMP inhibit Sp1-mediated PKG-I&agr; gene expression through separate mechanisms.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Although estradiol (E2) has been recognized to exert several vasculoprotective effects in several species, its effects in mouse vasomotion are unknown, and consequently, so is the estrogen receptor subtype mediating these effects. We investigated the effect of E2(80 &mgr;g/kg/day for 15 days) on NO production in the thoracic aorta of ovariectomized C57Bl/6 mice compared with those given placebo. E2increased basal NO production. In contrast, the relaxation in response to ATP, to the calcium ionophore A23187, and to sodium nitroprusside was unaltered by E2, whereas acetylcholine-elicited relaxation was decreased. The abundance of NO synthase I, II, and III immunoreactive proteins (using Western blot) in thoracic aorta homogenates was unchanged by E2. To determine the estrogen receptor (ER) subtype involved in these effects, transgenic mice in which either the ER&agr; or ER&bgr; has been disrupted were ovariectomized and treated, or not, with E2. Basal NO production was increased and the sensitivity to acetylcholine decreased in ER&bgr; knockout mice in response to E2, whereas this effect was abolished in ER&agr; knockout mice. Finally, these effects of E2on vasomotion required long-term and/or in vivo exposure, as short-term incubation of aortic rings with 10 nmol/L E2in the isolated organ chamber did not elicit any vasoactive effects. In conclusion, this study demonstrates that ER&agr;, but not ER&bgr;, mediates the beneficial effect of E2on basal NO production.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Extracellular adenosine production by the GPI-anchored Ecto-5′-Nucleotidase (Ecto-5′-Nu) plays an important role in the cardiovascular system, notably in defense against hypoxia. It has been previously suggested that HMG-CoA reductase inhibitors (HRIs) could potentiate the hypoxic stimulation of Ecto-5′Nu in myocardial ischemia. In order to elucidate the mechanism of Ecto-5′-Nu stimulation by HRIs, Ecto-5′-Nu activity and expression were determined in an aortic endothelial cell line (SVAREC) incubated with lovastatin. Lovastatin enhanced Ecto-5′-Nu activity in a dose-dependent manner. This increase was not supported by de novo synthesis of the enzyme because neither the mRNA content nor the total amount of the protein were modified by lovastatin. By contrast, lovastatin enhanced cell surface expression of Ecto-5′-Nu and decreased endocytosis of Ecto-5′-Nu, as evidenced by immunostaining. This effect appeared unrelated to modifications of cholesterol content or Ecto-5′-Nu association with detergent-resistant membranes. The effect of lovastatin was reversed by mevalonate, the substrate of HMG-CoA reductase, by its isoprenoid derivative, geranyl-geranyl pyrophosphate, and by cytotoxic necrotizing factor, an activator of Rho-GTPases. Stimulation of Ecto-5′-Nu by lovastatin enhanced the inhibition of platelet aggregation induced by endothelial cells. In conclusion, lovastatin enhances Ecto-5′-Nu activity and membrane expression in endothelial cells. This effect seems independent of lowering cholesterol content but could be supported by an inhibition of Ecto-5′-Nu endocytosis through a decrease of Rho-GTPases isoprenylation.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Glycoprotein IIb/IIIa (GP IIb/IIIa) inhibitors were shown recently to facilitate the rate and the extent of pharmacological thrombolysis. However, their synergistic potential with rtPA in dissolving thrombotic vaso-occlusions is not fully understood. We have therefore developed a dynamic and structural approach for analysis of fibrinolysis to assess the inhibiting effect of platelets and the facilitating effect of GPIIb/IIIa inhibitors in dissolving platelet-rich clots (PRCs). Fluorescent rtPA was used to study the architecture of PRCs, to follow the progression of the rtPA binding front, and to measure the lysis-front velocity using confocal microscopy. Fibrinolysis resistance of PRCs was related to a reduction of both rtPA binding and lysis-front velocities of platelet-rich areas compared with platelet-poor areas (2.4±0.2 versus 3.5±0.4 &mgr;m/min for rtPA binding velocity,P=0.04, and 1.2±0.6 versus 2.8±0.2 &mgr;m/min for lysis-front velocity,P=0.008, in platelet-rich and platelet-poor areas, respectively). Fibrinolysis appeared heterogeneous, leaving platelet-rich areas un-lysed. Adding pharmacological concentrations of abciximab (0.068 &mgr;mol/L) or eptifibatide (1 &mgr;mol/L) before clotting decreased the average surface of platelet-rich areas by 64% (P=0.0005) and 72% (P=0.0007), respectively. The resulting equalization of rtPA binding rate and rtPA binding-front velocity between platelet-rich and platelet-poor areas led to a 3-fold increase of the lysis-front velocity in platelet-rich areas of either abciximab-PRC (P=0.006) or eptifibatide-PRC (P=0.03). The overall lysis rate of treated-PRC was increased by 74% compared with control-PRC (P<0.01). These results demonstrate that fibrinolysis resistance of PRCs is related primarily to the heterogeneity in the clot structure between platelet-rich and platelet-poor areas. GP IIb/IIIa inhibitors facilitate the rate and the extent of fibrinolysis by improving rtPA binding velocity and, subsequently, the lysis rate in platelet-rich areas. These findings provide new insights on the synergistic potential of GP IIb/IIIa inhibitors and fibrinolytic agents.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Phosphorylation of Na channels has been suggested to increase their Ca permeability. Termed “slip-mode conductance” (SMC), this hypothesis predicts that Ca influx via protein kinase A (PKA)-modified Na channels can induce sarcoplasmic reticulum (SR) Ca release. We tested this hypothesis by determining if SR Ca release is graded withINain the presence of activated PKA (with Isoproterenol, ISO). Vm,Im, and [Ca]iwere measured in feline (n=26) and failing human (n=19) ventricular myocytes. Voltage steps from −70 through −40 mV were used to gradeINa. Na channel antagonists (tetrodotoxin), L-type Ca channel (ICa,L) antagonists (nifedipine, cadmium, verapamil), and agonists (Bay K 8644, FPL 64176) were used to separate SMC fromICa,L. In the absence of ISO,INawas associated with SR Ca release in human but not feline myocytes. After ISO, gradedINawas associated with small amounts of SR Ca release in feline myocytes and the magnitude of release increased in human myocytes.INa-related SR Ca release was insensitive to tetrodotoxin (n=10) but was blocked by nifedipine (n=10) and cadmium (n=3). SR Ca release was induced over the same voltage range in the absence of ISO with Bay K 8644 and FPL 64176 (n=9). Positive voltage steps (to 0 mV) to fully activate Na channels (SMC) in the presence of ISO and Verapamil only caused SR Ca release when block ofICa,Lwas incomplete. We conclude that PKA-mediated increases inICa,Land SR Ca loading can reproduce many of the experimental features of SMC.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

This study demonstrates that caveolae, omega-shaped membrane invaginations, are involved in cardiac sodium channel regulation by a mechanism involving the &agr; subunit of the stimulatory heterotrimeric G-protein, G&agr;s, via stimulation of the cell surface &bgr;-adrenergic receptor. Stimulation of &bgr;-adrenergic receptors with 10 &mgr;mol/L isoproterenol in the presence of a protein kinase A inhibitor increased the whole-cell sodium current by a “direct” cAMP-independent G-protein mechanism. The addition of antibodies against caveolin-3 to the cell’s cytoplasm via the pipette solution abrogated this direct G protein-induced increase in sodium current, whereas antibodies to caveolin-1 or caveolin-2 did not. Voltage-gated sodium channel proteins were found to associate with caveolin-rich membranes obtained by detergent-free buoyant density separation. The purity of the caveolar membrane fraction was verified by Western blot analyses, which indicated that endoplasmic/sarcoplasmic reticulum, endosomal compartments, Golgi apparatus, clathrin-coated vesicles, and sarcolemmal membranes were excluded from the caveolin-rich membrane fraction. Additionally, the sodium channel was found to colocalize with caveolar membranes by immunoprecipitation, indirect immunofluorescence, and immunogold transmission electron microscopy. These results suggest that stimulation of &bgr;-adrenergic receptors, and thereby G&agr;s, promotes the presentation of cardiac sodium channels associated with caveolar membranes to the sarcolemma.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends on the integrity of the carboxyl terminal (CT) domain. Experiments usingXenopusoocytes show that truncation of the CT domain alters the time course for current inactivation; however, correlation with the behavior of single Cx43 channels has been lacking. Furthermore, whereas chemical gating is associated with a “ball-and-chain” mechanism, there is no evidence whether transjunctional voltage regulation for Cx43 follows a similar model. We provide data on the properties of transjunctional currents from voltage-clamped pairs of mammalian tumor cells expressing either wild-type Cx43 or a mutant of Cx43 lacking the carboxyl terminal domain (Cx43M257). Cx43 transjunctional currents showed bi-exponential decay and a residual steady-state conductance of approximately 35% maximum. Transjunctional currents recorded from Cx43M257 channels displayed a single, slower exponential decay. Long transjunctional voltage pulses caused virtual disappearance of the residual current at steady state. Single channel data revealed disappearance of the residual state, increase in the mean open time, and slowing of the transition times between open and closed states. Coexpression of CxM257 with Cx43CT in a separate fragment restored the lower conductance state. We propose that Cx43CT is an effector of fast voltage gating. Truncation of Cx43CT limits channel transitions to those occurring across the higher energy barrier that separates open and closed states. We further propose that a ball-and-chain interaction provides the fast component of voltage-dependent gating between CT domain and a receptor affiliated with the pore.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID