ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Selective troponin I (TnI) modification has been demonstrated to be in part responsible for the contractile dysfunction observed with myocardial ischemia/reperfusion injury. We have isolated and characterized modified TnI products in isolated rat hearts after 0, 15, or 60 minutes of ischemia followed by 45 minutes of reperfusion using affinity chromatography with cardiac troponin C (TnC) and an anti-TnI antibody, immunological mapping, reversed-phase high-performance liquid chromatography, and mass spectrometry. Rat cardiac TnI becomes progressively degraded from 210 amino acid residues to residues 1-193, 63-193, and 73-193 with increased severity of injury. Degradation is accompanied by formation of covalent complexes between TnI 1-193 and, respectively, TnC residues 1-94 and troponin T (TnT) residues 191-298. The covalent complexes are likely a result of isopeptide bond formation between lysine 193 of TnI and glutamine 191 of TnT by the cross-linking enzyme transglutaminase. With severe ischemia, cellular necrosis results in specific release of TnI 1-193 into the reperfusion effluent and TnT degradation in the myocardium (25-, 27-, and 33-kDa products). Two-dimensional electrophoresis demonstrated that phosphorylation of TnI prevents ischemia-induced degradation. This study characterized the modified TnI products in isolated rat hearts reperfused after a brief or severe period of ischemia, revealing the progressive nature of TnI degradation, changes in phosphorylation, and covalent complexes with ischemia/reperfusion injury. Finally, we propose a model for ischemia/reperfusion injury in which the extent of proteolytic and transglutaminase activities ultimately determines whether apoptosis or necrosis is achieved. (Circ Res. 1999;84:9-20.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

50-fold induction of inducible NO synthase mRNA and the release of large amounts (5 to 8 nmol/[micro sign]g protein) of NO metabolites (NOx) into the medium. Cell death was completely blocked by an NO synthase inhibitor and attenuated by antioxidants (N-acetylcysteine and DTT) and the caspase inhibitor ZVAD-fmk. Cytokines also mediated an NO-dependent, sustained increase in myocyte expression of the Bcl-2 homologs Bak and Bcl-x(L). The NO donor S-nitrosoglutathione also induced apoptosis and cell levels of Bak, but not of Bcl-x(L). All effects of cytokines, including poly(ADP-ribose) polymerase cleavage, could be attributed to interleukin-1 beta; interferon-gamma and tumor necrosis factor-alpha had no independent effects on apoptosis or on NOx production. We conclude that cytokine toxicity to neonatal cardiac myocytes results from the induction of NO and subsequent activation of apoptosis, at least in part through the generation of oxygen free radicals. The rate and extent of this apoptosis is modulated by alterations in the cellular balance of Bak and Bcl-x(L), which respond differentially to cytokine-induced and exogenous NO and by the availability of oxidant species. (Circ Res. 1999;84:21-33.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The stimulatory GTP-binding protein Gs alpha transmits signals from catecholamine receptors to activate adenylyl cyclase and thereby initiate a cascade leading to cardiac chronotropy and inotropy. Transgenic mice overexpressing the Gs alpha subunit (Gs alpha) selectively in their hearts exhibit increased cardiac contractility in response to beta-adrenergic receptor stimulation. However, with aging, these mice develop a cardiomyopathy. This study sought morphological and biochemical evidence that overexpression of Gs alpha is associated with increased myocyte apoptosis in the older animals and to determine whether such overexpression can promote apoptosis of isolated neonatal cardiac myocytes exposed to beta-adrenergic receptor agonists. In the hearts of 15- to 18-month-old Gs alpha transgenic mice, histochemistry and electron microscopy illustrated the existence of numerous myocytes with abnormal nuclei embedded in collagen-rich connective tissue. Terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling (TUNEL, for in situ labeling of DNA breaks) demonstrated that [almost equal to]0.6% of myocyte nuclei contained fragmented DNA. Agarose gel electrophoresis provided further biochemical evidence of apoptosis by showing internucleosomal DNA fragmentation. Cultured cardiac myocytes from newborn Gs alpha transgenic mice showed increased TUNEL staining and internucleosomal DNA fragmentation compared with wild-type controls when treated with the beta-agonist isoproterenol. Thus, enhanced activation of beta-adrenergic signaling by overexpression of Gs alpha in the hearts of transgenic mice induces apoptosis of cardiac myocytes. This represents a potential mechanism that may contribute to the development of cardiomyopathy in this model. (Circ Res. 1999;84:34-42.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Transgenic mouse models have been developed to manipulate beta-adrenergic receptor (beta AR) signal transduction. Although several of these models have altered beta AR subtypes, the specific functional sequelae of beta AR stimulation in murine heart, particularly those of beta2-adrenergicreceptor (beta2AR) stimulation, have not been characterized. In the present study, we investigated effects of beta (2) AR stimulation on contraction, [Ca2+]itransient, and L-type Ca2+currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human beta2AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous beta2AR activation. In contrast, beta2AR stimulation by zinterol or isoproterenol plus a selective beta1-adrenergicreceptor (beta1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, beta2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to beta2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac beta2AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced beta2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [gamma-(32) P]GTP azidoanilide into alpha subunits of Gi2and Gi3after beta2AR stimulation by zinterol or isoproterenol plus the beta1AR blocker CGP 20712A. This effect to activate Giproteins was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) beta2ARs in murine cardiac myocytes couple to concurrent Gsand Gisignaling, resulting in null inotropic response, unless the G (i) signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to beta2AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of beta (2) AR-coupled Giproteins; and (3) spontaneous beta2AR activation may differ from agonist-stimulated beta2AR signaling. (Circ Res. 1999;84:43-52.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

NO- and prostanoid-independent relaxations are generally assumed to be mediated by an endothelium-derived hyperpolarizing factor (EDHF) that has been postulated to be an arachidonic acid metabolite. Recent evidence also suggests that direct heterocellular gap junctional communication (GJC) between endothelium and smooth muscle contributes to NO-independent relaxations. In the present study we have investigated the contribution of phospholipase A2(PLA2)-linked metabolites and GJC to EDHF-type relaxations in rabbit mesenteric artery. In isolated rings preconstricted with 10 [micro sign]mol/L phenylephrine in the presence of NG-nitro-L-argininemethyl ester (L-NAME) and indomethacin, acetylcholine (ACh) and the Ca2+ionophore A23187 evoked relaxations that were markedly attenuated by the Ca2+-dependentPLA2inhibitors 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (3 [micro sign]mol/L) and arachidonyl trifluoromethyl ketone (3 [micro sign]mol/L), but were potentiated by the sulfhydryl agent thimerosal (300 nmol/L). In intact rings, relaxations to ACh were attenuated synergistically by L-NAME and Gap 27 peptide, an inhibitor of GJC, whereas ACh-evoked relaxations of "sandwich" preparations were unaffected by the peptide but were abolished by L-NAME. In both ring and sandwich preparations A23187-induced relaxations were attenuated by inhibition of PLA2but were insensitive to L-NAME and Gap 27 peptide. We conclude that EDHF-type relaxations of rabbit mesenteric artery to ACh and A23187 depend on a common pathway that involves activation of PLA (2). In the case of ACh, relaxation requires transfer of a factor or factors from the endothelium to smooth muscle via gap junctions, whereas A23187 permits release directly into the extracellular space. (Circ Res. 1999;84:53-63.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Platelet-derived growth factor-alpha receptor (PDGF-alpha R) expression is markedly elevated in cultured vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) when compared with normotensive rat strains, Sprague-Dawley, Wistar, and Wistar-Kyoto rats (WKY). This "almost-all-or-none" type of differential expression strongly suggests that PDGF-alpha R or its transcription-regulating mechanisms or factors are significantly related to genetic hypertension. To evaluate the role of PDGF-alpha R in vascular remodeling and hypertension, we have investigated the underlying molecular mechanism. We have recently shown that the regulatory domain responsible for this difference is localized to the PDGF-alpha R promoter region between -246 and -139, which contains an enhancer core sequence specific for CCAAT-enhancer binding proteins (C/EBPs). We defined the roles of this element for hypertensive strain-specific PDGF-alpha R gene transcription. DNA-protein binding studies by competition in electromobility shift and supershift assays revealed that 2 members, C/EBP-beta and C/EBP-delta, are mainly responsible for DNA-protein complex formation; the former acts as a transcriptional repressor and the latter as an activator of the PDGF-alpha R gene, respectively. Western or Northern blot analyses supported evidence for high expression of C/EBP-delta seen only in SHR-derived VSMCs. Furthermore, forced expression of C/EBP-delta transactivated the transcriptional efficiency of the PDGF-alpha R gene even in WKY-derived VSMCs, whereas that of C/EBP-beta had an opposite effect in SHR-derived VSMCs. These findings indicate that differential expression of members of the C/EBP family, mainly C/EBP-delta and possibly C/EBP-beta, are responsible for the strain-specific gene transcription of PDGF-alpha R in VSMCs. (Circ Res. 1999;84:64-73.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Mast cells are present in the human arterial intima. To study whether mast-cell degranulation influences the rate of proliferation of smooth muscle cells, we cocultured sensitized (IgE-bearing) rat serosal mast cells and rat aortic smooth muscle cells (SMCs). When sensitized mast cells were stimulated to degranulate with antigen, the rate of proliferation of the cocultured SMCs decreased sharply. This inhibitory effect was found to be due mainly to the very high molecular weight (M (r)) heparin proteoglycans (average Mr750 000) released from the stimulated mast cells. When the heparin proteoglycans were purified from mast-cell granule remnants and added to the SMC culture, they were found to block the cell cycle at the G0[right arrow]S transition and the exit from the G2/Mphase, their inhibitory effect resembling that of commercial heparin. However, in contrast to the reported dependence of the inhibitory effect of commercial heparin on the release of transforming growth factor-beta from serum, the inhibitory effect of the mast cell-derived heparin proteoglycans in the presence of serum was not transforming growth factor-beta dependent. Moreover, the effect of the mast cell-derived heparin proteoglycans was more efficient than that of commercial heparins of high (average Mr15 000) and low (average Mr5000) molecular weight. We also purified heparin glycosaminoglycans (average Mr75 000) from the mast cell-derived heparin proteoglycans and found that they also inhibited SMC growth efficiently, although less strongly than their parent heparin proteoglycans. These results reveal, for the first time, that mast cells are able to regulate SMC growth. Thus, activated mast cells, by releasing heparin proteoglycans, possibly participate in the regulation of SMC growth in the human arterial intima, the site of atherogenesis. (Circ Res. 1999;84:74-83.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Endothelial thrombomodulin plays a critical role in hemostasis by binding thrombin and subsequently converting protein C to its active form, a powerful anticoagulant. Thrombomodulin thus represents a central mechanism by which patency is maintained in normal vessels. However, thrombomodulin expression decreases in perturbed endothelial cells, predisposing to thrombotic occlusion. An adenoviral construct expressing thrombomodulin (Adv/RSV-THM) was created and functionally characterized in vitro and in vivo. The impact of local overexpression of thrombomodulin on in vivo thrombus formation was subsequently examined in a stasis/injury model of arterial thrombosis. The construct prevented arterial thrombosis formation in all animals, while viral and nonviral controls typically developed occluding thrombi. By histological analysis, nonviral controls exhibited intravascular thrombus occluding a mean of 70.52 +/- 3.72% of available lumen, while viral controls reached 86.85 +/- 2.82% thrombotic occlusion; in contrast, Adv/RSV-THM reduced thrombosis to 28.61 +/- 3.31% of lumen in cross section. No significant intima-to-media ratio was observed in the thrombomodulin group relative to controls. Local infiltration of granulocytes and macrophages significantly decreased in the Adv/RSV-THM group relative to controls, while neutrophilic infiltration increased in viral controls relative to nonviral controls. This construct thus offers a viable technique for promoting a locally thromboresistant small-caliber artery, without the inflammatory damage that has limited many other adenoviral applications. (Circ Res. 1999;84:84-92.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID