ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Long-term nitroglycerin (NTG) treatment has been shown to be associated with cross-tolerance to endothelium-dependent vasodilators. It may involve increased production of reactive oxygen species (such as superoxide, O2·−) that rapidly inactivate the nitric oxide (NO) released from the endothelial cells. It remains to be elucidated, however, whether long-term treatment with NTG alters the activity and expression of the endothelial NO synthase (NOS III) and whether this enzyme can contribute to O2·−formation. We studied the influence of long-term NTG treatment on the expression of NOS III as assessed by RNase protection assay and Western blot. Tolerance was measured ex vivo in organ chamber experiments with rat aortic rings. O2·−and NO formation were quantified using lucigenin- andCypridinaluciferin analog–enhanced chemiluminescence as well as electron spin resonance (ESR) spectroscopy. Treatment of Wistar rats with NTG (Alzet osmotic minipumps, NTG concentration 10 &mgr;g · kg−1· min−1) for 3 days caused marked tolerance, cross-tolerance to the endothelium-dependent vasodilator acetylcholine, and a significant increase in O2·−-induced chemiluminescence. Tolerance was associated with a significant increase in NOS III mRNA to 236±28% and NOS III protein to 239±17%. In control vessels, the NOS inhibitorNG-nitro-L-arginine (L-NNA) increased the O2·−-mediated chemiluminescence, indicating that basal production of endothelium-derived NO depresses the baseline chemiluminescence signal. In the setting of tolerance, however, L-NNA decreased steady-state O2·−levels, indicating the involvement of NOS III in O2·−formation. Likewise, A23187-induced, NOS III–mediated O2·−production was more pronounced in tolerant than in control vessels. Vascular NO bioavailability as assessed with ESR spectroscopy using iron-thiocarbamate as a trap for NO was significantly reduced in tolerant vessels. Pretreatment of tolerant tissue in vitro with the protein kinase C (PKC) inhibitors reduced basal and stimulated NOS III–mediated O2·−production and partially reversed vascular tolerance. These findings suggest that NTG treatment increases the expression of a dysfunctional NOS III gene, leading to increased formation of O2·−and decreased vascular NO bioavailability. Normalization of NOS III–mediated O2·−production and improvement of tolerance with PKC inhibition suggests an important role for PKC isoforms in mediating vascular dysfunction caused by long-term NTG treatment. The full text of this article is available at http://www.circresaha.org.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Doxorubicin (DOX)–induced cardiomyopathy has been found to be associated with impaired Ca2+handling in the sarcoplasmic reticulum (SR), leading to reduced cardiac function. We have recently demonstrated that expression of mRNA encoding sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2), a major Ca2+transport protein in SR, is markedly decreased in DOX-treated hearts. To extend this observation, we have dissected the molecular mechanisms by which DOX downregulates SERCA2 gene transcription. Using cultured rat neonatal cardiac myocytes, we found that the antioxidantN-acetylcysteine blocked the DOX-induced decrease in SERCA2 mRNA levels, as well as the DOX-induced increase in H2O2concentration; thus, H2O2is an intracellular mediator of DOX activity. Using a luciferase reporter assay, we found that the sequence from −284 to −72 bp in the 5′ flanking region of the SERCA2 gene has a DOX-responsive element. Although several transcription factors have putative binding motifs in this region of the SERCA2 gene, only the expression of Egr-1 mRNA and the binding of Egr-1 protein to the 5′ regulatory sequence of SERCA2 gene increased markedly after DOX administration. We also found that overexpression of Egr-1 was associated with a significant reduction in SERCA2 gene transcription. In addition, Egr-1 antisense oligonucleotides blocked the DOX-induced reduction in SERCA2 mRNA, suggesting that Egr-1 is a transcriptional inhibitor of the SERCA2 gene in DOX-induced cardiomyopathy. We observed activation of 3 mitogen-activated protein kinases (MAPKs), p44/42 MAPK, p38 MAPK, and stress-activated MAPK/Jun N-terminal kinase, by DOX, but only a specific inhibitor of the p44/42 MAPK kinase suppressed the effects of DOX on Egr-1 and SERCA2 mRNA expression. These findings indicate that reactive oxygen intermediates, the transcription factor Egr-1, and p44/42 MAPK are critical elements in the transcriptional regulation of the SERCA2 gene in response to DOX.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

We have shown that leukocytes retract their pseudopods and detach from substrates after exposure to physiological fluid shear stresses (≈1.5 dyn/cm2). In inflammation, however, pseudopod projection during spreading and firm adhesion on endothelium is observed even in microvessels with normal blood flow and fluid shear stresses. Thus, we examined mechanisms that may serve to regulate the shear stress response of circulating leukocytes. In the presence of inflammatory mediators (platelet-activating factor [PAF] f-met-leu-phe), a subgroup of cells ceases to respond to shear stress. cGMP analogs and nitric oxide (NO) donors enhance the shear stress response and reverse the inhibitory effect of inflammatory mediators on the shear stress response, whereas depletion of cGMP leads to cessation of the shear stress response even in unstimulated leukocytes. The ability of cGMP to enhance the shear stress response is not associated with CD18 expression, because cGMP has no effect on CD18 expression in response to shear stress. The shear stress response of leukocytes in endothelial nitric oxide synthase (−/−) mice, in which NO level in blood is decreased, is attenuated compared with that in wild-type mice. In rat mesentery venules stimulated by PAF under normal blood flow, a cGMP analog diminishes pseudopod projection of leukocytes, whereas inhibition of NO leads to enhanced pseudopod projection and spreading. The evidence suggests that inflammatory mediators suppress the shear stress response of leukocytes leading to spreading even under normal physiological shear stress, whereas cGMP may serve to maintain shear stress response even in inflammation. The full text of this article is available at http://www.circresaha.org.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Insulin-like growth factor–I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation and directed migration. The mitogenic and chemotactic actions of IGF-I are mediated through the IGF-I receptor, but how the activation of the IGF-I receptor leads to these biological responses is poorly understood. In this study, we examined the role of phosphatidylinositol 3-kinase (PI3 kinase) in mediating the mitogenic and chemotactic signals of IGF-I. IGF-I treatment resulted in a significant increase in phosphotyrosine-associated PI3 kinase activity in cultured primary VSMCs. To determine whether insulin receptor substrate (IRS)–1, -2, or both are involved in IGF-I signaling in VSMCs, cell lysates were immunoprecipitated with either an anti-IRS-1 or an anti-IRS-2 antibody, and the associated PI3 kinase activity was determined. IGF-I stimulation resulted in a significant increase in IRS-1– but not IRS-2–associated PI3 kinase activity, suggesting that IGF-I primarily utilizes IRS-1 to transmit its signal in VSMCs. The IGF-I–induced increase in IRS-I–associated PI3 kinase activity was concentration dependent. At the maximum concentration (50 ng/mL), IGF-I induced a 60-fold increase. This activation occurred within 5 minutes and was sustained at high levels for at least 6 hours. IGF-I also caused a concentration-dependent and long-lasting activation of protein kinase B (PKB/Akt). Inhibition of PI3 kinase activation by LY294002 or wortmannin abolished IGF-I–stimulated VSMC proliferation and reduced IGF-I–directed VSMC migration by ≈60%. These results indicate that activation of PI3 kinase is required for both IGF-I–induced VSMC proliferation and migration.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Angiopoietin-1 (Ang1) is a strong apoptosis survival factor for endothelial cells. In this study, the receptor/second messenger signal transduction pathway for the antiapoptotic effect of Ang1 on human umbilical vein endothelial cells was examined. Pretreatment with soluble Tie2 receptor, but not Tie1 receptor, blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of Tie2 and the p85 subunit of phosphatidylinositol 3′-kinase (PI 3′-kinase) and increased PI 3′-kinase activity in a dose-dependent manner. The PI 3′-kinase–specific inhibitors wortmannin and LY294002 blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of the serine-threonine kinase Akt at Ser473 in a PI 3′-kinase–dependent manner. Expression of a dominant-negative form of Akt reversed the Ang1-induced antiapoptotic effect. Ang1 mRNA and protein were present in vascular smooth muscle cells but not in endothelial cells. Cultured vascular smooth muscle cells, but not human umbilical vein endothelial cells, secreted Ang1. These findings indicate that the Tie2 receptor, PI 3′-kinase, and Akt are crucial elements in the signal transduction pathway leading to endothelial cell survival induced by the paracrine activity of Ang1.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Microtubules have been shown to alter contraction in cardiac myocytes through changes in cellular stiffness. However, an effect on excitation-contraction coupling has not been examined. Here we analyze the effects of microtubule disruption by 1 &mgr;mol/L colchicine on calcium currents (ICa) and [Ca2+]itransients in rat ventricular myocytes.ICawas studied using the whole-cell patch-clamp technique. Colchicine treatment increasedICadensity (peak values, −4.6±0.4 and −9.1±1.3 pA/pF in 11 control and 12 colchicine-treated myocytes, respectively;P<0.05).ICainactivation was well fitted by a biexponential function. The slow component of inactivation was unchanged, whereas the fast component was accelerated after colchicine treatment (at −10 mV, 11.8±1.0 versus 6.7±1.0 ms in control versus colchicine-treated cells;P<0.005). [Ca2+]itransients were analyzed by fluo-3 epifluorescence simultaneously withICa. Peak [Ca2+]itransients were significantly increased in cardiac myocytes treated with colchicine. The values of F/F0at 0 mV were 1.1±0.02 in 9 control cells and 1.4±0.1 in 11 colchicine-treated cells (P<0.05). &bgr;-Adrenergic stimulation with 1 &mgr;mol/L isoproterenol increased bothICaand [Ca2+]itransient in control cells. However, no significant change was induced by isoproterenol on colchicine-treated cells. Colchicine and isoproterenol effects were similar and not additive. Inhibition of adenylyl cyclase by 200 &mgr;mol/L 2′-deoxyadenosine 3′-monophosphate blunted the colchicine effect. We suggest that &bgr;-adrenergic stimulation and microtubule disruption share a common pathway to enhanceICaand [Ca2+]itransient.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID