摘要:

Laminar shear stress exerts important effects on endothelial cell (EC) function and inhibits apoptosis of ECs induced by various stimuli. The mechanism by which hemodynamic forces, such as shear stress, are transduced into cellular signaling is still not known. Located at the cell surface, integrins, which are required for cell adhesion and cell survival, are potential mechanotransducers. Therefore, we investigated the effect of shear stress on integrin expression in ECs. Shear stress time-dependently increased the mRNA expression of the fibronectin receptor subunits &agr;5and &bgr;1with a maximum at 6 hours (283±41% and 215±27% of control, respectively). In addition, the protein levels of the fibronectin receptor subunits &agr;5and &bgr;1were enhanced with a maximum at 12 hours of shear stress exposure (343±53% and 212±38% of control, respectively). The shear stress–induced upregulation of integrins is independent of nitric oxide. Furthermore, we confirmed the enhanced functional activity of &agr;5&bgr;1integrin expression by FACS analysis. As a functional consequence, human umbilical vein ECs, which were preexposed to shear stress, revealed a significantly increased attachment (178±10% of static controls) and a more pronounced extracellular signal–regulated kinase 1 and 2 activation in response to cell attachment. Finally, we demonstrated that shear stress requires RGD-sensitive integrins to mediate its antiapoptotic effect. Taken together, these results define a novel mechanism by which shear stress may exert its atheroprotective effects via upregulation of integrins to support EC adhesion and survival.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Defective excitation-contraction coupling in heart failure is generally associated with both a reduction in sarcoplasmic reticulum (SR) Ca2+uptake and a greater dependence on transsarcolemmal Na+-Ca2+exchange (NCX) for Ca2+removal. Although a relative increase in NCX is expected when SR function is impaired, few and contradictory studies have addressed whether there is an absolute increase in NCX activity. The present study examines in detail NCX density and function in left ventricular midmyocardial myocytes isolated from normal or tachycardic pacing–induced failing canine hearts. No change of NCX current density was evident in myocytes from failing hearts when intracellular Ca2+([Ca2+]i) was buffered to 200 nmol/L. However, when [Ca2+]iwas minimally buffered with 50 &mgr;mol/L indo-1, Ca2+extrusion via NCX during caffeine application was doubled in failing versus normal cells. In other voltage-clamp experiments in which SR uptake was blocked with thapsigargin, both reverse-mode and forward-mode NCX currents and Ca2+transport were increased >2-fold in failing cells. These results suggest that, in addition to a relative increase in NCX function as a consequence of defective SR Ca2+uptake, there is an absolute increase in NCX function that depends on [Ca2+]iin the failing heart.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

The GATA-6 transcription factor is reported to be expressed in vascular myocytes. Because glomerular mesangial cells (GMCs) and vascular myocytes have similar properties, we examined whether GATA-6 was expressed in cultured GMCs and whether overexpression of GATA-6 induced cell cycle arrest in GMCs, using a recombinant adenovirus that expresses GATA-6 (Ad GATA-6). GATA-6 expression in GMCs was downregulated when quiescent GMCs were stimulated by serum to reenter the cell cycle. [3H]thymidine uptake was inhibited in GMCs infected with Ad GATA-6 in a dose- and time-dependent manner. The expression of cyclin A protein was decreased and that of the cyclin-dependent kinase inhibitor p21cip1was increased in GMCs infected with Ad GATA-6. Although the expression of p21cip1transcripts did not change remarkably, p21cip1protein was stabilized in GMCs infected with Ad GATA-6, suggesting a post-transcriptional regulation of p21cip1expression. Northern blot analysis showed that expression of the cyclin A transcript was decreased in Ad GATA-6–infected cells, whereas this decrease of cyclin A was not observed in GMCs derived from p21cip1null mice. Our results demonstrate that GATA-6 is endogenously expressed in GMCs and that overexpression of GATA-6 can induce cell cycle arrest. Our results also show that GATA-6–induced cell cycle arrest is associated with inhibition of cyclin A expression and p21cip1upregulation. Finally, our results indicate that the GATA-6–induced suppression of cyclin A expression depends on the presence of p21cip1.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Cardiac G protein–coupled receptors that couple to G&agr;sand stimulate cAMP formation (eg, &bgr;-adrenergic, histamine, serotonin, and glucagon receptors) play a key role in cardiac inotropy. Recent studies in rodent cardiac myocytes and transfected cells have revealed that one of these receptors, the &bgr;2-adrenergic receptor (AR), also couples to the inhibitory G protein G&agr;i(activation of which inhibits cAMP formation). If &bgr;2ARs could be shown to couple to G&agr;iin the human heart, it would have important ramifications, because levels of G&agr;iincrease with age and in failing human heart. Therefore, we investigated whether &bgr;2ARs in the human heart activate G&agr;i. By photoaffinity labeling human atrial membranes with [32P]azidoanilido-GTP, followed by immunoprecipitation with antibodies specific for G&agr;i, we found that G&agr;iis activated by stimulation of &bgr;2ARs but not of &bgr;1ARs. In addition, we found that other G&agr;s-coupled receptors also couple to G&agr;i, including histamine, serotonin, and glucagon. When coupling of these receptors to G&agr;iis disrupted by pertussis toxin, their ability to stimulate adenylyl cyclase is enhanced. These data provide the first evidence that &bgr;2AR and many other G&agr;s-coupled receptors in human atrium also couple to G&agr;iand that abolishing the coupling of these receptors to G&agr;iincreases the receptor-mediated adenylyl cyclase activity.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

The rate of vascular smooth muscle cell protein synthesis and cellular hypertrophy in response to angiotensin II (Ang II) is dependent on activation of protein tyrosine kinases (PTKs) and both the extracellular signal–regulated kinase (ERK) 1/2 and p70S6Kpathways. One potential PTK that may regulate these signaling cascades is focal adhesion kinase (FAK), a nonreceptor PTK associated with focal adhesions. We used an actin depolymerizing agent, cytochalasin D (Cyt-D), and a replication-defective adenovirus encoding FAK-related nonkinase (FRNK), an inhibitor of FAK-dependent signaling, as tools to assess whether FAK was upstream of the ERK1/2 and/or the p70S6Kpathways. Cyt-D reduced basal FAK phosphorylation and blocked Ang II–dependent FAK phosphorylation in a dose-dependent manner. Confocal microscopy indicated that Cyt-D induced actin filament disruption and FAK delocalization from focal adhesions. Cyt-D also reduced Ang II–induced ERK1/2 activation, but p70S6Kactivation was relatively unaffected. Cyt-D reduced basal protein synthetic rate and substantially reduced the Ang II–induced increase in protein synthesis. Similarly, FRNK overexpression blocked Ang II–induced FAK phosphorylation and ERK1/2 activation, but not p70S6Kphosphorylation, and markedly inhibited protein synthesis. This is the first report to demonstrate that FAK is a critical component of the signal transduction pathways that mediate Ang II–induced ERK1/2 activation, c-fosinduction, and enhanced protein synthesis in vascular smooth muscle cells.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID