摘要:

Abstract—Recent work on isolated sinoatrial node cells from rabbit has suggested that sarcoplasmic reticulum Ca2+release plays a dominant role in the pacemaker potential, and ryanodine at a high concentration (30 &mgr;mol/L blocks sarcoplasmic reticulum Ca2+release) abolishes pacemaking and at a lower concentration abolishes the chronotropic effect of &bgr;-adrenergic stimulation. The aim of the present study was to test this hypothesis in the intact sinoatrial node of the rabbit. Spontaneous activity and the pattern of activation were recorded using a grid of 120 pairs of extracellular electrodes. Ryanodine 30 &mgr;mol/L did not abolish spontaneous activity or shift the position of the leading pacemaker site, although it slowed the spontaneous rate by 18.9±2.5% (n=6). After ryanodine treatment, &bgr;-adrenergic stimulation still resulted in a substantial chronotropic effect (0.3 &mgr;mol/L isoproterenol increased spontaneous rate by 52.6±10.5%, n=5). In isolated sinoatrial node cells from rabbit, 30 &mgr;mol/L ryanodine slowed spontaneous rate by 21.5±2.6% (n=13). It is concluded that sarcoplasmic reticulum Ca2+release does not play a dominating role in pacemaking in the sinoatrial node. The full text of this article is available at http://www.circresaha.org.

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Abstract—While a diversity of cell types and distribution within the sinoatrial node and cell-cell interactions add complexity to a complete elucidation of the heart’s pacemaker function, it has become clear that cyclic variation of submembrane [Ca2+] and activation of the Na+-Ca2+exchanger during diastolic depolarization (DD) act in concert with ion channels to confer on sinoatrial node cells (SANCs) their status of dominance with respect to pacemaker function. Studies using confocal microscopy indicate that subsarcolemmal Ca2+release via ryanodine receptors occurs not only in response to the action potential (AP) upstroke, but also during the DD, and this is augmented by &bgr;-adrenergic receptor (&bgr;-AR) stimulation. Spontaneous APs simulated by mathematical SANC models beat at a faster rate when this subsarcolemmal Ca2+waveform measured under &bgr;-AR stimulation is introduced into the modeling scheme. Thus, in future investigation of pacemaker functioning in health, disease, and disease therapies the “bar ought to be raised” to embrace the impact of cyclic variation in submembrane [Ca2+] on pacemaker function. The full text of this article is available at http://www.circresaha.org.

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Biological pacemakers were recently created by genetic suppression of inward rectifier potassium current,IK1, in guinea pig ventricular cells. We simulated these cells by adjustingIK1conductance in the Luo-Rudy model of the guinea pig ventricular myocyte. After 81%IK1suppression, the simulated cell reached steady state with pacemaker period of 594 ms. Pacemaking current is carried by the Na+-Ca2+exchanger,INaCa, which depends on the intracellular calcium concentration [Ca2+]i. This [Ca2+]idependence suggests responsiveness (increase in rate) to &bgr;-adrenergic stimulation (&bgr;AS), as observed experimentally. Simulations of &bgr;AS demonstrate such responsiveness, which depends onINaCaexpression. However, a simultaneous &bgr;AS-mediated increase in the slow delayed rectifier,IKs, limits &bgr;AS sensitivity.

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Abstract—In mammalian organs under normoxic conditions, O2concentration ranges from 12% to <0.5%, with O2≈14% in arterial blood and <10% in the myocardium. During mild hypoxia, myocardial O2drops to ≈1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of Po2results in perceived hyperoxia. We hypothesized that O2, even in marginal relative excess of the Po2to which cardiac cells are adjusted, results in activation of specific signal transduction pathways that alter the phenotype and function of these cells. To test this hypothesis, cardiac fibroblasts (CFs) isolated from adult murine ventricle were cultured in 10% or 21% O2(hyperoxia relative to the Po2to which cells are adjusted in vivo) and were compared with those cultured in 3% O2(mild hypoxia). Compared with cells cultured in 3% O2, cells that were cultured in 10% or 21% O2demonstrated remarkable reversible G2/M arrest and a phenotype indicative of differentiation to myofibroblasts. These effects were independent of NADPH oxidase function. CFs exposed to high O2exhibited higher levels of reactive oxygen species production. The molecular signature response to perceived hyperoxia included (1) induction of p21, cyclin D1, cyclin D2, cyclin G1, Fos-related antigen-2, and transforming growth factor-&bgr;1, (2) lowered telomerase activity, and (3) activation of transforming growth factor-&bgr;1 and p38 mitogen-activated protein kinase. CFs deficient in p21 were resistant to such O2sensitivity. This study raises the vital broad-based issue of controlling ambient O2during the culture of primary cells isolated from organs.

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Abstract—Endothelial cell proteinase activated receptors (PARs) belong to a family of heterotrimeric G protein-coupled receptors that are implicated in leukocyte accumulation and potentiation of reperfusion injury. We characterized the effect and the signal transduction pathways recruited after stimulation of endothelial PAR2. We used von Willebrand Factor (vWF) release and monolayer permeability to peroxidase to report Weibel-Palade body (WPB) exocytosis and pore formation, respectively. Human umbilical vein endothelial cells (HUVECs) were stimulated with the selective PAR2agonist peptide SLIGRL-NH2or PAR1agonist peptide TFLLR-NH2. PAR2stimulation resulted in WPB exocytosis like PAR1stimulation but, unlike PAR1, failed to increase monolayer permeability. BAPTA-AM inhibited PAR2-induced exocytosis, indicating a PAR2calcium-dependent signal in ECs. Moreover, PAR2-like PAR1-stimulated exocytosis requires actin cytoskeleton remodeling, because vWF release is inhibited if the cells were pretreated with Jasplakinolide. Rho-GTPase activity is required for PAR-stimulated exocytosis, because inactivation of this family of actin-regulatory proteins withClostridium difficiletoxin B blocked exocytosis. Expression of dominant-negative mutant Cdc4217Ninhibited exocytosis whereas neither dominant-negative Rac17Nexpression nor C3 exotoxin treatment affected vWF release. PAR2stimulated RhoA-GTP weakly compared with the PAR1agonist. We conclude that both PAR2and PAR1elicit WP body exocytosis in a calcium and Cdc42 GTPase-dependent manner. In contrast, the differential effect of PAR1versus PAR2activation to increase monolayer permeability correlates with weak RhoA activation by the PAR2agonist.

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Abstract—We tested the hypothesis that steady laminar shear stress activates the glucocorticoid receptor (GR) and its transcriptional signaling pathway in an effort to investigate the potential involvement of GR in shear stress-induced antiatherosclerosis actions in the vasculature. In both bovine aortic endothelial cells (BAECs) and NIH3T3 cells expressing GFP-GR chimeric protein, wall shear stress of 10 or 25 dynes/cm2caused a marked nuclear localization of GFP-GR within 1 hour to an extent comparable to induction with 25 &mgr;mol/L dexamethasone. The shear mediated nuclear localization of GFP-GR was significantly reduced by 25 &mgr;mol/L of the MEK1 inhibitor (PD098059) or the PI 3-kinase inhibitor (LY294002). Also, Western blots demonstrated translocation of endogenous GR into nucleus of sheared BAECs. Promoter construct studies using glucocorticoid response element (GRE)-driven expression of secreted alkaline phosphatase (SEAP) indicated that BAECs exposed to shear stress of 10 and 25 dynes/cm2for 8 hours produced >9-fold more SEAP (n=6;P<0.005) than control cells, a level comparable to that observed with dexamethasone. Shear stress enhanced SEAP expression at 6 hours was reduced 50% (n=5;P<0.005) by MEK1/2 or PI 3-kinase inhibitors, but not by the NO inhibitor, L-NAME. Finally, in human internal mammary artery, endothelial GR is found to be highly nuclear localized. We report a new shear responsive transcriptional element, GRE. The finding that hemodynamic forces can be as potent as high dose glucocorticoid steroid in activating GR and GRE-regulated expression correlates with the atheroprotective responses of endothelial cells to unidirectional arterial shear stress.

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Abstract—Hemodynamic shear stress elicits a rise in endothelial [Ca2+]i, which may serve as a key second messenger to regulate many flow-associated physiological and biochemical processes. In the present study, we used Mn2+quenching of fluorescent dye Fluo3 as an assay to investigate the Ca2+influx of rat aortic endothelial cells in response to flow. We found that the Ca2+signaling in response to flow could be greatly influenced by the status of intracellular Ca2+stores. Depletion of intracellular Ca2+stores by thapsigargin (4 &mgr;mol/L) or cyclopiazonic acid (10 &mgr;mol/L) drastically sensitized the Ca2+influx in response to flow. Ca2+-mobilizing agonist bradykinin (100 nmol/L) or ATP (100 &mgr;mol/L) had similar sensitizing effect. The effect of bradykinin or ATP was blocked byXestospongin Cand U73122, suggesting that the sensitization was related to the IP3-mediated store depletion. On the other hand, the Mn2+quenching in response to flow was greatly reduced by ochratoxin A (100 nmol/L), an agent that could increase the filling state of intracellular Ca2+stores. In addition, we found that depletion-sensitized Ca2+influx in response to flow was mediated by a PKG-inhibitable cation channel and that the influx was affected by membrane potential and K+channel activity. In conclusion, the present study argues for a critical role of intracellular Ca2+status in determining the Ca2+signaling in response to flow and it provides a general mechanistic explanation for the stimulatory role of blood-borne agonists on flow-induced Ca2+influx.

ISSN:0009-7330    

出版商:OVID    

年代:2003

数据来源: OVID