摘要:

Myocardial stretch is a well-known stimulus that leads to hypertrophy. Little is known, however, about the intracellular pathways involved in the transmission of myocardial stretch to the cytoplasm and nucleus. Studies in neonatal cardiomyocytes demonstrated stretch-induced release of angiotensin II (Ang II). Because intracellular alkalinization is a signal to cell growth and Ang II stimulates the Na+/H+exchanger (NHE), we studied the relationship between myocardial stretch and intracellular pH (pHi). Experiments were performed in cat papillary muscles fixed by the ventricular end to a force transducer. Muscles were paced at 0.2 Hz and superfused with HEPES-buffered solution. pHiwas measured by epifluorescence with the acetoxymethyl ester form of the pH-sensitive dye 2[prime],7[prime]-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM). Each muscle was progressively stretched to reach maximal developed force (Lmax) and maintained in a length that was [tilde operator]92% Lmax(Li). During the "stretch protocol," muscles were quickly stretched to Lmaxfor 10 minutes and then released to Li; pHisignificantly increased during stretch and came back to the previous value when the muscle was released to Li. The increase in pHiwas eliminated by (1) specific inhibition of the NHE (EIPA, 5 [micro sign]mol/L), (2) AT1-receptorblockade (losartan, 10 [micro sign]mol/L), (3) inhibition of protein kinase C (PKC) (chelerythrine, 5 [micro sign]mol/L), (4) blockade of endothelin (ET) receptors with a nonselective (PD 142,893, 50 nmol/L) or a selective ETAantagonist (BQ-123, 300 nmol/L). The increase in pHiby exogenous Ang II (500 nmol/L) was also reduced by both ET-receptor antagonists. Our results indicate that after myocardial stretch, pHiincreases because of stimulation of NHE activity. This involves an autocrine-paracrine mechanism in which protein kinase C, Ang II, and ET play crucial roles. (Circ Res. 1998;83:775-780.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

It was the aim of this study to characterize the spread of activation at the cellular level in cardiac tissue during conduction slowing, a key element of reentrant arrhythmias; therefore, activation patterns were assessed at high spatiotemporal resolution in narrow (70 to 80 [micro sign]m) and wide (230 to 270 [micro sign]m) linear strands of cultured neonatal rat ventricular myocytes, using multiple site optical recording of transmembrane voltage. Slow conduction was induced by graded elevation of [K+]o, by applying tetrodotoxin, or by exposing the preparations to the gap junctional uncouplers palmitoleic acid or 1-octanol. The main findings of the study are 4-fold: (1) gap junctional uncoupling reduced conduction velocity (range, 37 to 47 cm/s under control conditions) to a substantially larger extent before block (<or=to1 cm/s; ultra-slow conduction) than did a reduction of excitability (range, [tilde operator]10 to 15 cm/s); (2) activation wavefronts during uncoupling meandered within the boundaries of the preparations, resulting in a pronounced additional slowing of conduction in wide cell strands; (3) at the cellular level, propagation during uncoupling-induced ultra-slow conduction was sustained by sequentially activated tissue patches, each of which consisted of a few cells being activated simultaneously; and (4) depending on the uncoupler used, maximal action potential upstroke velocities during ultra-slow conduction were either slightly (palmitoleic acid) or highly (1-octanol) depressed. Thus, depolarizing inward currents, the spatial pattern and degree of gap junctional coupling, and geometrical factors all contribute in a concerted manner to conduction slowing, which, at its extreme (0.25 cm/s measured over 1 mm), can reach values low enough to permit, theoretically, reentrant excitation to occur in minuscule areas of cardiac tissue (much < 1 mm2). (Circ Res. 1998;83:781-794.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

In cardiac tissue, functional or structural current-to-load mismatches can induce local slow conduction or conduction block, which are important determinants of reentrant arrhythmias. This study tested whether spatially repetitive mismatches result in a steady-state slowing of conduction. Patterned growth of neonatal rat heart cells in culture was used to design unbranched cell strands or strands releasing branches from either a single point or multiple points at periodic intervals. Electrical activation was followed optically using voltage-sensitive dyes under control conditions and in elevated [K+]o(5.8 and 14.8 mmol/L, respectively; in the latter case, propagation was carried by the L-type Ca2+current). Preparations with multiple branch points exhibited discontinuous and slow conduction that became slower with increasing branch length and/or decreasing inter-branch distance. Compared with unbranched strands, conduction was maximally slowed by 63% under control conditions (from 44.9 +/- 3.4 to 16.7 +/- 1.0 cm/s) and by 93% in elevated [K+]o(from 15.7 +/- 2.3 to 1.1 +/- 0.2 cm/s). Local activation delays induced at a single branch point were significantly larger than the delays per branch point in multiple branching structures. Also, selective inactivation of inward currents in the branches induced conduction blocks. These 2 observations pointed to a dual role of the branches in propagation: whereas they acted as current sinks for the approaching activation thus slowing conduction ("pull" effect), they supplied, once excited, depolarizing current supporting downstream activation ("push" effect). This "pull and push" action resulted in a slowing of conduction in which the safety was largely preserved by the "push" effect. Thus, branching microarchitectures might contribute to slow conduction in tissue with discontinuous geometry, such as infarct scars and the atrioventricular node. (Circ Res. 1998;83:795-805.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

We recently have reported that suppression of the slowly inactivating component of the outward current, Islow, in ventricular myocytes of transgenic mice (long QT mice) overexpressing the N-terminal fragment and S1 segment of Kv1.1 resulted in a significant prolongation of action potential duration and the QT interval. Here we describe the detailed biophysical properties and physiological role of I (slow) by applying the whole-cell patch-clamp technique at both room temperature and 37[degree sign]C. This current activates rapidly with time constants ranging from 3.8 +/- 0.8 ms at -20 mV to 2.1 +/- 0.5 ms at 50 mV at room temperature. The half-activation voltage and slope factor are -12.5 +/- 2.6 mV and 7.7 +/- 1.0 mV, respectively. The inactivation of this current is slow compared with the fast inactivating component Ito, with time constants of [tilde operator]100 ms at 37[degree sign]C. The steady-state inactivation of Islowis not temperature-dependent, with half-inactivation voltages and slope factors of -35.1 +/- 1.3 and -5.4 +/- 0.4 mV at 37[degree sign]C, and -37.6 +/- 1.8 and -5.8 +/- 0.6 mV at room temperature. Double exponentials were required to describe the time-dependent recovery of Islowfrom steady-state inactivation, with time constants of 233 +/- 34 and 3730 +/- 702 ms at 37[degree sign]C, and 830 +/- 240 and 8680 +/- 2410 ms at room temperature. Islowis highly sensitive to 4-aminopyridine but is insensitive to tetraethylammonium, alpha-dendrotoxin, and E-4031. Stimulation with action-potential waveforms under voltage-clamp mode revealed that this current plays an important role in the early and middle phases of repolarization of the cardiac action potential. We conclude that the biophysical properties and pharmacological profiles of Isloware similar to those of Kv1.5-encoded currents. (Circ Res. 1998;83:806-814.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Interferon (IFN)-gamma and IFN-alpha/beta induction of the transporter associated with antigen processing-1 (TAP1) promoter was compared in HeLa cells and endothelial cells (ECs). In HeLa cells, IFN-gamma acts through Stat1 alpha/Stat1 alpha homodimers binding to the gamma activating sequence (GAS) and IFN-alpha/beta acts through Stat1/Stat2/p48 binding to the IFN-stimulated response element (ISRE). In ECs, however, IFN-gamma and IFN-alpha/beta act through both the GAS and ISRE. The basis of the IFN signaling crossover in ECs was investigated. HeLa and ECs contain similar ratios of Stat1 alpha to Stat2 proteins, and IFN-alpha/beta also activates the same Janus kinases (JAKs) (Jak1 and tyrosine kinase (Tyk) 2 but not Jak2). However, IFN-alpha/beta activates more Stat1 alpha than does IFN-gamma in ECs, whereas the reverse occurs in HeLa, and expression of the IFN-alpha/beta receptor-associated phosphatase SHP-1 is much lower in ECs than HeLa cells. Overexpression of SHP-1 in ECs blocks IFN-alpha/beta signaling through GAS, and expression of a dominant negative SHP-1 in HeLa cells permits IFN-alpha/beta signaling through GAS, demonstrating a role for SHP-1 in regulating crossovers between the IFN-alpha/beta and IFN-gamma signaling pathways. (Circ Res. 1998;83:815-823.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Activation of the Na+/H+exchanger isoform-1 (NHE-1) by angiotensin II is an early signal transduction event that may regulate vascular smooth muscle cell (VSMC) growth and migration. Many signal transduction events stimulated by angiotensin II are mediated by the mitogen-activated protein (MAP) kinases. To define their roles in angiotensin II-mediated NHE-1 activity, VSMCs were treated with angiotensin II and the activities of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) were measured. Angiotensin II rapidly (peak, 5 minutes) activated p38 and ERK1/2, whereas JNK was activated more slowly (peak, 30 minutes). Because angiotensin II stimulated Na+/H+exchange within 5 minutes, the effects of p38 and ERK1/2 antagonists on Na+/H+exchange were studied. The MEK-1 inhibitor PD98059 decreased ERK1/2 activity and Na+/H+exchange stimulated by angiotensin II. In contrast, the specific p38 antagonist SKF-86002 increased Na+/H+exchange. Two mechanisms were identified that may mediate the effects of p38 and SKF-86002 on angiotensin II-stimulated Na+/H+exchange. First, angiotensin II activation of ERK1/2 was increased 1.5- to 2.5-fold (depending on assay technique) in the presence of SKF-86002, demonstrating that p38 negatively regulates ERK1/2. Second, the ability of angiotensin II-stimulated MAP kinases to phosphorylate a glutathione S-transferase fusion protein containing amino acids 625 to 747 of NHE-1 in vitro was analyzed. The relative activities of endogenous immunoprecipitated p38, ERK1/2, and JNK were 1.0, 2.0, and 0.05 versus control, respectively suggesting that p38 and ERK1/2, but not JNK, may phosphorylate NHE-1 in VSMC. These data indicate important roles for p38 and ERK1/2 in angiotensin II-mediated regulation of the Na+/H+exchanger in VSMC. (Circ Res. 1998;83:824-831.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis that stimulates proliferation, migration, and proteolytic activity of endothelial cells. Although the mitogenic activity of VEGF is endothelial cell specific, recent reports indicate VEGF is able to stimulate chemotaxis and tissue factor production in monocytes. VEGF-stimulated activity in monocytes is mediated by the VEGF receptor flt-1. The purpose of the present study was to investigate the effects of VEGF on another major cell type in the vascular wall, namely, the vascular smooth muscle cell (SMC). Using cultured cells, we showed that VEGF has a minimal mitogenic effect on SMCs, which is in accordance with published data. However, VEGF treatment significantly enhanced production of matrix metalloproteinase (MMP)-1, -3, and -9 by human SMCs. The upregulation of MMP-1 and MMP-9 was pronounced, and the stimulation for MMP-3 was less prominent. Stimulation could be demonstrated at both protein and mRNA levels, as reflected by ELISA, zymography, and Northern blot analysis. To explore the signal transduction pathway for the effect of VEGF on SMCs, we studied the expression of 2 high-affinity VEGF receptors, the kinase insert domain-containing receptor (KDR) and flt-1, in human SMCs. Both reverse transcriptase-polymerase chain reaction and immunoblotting revealed the expression of flt-1. Immunoprecipitation followed by immunoblotting illustrated phosphorylation of the flt-1 receptor after VEGF treatment. Similar methodology failed to detect expression of KDR in human SMCs. These data suggest the role of flt-1 in mediating VEGF-stimulated MMP expression of SMCs. The physiological relevance of MMP upregulation was studied by examining VEGF-stimulated SMC migration through 2 synthetic extracellular matrix barriers, Matrigel and Vitrogen. Our results indicate that VEGF treatment accelerated SMC migration through both barriers, and that this response was blocked by MMP inhibition in Matrigel, which supports a permissive role of MMP in SMC migration. These data are the first to show a direct effect of VEGF on SMCs. SMC-derived MMPs may be an additional source of proteases to digest vascular basement membrane, which is a crucial step in the initial stage of angiogenesis. The MMPs may also contribute to SMC migration in angiogenesis and atherogenesis. (Circ Res. 1998;83:832-840.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating Gq-protein-coupledAT1receptors, which leads to elevation of cytosolic Ca2+([Ca2+]i) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. To assess the link between these Ang II-induced signaling events, we examined the effect of Ang II on the proline-rich tyrosine kinase (PYK2), previously found to be activated by a variety of stimuli that increase [Ca (2+)]ior activate PKC. PYK2 distribution was demonstrated in rat aortic tissue and in cultured VSMC by immunohistochemistry, revealing a cytosolic distribution distinct from smooth muscle alpha-actin, focal adhesion kinase, or paxillin. The involvement of PYK2 in Ang II signaling was measured by immunoprecipitation and immune complex kinase assays. Treatment of quiescent VSMC with Ang II resulted in a concentration- and time-dependent increase in PYK2 tyrosine phosphorylation and kinase activity in PYK2 immunoprecipitates. PYK2 phosphorylation was inhibited by AT1receptor blockade and was attenuated by downregulation of PKC or the chelation of [Ca2+]i. Treatment with either phorbol ester or Ca2+ionophore also increased PYK2 phosphorylation, suggesting that PKC activation and/or increased [Ca2+] (i) are both necessary and sufficient to activate PYK2. Activation of PYK2 by Ang II was also associated with increased PYK2-src complex formation, suggesting that PYK2 activation represents a potential link between Ang II-stimulated [Ca2+] (i) and PKC activation with downstream signaling events such as mitogen-activated protein kinase activation involved in the regulation of VSMC growth. (Circ Res. 1998;83:841-851.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Angiopoietin-2 (Ang2) is a ligand for the endothelial cell tyrosine kinase receptor Tie2 and counteracts blood vessel maturation/stability mediated by angiopoietin-1 (Ang1), the other known ligand of Tie2. Using degenerate oligonucleotides and reverse transcriptase-polymerase chain reaction, we have screened bovine microvascular endothelial (BME), aortic, lymphatic, pulmonary artery, and transformed fetal aortic endothelial cells, as well as rat smooth muscle cells for Ang1 and Ang2 expression. Except for high Ang2 mRNA levels found in BME cells, none of the endothelial cell types studied expressed appreciable levels of Ang1 or Ang2 mRNAs, whereas smooth muscle cells expressed both Ang1 and Ang2. BME cell Ang2 mRNA levels were increased by vascular endothelial growth factor (1.9- to 2.9-fold), basic fibroblast growth factor (1.6- to 2-fold), both cytokines in combination (2.9- to 4-fold), and hypoxia (3.1- to 5.6-fold) and were decreased by Ang1 (31% to 70%) or transforming growth factor-beta1(64% to 81%). Ang2 also decreased (60% to 82%) BME cell Ang2 mRNA. mRNA levels for the Tie1 or Tie2 receptors were only slightly modulated under the conditions described above. These findings suggest that the angiogenic effect of a number of regulators may be achieved in part through the regulation of an autocrine loop of Ang2 activity in microvascular endothelial cells. (Circ Res. 1998;83:852-859.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

In human heart, we detected mRNAs and proteins for C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9 with the use of reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemical techniques. We found an upregulation of both mRNAs and proteins in areas of recent and old myocardial infarctions. In both situations, the classical complement pathway was activated, with C4d, C3d, and the membrane attack complex (C5b-9) being deposited on damaged cardiac myocytes. These activated complement components were also identified on Western blots of infarcted tissue. Complement mRNAs in infarcted heart tissue were higher than those in liver, and liver complement mRNAs were not upregulated in cases with infarcted hearts. Our results establish that (1) complement proteins are endogenously produced by human heart; (2) the classical complement pathway is fully activated after myocardial infarction; (3) complement activation is directly involved in myocardial damage after ischemic insults; and (4) damage from complement activation may be chronically sustained. These data suggest that inhibition of the complement system should be effective in treating myocardial infarction. (Circ Res. 1998;83:860-869.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID