摘要:

A distal transcriptional enhancer has been previously reported upstream of the mouse renin gene. A homologous sequence is also present upstream of the human renin gene, but the mouse and human renin enhancers differ markedly in their ability to activate transcription of a renin promoter. Although the 2 enhancers share high homology in their 202-bp promoter distal portions, their 40-bp proximal portions are heterogeneous. Chimeric enhancers were used to test the role of the 40-bp segment (m40) of the enhancer by using transient transfection analysis in mouse kidney renin-expressing As4.1 cells. Deletion of m40 from the mouse renin enhancer or its addition to the human renin enhancer did not significantly change transcriptional activity when placed close to a mouse or human renin promoter. However, when placed further upstream of a renin promoter, the same deletion and substitution markedly altered enhancer activity. Electrophoretic gel mobility shift analysis identified 2 elements, a and b, in m40 that specifically bound nuclear proteins from As4.1 cells. Mutagenesis and transient transfection analysis revealed that element b accounts for the function of m40 and that element a antagonizes the positive influence of element b. Gel competition and supershift analysis revealed that nuclear factor-Y, a ubiquitous CAAT-box binding protein, binds to element a. Sequence analysis revealed that the human renin enhancer has a natural loss-of-function mutation in element b that affects its ability to transactivate when placed far upstream of a promoter. Reversion of the human renin element b to match the mouse sequence restored transactivation of the enhancer in mouse As4.1 cells. These data suggest that element b cooperates with the rest of the enhancer to maintain full enhancer activity, whereas element a may regulate enhancer activity. Sequence differences in these elements may explain the functional differences in the mouse and human renin enhancer sequences.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Hypoxic pulmonary vasoconstriction is initiated by inhibiting one or more voltage-gated potassium (Kv) channel in the vascular smooth muscle cells (VSMCs) of the small pulmonary resistance vessels. Although progress has been made in identifying which Kv channel proteins are expressed in pulmonary arterial (PA) VSMCs, there are conflicting reports regarding which channels contribute to the native O2-sensitive K+current. In this study, we examined the effects of hypoxia on the Kv1.2, Kv1.5, Kv2.1, and Kv9.3 α subunits expressed in mouse L cells using the whole-cell patch-clamp technique. Hypoxia (PO2=≈30 mm Hg) reversibly inhibited Kv1.2 and Kv2.1 currents only at potentials more positive than 30 mV. In contrast, hypoxia did not alter Kv1.5 current. Currents generated by coexpression of Kv2.1 with Kv9.3 α subunits were reversibly inhibited by hypoxia in the voltage range of the resting membrane potential (EM) of PA VSMCs (≈28% at −40 mV). Coexpression of Kv1.2 and Kv1.5 α subunits produced currents that displayed kinetic and pharmacological properties distinct from Kv1.2 and Kv1.5 channels expressed alone. Moreover, hypoxia reversibly inhibited Kv1.2/Kv1.5 current activated at physiologically relevant membrane potentials (≈65% at −40 mV). These results indicate that (1) hypoxia reversibly inhibits Kv1.2 and Kv2.1 but not Kv1.5 homomeric channels, (2) Kv1.2 and 1.5 α subunits can assemble to form an O2-sensitive heteromeric channel, and (3) only Kv1.2/Kv1.5 and Kv2.1/Kv9.3 heteromeric channels are inhibited by hypoxia in the voltage range of the PA VSMC EM. Thus, these heteromeric channels are strong candidates for the K+channel isoforms initiating hypoxic pulmonary vasoconstriction.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Thyroid hormone regulation of the cardiac pacemaker gene, the hyperpolarization-activated cyclic nucleotide-gated channel gene (HCN2), was studied in rats by Northern analysis. Thyroid hormone administration to hypothyroid rats resulted in a doubling of the HCN2/β-actin mRNA ratio. A smaller, not statistically significant, increase in HCN2 mRNA occurred when euthyroid animals were made hyperthyroid. A single large dose of L-triiodothyronine given to hypothyroid rats caused a 4.7-fold increase in myocardial HCN2 mRNA expression level and only a 2.3-fold increase in the β-actin mRNA level. Although the rat HCN2 promoter has not been cloned, we identified a consensus thyroid hormone response element in the promoter sequence of the human HCN2 gene. Therefore, the increase in rat HCN2 mRNA is likely due to L-triiodothyronine stimulation of HCN2 gene transcription. The results suggest that the regulation of heart rate by thyroid hormone may be explained, at least in part, by the positive effect of this hormone on HCN2 gene expression.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Endothelial cells exhibit profound changes in cell shape in response to altered shear stress that may require disassembly/reassembly of adherens junction protein complexes that mediate cell-cell adhesion. To test this hypothesis, we exposed confluent porcine aortic endothelial cells to 15 dyne/cm2of shear stress for 0, 8.5, 24, or 48 hours, using a parallel plate flow chamber. Cells were fixed and stained with antibodies to vascular endothelial (VE) cadherin, α-catenin, β-catenin, or plakoglobin. Under static conditions, staining for all proteins was intense and peripheral, forming a nearly continuous band around the cells at cell-cell junctions. After 8.5 hours of shear stress, staining was punctate and occurred only at sites of continuous cell attachment. After 24 or 48 hours of shear, staining for VE-cadherin, α-catenin, and β-catenin was intense and peripheral, forming a band of "dashes" (adherens plaques) that colocalized with the ends of stress fibers that inserted along the lateral membranes of cells. Staining for plakoglobin was not observed after 24 hours of shear stress, but returned after 48 hours. Western blot analysis indicated that protein levels of VE-cadherin, α-catenin, and plakoglobin decreased, whereas β-catenin levels increased after 8.5 hours of shear stress. As cell shape change reached completion (24 to 48 hours), all protein levels were upregulated except for plakoglobin, which remained below control levels. The partial disassembly of adherens junctions we have observed during shear induced changes in endothelial cell shape may have important implications for control of the endothelial permeability barrier and other aspects of endothelial cell function.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Oxidized LDL (oxLDL) (0.1 mg/mL) increased [Ca2+]iin vascular smooth muscle cells (VSMCs) within 5 to 10 seconds of incubation. This increase was mediated via an inositol 1,4,5-trisphosphate (IP3)-dependent release of Ca2+from the sarcoplasmic reticulum. However, atherosclerosis is a gradual process in which VSMCs are more likely exposed to low concentrations of oxLDL over extended periods rather than acute exposures. It is very possible, therefore, that lower [oxLDL] and longer exposure times may induce a very different response with regard to regulation of [Ca2+]i. VSMCs were incubated with 4- to 100-fold lower [oxLDL] for up to 6 days. The conditions were not cytotoxic. Basal [Ca2+]iwas not altered. Surprisingly, however, after chronic exposure to oxLDL, a brief addition of oxLDL (0.1 mg/mL) or norepinephrine failed to elicit the expected rise in Ca2+i. Because the acute effects of oxLDL on control cells were mediated through an IP3-dependent pathway, we investigated the integrity of the VSMC IP3receptors. Immunocytochemical analysis and Western blots revealed a depression in the density of IP3receptors after chronic exposure of VSMCs to oxLDL. These changes in IP3receptors have significance under atherosclerotic conditions as well. Immunocytochemical analysis revealed a decrease in IP3receptor density in the medial layer under atherosclerotic plaques in situ. Our data, therefore, demonstrate a striking difference between the acute and chronic effects of oxLDL on VSMC calcium. Whereas acute exposure to oxLDL stimulates [Ca2+]i, chronic exposure results in depressed Ca2+transients, apparently through a decrease in IP3receptor density. These changes have functional implications for the atherosclerotic vessel in vivo, and our data implicates oxLDL in this process.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The role of reactive oxygen species, such as superoxide anions (O2•−) and hydrogen peroxide (H2O2), in modulating vascular smooth muscle cell proliferation and viability is controversial. To investigate the role of endogenously produced H2O2, rat aortic smooth muscle cells were infected with adenoviral vectors containing cDNA for human catalase (AdCat) or a control gene, β-galactosidase (AdLacZ). Infection with AdCatresulted in dose-dependent increases in intracellular catalase protein, which was predominantly localized to peroxisomes. After infection with 100 multiplicity of infection (MOI) of AdCat,cellular catalase activity was increased by 50- to 100-fold, and intracellular H2O2concentration was reduced, as compared with control. Infection with AdCatreduced [3H]thymidine uptake, an index of DNA synthesis, in cells maintained in medium supplemented with 2% serum (0.37±0.09 disintegrations per minute per cell [AdLacZ] versus 0.22±0.08 disintegrations per minute per cell [AdCat],P<0.05). Five days after infection with 100 MOI of AdCat,cell numbers were reduced as compared with noninfected or AdLacZ-infected cells (157 780±8413 [AdCat],P<0.05 versus 233 700±3032 [noninfected] or 222 410±5332 [AdLacZ]). Furthermore, the number of apoptotic cells was increased 5-fold after infection with 100 MOI of AdCatas compared with control. Infection with AdCatresulted in induction of cyclooxygenase (COX)-2, and treatment with a COX-2 inhibitor overcame the AdCat-induced reduction in cell numbers. These findings indicate that overexpression of catalase inhibited smooth muscle proliferation while increasing the rate of apoptosis, possibly through a COX-2-dependent mechanism. Our results suggest that endogenously produced H2O2importantly modulates survival and proliferation of vascular smooth muscle cells.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Endothelial dysfunction, as observed in hypertension and atherosclerosis, is associated with a reduction in the bioavailability of endothelium-derived nitric oxide (NO). We tested the hypothesis that alterations in the soluble guanylyl cyclase (sGC) pathway may also contribute to the pathogenesis of hypertension. Therefore, we investigated the expression and activity of sGC in young (6 weeks) and aging (17 months) spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto rats (WKY). Endothelium-independent relaxation of aortic rings in response to the sGC activator YC-1 was attenuated in SHR, and expression of both α1and β1subunits of heterodimeric sGC and the basal contents of cGMP were reduced specifically in SHR aorta. Moreover, mRNA expression of the cGMP receptor and effector protein cGMP-dependent protein kinase type Iα (cGKIα) was also reduced. Interestingly, downregulation of both sGC and cGKIα expression was observed in young, ie, normotensive SHR, whereas impairment of the endothelium-independent relaxation was found only in aging SHR. Accordingly, similar cGMP levels were reached in response to YC-1 in young SHR and young WKY, suggesting a compensatory increased sensitivity or effectiveness of the sGC pathway in young SHR. In aging SHR, however, increased sensitivity to YC-1 no longer compensated for the impairment of endothelium-independent relaxation, suggesting that other mechanisms were involved. In fact, endothelium-independent relaxations were partially restored by superoxide dismutase, suggesting a pathophysiological role of superoxide production, particularly at later disease stages. Thus, tissue-specific downregulation of components of the sGC/cGMP pathway is an early event in the pathogenesis of hypertension.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Src tyrosine kinases have been shown to mediate cellular responses to stress in noncardiac cells. However, the effect of myocardial ischemia on Src tyrosine kinases is unknown. Furthermore, the identity of the tyrosine kinase(s) involved in the genesis of ischemic preconditioning (PC) remains obscure. Here, we present the first evidence that ischemic PC (6 cycles of 4-minute coronary occlusion and 4-minute reperfusion) induces selective activation of 2 members of the Src family of tyrosine kinases, Src and Lck, in the heart of conscious rabbits. The activation of Src in the particulate fraction was not evident at 5 minutes after ischemic PC but became apparent at 30 minutes (+119% versus control), whereas the activation of Lck in the particulate fraction was apparent both at 5 minutes (+103% versus control) and at 30 minutes (+89%) after ischemic PC. The activity of the other 5 members of the Src tyrosine kinases expressed in the rabbit heart (Fyn, Fgr, Yes, Lyn, and Blk) was not affected by ischemic PC. Ischemic PC had no effect on the activity of epidermal growth factor receptor kinases, either at 5 or at 30 minutes. The activation of Src and Lck was completely abrogated by the tyrosine kinase inhibitor lavendustin A, given at doses that have previously been shown to block the protective effect of ischemic PC in this same conscious rabbit model, suggesting that Src and Lck kinases are essential for the development of ischemic PC. The activity of the &epsis; isoform of protein kinase C (PKC) in the particulate fraction increased at 5 minutes (+72%) and at 30 minutes (+67%) after ischemic PC. Pretreatment with lavendustin A had no effect on the activation of PKC&epsis;, whereas pretreatment with the PKC inhibitor chelerythrine (given at doses that have previously been shown to block ischemic PC) blocked not only the activation of PKC&epsis; but also that of Src and Lck, indicating that Src and Lck are downstream of PKC&epsis; in the signaling cascade of ischemic PC. This study identifies a new component of the signaling mechanism of ischemic PC. The results support the concept that, in conscious rabbits, 2 specific members of the Src family of tyrosine kinases, Src and Lck, play an important role in the genesis of late PC by serving as downstream elements of PKC-mediated signal transduction.

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Viral myocarditis is an important cause of heart failure and dilated cardiomyopathy. T lymphocytes are implicated in myocardial damage in murine models of coxsackievirus B3 (CVB3) myocarditis. We used knockout mice lacking CD4 (CD4−/−), CD8 (CD8−/−), both coreceptors (CD4−/−CD8−/−), or the T-cell receptor β chain (TCRβ−/−) to address the contribution of T-cell subpopulations to host susceptibility to CVB3 myocarditis. Severity of disease was magnified in CD8−/−mice but attenuated in CD4−/−mice, consistent with a pathogenic role for CD4+lymphocytes. Elimination of both CD4 and CD8 molecules from T lymphocytes by genetic knockout better protected mice from myocarditis, demonstrating that both CD4+and CD8+T cells contribute to host susceptibility. The same benefit occurred in TCRβ−/−mice, with prolonged survival and minimal myocardial disease observed after CVB3 infection. Elevated interferon-γ and decreased tumor necrosis factor-α expression are associated with attenuated myocardial damage in CD4−/−CD8−/−mice. These results show that the presence of TCRαβ+T cells enhances host susceptibility to myocarditis. The severity of myocardial damage and associated mortality are dependent on the predominant T-cell type available to respond to CVB3 infection. One mechanism by which CD4+and CD8+T-cell subsets influence the pathogenesis of myocarditis may involve specific cytokine expression patterns.

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