摘要:

&NA;To investigate mechanisms underlying the agonist‐induced desensitization of the type 1A angiotensin II receptor (AT1A‐R), we have stably expressed in Chinese hamster ovary (CHO) cells the wild‐type receptor and truncated mutants lacking varying lengths of the cytoplasmic tail. Assay of inositol 1,4,5‐trisphosphate (IP (3)) formation in response to agonist demonstrated that the truncated mutants T318, T328, and T348 lacking the last 42, 32, or 12 amino acid residues, respectively, couple with Gqprotein with an efficiency similar to that of full‐length receptors, whereas coupling of Gqprotein was abolished in the T310 truncated mutant devoid of the carboxyl‐terminal 50 amino acids. Exposure of CHO/AT1A‐Rcells expressing the wild‐type AT1A‐Rto angiotensin II resulted in rapid and dose‐dependent homologous desensitization of receptor‐mediated IP3formation, which was independent of the receptor internalization. Mastoparan, an activator of G protein‐coupled receptor kinase (GRK), induced desensitization of the AT1A‐R. The agonist‐induced desensitization of the receptor was largely prevented by heparin, a potent inhibitor of GRK, whereas it was only partially attenuated by a protein kinase C (PKC)‐specific inhibitor. The homologous or heterologous desensitization of the receptor was greatly impaired in the truncated mutants T318 and T328, lacking the Ser/Thr‐rich (13 or 12 Ser/Thr residues) cytoplasmic tail of the AT1A‐R. Deletion of the last two Ser residues, including one PKC consensus site in the receptor tail, prevented only phorbol 12‐myristate 13‐acetate‐induced desensitization by 30%. Moreover, we found an agonist‐induced translocation of a heparin‐sensitive kinase activity. The angiotensin II‐stimulated heparin‐sensitive kinase could phosphorylate a thioredoxin fusion protein containing the entire AT1A‐Rcytoplasmic tail (N295to E359), which lacks consensus phosphorylation sites for GRK1, GRK2, and GRK3. The heparin‐sensitive kinase may not be GRK2, GRK3, or GRK6 expressed in CHO/AT1A‐Rcells, since angiotensin II did not induce translocation of these receptor kinases. Potential Ser/Thr phosphorylation sites located between S328and S347in the cytoplasmic tail of AT1A‐Rseem to play a critical role in the heterologous and homologous desensitization of the receptor. A heparin‐sensitive kinase other than GRK2, GRK3, or GRK6 may be involved in the agonist‐induced homologous desensitization of the AT1A‐R. (Circ Res. 1998;82:523‐531.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Low and oscillatory shear stresses are major features of the hemodynamic environment of sites opposite arterial flow dividers that are predisposed to atherosclerosis. Atherosclerosis is a focal inflammatory disease characterized initially by the recruitment of mononuclear cells into the arterial wall. The specific characteristics of the hemodynamic environment that facilitate the generation of arterial inflammatory responses in the presence of, for example, hyperlipidemia are unknown. We show here that prolonged oscillatory shear stress induces expression of endothelial cell leukocyte adhesion molecules, which are centrally important in mediating leukocyte localization into the arterial wall. Vascular cell adhesion molecule‐1 was upregulated an average 9‐fold relative to endothelial monolayers in static culture. Intercellular adhesion molecule‐1 and E‐selectin exhibited 11‐fold and 7.5‐fold increases, respectively. Upregulation of these adhesion molecules was associated with enhanced monocyte adherence. Cytokine stimulation of surface vascular cell adhesion molecule‐1 was maximally induced after 6 and 8 hours of cytokine incubation. Oscillatory shear stress for these time periods elicited respective vascular cell adhesion molecule‐1 levels of 16% and 30% relative to those observed for cytokine stimulation. Surface intercellular adhesion molecule‐1 induction by cytokine stimulation for 24 hours was found to be approximately five times the level detected after 24 hours of oscillatory shear stress. Experiments performed in the presence of the antioxidant N‐acetylcysteine demonstrated that the expression of vascular cell adhesion molecule‐1 could be almost totally abolished, whereas that of intercellular adhesion molecule‐1 was typically reduced by [approximate]70%. These results imply that oscillatory shear stress per se is sufficient to stimulate mononuclear leukocyte adhesion and, presumptively, migration into the arterial wall. These results further indicate that atherosclerotic lesion initiation is likely related, at least in part, to unique signals generated by oscillatory shear stress and that the mechanism of upregulation is, to some extent, redox sensitive. (Circ Res. 1998;82:532‐539.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Glycosphingolipids (GSLs) and their metabolites play important roles in a variety of biological processes. We have previously reported that lactosylceramide (LacCer), a ubiquitous GSL, stimulates NADPH oxidase‐dependent superoxide generation by aortic smooth muscle cells and their consequent proliferation. We postulated that LacCer may upregulate adhesion molecules on human polymorphonuclear leukocytes (hPMNs), perhaps also via NADPH oxidase‐dependent reactive oxygen metabolite (ROM) generation. Incubation of hPMNs with LacCer upregulated CD11b/CD18 (Mac‐1) and CD11c/CD18, as determined by fluorescence‐automated cell sorting. LacCer also stimulated these hPMNs to generate superoxide via NADPH oxidase, as determined by lucigenin‐enhanced chemiluminescence. However, the upregulation of Mac‐1 by LacCer did not itself appear to be mediated by ROMs, since neither an antioxidant nor an NADPH oxidase inhibitor substantially inhibited the Mac‐1 upregulation. However, this Mac‐1 upregulation was significantly inhibited by two disparate phospholipase A2(PLA2) inhibitors. Moreover, LacCer induced arachidonic acid metabolism, which was inhibited by the PLA2inhibitors, but not by an NADPH oxidase inhibitor. To evaluate the effect of LacCer on hPMN adhesion to endothelium, hPMNs stimulated with LacCer were allowed to adhere to unstimulated human endothelial cell monolayers. LacCer stimulated hPMN adhesion to endothelial cells, which was blocked by anti‐CD18 and by the PLA2inhibitors. We conclude that LacCer stimulates both Mac‐1 upregulation and superoxide generation in hPMNs but that ROMs are not the upstream signal for Mac‐1 upregulation. This mechanism may well be relevant to acute endothelial injury in inflammation and other pathological conditions. (Circ Res. 1998;82:540‐547.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;The regulation of vascular smooth muscle cell (VSMC) proliferation by the fibronectin matrix was tested by treating human umbilical artery smooth muscle cells (HUASMCs) with a recombinant fragment of fibronectin (protein III1‐C) that has previously been shown to modulate fibronectin matrix assembly. III1‐Cinhibited HUASMC proliferation by 75% to 90%. The inhibition of growth was time dependent; III1‐Chad no effect on DNA synthesis after 0 to 5 hours of treatment but did have an effect at 24 hours and beyond. III1‐Cdid not stimulate apoptosis in these cells, indicating that the inhibition of proliferation was not due to an induction of programmed cell death. The effects of III1‐Con cell growth were only specific for normal diploid smooth muscle cells. III1‐Chad no effect on the proliferation of IMR‐90 fibroblasts, endothelial cells, NIH 3T3 cells, or the rat aortic smooth muscle cell line A7r5. However, III1‐Cdid inhibit proliferation by primary rat aortic smooth muscle cells. An analysis of HUASMC fibronectin receptor (integrin alpha 5 beta 1) distribution revealed that III1‐Cdid not inhibit alpha 5 beta 1 localization to focal contacts. Moreover, III1‐Chad no effect on the relative expression levels of seven different integrin subunits on HUASMCs. However, III1‐Cdid inhibit fibronectin matrix assembly by rat aortic smooth muscle cells, HUASMCs, A7r5 cells, IMR‐90 cells, and endothelial cells. An analysis of fibronectin synthesis indicated that the inhibition of fibronectin matrix assembly by III1‐Cwas not due solely to a decrease in fibronectin synthesis. Finally, treatment of HUASMCs with anti‐fibronectin monoclonal antibody L8 (which is known to inhibit fibronectin matrix assembly) also decreased the rate of HUASMC DNA synthesis. These results demonstrate that III1‐Cinhibits VSMC proliferation and suggest that this effect may be mediated by the inhibition of fibronectin matrix assembly. (Circ Res. 1998;82:548‐556.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Cyclic nucleotides are known to modify voltage‐gated (L‐type) Ca (2+) channel activity in vascular smooth muscle cells, but the exact mechanism(s) underlying these effects is not well defined. The purpose of the present study was to investigate the modulatory role of the cAMP‐ and cGMP‐dependent protein kinase (PKA and PKG, respectively) pathways in Ca2+channel function by using both conventional and perforated‐patch‐clamp techniques in rabbit portal vein myocytes. The membrane‐permeable cAMP derivative, 8‐bromo cAMP (0.1 to 10 [micro sign]mol/L), significantly increased (14% to 16%) peak Ba2+currents, whereas higher concentrations (0.05 to 0.1 mmol/L) decreased Ba2+currents (23% to 31%). In contrast, 8‐bromo cGMP inhibited Ba2+currents at all concentrations tested (0.01 to 1 mmol/L). Basal Ca2+channel currents were significantly inhibited by the PKA blocker 8‐Bromo‐2[prime]‐O‐monobutyryladenosine‐3[prime],5[prime]‐monophosphorothioate, Rp‐isomer (Rp 8‐Br‐MP cAMPS, 30 [micro sign]mol/L) and enhanced by the PKG inhibitor beta‐Phenyl‐1,N (2‐etheno‐8‐bromoguanosine‐3)[prime],5[prime]‐monophosphorothioate, Rp‐isomer (Rp‐8‐Br PET cGMPS, 10 nmol/L). In the presence of Rp 8‐bromo PET cGMPS (10 to 100 nmol/L), both 8‐bromo cAMP (0.1 mmol/L) and 8‐bromo cGMP (0.1 mmol/L) enhanced Ba2+currents (13% to 39%). The excitatory effect of 8‐bromo cGMP was blocked by Rp 8‐bromo MB‐cAMPS. Both 8‐bromo cAMP (0.05 mmol/L) and forskolin (10 [micro sign]mol/L) elicited time‐dependent effects, including an initial enhancement followed by suppression of Ba2+currents. Ba2+currents were also enhanced when cells were dialyzed with the catalytic subunit of PKA. This effect was reversed by the PKA blocker KT 5720 (200 nmol/L). Our results suggest that cAMP/PKA stimulation enhances and cGMP/PKG stimulation inhibits L‐type Ca2+channel activity in rabbit portal vein myocytes. Our results further suggest that both cAMP and cGMP have a primary action mediated by their own kinase as well as a secondary action mediated by the opposing kinase. (Circ Res. 1998;82:557‐565.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;In recent years, significant progress has been made toward understanding skeletal muscle development. However, the mechanisms that regulate smooth muscle development and differentiation are presently unknown. To better understand smooth muscle‐specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a highly specific marker of differentiated smooth muscle cells. The goal of the present study was to isolate and characterize the mouse SMHC gene promoter, since the mouse promoter would be particularly suited for in vivo promoter analyses in transgenic mice and would serve as a tool for targeting genes of interest into smooth muscle cells. We report here the isolation and characterization of the mouse SMHC promoter and its 5[prime] flanking region. DNA sequence analysis of a 2.6‐kb portion of the promoter identified several potential binding sites for known transcription factors. Transient transfection analysis of promoter deletion constructs in primary cultures of smooth muscle cells showed that the region between ‐1208 and ‐1050 bp is critical for maximal SMHC promoter activity. A comparison of SMHC promoter sequences from mouse, rat, and rabbit revealed the presence of a highly conserved region located between ‐967 and ‐1208 bp. This region includes three CArG/CArG[star, filled]‐like elements, two SP‐1 binding sites, a NF‐1‐like element, an Nkx2‐5 binding site, and an Elk‐1 binding site. Gel mobility shift assay and DNase I footprinting analyses show that all three CArG/CArG[star, filled]‐like elements can form DNA‐protein complexes with nuclear extract from vascular smooth muscle cells. Protein binding to the CArG[star, filled] elements can be competed out by either serum response element or by an authentic CArG element from the cardiac alpha‐actin gene. Using a serum response factor (SRF) antibody, we demonstrate that SRF is part of the protein complex. In addition, we show that cotransfection with the SRF dominant‐negative mutant expression vector abolishes SMHC promoter activity, suggesting that SRF protein plays a critical role in SMHC gene regulation. (Circ Res. 1998;82:566‐575.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Adhesion molecules mediate inflammatory myocardial injury after ischemia/reperfusion. Cytokine release and hypoxia are features of acute ischemia that may influence expression of these molecules. Accordingly, we studied intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) responses to cytokines and acute hypoxia in cultured myocardial cells. Northern blot analysis and immunoassay showed that the proinflammatory cytokines interleukin‐1 beta (IL‐1 beta) and tumor necrosis factor‐alpha stimulated concentration‐dependent increases in ICAM and VCAM mRNA and protein. In both cardiac myocytes and fibroblasts, pretreatment with a specific inhibitor of nuclear transcription factor‐kappa B (NF‐kappa B) prevented cytokine induction of both molecules. We also found that inhibition of tyrosine kinase and p38/RK (stress‐activated protein kinase) pathways prevented IL‐1 beta‐induced ICAM and VCAM protein synthesis, whereas extracellular signal‐regulated protein kinase (ERK1/ERK2) inhibition did not. Neither hypoxia (0% O2for 6 hours) alone nor hypoxia/reoxygenation had any significant effect on ICAM and VCAM mRNA. However, hypoxia did enhance IL‐1 beta‐induced ICAM mRNA expression in myocytes. As a possible mechanism of this synergistic action on CAM expression, hypoxia induced a time‐dependent increase in the DNA binding activity of both NF‐kappa B and activator protein‐1 (AP‐1), two transcription factors important for cell adhesion molecule expression. In contrast to the enhanced ICAM mRNA induced by IL‐1 beta during hypoxia, however, protein levels for this adhesion molecule were unchanged beyond IL‐1 beta‐stimulated levels, suggesting posttranscriptional and/or posttranslational control mechanisms. We conclude that cytokines regulate ICAM and VCAM mRNA and protein in both cardiac myocytes and fibroblasts. Furthermore, adhesion molecule induction requires translocation of at least two transcription factors, NF‐kappa B and AP‐1. (Circ Res. 1998;82:576‐586.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Although a substantial coronary angiogenesis occurs after thyroid hormone treatment, its regulation and relationship to cardiac hypertrophy are not understood. This study was designed to determine (1) the onset of capillary proliferation, (2) the sites of capillary proliferation, and (3) whether basic fibroblast growth factor (bFGF) upregulation occurs in response to thyroxine administration. Male Sprague‐Dawley rats were injected daily with L‐thyroxine (T4, 0.2 mg/kg SC). Bromodeoxyuridine labeling of capillary endothelial cells increased during the first 24 hours of treatment and peaked after 2 days of treatment. Northern blot analysis revealed a slight increase in bFGF mRNA during this period, followed by a doubling of expression by 48 hours, at which time bFGF protein was also increased. In situ hybridization, used to localize bFGF mRNA, showed an increase in transcripts within 24 hours after T4. This enhancement was uniform in the epimyocardium and endomyocardium. Histochemical analysis (double staining for alkaline phosphatase and dipeptidyl peptidase) of frozen sections, used to discriminate capillary profiles as arteriolar and venular, respectively, showed that growth occurred in the latter, since the percentage of capillary profiles positive for dipeptidyl peptidase was higher than the control value after 4 days of T4administration. These data indicate that in the thyroxine model of cardiac hypertrophy (1) capillary DNA synthesis occurs after a single injection of thyroxine, (2) capillary growth coincides with an upregulation in bFGF mRNA and increase in bFGF protein, and (3) proliferation occurs in the venular capillaries. (Circ Res. 1998;82:587‐593.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Recently, insulin‐like growth factor‐1 (IGF‐1) has been claimed to positively influence the cardiac performance of the decompensated heart. On this basis, the effects of constitutive overexpression of IGF‐1 on the mechanical behavior of myocytes were examined in transgenic mice in which the cDNA for the human IGF‐1B was placed under the control of a rat alpha‐myosin heavy chain promoter. In mice heterozygous for the transgene and in nontransgenic littermates at 2.5 months of age, the alterations in Ca2+sensitivity of tension development, unloaded shortening velocity, and sarcomere compliance were measured in skinned myocytes. The quantities and state of phosphorylation of myofilament proteins in these enzymatically dissociated ventricular myocytes were also examined. The overexpression of IGF‐1 was characterized by a nearly 15% reduction in myofilament isometric tension at submaximum Ca2+levels in the physiological range, whereas developed tension at maximum activation was unchanged. In contrast, unloaded velocity of shortening was increased 39% in myocytes from transgenic mice. Moreover, resting tension in these cells was reduced by 24% to 33%. Myocytes from nontransgenic mice pretreated with IGF‐1 failed to reveal changes in myofilament Ca2+sensitivity and unloaded velocity of shortening. The quantities of C protein, troponin I, and myosin light chain‐2 were comparable in transgenic and nontransgenic mice, but their endogenous state of phosphorylation increased 117%, 100%, and 100%, respectively. Troponin T content was not altered, and myosin isozymes were essentially 100% V1in both groups of mice. In conclusion, constitutive overexpression of IGF‐1 may influence positively the performance of myocytes by enhancing shortening velocity and cellular compliance. (Circ Res. 1998;82:594‐603.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

&NA;Electrical coupling of pacemaker cells at gap junctions appears to play an important role in sinus node function. Although the major cardiac gap junction protein, connexin43 (Cx43), is expressed abundantly in atrial and ventricular muscle, its expression in the sinus node has been a subject of controversy. The objectives of the present study were to determine whether Cx43 is expressed by sinus node myocytes, to characterize the spectrum of connexin expression phenotypes in sinus node pacemaker cells, and to define the spatial distribution of different connexin phenotypes in the intact sinus node. To fulfill these objectives, we performed high‐resolution immunohistochemical analysis of disaggregated adult canine sinus node preparations. Using enhanced tissue preservation and antigen retrieval techniques, we also performed immunohistochemical studies on sections of intact canine sinus node tissue. Analysis of disaggregated sinus node preparations revealed three populations of pacemaker cells distinguished on the basis of connexin immunohistochemical phenotype: [approximate]55% of cells expressed only connexin40 (Cx40); 30% to 35% of cells expressed Cx43, connexin45 (Cx45), and Cx40; and the remaining cells had no detectable connexin expression. In immunostained sections of intact sinus node, Cx43‐ and Cx45‐positive cells were limited in their distribution and were observed in discrete bundles that appeared to abut atrial myocytes. In contrast, Cx40 immunoreactive signal was widely distributed in the sinus node region. These results indicate that subsets of pacemaker cells express distinct connexin phenotypes. Differential expression of connexins could create regions within the sinus node with different conduction properties, thereby contributing to the nonuniform conduction properties seen in this tissue. (Circ Res. 1998;82:604‐612.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID