摘要:

It has been suggested that phosphorylation of a 40S ribosomal protein, S6, regulates protein synthesis. Two distinct families of S6 kinase have been identified, the rsk-encoded 85- to 92-kD S6 kinase (RSK) and the 70- or 85-kD S6 kinase (p70 sup S6K). We have previously shown that hypertrophic stimuli, such as angiotensin II (Ang II), rapidly activate RSK in cardiac myocytes. However, RSK and p70S6Kare regulated by distinct mechanisms, and p70S6K, but not RSK, is the physiological S6 kinase in vivo in other cell types. Using cultured neonatal rat ventricular myocytes, we examined whether Ang II activates p70S6Kand investigated the effect of rapamycin, a potent yet indirect inhibitor of p70S6K, on the Ang II-induced hypertrophic response. Immunoblot analyses indicate that cardiac myocytes express the 70- and 85-kD forms of p70S6K. Ang II caused a rapid and sustained activation of p70 sup S6K through the type I Ang II receptor. Rapamycin inhibited Ang II-induced activation of p70S6Kin a dose-dependent manner, with an IC50of 0.14 ng/mL (0.15 nmol/L). Rapamycin did not inhibit Ang II-induced activation of tyrosine kinase, mitogen-activated protein kinase, RSK, and protein kinase C. The effect of rapamycin is unlikely to be mediated by its effect on p34cdc2and p33cdk2because Ang II did not activate these cell cycle-dependent kinases in cardiac myocytes. In contrast, a dose-dependent inhibition of p70S6Kby rapamycin is very closely correlated with its inhibition of the Ang II-induced increase in protein synthesis. Interestingly, rapamycin did not affect the Ang II-induced activation of specific gene expression, including the immediate-early gene c-fos and fetal type genes, such as atrial natriuretic factor and skeletal alpha-actin. Moreover, rapamycin did not suppress Ang II-induced phenotypic changes at the protein level, such as increased atrial natriuretic factor secretion, expression of beta-myosin heavy chain, and organization of actin into sarcomeric units. These results indicate that p70S6Kis activated by Ang II and that a rapamycin-sensitive signaling mechanism, most likely p70S6K, plays an essential role in the Ang II-induced increase in overall protein synthesis but not in Ang II-induced specific phenotypic changes in cardiac myocytes.(Circ Res. 1995;77:1040-1052.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

The angiotensin II type-1 (AT1) receptor, a G protein-coupled receptor, lacks intrinsic kinase activity. However, recent data show that angiotensin II (Ang II) stimulates tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), Stat91 (one of the signal transducers and activators of transcription), and paxillin in vascular smooth muscle cells. The tyrosine kinases responsible for these phosphorylation events are unknown. Src family kinases have been shown to phosphorylate PLC-gamma 1 and to be activated by G protein-coupled receptors. We hypothesized that pp60c-src associates with the AT1receptor and is activated after Ang II stimulation of smooth muscle cells. We immunoprecipitated pp60c-src from Ang II-stimulated vascular smooth muscle cells and measured pp60c-src activity by autophosphorylation and by phosphorylation of enolase. Both assays demonstrated an approximately threefold increase in pp60c-src activity within 1 minute. A similar increase in Ang II-stimulated pp60c-src activity was observed in Chinese hamster ovary cells transfected with the AT1receptor but not in untransfected cells. These data are the first to show that pp60c-src is activated by Ang II. To determine if pp60c-src associated with the AT1receptor, the AT1receptor was immunoprecipitated (with two different antibodies), and Western blots were performed with two different anti-pp60 sup c-src antibodies. No pp60c-src was detected. In addition, direct interaction between the AT1receptor and pp60c-src could not be demonstrated by using a glutathione S-transferase (GST)-AT1fusion protein to bind proteins from cell lysates stimulated by Ang II. In combination with recent findings that anti-pp60 sup c-src antibodies inhibit Ang II-mediated PLC-gamma 1 phosphorylation, our data suggest an important role for pp60c-src in Ang II signal transduction.(Circ Res. 1995;77:1053-1059.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

In the present study, cis elements in the 5 prime-flanking sequence (FS) of the rat atrial natriuretic factor (ANF) gene involved in regulating basal and alpha sub 1-adrenergic-inducible transcription were investigated. Truncation analyses using ANF-luciferase reporter constructs transfected into primary neonatal rat cardiac myocytes showed that an A/T-rich serum response element (SRE) at minus 114 bp of the ANF 5 prime-FS, which bound serum response factor (SRF), was required for basal and inducible transcription. In constructs composed of 134 bp of rat ANF 5 prime-FS driving luciferase (ANF-134Luc), mutations in the SRE at minus 114 bp disrupted SRF binding and ANF promoter activity. However, the same mutations in ANF-638Luc had little effect, suggesting a collaborating role for more distal sequences, such as the other SRE in ANF-638 at minus 406 bp. In ANF-638Luc, mutations in the SRE at minus 406 bp that disrupted SRF binding to that site decreased ANF reporter activity by only 25%; however, mutating both of the SREs completely blocked alpha1-adrenergic-inducible activity. Mutation analyses showed that an big fill circle big fill circle big fill circle (SP-1)-like site at minus 69 bp, shown previously to confer inducibility in reporters with 134 bp of ANF 5 prime-FS, was not required in ANF-638Luc. However, double mutants in the SP-1-like region and either SRE completely blocked alpha1-adrenergic-inducible ANF promoter activity. These findings emphasize that no single element is responsible for alpha1-adrenergic agonist-regulated ANF transcription but that the SREs at minus 114 and minus 406 bp and the SP-1-like sequence at minus 69 bp mediate the effect in collaboration.(Circ Res. 1995;77:1060-1069.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Previous studies showed that angiotensin II type-2 receptor (AT2) sites were increased when R3T3 cells were growth arrested and decreased when they were stimulated with fibroblast growth factor or serum. We examined the effects of several other growth factors on the expression of AT2mRNA to clarify the relation between the AT2receptor and growth factors. R3T3 cells were cultured in the medium containing 10% FCS until they were confluent, and then serum was removed. AT2mRNA was increased after serum was depleted, and the expression level reached a plateau after 2 days of serum depletion. The presence of serum (10%), fibroblast growth factor (10 ng/mL), or lysophosphatidic acid (1 mu mol/L) reduced the AT2mRNA expression. Phorbol ester (1 to 100 nmol/L) also suppressed the AT2mRNA expression in a dose-dependent manner. Interleukin-1 beta (1 ng/mL) enhanced the AT2mRNA expression 1.6-fold and the AT2receptor number 1.4-fold. Insulin (100 nmol/L) enhanced AT2mRNA expression 1.4-fold and the AT2receptor number 1.6-fold. These results suggest that AT2mRNA expression is modulated by multiple growth factors in both positive and negative directions. The presence of potential cis DNA elements that respond to interleukin-1 beta (CCAAT enhancer binding protein site), insulin [insulin response sequence of phospho(enol)pyruvate carboxykinase gene], and phorbol ester (AP-1 site) in the promoter region of the mouse AT2gene suggests that the effects of these growth factors and phorbol ester may be mediated via these cis DNA elements.(Circ Res. 1995;77:1070-1076.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

To evaluate the concept that localized delivery of angiogenic factors via virus-mediated gene transfer may be useful in the treatment of ischemic disorders, the replication-deficient adenovirus (Ad) vector AdCMV.VEGF165(where CMV is cytomegalovirus and VEGF is vascular endothelial growth factor) containing the cDNA for human VEGF165, a secreted endothelial cell-specific angiogenic growth factor, was constructed. Human umbilical vein endothelial cells (HUVECs) and rat aorta smooth muscle cells (RASMCs) infected with AdCMV.VEGF165(5 and 20 plaque-forming units [pfu] per cell) demonstrated VEGF mRNA expression and protein secretion into the supernatant. Furthermore, the conditioned medium from these cells enhanced vascular permeability in vivo. In contrast, neither VEGF mRNA nor secreted protein was found in uninfected HUVECs or RASMCs or in cells infected with the control vector AdCMV. beta gal (where beta gal is beta-galactosidase). Assessment of starved HUVECs at 14 days demonstrated sixfold more cells for AdCMV.VEGF165-infected HUVECs (20 pfu per cell) than for either infected or uninfected control cells. RASMC proliferation was unaffected by infection with AdCMV.VEGF165. When plated in 2% serum on dishes precoated with reconstituted basement membrane (Matrigel), HUVECs infected with AdCMV.VEGF sub 165 (20 pfu per cell) differentiated into capillary-like structures. Under similar conditions, both uninfected HUVECs and HUVECs infected with AdCMV. beta gal did not differentiate. To evaluate the ability of AdCMV.VEGF165to function in vivo, either AdCMV.VEGF165or AdCMV. beta gal (2 times 1010pfu) was resuspended in 0.5 mL Matrigel and injected subcutaneously into mice. Immunohistochemical staining demonstrated VEGF in the tissues surrounding the Matrigel plugs containing AdCMV.VEGF sub 165 up to 3 weeks after injection, whereas no VEGF was found in the control plugs with AdCMV. beta gal. Two weeks after injection, there was histological evidence of neovascularization in the tissues surrounding the Matrigel containing AdCMV.VEGF sub 165, whereas no significant angiogenesis was observed in response to AdCMV. beta gal. Furthermore, the Matrigel plugs with AdCMV.VEGF165demonstrated hemoglobin content fourfold higher than the plugs with AdCMV. beta gal. Together, these in vitro and in vivo studies are consistent with the concept that Ad vectors may provide a useful strategy for efficient local delivery of VEGF165in the treatment of ischemic diseases.(Circ Res. 1995;77:1077-1086.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

This study addresses the structural requirements for the intracellular processing and receptor binding properties of endothelin-1 (ET-1). Point mutants of preproendothelin-1 cDNA, with replacement of the codons for Lys (Ref. 9) of ET-1 by ones for Ala and Glu and of Ile (Ref. 20) and Trp (Ref. 21) by ones encoding Ala, were expressed in COS-7 cells. Competitive binding experiments on rat vascular smooth muscle cells (A-10), which were shown to be an ETAreceptor-rich cell line, between [sup 125 I]ET-1 and synthetic ET-1, wild-type recombinant ET-1, and recombinant [Ala (Ref. 9)]ET-1, [Glu (Ref. 9)]ET-1, [Ala (Ref. 20)]ET-1, and [Ala (Ref. 21)]ET-1 yielded Kivalues of 0.2 plus minus 0.02, 0.2 plus minus 0.02, 0.04 plus minus 0.01, 1.4 plus minus 0.2, 1.6 plus minus 0.2, and more than 50 nmol/L, respectively. In similar experiments with ETBreceptor-rich human Girardi heart cells, the corresponding values were 0.2 plus minus 0.03, 0.2 plus minus 0.03, 0.2 plus minus 0.04, 0.2 plus minus 0.06, 1.4 plus minus 0.4, and more than 50 nmol/L. The ETAreceptor-mediated contractile responses to [Glu (Ref. 9)]ET-1 and [Ala (Ref. 20)]ET-1, measured by using canine coronary artery rings, were decreased approximately fourfold to fivefold compared with the response produced by synthetic or wild-type recombinant ET-1, whereas [Ala (Ref. 9)]ET-1 was found to be more potent, and [Ala (Ref. 21)]ET-1 did not produce any contraction. These results demonstrate that Ile (Ref. 20) and Trp (Ref. 21) are involved in binding to both receptor subtypes. Of considerable interest was the observation that [Glu (Ref. 9)]ET-1 also blunts the ETAreceptor subtype-mediated contractile response to ET-1 stimulus.(Circ Res. 1995;77:1087-1094.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Platelet-derived growth factor (PDGF)-induced smooth muscle cell (SMC) fibrinolysis is necessary for SMC migration. In order to determine whether the T-cell lymphokines interleukin-4 (IL-4) and gamma interferon (gamma-IFN) affect SMC fibrinolysis and migration, we examined the effects of human recombinant IL-4 and gamma-IFN on human aortic SMC tissue-type plasminogen activator (TPA), urokinase-type plasminogen activator (UPA), and plasminogen activator inhibitor type-1 (PAI-1) antigen production, as determined by enzymelinked immunosorbent assays. Although IL-4 had no direct effect on SMC TPA antigen, IL-4 potentiated SMC TPA antigen levels and activity in conditioned media and cellular lysates in media containing 2% fetal bovine serum but did not change UPA or PAI-1 production. gamma-IFN attenuated IL-4 augmentation of SMC TPA antigen production in conditioned media, although gamma-IFN itself had no direct effects on SMC TPA and PAI-1 antigen production. IL-4 augmented PDGF induction of SMC TPA antigen. gamma-IFN inhibited PDGF induction of SMC TPA antigen and IL-4 potentiation of this process. gamma-IFN diminished the promigratory effects of both IL-4 and PDGF on in vitro SMC migration. Tranexamic acid, a plasmin inhibitor, abrogated the stimulation of SMC migration by IL-4. Therefore, IL-4 and gamma-IFN modulate the induction of SMC TPA and SMC migration by 2% fetal bovine serum and PDGF.(Circ Res. 1995;77:1095-1106.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

We have previously shown that aortic organ cultures from 1- to 3-day-old chickens initially mimic the high levels of elastin production seen in vivo. However, more prolonged incubation of these tissues results in decreased synthesis of elastin. In the present study, we demonstrate that decreased production of elastin in these aortic organ cultures is selective for elastin compared with collagen and is correlated with decreased steady state levels of mRNA for elastin. These decreases in steady state levels of elastin mRNA are due at least in part to a rapid and selective destabilization of mRNA for elastin, the half-life of which falls from approximate equal 25 hours in fresh aortic tissues to approximate equal 15 hours after incubation for only 8 hours. Destabilization of elastin mRNA can be prevented by incubation in the presence of blockers of DNA transcription (5,6-dichlorobenzimidazole riboside and actinomycin D) and mRNA translation (cycloheximide). Furthermore, the half-life of aortic elastin mRNA decreases from approximate equal 25 hours in the 1-day-old chicken to approximate equal 7 hours in the 8-week-old chicken, demonstrating that destabilization of mRNA is an important contributing factor in the decline in production of aortic elastin taking place during normal postnatal growth.(Circ Res. 1995;77:1107-1113.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

K sup plus channel openers (PCOs), such as pinacidil, elicit vasodilation primarily by hyperpolarization-induced inhibition of L-type Ca2plus channel activation. The physiological role of other mechanisms suggested to contribute to PCO-induced vasodilation is not well established. In the renal microcirculation, L-type Ca2plus channels play a prominent role in vasocon-striction of the afferent arteriole (AA) but are absent or physiologically silent in the efferent arteriole (EA). Thus, L-type Ca2plus channel-dependent and -independent mechanisms can readily be distinguished in this model. In the present study, we found that pinacidil potently inhibited Bay K 8644-induced AA vasoconstriction. Pinacidil also preferentially inhibited angiotensin II-induced AA vasoconstriction (approximately ninefold greater potency than EA). These results are consistent with an AA effect of pinacidil on L-type Ca2plus channel activation. Unexpectedly, 10 mu mol/L pinacidil inhibited AA and EA responses to similar extents (84 plus minus 10% and 71 plus minus 9%, respectively). In both AAs and EAs, glibenclamide restored normal reactivity, indicating an involvement of the ATP-sensitive Kpluschannels. In the EA, however, pretreatment with diltiazem did not alter the effects of pinacidil. Nevertheless, 45 mmol/L KCl reversed the EA actions of pinacidil, indicating an essential requirement for a normal Kplusgradient. These findings suggest that the EA actions of pinacidil involve alterations in membrane potential but not changes in L-type Ca2plus channel activity. Overall, our findings do support the premise that L-type Ca2plus channel modulation is involved in PCO-induced vasodilation in the renal microcirculation. The EA actions of pinacidil, however, suggest important additional vasodilatory mechanisms that also involve ATP-sensitive Kpluschannel-induced hyperpolarization but are independent of L-type Ca2plus channel modulation.(Circ Res. 1995;77:1114-1120.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

The role of mononuclear leukocytes for the migration of smooth muscle cells (SMCs) during intimal thickening was investigated in the rabbit model of electrically stimulated carotid artery. The approach was to inhibit leukocyte entry into the arterial intima with antibodies against the adhesion molecules very late activation antigen-4 (VLA-4) and CD11/CD18. In electrically stimulated control rabbits treated either with saline or a nonspecific antibody, all types of granulocytes, monocytes, and lymphocytes migrated across an intact endothelium into the acellular subendothelial space, followed by the movement of SMCs from the media into the intima within 36 hours of applying electrical current. Treatment of the rabbits with monoclonal antibody (mAb) HP1/2 directed toward the alpha4subunit (CD49d) of VLA-4 inhibited mononuclear leukocyte invasion (consisting of monocytes and lymphocytes) by approximate equal 70% compared with the IgG-treated control rabbits and completely abolished the minimal influx of basophils and eosinophils after 36 hours. Neutrophil infiltration, however, remained unaffected by anti-VLA-alpha4treatment. Under these conditions, SMC migration across the internal elastic lamina was reduced by 50%. The use of mAb HP1/2 together with mAb 60.3 (directed to the beta2chain of CD11/CD18) completely abolished the influx of monocytes, lymphocytes, and all types of granulocytes into the arterial intima. This complete blockade of leukocyte infiltration resulted in a 70% reduction of intimal SMC accumulation. Together with our previous findings excluding neutrophils as stimulators of SMC migration, the present results indicate that mononuclear leukocytes promote lesion development by stimulating SMC migration.(Circ Res. 1995;77:1121-1128.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID