摘要:

Many hypertrophic stimuli such as angiotensin II (Ang II) activate phospholipases through G protein-coupled receptors in cardiac myocytes. However, it is not known whether these stimuli also activate the tyrosine phosphorylation-dependent signaling pathway, which plays an essential role in growth factor-induced mitogenic responses in other cell types. Serine/threonine kinases such as mitogen-activated protein (MAP) kinases and 90-kD S6 kinase (RSK) are activated in response to many growth stimuli and are important downstream signaling pathways of tyrosine kinases. Therefore, we examined whether Ang II activates these protein kinases in primary cultures of cardiac myocytes and fibroblasts from neonatal rats. Ang II rapidly induced tyrosine phosphorylation of multiple proteins, including 42-, 44-, 75- to 80-, and 120- to 130-kD proteins, in both cardiac myocytes and fibroblasts. This was accompanied by an increase in tyrosine kinase activity. The 42- and 44-kD proteins were immunologically related to an extracellular signal-regulated kinase family (MAP kinases). Ang II rapidly increased kinase activity of MAP kinases and their downstream kinase, RSK. The Ang II-induced tyrosine phosphorylation and activation of MAP kinases and RSK were AT sub 1 receptor-mediated. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate or an increase in intracellular Ca2plus by the Ca2plus ionophore A23187 was sufficient to cause tyrosine phosphorylation of multiple proteins and activation of MAP kinase and RSK. Although downregulation of PKC did not suppress Ang II-induced activation of MAP kinase and RSK, chelating intracellular Ca2plus by BAPTA-AM completely abolished Ang II-induced activation of these kinases. Activation of MAP kinases and RSK was also observed in myocytes stimulated with other agonists for Gqproteincoupled receptors, such as phenylephrine, norepinephrine, and endothelin 1, but not with agonists to Gsprotein-coupled receptors, such as isoproterenol. These results suggest that Ang II and other hypertrophic stimuli, known to act through Gqprotein-coupled receptors, rapidly cause tyrosine phosphorylation of several intracellular substrates through activation of tyrosine kinase and activate MAP kinases and RSK in cardiac myocytes as well as in cardiac fibroblasts. Furthermore, intracellular Ca2plus, rather than PKC, seems to be critical for Ang II-induced activation of these protein kinases in cardiac myocytes.(Circ Res. 1995;76:1-15.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Human monocytes express the important procoagulant protein, tissue factor (TF), after stimulation by a variety of agents, including bacterial lipopolysaccharide (LPS). Monocyte TF expression may contribute to intravascular coagulation in a number of disease states. The present studies show that monocytic cell TF expression can be inhibited by several agents known to block cellular Kpluschannels. Exposure of human peripheral blood to 100 ng/mL LPS for 2 hours led to pronounced TF procoagulant activity associated with the mononuclear cell fraction. This was inhibited by 4-aminopyridine (2 mmol/L), tetraethylammonium chloride (10 mmol/L), and apamin (1 mu mol/L). In contrast, charybdotoxin (100 nmol/L) was inactive. More detailed studies were carried out in cultured human monocytic tumor THP-1 cells. These cells exhibited low but detectable levels of TF mRNA, measured by reverse transcription and polymerase chain reaction; cell surface procoagulant activity, measured by a plasma clotting assay; and cell homogenate TF antigen, measured by immunoassay. Exposure of THP-1 cells to 1 mu g/mL LPS led to threefold to fivefold increases in all three parameters. Basal and LPS-induced levels of all three parameters were reduced in a dose-dependent manner by 4-aminopyridine (I50, 1 mmol/L) and tetraethylammonium chloride (I sub 50, 20 mmol/L) but not by apamin or charybdotoxin. Expression of TF activity was also inhibited by glibenclamide, an inhibitor of ATP-dependent Kpluschannels (I50, 25 mu mol/L). These results suggest that facilitation of TF synthesis may be an important role for Kpluschannels in monocytes.(Circ Res. 1995;76:16-20.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) are important features of atherosclerosis. The molecular mechanisms are largely unclear; however, protein kinase C (PKC) is a key enzyme in the intracellular signaling pathways that mediate this process. We studied the activity and immunoreactivity of PKC-alpha in primary cultures of VSMCs from rat aortas under different conditions of growth and differentiation. PKC-alpha was determined under the following conditions: (1) during the growth phase and after confluence of cultured (passages 1 through 3) VSMCs, (2) before and after induction of differentiation in VSMCs by retinoic acid, and (3) in primary cultures of VSMCs from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats during early passages. PKC activity was measured by in vitro substrate phosphorylation. PKC-alpha immunoreactivity was assessed by Western blot using specific polyclonal antibodies and by immunostaining with confocal microscopy. Cell proliferation was measured by direct count. The cell phenotype was characterized by immunostaining and Western blot for alpha-actin and desmin. PKC-alpha expression and PKC activity during VSMC growth showed a decrease during rapid growth and an increase in confluent cells. This pattern was associated with the respective changes in cell differentiation. Retinoic acid induced an increase in PKC-alpha expression together with a more differentiated phenotype. Subcultured, rapidly growing VSMCs from SHR showed a decreased PKC-alpha expression compared with cells from WKY rats. To establish cause and effect, we next microinjected either PKC-alpha or inactivated material directly into dedifferentiated cells. We found that cells injected with active PKC-alpha expressed increased amounts of actin compared with control cells. We identified a close correlation between PKC-alpha and actin immunofluorescence. We conclude that PKC-alpha is downregulated in rapidly growing VSMCs. Our findings demonstrate an inverse association between PKC-alpha expression and VSMC differentiation. They suggest a role for downregulation of PKC-alpha in the proliferative response of these cells.(Circ Res. 1995;76:21-29.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

The objective of this study was to assess the potential contribution of hydrogen peroxide (H sub 2 O sub 2) to the leukocyte-endothelial cell adhesion and increased microvascular permeability observed in rat mesenteric venules after inhibition of nitric oxide synthesis with NG-nitro-L-arginine methyl ester (L-NAME). Leukocyte adherence and emigration and leakage of fluorescein isothiocyanate-labeled albumin were monitored in postcapillary venules before and after exposure of the tissue to L-NAME. H2O2production in mesenteric tissue was monitored by using dihydrorhodamine 123 (DHR), the H2O2-sensitive fluorochrome. L-NAME elicited a rapid increase in both the rate of albumin extravasation and oxidation of DHR, which was followed by an increased adherence and emigration of leukocytes in postcapillary venules. Treatment with either catalase or dimethylthiourea attenuated the L-NAME-induced oxidative stress, albumin leakage, and leukocyte-endothelial cell adhesion. Oxidation of DHR was enhanced in animals treated with either 3-amino-1,2,4-triazole (ATZ), an inhibitor of endogenous catalase, or a combination of ATZ and maleic acid diethyl ester, which depletes intracellular glutathione. Animals receiving a CD11/CD18-specific antibody to prevent leukocyte adhesion/emigration exhibited a reduced oxidation of DHR in response to L-NAME. These findings indicate that most of the H2O2(and secondarily derived oxidants) generated in mesenteric tissue exposed to an inhibitor of nitric oxide production is due to accumulation of activated leukocytes.(Circ Res. 1995;76:30-39.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

The physiological function of beta sub 2-adrenergic receptors in the neonatal and adult heart is incompletely understood, and possible age-dependent differences in beta2-receptor actions have not been considered. We used isoproterenol (mixed beta1-and beta2-receptor agonist) and zinterol (beta2-selective agonist) to compare beta-receptor subtype actions in neonatal and adult rat ventricular myocytes. When delivered as a bolus at a final concentration of 10minus7 mol/L, both isoproterenol and zinterol increased the amplitude and hastened the kinetics of the calcium and cell-shortening transients in neonatal myocytes. Under identical experimental conditions, isoproterenol increased the amplitude and accelerated the kinetics of the calcium transient and the twitch in adult myocytes, whereas zinterol did not. In the presence of CGP 20712A (beta1-receptor blocker), a 100-fold higher concentration of zinterol increased the amplitude but prolonged the duration of the twitch in adult myocytes. To probe the mechanism for this age-dependent difference in beta2-receptor responsiveness, we compared beta-receptor expression and stimulation of cAMP accumulation in neonatal and adult myocytes. beta-Receptor density was 44 339 plus minus 5178 sites per cell in neonatal myocytes and 186 346 plus minus 13 356 sites per cell in adult myocytes; the relative proportion of beta2-receptors was comparable in each (16.7 plus minus 2.3% and 16.9 plus minus 0.9%, respectively). Isoproterenol induced a large increase in cAMP accumulation in neonatal and adult myocytes (20.0 plus minus 1.0- and 20.6 plus minus 2.6-fold over basal). In contrast, zinterol evoked a substantial increase in cAMP accumulation in neonatal myocytes but only a minor increase in adult myocytes. These studies provide evidence that at low agonist concentrations, beta2-receptor activation contributes to the positive inotropic response by increasing cAMP and increasing the amplitude and hastening the kinetics of the twitch in neonatal, but not adult, myocytes. Moreover, these results suggest that age-dependent differences in beta2-receptor coupling to more distal elements in the signaling cascade can influence myocyte beta2-receptor responsiveness.(Circ Res. 1995;76:40-52.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Evidence in rat skeletal muscle suggests that local metabolic control of blood flow is facilitated by the reliance on alpha sub 2D-adrenergic receptors (ARs) for constriction of arterioles, together with the strong sensitivity of this constriction to inhibition by hypoxia. The present study examined the role of ATP-sensitive K sup plus (KATP) channels in the selective interaction between alpha2D-ARs and hypoxia. Arterioles from rat cremaster muscle that possess both alpha1D(alpha1A/D)- and alpha2D-AR subtypes were microcannulated, pressurized, and isolated in a tissue bath for measurement of changes in lumen diameter. Three studies first examined whether stimulation of alpha2D- and alpha1D-ARs involves inhibition of the KATPchannel. Concentration-dependent constriction by the KATPantagonists glibenclamide (GLB, 0.01 to 10 mu mol/L) and disopyramide (0.001 to 1 mmol/L) were abolished during alpha2Dstimulation but unaffected during alpha1Dstimulation. Activation of the KATPchannel by cromakalim inhibited alpha2Dconstriction with greater potency than alpha1D(EC50, 7.0 plus minus 0.2 versus 6.3 plus minus 0.1). Finally, GLB (0.5 mu mol/L) abolished dose-dependent alpha2Dconstriction, whereas alpha1Dwas unaffected. These data suggest that alpha2Dbut not alpha1Dstimulation is ``coupled'' with closure of the KATPchannel, leading to depolarization and contraction of vascular smooth muscle. In a second series, hypoxic (PO2, 6 mm Hg) inhibition of intrinsic smooth muscle tone was completely reversed by 0.1 mu mol/L GLB, concentration-dependent GLB constriction was enhanced during hypoxia, and hypoxia reversed GLB constriction. These data confirm reports by others that hypoxia potentiates the activation of K sub ATP channels, leading to hyperpolarization and relaxation. Finally, GLB constriction, which was abolished by concomitant alpha2Dstimulation, was completely restored by simultaneous activation of KATPchannels with hypoxia. These findings suggest that the sensitivity of alpha2D-AR constriction to inhibition by hypoxia arises through ``antagonistic coupling'' between these two stimuli, by which the alpha2D-AR inhibits and hypoxia activates KATPchannels.(Circ Res. 1995;76:53-63.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

The ADP-ATP carrier of the inner mitochondrial membrane is an autoantigen in myocarditis and dilated cardiomyopathy. Sera of patients with these diseases contain carrier-specific autoantibodies that inhibit the transmembrane nucleotide transport on isolated mitochondria. Guinea pigs immunized with the isolated ADP-ATP carrier protein also generate specific carrier-inactivating antibodies. In this study, we measured the cardiac function of guinea pigs immunized with the ADP-ATP carrier by determining the external heart work (EHW) of their isolated perfused spontaneously beating hearts stimulated by 4.0 mmol/L calcium and aortic ligature. Further, the electrogenic transport activity of the ADP-ATP carrier was estimated by calculating the cytosolic-mitochondrial difference of the phosphorylation potential of ATP [Delta G(cyt-mit)] in the freeze-clamped isolated hearts by nonaqueous fractionation. The EHW of immunized guinea pigs was seen to be reduced by 54% (P less than .005) compared with nonimmunized control guinea pigs, and Delta G(cyt-mit) declined from 4.9 kJ/mol ATP in nonimmunized control hearts to 2.3 kJ/mol ATP in the hearts of the immunized guinea pigs (P less than .005). The decisive result of this study, however, is the close relation observed between the magnitude of reduction of Delta G(cyt-mit) and the size of the decrease in EHW (r equals .87). Therefore, it seems plausible that antibody-mediated carrier dysfunction (creating the observed imbalance in myocardial energy metabolism) is responsible for the impairment of cardiac function. Our data support the hypothesis that immunopathic mechanisms in myocarditis and dilated cardiomyopathy can trigger subsequent heart failure. The underlying pathophysiological reason seems to be a metabolic disorder initiated by the antibody-mediated inactivation of the ADP-ATP carrier.(Circ Res. 1995;76:64-72.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Catecholamines have been implicated in the phenomenon of ischemic preconditioning. We have previously demonstrated that ischemic preconditioning against postischemic mechanical dysfunction in the isolated rat heart is mediated by the alpha1-adrenergic receptor. The purpose of this study was to delineate the signal transduction of preconditioning distal to the alpha1-adrenergic receptor. Our results suggest that (Ref. 1) transient ischemia and alpha1-adrenergic receptor-induced preconditioning is inhibited by protein kinase C (PKC) antagonists, (Ref. 2) functional protection against global ischemia/reperfusion injury can be induced by infusion of diacylglycerol, the second messenger of the alpha1-adrenergic pathway, and (Ref. 3) transient ischemia and alpha1-adrenergic preconditioning are both characterized by similar translocation of PKC-delta to the sarcolemma of myocardium. These findings suggest that PKC is an effector of preconditioning in the isolated rat heart.(Circ Res. 1995;76:73-81.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Recent evidence suggests a cardioprotective effect of adenosine in myocardial ischemia and reperfusion. The present study was undertaken to determine (1) whether adenosine attenuates myocardial stunning, (2) if so, whether the beneficial effect of adenosine takes place during ischemia or after reperfusion, and (3) whether adenosine preconditions against myocardial stunning. A total of 93 dogs were used. In phase A of the study, open-chest dogs undergoing a 15-minute occlusion of the left anterior descending coronary artery followed by 4 hours of reperfusion received an intracoronary infusion of either saline (group I [control], n equals 14), 2 mg/min adenosine from 30 minutes before occlusion until 1 hour after reperfusion (group II, n equals 10), or 2 mg/min adenosine from 2 minutes before reperfusion until 1 hour after reperfusion (group III, n equals 11). Regional myocardial function (assessed as systolic wall thickening) was similar in the three groups at baseline and during ischemia. After reperfusion, dogs treated with adenosine before, during, and after ischemia (group II) demonstrated a significant improvement in the recovery of function that persisted throughout the 4 hours of reperfusion. In contrast, in dogs treated only during the reperfusion period (group III), the recovery of function was not statistically different from that in control dogs. The enhanced recovery effected by adenosine in group II could not be ascribed to differences in ischemic zone size, collateral flow during occlusion, coronary flow after reperfusion, arterial pressure, heart rate, or other hemodynamic variables. In phase B of the study, dogs received an intracoronary infusion of either saline (group IV [control], n equals 6) or adenosine (4 mg/min from 40 to 10 minutes before occlusion [group V, n equals 6]). Despite pretreatment with adenosine, the recovery of function in group V was indistinguishable from that in the control group. This study demonstrates that (1) continuous administration of adenosine before, during, and after ischemia results in a significant and sustained attenuation of myocardial stunning; (2) this improved recovery of function cannot be attributed to nonspecific variables, such as collateral flow during coronary occlusion, coronary flow after reperfusion, or other hemodynamic factors, and therefore reflects a direct cardioprotective action of adenosine; (3) the protection against stunning is lost or markedly diminished if adenosine is given only at reperfusion; and (4) administration of adenosine before ischemia does not precondition the myocardium against the stunning induced by a 15-minute occlusion. The failure of adenosine to produce a beneficial effect when given only at reperfusion indicates that in the 15-minute occlusion model, the nucleoside acts primarily by decreasing the severity of ischemic injury rather than by mitigating reperfusion injury.(Circ Res. 1995;76:82-94.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

We sought to determine the role of adenosine in the sustained but reversible decrease in cardiac neurotransmission that occurs after brief ischemia. Adult mongrel dogs were anesthetized and instrumented for measurements of heart rate, arterial pressure, and left anterior descending coronary artery (LAD) and left circumflex coronary artery (LCX) flow velocities. Changes in coronary vascular resistance were measured during bilateral stimulation of the stellate ganglia. After beta-adrenergic blockade and bilateral vagotomy, stellate stimulation increased coronary vascular resistance in the LAD and LCX beds 28 plus minus 2% and 30 plus minus 3%, respectively. After a 15-minute infusion of adenosine into the LAD, the peak increase in LAD resistance was significantly reduced (18 plus minus 2%) compared with LCX (34 plus minus 5%) and control (P less than. 05, n equals 6) resistance. The LAD response after infusion of the vasodilator papaverine was unchanged (n equals 6). Intracoronary infusion of adenosine deaminase (n equals 10) but not vehicle (n equals 5) into the LAD during a 15-minute LAD occlusion prevented the attenuation in constriction to stellate stimulation. We conclude that adenosine, exogenously infused or endogenously produced, is capable of reducing cardiac neurotransmission.(Circ Res. 1995;76:95-101.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID