摘要:

G proteins are critically important mediators of many signal transduction systems. In the present study, we investigated the effect of direct activation of pertussis toxin (PTX)-sensitive G protein (GPTX130 micro m). Topically applied mastoparan (G protein activator, 10, 30, and 100 micro mol/L) produced homogeneous microvascular dilation in a concentration-dependent manner (10 micro mol/L, 7.9 +/- 2.0%; 30 micro mol/L, 10.3 +/- 2.4%; and 100 micro mol/L, 16.7 +/- 4.5% in small microvessels; 10 micro mol/L, 5.3 +/- 1.2%; 30 micro mol/L, 9.8 +/- 2.5%; and 100 micro mol/L, 15.5 +/- 3.9% in large microvessels). These dilations were reversed to constriction by pretreatment with PTX (300 ng/mL, 2 hours) in both microvessel groups. Blockade of nitric oxide production by Nomega-nitro-L-arginine (LNNA, 300 micro mol/L) offset the mastoparan-induced dilation in large microvessels but not in small microvessels. Cosuperfusion of glibenclamide (10 micro mol/L) with LNNA produced constriction of all sizes of microvessels in response to mastoparan, whereas charybdotoxin (10 nmol/L) did not affect the mastoparan effect. Pretreatment with glibenclamide alone reversed mastoparan dilation to constriction in small microvessels, whereas it only offset the dilation without producing constriction in large microvessels. We conclude that the activation of GPTXproduces homogeneous coronary arterial microvascular dilation and that the underlining mechanisms of the dilation are vessel size dependent. The L-arginine-nitric oxide pathway mediates the dilation only in large microvessels, whereas ATP-sensitive K sup + channel activation plays a central role in the dilation of small microvessels when GPTXis directly activated. ATP-sensitive K sup + channels are also involved in the dilation of large microvessels in a synergistic fashion with nitric oxide production. (Circ Res. 1997;80:1-10.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Excessive NO produced by an inducible NO synthase (iNOS) has been implicated in many types of immune-associated disorders of the cardiovascular system, but it remains to be determined whether NO plays a role in myocarditis. Thus, the significance of iNOS expression in the development of experimental autoimmune myocarditis (EAM), an animal model of human giant cell myocarditis, was investigated. Lewis rats were immunized with cardiac myosin and were killed 7, 14, 21, 28, and 49 days after immunization. The development of severe myocarditis was observed on days 14, 21, and 28 in association with significant deterioration of hemodynamics determined by cardiac catheterization, which peaked on day 21. In parallel with histological severity of myocarditis and deterioration of cardiac performance, iNOS activity in the heart measured by [sup 14 C]L-citrulline formation was markedly increased on days 14, 21, and 28. The expression of iNOS was confirmed by immunoblotting and was localized to the infiltrating inflammatory cells found in the vicinity of necrotic myocytes by immunohistochemical analysis. Aminoguanidine, a selective inhibitor of iNOS, significantly decreased the iNOS activity (1.04 +/- 0.37 compared with 29.1 +/- 8.62 pmol [center dot] min sup -1 [centered dot] mg protein sup -1 in untreated myosin-immunized rats, P<.01) and effectively attenuated histopathological changes of EAM on day 21. Hemodynamic parameters were also improved from 64 +/- 3 to 89 +/- 3 mm Hg for mean blood pressure, from 80 +/- 2 to 113 +/- 4 mm Hg for left ventricular systolic pressure, from 7.8 +/- 0.3 to 3.2 +/- 0.3 mm Hg for left ventricular end-diastolic pressure, from 2867 +/- 137 to 4180 +/- 102 mm Hg/s for +dP/dt, and from 2717 +/- 132 to 4180 +/- 184 mm Hg/s for -dP/dt (P<.01). The values after aminoguanidine treatment were not significantly different from the control values. These results suggest an important role for NO in mediating pathophysiological changes in myocarditis of autoimmune origin. (Circ Res. 1997;80:11-20.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Endothelium-dependent relaxation of porcine coronary arteries is attenuated under hypoxic conditions. Recent evidence also indicates that pHimay modulate the release of the endothelium-derived relaxing factor. We tested the hypothesis that hypoxia-induced attenuation of endothelium-dependent relaxation is mediated by alterations in pHi. We developed a novel method for loading surface cells, whereby endothelial cell pHicould be measured in situ on the intact porcine coronary artery. Endothelial cells of arterial ring segments were selectively loaded with the fluorescent indicator BCECF-AM. Differential loading of the endothelial cell layer was verified by confocal microscopy. pHiof the endothelial cells in situ and of endothelium-denuded arteries was measured with a Photon Technology International spectrofluorimeter. The functional integrity of the endothelium was assessed by the endothelium-dependent relaxation to substance P in a paired adjacent ring. In the experimental protocol for pHimeasurements, preparations were perfused with a physiological bicarbonate buffer (pH 7.4), stimulated with KCl (29 mmol/L), and then subjected to hypoxia and reoxygenation. The mean basal pHiin endothelial cells on the intact six arteries was 6.92 +/- 0.07. Addition of KCl to the perfusion medium decreased (P=.025) pHito 6.79 +/- 0.07. Subsequent bubbling with N2increased (P=.009) pHito 7.00 +/- 0.06, which was reversed by reoxygenation. In contrast to the in situ endothelium, pHiof the smooth muscle was not significantly altered from its basal value of 7.24 +/- 0.06 (n=5) by either KCl or hypoxia. This differential behavior corroborated the confocal data indicating differential dye loading. These data thus suggest that oxygen-sensitive alterations in pHimay be an important mechanism of signal transduction in endothelial cells. (Circ Res. 1997;80:21-27.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Although fibroblast growth factor-2 (FGF-2) participates in the response to vascular injury, the role of cellular deformation in FGF-2 release is incompletely understood. To test the hypothesis that mechanical strain tightly controls FGF-2 release, a novel device was used to impose homogeneous and uniform biaxial strain to human vascular smooth muscle cells. Release of FGF-2 increased with the number of cycles of strain (14%, 1 Hz); 1, 9, and 90 cycles of strain, respectively, released 0.55 +/- 0.06%, 2.9 +/- 0.3%, and 5.5 +/- 1.3% of the total cellular FGF-2 (versus 0.00 +/- 0.40% for control, P<.05), but release was not further increased for strain of 90 to 90 000 cycles. Mechanical release of FGF-2 depended on both the frequency and amplitude of deformation. For example, strain (90 cycles, 1 Hz) at 4% amplitude released only 0.1 +/- 0.1% of the total FGF-2, but strain at 14% and 33% amplitudes, respectively, released 5.7 +/- 0.5% and 19.0 +/- 3.0% of the FGF-2 cellular pool (P<.05), suggesting a strain amplitude threshold for FGF-2 release. Injury to a subpopulation of cells increased with the frequency and amplitude of strain, but cells were not injured by strains below 10% amplitude. Strain following pretreatment with heparin released 12.6 +/- 1.6% of the total FGF-2 (versus 15.8 +/- 0.9% for strain alone, P<.05), indicating that most FGF-2 was liberated from the nuclear or cytoplasmic pools and not from low-affinity extracellular receptors. Conversely, strain in the presence of heparin released 25.2 +/- 3.5% of the total FGF-2 (versus 15.6 +/- 2.6% for strain alone, P<.05). Thus, cellular strain closely modulates the release of intracellular FGF-2 from human vascular smooth muscle cells, but FGF-2 release is negligible in response to the smaller strains that occur in the normal artery. In addition, larger mechanical strains lead to transfer of intracellular FGF-2 to the extracellular low-affinity receptors, where FGF-2 may be displaced by heparin. These observations provide insight into the mechanisms by which deforming vascular injury, such as that produced by arterial interventions, may elicit a proliferative response. (Circ Res. 1997;80:28-36.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Oxidation converts low-density lipoprotein (LDL) into a cytotoxin in vitro. Oxidized LDL exists in vivo in atherosclerotic lesions and possibly in plasma. Many cell functions are altered in vitro by oxidized LDL, but few have been examined in vivo. To test whether oxidized LDL could injure endothelial cells and alter endothelial permeability to macromolecules in vivo, we infused oxidized LDL, native LDL, or their solvent intravenously into rats. Subsequently, endothelial cell injury and proliferation were measured, and the transport into the aorta wall of the macromolecule horseradish peroxidase (HRP) was quantified. Transport data were analyzed using mathematical models of macromolecular transport; parameters were estimated by optimally fitting model-predicted HRP concentrations to experimental data. Compared with native LDL or solvent control infusion, oxidized LDL infusion increased (1) the number of injured aortic endothelial cells fivefold to sixfold at 36 hours, (2) proliferation of endothelial cells at 48 hours, (3) intimal and medial accumulations of HRP twofold to threefold at 48 hours, and (4) the permeability coefficient of the endothelium to HRP fourfold to fivefold at 48 hours. Hence, oxidized LDL administered in vivo can injure the endothelium, despite the presence of endogenous antioxidants, compromising the function of the endothelium as a permeability barrier. (Circ Res. 1997;80:37-44.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Recent studies suggest that superoxide production by the NADPH/NADH oxidase may be involved in smooth muscle cell growth and the pathogenesis of hypertension. We previously showed that angiotensin II (Ang II) activates a p22phoxbased NADPH/NADH oxidase in cultured rat vascular smooth muscle cells and in animals made hypertensive by infusion of Ang II. To investigate the mechanism responsible for this increased oxidase activity, we examined p22phox mRNA expression in rats made hypertensive by implanting an osmotic minipump that delivered Ang II (0.7 mg/kg per day). Blood pressure began to increase 3 days after the start of Ang II infusion and remained elevated for up to 14 days. Expression of p22phox mRNA in aorta was also increased after 3 days and reached a maximum increase of 338 +/- 41% by 5 days after pump implantation compared with the value after sham operation. This increase in mRNA expression was accompanied by an increase in the content of the corresponding cytochrome (twofold) and NADPH oxidase activity (179 +/- 11% of that in sham-operated rats 5 days after pump implantation). Treatment with the antihypertensive agents losartan (25 mg/kg per day) or hydralazine (15 mg/kg per day) inhibited this upregulation of mRNA levels and activity. Furthermore, infusion of recombinant heparin-binding superoxide dismutase decreased both blood pressure and p22phox mRNA expression. In situ hybridization of aortic tissue showed that p22phox mRNA was expressed in medial smooth muscle as well as in the adventitia. These findings suggest that Ang II-induced hypertension activates the NADPH/NADH oxidase system by upregulating mRNA levels of one or several components of this oxidase system, including the p22phox, and that the NADPH/NADH oxidase system is associated with the pathology of hypertension in vivo. (Circ Res. 1997;80:45-51.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Loss of myofilaments has been observed in both adaptive cardiac responses (ie, hypertrophy) as well as in chemotheraputic use of antineoplastic drugs with cardiotoxic side effects (ie, doxorubicin). An understanding of the degenerative process is a prerequisite for determining approaches to limit the cardiomyopathic changes associated with chronic heart disease or long-term chemotheraputic treatments. However, little is known about the specific events and molecular changes that initiate the degenerative process. To study this process, neonatal rat cardiomyocytes were treated with doxorubicin, which induced rapid and widespread thin-filament degeneration as observed by fluorescence confocal microscopy. which demonstrated deterioration of sarcomeric thin-filament structure. Changes in the spontaneous beating of cardiomyocytes corresponding with myofibrillar degeneration were apparent using differential interference contrast video microscopy. After finding induction of kinase activity by doxorubicin in cultured cardiomyocytes, the protective effects of specific inhibitors of kinase activity were assessed for their ability to inhibit doxorubicin-induced myofibrillar breakdown. Doxorubicin-induced changes appeared similar to the degeneration observed after treatment with a protein kinase activator (phorbol 12-myristate 13-acetate) or a serine-threonine protein phosphatase inhibitor (okadaic acid). Collectively, these results indicate that activation of protein kinase is an important event in the initiation of myofibrillar degeneration by doxorubicin. Further analyses of myofibrillar proteins with respect to biochemical modifications will be necessary to determine if phosphorylation events transmit signal(s) to initiate degeneration. (Circ Res. 1997;80:52-61.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

During static exercise, several reflex systems that increase sympathetic nerve activity, heart rate, arterial pressure, and cardiac output are activated. At rest, the renal circulation receives the most blood flow per tissue weight of any organ in the body. However, the renal circulatory response to static exercise has not been studied in humans because of technical limitations in methods for measuring rapid changes in renal blood flow. The aim of this study was to determine the renal blood flow response to static exercise in healthy humans and, specifically, to clarify the reflex mechanisms underlying this response. Renal cortical blood flow was measured using dynamic positron emission tomography and the blood flow agent oxygen-15 water. Graded handgrip exercise, posthandgrip circulatory arrest, and administration of intra-arterial adenosine were performed to clarify the mechanisms controlling renal blood flow during static exercise. The major new findings in this study are that in healthy humans (1) renal cortical blood flow decreases (basal versus handgrip, 4.4 +/- 0.1 versus 3.5 +/- 0.1 mL [center dot] min sup -1 [center dot] g sup -1; P=.008) and renal cortical vascular resistance increases (basal versus handgrip, 17 +/- 1 versus 26 +/- 2 U; P=.01) in response to static handgrip exercise; (2) central command and/or the mechanoreflex contributes importantly to the early decrease in renal blood flow (basal versus handgrip, 4.2 +/- 0.2 versus 3.5 +/- 0.3 mL [centered dot] min sup -1 [center dot] g sup -1; P=.04) and to the increase in renal cortical vascular resistance (basal versus handgrip, 20 +/- 1 versus 25 +/- 2 U; P=.04); (3) the muscle metaboreflex contributes to further decreases in renal blood flow (basal versus posthandgrip circulatory arrest, 4.3 +/- 0.1 versus 3.5 +/- 0.2 mL [centered dot] min sup -1 [center dot] g sup -1; P=.002) and increases in renal cortical vascular resistance (basal versus handgrip, 18 +/- 1 versus 25 +/- 3 U; P=.002); and (4) exogenous adenosine activates the muscle metaboreflex producing reflex renal vasoconstriction and decreased renal blood flow, which may implicate endogenous adenosine generated during ischemic exercise as a potential activator of the muscle metaboreflex during ischemic handgrip exercise. (Circ Res. 1997;80:62-68.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

The hypothesis that rat cardiomyocytes become susceptible to hypercontracture after anoxia/reoxygenation was investigated. The cells were gradually overloaded with Ca2+ after different periods of simulated ischemia (substrate-free anoxia, medium at pH 6.4) followed by 20 minutes of reoxygenation. The cytosolic Ca2+ concentration (measured with fura 2) at which the cells developed maximal hypercontracture (Camax) was used as an index for their susceptibility to hypercontracture (SH). SH was increased in cardiomyocytes after prolonged periods of simulated ischemia; ie, these cells developed hypercontracture at significantly lower cytosolic Ca2+ levels than did normoxic cells (Camax, 0.80 +/- 0.05 micro mol/L versus 1.27 +/- 0.05 micro mol/L; P<.01). To find the possible cause of increased SH, the influence of Ca2+ overload, acidosis, and protein dephosphorylation were studied. Prevention of cytosolic Ca2+ overload in anoxic cardiomyocytes or imitation of ischemic acidosis in normoxic cells did not influence Camax. In contrast, use of 10 micro mol/L cantharidin (inhibitor of protein phosphatases 1 and 2A) during anoxic superfusion prevented the reduction of Camax. Furthermore, treatment of normoxic cardiomyocytes with 20 mmol/L of the chemical phosphatase 2,3-butanedione monoxime reduced Camax. Therefore, prolonged simulated ischemia increases susceptibility of cardiomyocytes to hypercontracture. This seems to be due to protein dephosphorylation. (Circ Res. 1997;80:69-75.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Oxygen-derived free radicals have been reported to damage the sarcoplasmic reticulum (SR) Ca2+-ATPase, potentially contributing to cellular Ca2+ overload and myocardial damage after ischemia and reperfusion. To determine whether the ATP binding site on Ca2+-ATPase is involved in oxygen radical injury, SR vesicles containing bound Ca2+-ATPase were isolated from rabbit cardiac and skeletal muscle and exposed to a hydroxyl radical ([center dot]OH)-generating system consisting of H2O2and Fe3+-nitrilotriacetic acid in amounts that generate a magnitude of [center dot]OH similar to that which occurs in the reperfused heart. [center dot]OH exposure completely inhibited Ca2+-ATPase activity and SR45Ca uptake for both cardiac and skeletal muscle. In contrast, when the purified vesicles were premixed with 1 mmol/L ATP before exposure to [center dot]OH, complete protection was observed: there was no loss of ATPase activity or45Ca transport. No significant protection occurred with adenosine, sucrose, AMP, or ADP (1 mmol/L each). SDS-gel electrophoresis indicated that [center dot]OH did not damage the primary structure of the enzyme. Electron paramagnetic resonance spin-trapping experiments demonstrated that ATP did not scavenge [center dot]OH. These results suggest that [center dot]OH denatures the SR Ca2+-ATPase by directly attacking the ATP binding site, and occupation of the active site by ATP protects against [centered dot]OH-induced loss of enzymatic activity and SR Ca2+ transport. The depletion of ATP that occurs during ischemia may enhance the toxic effect of [center dot]OH at the time of reperfusion. (Circ Res. 1997;80:76-81.)

ISSN:0009-7330    

出版商:OVID    

年代:1997

数据来源: OVID