摘要:

Ouabain (1×10−7-3×10−5M) elicited concentration-dependent vasoconstriction in human placental arteries and veins. These responses, but not those produced by 10−6M 5-hydroxytryptamine, were increased after the removal of vascular endothelium. In placental arteries, the respective blockade of cyclooxygenase or lipoxygenase with indomethacin or 5,8,11,15-eicosatetraynoic acid as well as the inactivation of nitric oxide with phenidone or oxyhemoglobin and the inhibition of nitric oxide synthesis withNG-monomethyl l-arginine (all at 10−5M) did not mimic the effects of endothelial denudation on ouabain-evoked contractions. Bioassay experiments suggested that the above-mentioned endothelial effects are mediated by diffusible factors.86Rb+uptake, a method to measure sodium pump activity, was significantly reduced by the removal of endothelium. These results suggest the existence of an inhibitory modulation by the endothelium of the contractions induced by ouabain, likely mediated by a diffusible factor(s) released from these cells. The nature of this substance is unknown but is not related to prostaglandins or leukotrienes, and neither is it a nitric oxide-related compound. Its mechanism of action could be stimulating the activity of vascular sodium pump and/or antagonizing its inhibition by ouabain.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

This study was designed to investigate the mechanism of endothelin-1 (ET-1) contractions inStaphylococcusα-toxin-permeabilized vascular smooth muscle. Rabbit small mesenteric arteries permeabilized with α-toxin were mounted for isometric or isotonic force recording or were processed for determination of myosin light chain (MLC) phosphorylation levels. Addition of 100 nM ET-1 plus 10 μM GTP significantly enhanced myofilament Ca2+sensitivity as compared with the addition of Ca2+alone (EC50, 0.47 μM Ca2+for Ca2+alone and 0.13 μM Ca2+for ET-1 plus GTP). This enhanced sensitivity was reversed by GDPβS. ET-1-induced contractions were relaxed at a constant [Ca2+] by the addition of 30 μM cAMP or cGMP, demonstrating a direct effect of the cyclic nucleotides on contractile regulation. Inhibition of protein kinase C activity by 100 nM staurosporine relaxed ET-1 plus GTP-induced contractions, and pretreatment with 40 μM chelerythrine inhibited the ET-1 plus GTP increase in force. At 0.32 μM Ca2+, steady-state levels of shortening velocity were not increased by ET-1 plus GTP, although steady-state levels of MLC phosphorylation were significantly enhanced. The ET-1-induced increase in MLC phosphorylation was not altered by changes in [Ca2+], whereas the shortening velocity was Ca2+dependent, suggesting that the increase MLC phosphorylation level may be the result of protein kinase C, rather than MLC kinase, activation. These results are consistent with the hypothesis that ET-1 increases myofilament Ca2+sensitivity by a G protein-dependent pathway and subsequent activation of protein kinase C. We propose that protein kinase C increases myofilament Ca2+sensitivity during ET-1 stimulation either by phosphorylation of a thin-filament regulatory protein or by downregulation of the MLC phosphatase.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

The purpose of the present study was to determine whether sympathetic coronary vasoconstrictor responses are altered after brief ischemia and reperfusion. Adult mongrel dogs were anesthetized and instrumented for measurements of heart rate, arterial pressure, left ventricular pressure, left ventricular dP/dt, anterior myocardial wall thickening, and left circumflex coronary artery (LCX) and left anterior descending coronary artery (LAD) blood flow velocities. Changes in coronary vascular resistance were recorded during intravenous bolus doses of norepinephrine and bilateral electrical stimulation of the stellate ganglia. After β-adrenergic blockade and bilateral vagotomy, electrical stimulation of the stellate ganglia increased coronary vascular resistance in the LAD and LCX beds by 38±5% and 39±5%, respectively. After a 15-minute LAD occlusion, repeat electrical stimulation produced increases in coronary resistance of 16±3% and 45±8%, respectively (p<0.05 for the LAD before versus after the occlusion). The peak increase in coronary vascular resistance to two doses of norepinephrine was unchanged. After a shorter period of myocardial ischemia (7 minutes), similar increases in coronary resistance to stellate stimulation were observed before (27±4%) and after (26±6%) myocardial ischemia. The mechanism of this impaired sympathetic coronary vasoconstriction was further tested by examining the responses to bretylium and tyramine. Brief ischemia did not alter the coronary constrictor responses to either bretylium or tyramine, suggesting that mechanisms governing prejunctional release of norepinephrine are intact in the postischemic coronary arterial bed. The postischemic myocardium was characterized by mild reductions in left ventricular dP/dt and marked reductions in transmural myocardial wall thickening, characteristic of myocardial stunning. We conclude that after brief myocardial ischemia, coronary vasoconstriction to sympathetic activation is impaired, whereas constriction to direct receptor activation (norepinephrine) and stimulated prejunctional release of a neurotransmitter (bretylium or tyramine) remain intact. These data are consistent with the interpretation that sympathetic efferent neural conduction is impaired in regions of stunned myocardium.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Myocardial phospholipase D (PLD) is primarily localized at the sarcolemmal level and selectively hydrolyzes phosphatidylcholine to form phosphatidic acid as part of the signal transduction mechanisms for regulating Ca2+movements in the heart. Since the myocardial cell damage induced by oxidative stress is associated with abnormalities in Ca2+homeostasis and thiol status, we examined the thiol group dependence and the effects of oxidant species on this enzyme. Sarcolemmal membranes isolated from rat heart were exposed to several types of thiol group modifiers. Alkylation withN-ethylmaleimide or methyl methanethiosulfonate, mercaptide formation withp-chloromercuriphenylsulfonic acid, and thiol-disulfide exchange with 5,5'-dithio-bis(2-nitrobenzoate) depressed sarcolemmal PLD activity; in all cases the depression was prevented by dithiothreitol. At different concentrations ofN-ethylmaleimide the PLD depression correlated well (r=0.98) with the decrease in total thiol group content of the membrane. The enzyme activity was not affected by xanthine-xanthine oxidase, a superoxide anion-generating system, but was depressed by hydrogen peroxide (H2O2) in a concentration-dependent manner. This inhibitory effect was prevented by catalase as well as by dithiothreitol, but not by d-mannitol. The effect of a hydroxyl radical-generating system (Fenton reaction) could not be assessed because of an interfering direct inhibition by Fe2+. Dithiothreitol was also able to restore PLD activity in H2O2-pretreated membranes and to prevent a severe deactivation of the enzyme by hypochlorous acid (HOCl). Protection by glutathione and inhibition by its oxidized form were also observed. The results indicate that sarcolemmal PLD activity is inhibited by nonradical oxidants H2O2and HOCl through reversible modification of associated thiol groups, which are critical for the enzyme activity. Thus, this enzyme may be controlled by the glutathione redox status of the cardiac cell. The possible relevance for ischemia/reperfusion injury is considered.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

The increased capillary fluid filtration required to rapidly create edema in acute inflammation can be generated by increased negativity of the interstitial fluid pressure (Pif). This observation suggests that connective tissues can “actively” enhance capillary fluid filtration. We now show that in vivo blockade of β1-integrin adhesion receptors in rat skin causes local edema concomitant with increased negativity of Pif. Experiments were performed on the dorsal side of the hind paw, and Pifwas measured with sharpened glass capillaries (tip diameter, 3–7 μm) connected to a servo-controlled counterpressure system. Measurements were made after circulatory arrest had been induced with intracardiac potassium chloride in pentobarbital anesthesia. This procedure prevents the vascular phenomena of increased fluid and protein flux leading to edema formation, which in turn can increase Pifand therefore potentially mask an increased negativity of Pif. Control Pifaveraged −0.58±0.81 (mean±SD) mm Hg (n=37). Subdermal injection of 5 μl monospecific rabbit anti-rat integrin β1-subunit immunoglobulin G caused increased negativity of Pifto average values between −4 and −6 mm Hg within 10 minutes after injection. Subdermal injection of 0.9% oNaCl, preimmune immunoglobulin G, rat anti-fibronectin, and peptides with Arg-Gly-Asp and Arg-Gly-Glu sequences did not change Pifsignificantly. In another series of experiments, 5 μl anti-β1integrin immunoglobulin G was injected subdermally in rats with intact circulation and resulted in an increase in total tissue water corresponding to a doubling of the interstitial fluid volume in 10 minutes (p<0.05). Perturbation of β1-integrin function with an increased negativity of Pifdemonstrates a fundamentally new mechanism to control Pifand thereby transcapillary fluid transport. These findings demonstrate for the first time a novel and dynamic in vivo function of β1-integrins.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

The results of work by several investigators indicate that crossbridge attachment serves as a positive feedback mechanism that transiently increases the Ca2+affinity of troponin C (TnC) during each normal heartbeat. To monitor structural changes in the cardiac isoform of TnC (cTnC) associated with Ca2+binding and crossbridge attachment in muscle, we labeled cTnC with the sulfhydryl-specific fluorescent probe 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid (IAANS). When IAANS-labeled cTnC (cTnCIAANS) was substituted for endogenous TnC, the fluorescence intensity of cardiac and skeletal muscle preparations increased substantially during rigor crossbridge attachment in the absence of Ca2+(pCa 9.2). In cardiac muscle, the fluorescence signal increased the same amount in rigor and maximal activation, whereas in skeletal muscle, it was higher in rigor (rigor: cardiac and skeletal=1; pCa 4.0: cardiac=0.98±0.13, skeletal=0.59±0.05). This indicates that crossbridge attachment alone is capable of influencing the structure of cTnCIAANS. Because the relative fluorescence intensity of cTnCIAANSwas more sensitive to Ca2+than was force in both preparations (cardiac: pCasofluorescence=6.05±0.05, pCa50force=5.51±0.11; skeletal: pCa50fluorescence=5.94±0.13, pCaso force=5.65±0.14), we measured the Ca2+sensitivity of the strong crossbridge attachment (sinusoidal stiffness was measured by imposing 1 kHz at 0.1–0.2% muscle length) in rat trabeculae. Like fluorescence, stiffness was also more sensitive to Ca2+than force was (pCa50stiffness=5.49±0.05, pCasoforce=5.41±0.05,p<.0.05), and this suggests that strong attachment of cycling crossbridges in zero- or low-force states may also influence the conformation of cTnC. The results from a computer model of crossbridge attachment also support this interpretation.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Hypoxia is a potent coronary-vasodilating signal; its mechanisms are still controversial. We have assessed the possible role of nitric oxide (NO) in hypoxic coronary vasodilatation (HCVD) in isolated guinea pig hearts perfused at constant pressure. HCVD was elicited by a 1-minute 100% N2exposure; coronary flow doubled within 1 minute of hypoxia (early phase) and returned to baseline within 40 seconds after reoxygenation (late phase). The early phase of HCVD was associated with a rapid approximately eightfold increase in cGMP overflow, an indication of NO release. The specific NO synthase inhibitorNω-methyl-L-arginine (NMA, 0.1–1 mM) antagonized HCVD and the associated increase in cGMP spillover (maximum inhibition, ≈65%); excess arginine (1.2 mM) prevented both effects. The late phase of HCVD was associated with an increase in adenosine overflow and was attenuated by the adenosine receptor antagonist BW A1433 (1 μM; maximum inhibition, ≈45%). Indomethacin (10 μM) inhibited HCVD in spontaneously beating hearts by ≈35% but had no effect in hearts paced at faster rates. NMA and BW A1433 were more effective in combination than alone (maximum inhibition, ≈72%). However, irrespective of the concentrations used, there was no synergism among the anti-HCVD effects of NMA, BW A1433, and indomethacin, nor was HCVD completely inhibited by the antagonists, whether alone or in combination. Our findings indicate that NO is an important mediator of the early phase of HCVD, whereas additional mechanisms and/or factors, including adenosine and vasodilatatory prostaglandins, contribute to the late phase.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Cyclic AMP (cAMP)-dependent chloride channels modulate changes in resting membrane potential and action potential duration in response to autonomic stimulation in heart. A growing body of evidence suggests that there are marked similarities in the properties of the cAMP-dependent chloride channels in heart and cystic fibrosis transmembrane regulator (CFTR) chloride channels found in airway epithelia or in cells expressing the CFTR gene product. We isolated poly A+mRNA from rabbit ventricle and converted it to cDNA for amplification using the polymerase chain reaction (PCR). A fragment corresponding to the nucleotide-binding domain 1 (NBD1) of the CFTR transcript was cloned. Comparison of the amino acid sequence of NBD1 of human CFTR with the deduced sequence of the rabbit heart PCR product indicated 98% identity. Northern blot analysis, using the heart amplification product as a cDNA probe, demonstrated expression of homologous transcripts in human atrium, guinea pig and rabbit ventricle, and dog pancreas.Xenopusoocytes injected with poly A+mRNA extracted from rabbit and guinea pig ventricle or dog pancreas expressed robust time-independent chloride currents in response to an elevation of cAMP. We conclude that CFTR chloride channels are expressed in heart and are responsible for the observed cAMP-dependent chloride conductance.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

To determine whether membrane free cholesterol affects calcium currents in vascular smooth muscle cells, whole-cell patch clamp recordings were made before and after cholesterol enrichment of cells by exposure to cholesterol-rich liposomes. Exposure to cholesterol-rich liposomes resulted in a gradual increase in the L-type current over 20 hours and a plateau (73±7% increase over basal) between 20 and 32 hours. This effect was associated with a rightward shift in the inactivation potential and a decrease in the sensitivity to (—)-PN-202-791, a dihydropyridine antagonist. There was no change in the maximum L-type current stimulated by (+)-PN-202-791, a dihydropyridine agonist. Liposome exposure caused a small, transient increase in the T-type current (peak effect, 20 minutes). We conclude that membrane cholesterol has important effects on the L-type calcium current in vascular smooth muscle cells, which is most likely due to an alteration in channel functional state rather than an increase in channel expression.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Thrombin cleaves its receptor at arginine-41, resulting in the generation of a new receptor NH2-terminus with the sequence SFLLRNPNDKYEPF. This peptide (TRP-14) may signal a variety of thrombin's responses. We examined the effects of TRP-14 in inducing endothelial cell hyperadhesivity and neutrophil (PMN) adhesion to endothelial cell monolayers. Human umbilical vein endothelial cells (HUVECs) challenged with TRP-14 (10−4to 10−5M) produced concentration-dependent increases in endothelial adhesivity to PMN. In contrast, position 1 to 2 inverted peptide (FSLLRNPNDKYEPF) did not induce the response. The adhesion response was transient; that is, PMN adhesion increased within 15 minutes and decreased by 75 minutes after TRP-14 challenge of HUVECs. The transient endothelial adhesiveness paralleled the time course of P-selectin expression. TRP-14-induced release of P-selectin from intracel-lular stores may be a critical determinant of the response since treatment of endothelial cells with anti-P-selectin monoclonal antibody (mAb) G1 prevented the increase in PMN adhesion. Control nonneutralizing anti-P-selectin mAb S12 and mAb RR1/1 directed against intercellular adhesion molecule-1 (ICAM-1) on HUVECs were ineffective. The results indicate that the “tethered ligand” of the thrombin receptor created by the proteolytic action of thrombin on its receptor (i.e., TRP-14) signals increased endothelial adhesiveness by a P-selectin-dependent mechanism. Thrombin-induced PMN adhesion may involve formation of a new NH2-terminus of the endothelial thrombin receptor with the sequence SFLLRNPNDKYEPF followed by activation of endothelial second messenger pathways and the transient expression of P-selectin.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID