Protective effect of benidipine against the abnormal electrical activity in single ventricular myocytes of the guinea pig under simulated ischemic conditions and reperfusion

Induction of electrical abnormalities (EAs) under simulated ischemic conditions and after reperfusion was measured from single cardiac myocytes isolated from guinea pig ventricle using whole-cell voltage or current clamp with perforated patch variation. Conditions of simulated ischemia were produced by the exchange of medium from the standard one oxygenated with 95% O2-5% CO2 gas (pH 7.4) to the modified one, which contained no glucose, 8 mM K+ and 30 mM sodium-D,L-lactate and was gassed with 90% argon-10% CO2 (pH 6.6). Under the simulated ischemia for 20 min, EAs such as delayed afterdepolarization, early afterdepolarization, automatic activity or transient inward current were observed in about 37% of myocytes driven electrically at 1 Hz. Irreversible hypercontracture occurred in myocytes of 10% or less. Upon reperfusion with the standard solution, EAs and hypercontracture were observed in about 43% and 22% of cells, respectively. Glibenclamide-sensitive current was detected during ischemia, but tended to be enhanced during reperfusion. Amplitude of Ca2+ current and ATP-sensitive K+ current after reperfusion varied widely with time and from cell to cell. When myocytes were pretreated for 10 min with 10 nM benidipine, a 1,4-dihydropyridine derivative Ca2+ blocker, the incidence of EAs and hypercontracture was markedly reduced, suggesting the protective effect of benidipine against cardiac cell injury during ischemia and reperfusion.     

Preconditioning prevents the negative inotropic action of phenylephrine in rat isolated stunned papillary muscle.

The aim of the present study was to establish a model of ischemic preconditioning of rat isolated papillary muscle and to investigate its effect on the simulated ischemia-induced disturbances in contractility and responsiveness to isoproterenol and phenylephrine. Experiments were performed in rat left ventricle papillary muscle. The following parameters were measured: force of contraction (Fc), velocity of contraction (+dF/dt), velocity of relaxation (-dF/dt), time to peak contraction (ttp), and relaxation time at the level of 10% of total amplitude (tt10). After 60 min of simulated ischemia induced by the perfusion of isolated tissues with no-substrate solution aerated by 95% N2/5% CO2, all of the measured parameters were markedly decreased. There was not complete recovery of Fc, +dF/dt and -dF/dt after 60 min of reperfusion. Positive inotropic action of isoproterenol does not differ before and after simulated ischemia. In contrast, phenylephrine induces a positive inotropic action in non-treated, but a significant negative one in simulated ischaemia/reperfusion treated preparations. The latter effect of phenylephrine was reversed by chloroethylclonidine (CEC), a selective blocker of alpha1b-adrenoceptor, but not by WB-4101, a selective blocker of alpha1a-adrenoceptor. Ischemic preconditioning of rat isolated cardiac tissue induced by the 5 min perfusion with no-substrate solution, aerated by 95% N2/ 5% CO2, in the presence of fast electrical pacing (BCL shortened from 2000 ms to 700 ms) and 10 min reperfusion, significantly improves a recovery of the contractility and prevents phenylephrine negative inotropic action.     

Effects of trimetazidine on pHi regulation in the rat isolated ventricular myocyte.

1. We have examined the effects of trimetazidine (TMZ) on intracellular pH (pHi) regulation in rat isolated ventricular myocytes. pHi was recorded ratiometrically by use of the pH-sensitive fluoroprobe, carboxy-SNARF-1 (carboxy-seminaphtorhodafluor). 2. Following an intracellular acid load (induced by 10 mM NH4Cl removal), pHi recovery in HEPES-buffered Tyrode solution was significantly slowed down upon application of 0.3 mM TMZ only when myocytes were pretreated for 5 h 30 min (slowing by approximately 50%; P < 0.01). This effect of TMZ on pHi recovery was shown to be not only time- but also dose-dependent with a large, quickly reversible, effect obtained with 1 mM TMZ applied for 2-3 h (slowing by approximately 64%; P < 0.001). This slowing of pHi recovery was also associated with a decrease of the NH4+ removal-induced acidification. 3. Relationship between intracellular intrinsic buffering power (beta i) and pHi was assessed in absence or presence of TMZ (0.3 mM or 1 mM). As expected, beta i increased roughly linearly with a decrease in pHi in all cases. However, both concentrations of TMZ significantly increased beta i (by approximately 55 and 65% at pHi 7.1, respectively). 4. When Na+/H+ exchange was inhibited by dimethyl amiloride (DMA; 40 microM), trimetazidine (1 mM) did not change the H+ flux estimated at pHi 7.1 (0.31 +/- 0.03 mequiv l-1 min-1, n = 5, control, versus 0.30 +/- 0.025 mequiv l-1 min-1, n = 5, TMZ), ruling out any effect of TMZ on background acid loading. 5. Acid efflux carried by Na+/H+ exchange was significantly decreased only when myocytes were pretreated with 1 mM TMZ, for 2-3 h (JeH = 2.86 +/- 0.38 mequiv l-1 min-1, n = 26, control, versus 1.66 +/- 0.26 mequiv l-1 min-1, n = 10, TMZ, estimated at pHi 7.1; P < 0.05). 6. In conclusion, the present work demonstrates that, following an intracellular acid load in HEPES-buffered medium, trimetazidine slows down pHi recovery in rat isolated ventricular myocytes, primarily through an increase of beta i. An effect on Na+/H+ exchange is also detected but only after long-term incubation of the myocytes with TMZ.     

Protective effect of SM-20550, a selective Na+ - H+ exchange inhibitor, on ischemia-reperfusion-injured hearts

The protective effects of Na+ - H+ exchange inhibitors SM-20550 (SM) and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) against ischemia-reperfusion injury were investigated in guinea pig Langendorff hearts. The changes in intracellular pH (pHi), high-energy phosphates, and biologic intracellular active ions ([Na+]i and [Ca2+]i) were regarded using the 31P-NMR and specific fluorescent signals from the heart tissues together with simultaneous recordings of the left ventricular developed pressure (LVDP). The recovery rate of LVDP from ischemia (40 min) by reperfusion was 36.8% in the control experiments, whereas in the presence of SM 10(-7) M, a gradual increase to 75.9% (55.5% with 10(-8) M), in contrast to EIPA (10(-7) M), 47.5% was observed. SM 10(-7) M restored the ATP level by 70% in 40-min reperfusion, which was already higher than the control in the latter half (20-40 min) of the ischemic period. The recovery rate of phosphocreatine by pretreatment of the heart with SM 10(-7) M was 75% in 40 min reperfusion. The pHi estimated from Pi/phosphocreatine chemical shift became highly acidic in ischemic heart so that SM 10(-7) M caused slight but significant pHi reduction from control pHi of 5.89 to 5.75. The level returned to pHi at around 7.38 during 30-40 min reperfusion, and the recovery was significantly greater than the control pHi of 7.24. The fura-2 Ca2+ or SBFI-Na+ signals during Langendorff ischemia heart increased, and rapidly returned to the control level after the reperfusion. SM suppressed the [Na+]i or [Ca2+]i elevation induced in the late stage during ischemia, resulting in LVDP restoration after reperfusion; Diastolic Ca2+ in the end period of ischemia, SM 10(-7) M 194% versus drug-free 220.7%. Na+: SM 10(-7) M 121.6% versus drug-free 128.0%. The present results suggest that the selective Na+ - H+ exchange inhibitor SM is promising as a potent and specific protective agent against ischemia-reperfusion injuries with Ca2+ overload induced via Na+ - H+, Na+ - Ca2+ exchange.     

Effect of inosine on function and adenine nucleotide content of the isolated working rat heart: studies of postischemic reperfusion.

Inosine added to the perfusion medium for isolated working rat heart induced a dose-dependent coronary vasodilator and positive functional effect. Inosine increased coronary flow (ml/min) from 11.8 +/- 0.5 (control, n = 16) to 15.6 +/- 0.6 (0.1 mM, n = 10), to 18.2 +/- 1.1 (0.5 mM, n = 6), and to 18.4 +/- 0.8 (1.0 mM, n = 14). Left ventricular systolic pressure was increased (LVSP, mm Hg) from 72.5 +/- 1.1 (control) to 75.4 +/- 1.7 (0.1 mM) to 78.7 +/- 2.1 (0.5 mM) and to 82.6 +/- 1.5 (1.0 mM). and cardiac output (CO, ml/min) from 26.4 +/- 2.0 (control) to 29.6 +/- 2.3 (0.1 mM), to 30.2 +/- 3.5 (0.5 mM), and to 37.4 +/- 2.0 (1.0 mM). At the end of 30-min reperfusion after a 15-min period of global total ischemia (n = 9), the functional parameters were significantly reduced (coronary flow 8.5 +/- 0.4 ml/min, LVSP 63 +/- 1.9 mm Hg, CO 13.6 +/- 1.9 ml/min). When inosine was present in the perfusion medium during the entire experimental protocol, coronary flow and heart function were also enhanced in a dose-dependent manner. The content of cardiac ATP (mumol/g) was decreased at the end of the 15-min ischemic period (1.89 +/- 0.17, n = 7; control 3.85 +/- 0.05, n = 4), and inosine had no effect. At the end of the 30-min postischemic reperfusion period, the ATP pool had recovered to an appreciable extent (3.01 +/- 0.11, n = 9) and was higher when 1.0 mM inosine had been infused (3.44 +/- 0.11, n = 7). Thus, inosine increased coronary flow dose dependently and, as a consequence, the function of the isolated perfused working heart both under control conditions and during the postischemic reperfusion period.     

Verapamil和Mn~（2＋）对缺氧及缺氧复氧心肌细胞内pH的影响

采用荧光探针ＢＣＥＣＦ／ＡＭ结合计算机图像处理技术测定不同时间的缺氧和缺氧复氧单心肌细胞内ｐＨ的变化以及Ｃａ２＋通道阻滞剂Ｖｅｒａｐａｍｉｌ和Ｎａ＋－Ｃａ２＋交换抑制剂Ｍｎ２＋对其的影响。结果显示随着缺氧时间的延长，细胞内ｐＨ也逐渐降低。复氧开始４０ｍｉｎ内，细胞内ｐＨ并未恢复正常。Ｖｅｒａｐａｍｉｌ能减轻缺氧细胞内酸化程度并使其接近正常水平（Ｐ＞０．０５），却未能减轻缺氧复氧细胞内的酸化。无论是缺氧或缺氧复氧心肌细胞，Ｍｎ２＋均未能减轻细胞内的酸化程度。本实验结果提示缺氧和缺氧复氧时细胞内酸化途径并非完全一致。ＶｅｒａＰａｍｉｌ抑制缺氧细胞内ｐＨ下降是其保护缺氧心肌作用的机制之一。     

腺苷对模拟缺血再灌注豚鼠心室肌细胞动作电位的影响及其离子机制

目的　研究腺苷 (Ado)在模拟缺血缺氧时对豚鼠心室肌细胞动作电位 (AP)、L 型钙电流 (ICa.L)和ATP敏感性钾电流 (IK .ATP)的影响。方法　在离体豚鼠心室肌和酶解法分离的单个豚鼠心室肌细胞上 ,分别应用细胞内微电极和全细胞膜片钳技术记录动作电位和电流。结果　在模拟缺血缺氧状态下 ,Ado剂量依赖性的增大动作电位时程 (APD)的缩短 (P <0 0 5 ) ;抑制再灌注后APD恢复 ,而表现出迟后恢复。在缺血缺氧状态下 ,ICa.L受到抑制 ,Ado并不加重心肌细胞缺血缺氧时ICa.L的减小 ,而再灌注后ICa .L的恢复比较缓慢 ,但同对照组比较无差异。Ado可加速缺血缺氧时IK .ATP的激活 ,Ado(10 0 μmol·L-1)组在缺血缺氧时和再灌注后IK .ATP较对照组均明显增大。结论　在缺血缺氧状态下 ,Ado可增大APD的缩短 ,抑制再灌注后APD的恢复 ,其机制主要在于在缺血缺氧状态下Ado增大了IK .ATP。     

Experimental hyperlipidemia prevents the protective effect of ischemic preconditioning on the contractility and responsiveness to phenylephrine of rat-isolated stunned papillary muscle.

This study was designed to establish a hyperlipidemic diet (significant increase in the cholesterol and triglycerides blood levels, but without atherogenic changes in heart muscle and coronary vessels) and to investigate the influence of experimental hyperlipidemia on the effects of ischemic preconditioning (PC) of rat-isolated papillary muscle on the time course of contractility during simulated ischemia and reperfusion and responsiveness to phenylephrine under such a condition. The animals were divided in four experimental groups: standard diet-fed control group (SD), SD underwent ischemic preconditioning (SD-PC), hyperlipidemic diet-fed group (HLD) and HLD underOFFt PC (HLD-PC). Force of contraction (Fc), velocity of contraction (+dF/dt), and velocity of relaxation (-dF/dt) were measured. HLD preparations were more sensitive to ischemia then SD ones. PC, performed by 5-min perfusion with no-substrate solution gassing with 95% N2/5% CO2 in the presence of fast electrical stimulation, and 10 min of reperfusion with normal solution and rate of stimulation, significantly increased the resistance of isolated cardiac tissues to simulated ischemia in SD-PC group, but not in HLD-PC group. Negative inotropic action of phenylephrine occured in SD group of preparations after simulated-ischemia/reperfusion period was also prevented by PC. Therefore, we conclude that experimental hyperlipidemia significantly influenced the function of rat heart muscle including the higher sensitivity to ischemia and different reaction to the same PC procedure.     

Energy preserving effect of l-cis diltiazem in isolated ischemic and reperfused guinea pig hearts: a 31P-NMR study

We determined the effect of 1-cis diltiazem, the enantiomer of diltiazem (d-cis isoform), on the energy metabolism of isolated guinea pig hearts during ischemia-reperfusion. We used 31P-NMR to measure the high-energy phosphate content and intracellular pH (pHi) during global ischemia for 30 min followed by reperfusion for 30 min. Before ischemia, the left ventricular developed pressure (LVDP) was reduced less by 10 microM l-cis diltiazem than by 3 microM diltiazem or 500 nM nifedipine. However, 10 microM l-cis diltiazem preserved the intracellular ATP content during ischemia and reperfusion, reduced the end-diastolic pressure increase during ischemia and reperfusion, and restored LVDP after reperfusion. Nifedipine at 50 nM, which reduced the LVDP more than 10 microM l-cis diltiazem, showed no cardioprotective effect. Ten micromolar l-cis diltiazem and 3 microM diltiazem, but neither 50 nor 500 nM nifedipine, reduced the pHi decrease that occurred 25 or 30 min after the onset of ischemia. Therefore, l-cis diltiazem has a cardioprotective effect on ischemic and reperfused myocardium and is less cardiodepressive than diltiazem and nifedipine. The effect of l-cis diltiazem during ischemia and reperfusion involves energy preservation, which is probably independent of its Ca2+-channel blocking action.     

Chrysanthemum morifolium attenuated the reduction of contraction of isolated rat heart and cardiomyocytes induced by ischemia/reperfusion

The present study was aimed to investigate the effect of Chrysanthemum morifolium Ramat. (CM) on isolated rat heart and ventricular myocytes during ischemia/anoxia and reperfusion/reoxygenation. The ischemia/reperfusion injury was induced by ligation the left artery descending coronary of isolated rat heart for 30 min followed by 30 min reperfusion with Langendorff equipment. Cell contraction in enzymatically isolated ventricular myocytes was determined by a video tracking system. The results showed CM (0.25 g/L to 1.0 g/L) increased left ventricular developed pressure (LVDP), +/- dp/dt(max), LVDP x HR and coronary flow (CF) and decreased heart rate (HR) in dose dependent manner. CM (0.5 g/L) attenuated the reduction of LVDP, +/- dp/dt(max) and CF caused by ischemia/reperfusion. CM (0.25 g/L to 1.0 g/L) increased peak velocity of cell shortening/relengthening (+/- dL/dt(max)) and contraction amplitude (dL) of isolated ventricular myocytes in a dose-dependent way under control condition, but without significant effect on end-diastolic cell length (L0). Under anoxia 5 min followed by 10 min reoxygenation, CM attenuated the reduction in contractile parameters. The results suggest that CM processes cardioprotective effect during ischemia/anoxia and reperfusion/reoxygenation in the isolated rat heart and the ventricular myocytes.     

The effect of amrinone on LDH release and perfusion pressure in isolated ischemic rabbit hearts

Isolated rabbit hearts were perfused normoxically or ischemically using the technique of Langendorff. The hearts were perfused with a modified Krebs-Henseleit solution, perturbated with 95% O2/5% CO2 or in case of ischemia with N2 which replaced the O2. The perfusion rate was 25 ml/min under normoxic and 2.5 ml/min under ischemic conditions. The oxygen pressure was about 65 kPa in the normoxic and about 6 kPa in the ischemic medium. Reperfusion of ischemic hearts was realized in some cases normoxically. During the ischemic perfusion and reperfusion LDH was released time depended into the perfusion medium and the aortic inflow pressure increased. The LDH release and the increase of the inflow pressure are strong correlated. Both parameters direct to ischemic myocardial lesions. Amrinone applied as bolus in the start period of ischemia or as an infusion during the ischemic and reperfusion time limited the release of LDH and the increase of the inflow pressure. The results were discussed as a direct protecting effect of amrinone against a myocardial ischemic lesion, which is in correspondence with the improving effects of amrinone on the ischemia caused heart failure of canines in vivo, shown by Campbell et al.     

触发活性在实验性心肌“缺血”和再灌注心律失常中的作用

离体豚鼠心室肌模拟“缺血”和再灌注的细胞电生理实验表明:(1)迟后除极电位(DAD)在心肌“缺血”时的发生率仅为16.7%,再灌注时增加到50%。如“缺血”台氏液中加入异丙肾素或增加钙含量,对缺血时的DAD 发生率无影响,再灌注时则分别增加到72.3%和57.1%,并见DAD 引发的持续触发活性;(2)早后除极电位(EAD)在心肌“缺血”时的发生率为8.3%,再灌注时为16.7%,且不受“缺血”台氏液中异丙肾素或高钙的影响,均未引发触发活性。     

Opening the calcium-activated potassium channel participates in the cardioprotective effect of puerarin

The aim of the present study was to determine whether the effective cardioprotection conferred by puerarin against ischemia and reperfusion is mediated by the calcium-activated potassium channel. Hearts isolated from male Sprague-Dawley rats were perfused on a Langendorff apparatus and subjected to 30 min of global ischemia followed by 120 min of reperfusion. The production of formazan, which provides an index of myocardial viability, was measured by absorbance at 550 nm, and the level of lactate dehydrogenase (LDH) in the coronary effluent was determined. Pretreatment with puerarin at 0.24 mmol/l for 5 min before ischemia increased myocardial formazan content and reduced LDH release during reperfusion. Administration of paxilline (1 micromol/l), an antagonist of the calcium-activated potassium channel, attenuated the protective effects of puerarin. In isolated ventricular myocytes, pretreatment with puerarin prevented simulated ischemia and reperfusion injury, hydrogen peroxide-induced cell death and the release of reactive oxygen species. Paxilline and chelerythrine (a protein kinase C inhibitor) both attenuated the effects of puerarin. These findings indicate that puerarin protects the myocardium against ischemia and reperfusion injury via opening the calcium-activated potassium channel and activating protein kinase C.     

Protection of the reperfused ischemic isolated rat heart by phosphatidylcholine.

We examined phosphatidylcholine (PC) effects on the isolated rat heart subjected to low- or zero-flow ischemia followed by reperfusion. Untreated hearts subjected to 30 min of low-flow ischemia recovered 15% contractility following reperfusion compared to time-control hearts. Phosphatidylcholine (0.005%) addition either 10 or 20 min before ischemia significantly enhanced recovery to approximately 61% and reduced the incidence of arrhythmias during ischemia and reperfusion. Contracture during ischemia and reperfusion was significantly reduced when PC was added 20 min before ischemia. Phosphatidylcholine was ineffective when administered at the time of reperfusion except for a moderate reduction in arrhythmia development. Phosphatidylcholine also produced a salutary effect when added 20 min prior to zero-flow ischemia. Subsarcolemmal mitochondria (SLM) and, to a much lesser degree, interfibrillar mitochondria (IFM) of untreated hearts subjected to low-flow ischemia and reperfusion exhibited depressed oxidative phosphorylation which was prevented by PC. Both mitochondrial populations exhibited a marked depression in ADP/ATP translocase activity; however, this was generally unaffected by PC. Subsarcolemmal mitochondria but not IFM of zero-flow ischemic reperfused hearts also exhibited significantly depressed oxidative phosphorylation, which was unaffected by PC. Zero-flow ischemia produced a rapid and total cessation of contractility. Both populations exhibited a substantial PC-insensitive reduction in translocase activity. Our results demonstrate, for the first time, a protection by PC on the reperfused ischemic heart. The PC-induced protection following low-flow but not zero-flow ischemia is associated with improved SLM oxidative phosphorylation suggesting dissimilar contribution of mitochondria to reperfusion-associated myocardial injury.     

ACE-inhibition attenuates cardiac cell damage and preserves release of NO in the postischemic heart

Cardioprotective effects of angiotensin-converting enzyme (ACE) inhibition have been demonstrated in postischemic reperfusion. This occurred via bradykinin and indirect evidence suggested mediation by nitric oxide (NO), which probably acts as a radical scavenger. To test this hypothesis, we measured release of lactate dehydrogenase (LDH) from isolated guinea pig hearts (constant flow perfusion, 37 degrees C) as a marker of cellular damage, before and after global ischemia (15 min), and we investigated the release of NO during reperfusion, both, without and with ACE inhibition. The main catabolites of NO, nitrate and nitrite, were also quantified. Coronary perfusion pressure (CPP) indicated coronary resistance changes. Cilazaprilat (CIL, 10 microM) was used for inhibition of ACE. Marked and protracted cellular damage occurred during reperfusion in the control group, myocardial LDH release rising nearly 10-fold from 1.5 mU/ml (basal level) to 14 mU/ml during acute reperfusion, then declining to 7 mU/ml after 5 min. ACE inhibition mitigated the acute rise of LDH (9 mU/ml), and reduced its release to preischemic values already after 3 min of reperfusion. Postischemic NO release in the 2nd min of reperfusion was about 40% of the preischemic value (approx. 200 nM) in untreated hearts, while there was 70% recovery after ACE inhibition. After 25 min, NO had recovered to 69% in controls vs. 100% with CIL. Coronary venous nitrate + nitrite was not infringed during early reperfusion (2nd min). After 25 min, nitrate + nitrite had decreased in controls (about 75% of preischemic values), but increased to 110% with CIL. In control hearts, CPP rose continuously from the 10th to the 25th min of reperfusion (from 39 to 55 mmHg), indicating progressive vasoconstriction. CIL significantly attenuated this effect. The results suggest that NO might be consumed during early reperfusion in the act of detoxifying radicals. In control hearts, "endothelial stunning" takes place. Concerning NO production and vasodilatory tone, ACE-inhibition augments postischemic NO release and mitigates disturbances caused by ischemia and reperfusion.     

Acidosis-induced p38 MAPK activation and its implication in regulation of cardiac contractility

AIM: To determine the possible role of pH in mediating activation of p38 mitogen-activated protein kinase (MAPK) and the consequent function of activated p38 MAPK in regulating cardiac contractility. METHODS: Adult rat cardiomyocytes were isolated and cultured. Low pH media was used to induce intracellular acidosis and contraction of single cardiomyocyte was measured. RESULTS: Phosphorylation of p38 MAPK was increased during ischemia, and pHi was decreased. Intracellular acidosis activated p38 MAPK to a similar level as ischemia. Inhibition of p38 MAPK activation by SB203580, a specific inhibitor of p38 MAPK, reversed acidosis-mediated reduction of myocyte contractility. CONCLUSION: In adult rat cardiomyocytes, intracellular acidification activated p38 MAPK and decreased cardiac contractility. Pretreatment of cardiomyocytes with SB203580 completely blocked p38 MAPK activation and partially reversed acidosis-mediated decline of cardiac contractility.     

Experimental conditions determine effects of ascorbic acid on reperfusion injury: comparison of tissue damage with hemodynamic parameters in rat isolated hearts.

Restoration of coronary blood flow after myocardial ischemia is always a matter of urgency, but the resulting surgical or drug-induced reperfusion of ischemic tissue is often associated with myocardial functional disturbances and tissue injury. The present study was carried out to select experimental conditions under which optimal effects of antioxidants can be observed on the adverse effects of reperfusion of ischemic myocardium. The release of lactate dehydrogenase (LDH) and changes in hemodynamic parameters were compared in two models of cardiac reperfusion injury in rat isolated hearts. LDH release from electrically-stimulated hearts perfused under constant flow and with initial (5 min) reperfusion in calcium-free buffer was greater than that from hearts perfused under constant pressure in which ischemia was induced by reduced flow. Combined SOD+catalase was a weak inhibitor of LDH release in both models, ascorbic acid being more potent under constant pressure than under constant flow conditions. A longer ischemic period enhanced the inhibitory effect of ascorbate. Contractility and ventricular end-diastolic pressure recovered slowly during perfusion under constant flow and brief calcium removal, but remained unphysiological under constant pressure. SOD+catalase had no effect on hemodynamic parameters. Ascorbic acid exacerbated ischemia+reperfusion-induced changes in contractility, ventricular pressure, heart rate and coronary flow under constant pressure, but facilitated recovery of contractility on reperfusion under constant flow and brief calcium removal. In studies on antioxidants, different experimental conditions appear to be necessary to observe beneficial effects on tissue damage on the one hand and on hemodynamics on the other. Mild to moderate ischemia, with sustained pacemaker activity, appears to be the condition under which antioxidants provide hemodynamic improvement. In isolated rat hearts, biochemical parameters of tissue damage may be misleading for the effects of antioxidants.     

Enhancement of intracellular Cl- concentrations induced by extracellular ATP in guinea pig ventricular muscle

We investigated effects of extracellular ATP on intracellular chloride activities ([Cl-]i) and possible contribution of the Cl--HCO3- exchange to this increase in [Cl-]i in isolated guinea pig ventricular muscles. The [Cl-]i and intracellular pH (pHi) were recorded in quiescent ventricular muscles using double-barreled ion-selective microelectrode techniques. MgATP at a concentration higher than 0.1 mM, induced an increase in [Cl-]i, and this increase in [Cl-]i was dependent on the concentration of ATP but not on the concentration of magnesium ions present in the perfusion solution. NaADP, but not NaAMP, at a concentration of 0.5 mM induced a similar increase in [Cl-]i as that induced by MgATP. However, the NaADP-induced increase in [Cl-]i was transient and gradually returned to the control level even though NaADP was continuously present. Furthermore, ATP also triggered a transient acidification of pHi, and both increases in [Cl-]i and intracellular H+ induced by ATP were prevented when preparations were pretreated with stilbene derivatives, SITS and DIDS, or perfused with a Cl--free solution. Our findings showed that the increased extracellular ATP concentrations might trigger an increase in [Cl-]i in ventricular muscles. In light of previous studies showing that cardiac ischemia induced increases in extracellular nucleotide concentrations and [Cl-]i in ventricular muscles, we propose that ischemia-induced accumulation of ATP concentration in the extracellular space may be an important factor to trigger increment of [Cl-]i during ischemic conditions.     

Interaction between cyclic GMP protein kinase and cyclic AMP may be diminished in stunned cardiac myocytes

We tested the hypothesis that the importance of the negative functional effects of the cyclic GMP protein kinase would be reduced in stunned (simulated ischemia/reperfusion) cardiac myocytes. Ventricular cardiac myocytes were isolated from New Zealand white rabbits (N=7). Myocytes were studied at baseline and after simulated ischemia (15 min of 95% N(2)-5% CO(2) at 37 degrees C) followed by simulated reperfusion (reoxygenation). Cell shortening was studied with a video edge detector; O(2) consumption was measured using O(2) electrodes. Protein phosphorylation was measured autoradiographically after gel electrophoresis. Functional and metabolic data were acquired after: (1) 8-(4-chlorophenylthio)guanosine-3',5'-monophosphate (PCPT, cGMP protein kinase agonist) 10(-7) or 10(-5) M; (2) 8-Br-cAMP 10(-5) M followed by PCPT 10(-7) or 10(-5) M; (3) beta-phenyl-1, N(2)-etheno-8-bromoguanosine-3',5'-monophosphorothioate, SP-isomer (SP, cGMP protein kinase agonist) 10(-7) or 10(-5) M; (2) 8-Br-cAMP 10(-5) M followed by SP 10(-7) or 10(-5) M. At baseline, percent of shortening (Pcs) and maximal rate of shortening (Rs) were significantly lower in the stunned myocytes (Pcs: 5.0+/-0.2% control vs. 3.8+/-0.3 stunned; Rs: 64.8+/-5.9 microm/s control vs. 46.9+/-4.8 stunned). In both groups, PCPT and SP dose-dependently decreased Pcs and Rs. The effects were slightly, but not significantly, less in stunned myocytes. 8-Br-cyclic AMP significantly increased function in control, but not stunned myocytes (Pcs, 4.5+/-0.5 to 6.2+/-0.8 control vs. 3.1+/-0.2 to 3.6+/-0.2 stunned). The negative functional effects of PCPT and SP were diminished after 8-Br-cyclic AMP in control (from -39% to-29%) and diminished significantly more in the stunned myocytes (-19%). PCPT and cyclic AMP phosphorylated similar protein bands. In stunned myocytes, three (22, 31 and 53 kDa) bands were enhanced less by PCPT.     

去甲肾上腺素和多巴酚丁胺联用与多巴胺单用对感染性休克绵羊内脏灌注的比较

AIM: To compare the effect of norepinephrine-dobutamine with dopamine alone on splanchnic perfusion in sheep with septic shock. METHODS: Twenty sheep with septic shock induced by lipopolysaccharides were divided into two groups. When systolic pressure decreased by 5.3 kPa, basic values of hemodynamic parameters and intestinal intramucosal pH (pHi) were recorded. Each group was randomized to receive an intravenous infusion of norepinephrine-dobutamine or dopamine, and titrated to obtain mean arterial pressure (MAP) > 12 kPa with an optimal cardiac preload. Hemodynamic parameters and mucosal pHi were repeated at 1, 2, 3, and 4 h after basic measurement. RESULTS: After norepinephrine-dobutamine or dopamine infusion, MAP, cardiac output, and oxygen delivery increased in all animals compared with basic values in both groups (P < 0.05). Compared with baseline values, lactate concentrations decreased at 3 h and 4 h [from (4 +/- 2) mmol/L to (2 +/- 1) mmol/L] in the norepinephrine-dobutamine group (P < 0.05 ). Arterial lactate concentrations had no change in dopamine group, but arterial pH decreased from 7.40 +/- 0.05 to 7.26 +/- 0.06 at 1 h (P < 0.05 ). No difference in pHi was found in dopamine group, but in the norepinephrine-dobutamine group, compared with baseline, pHi increased from 7.19 +/- 0.04 to 7.36 +/- 0.07 at 3 h (P < 0.05). CONCLUSION: Both norepinephrine-dobutamine and dopamine alone could improve systemic hemodynamics in sheep with septic shock, but norepinephrine-dobutamine was better than dopamine on splanchnic perfusion.