Ischemic and anesthetic preconditioning reduces cytosolic [Ca2+] and improves Ca(2+) responses in intact hearts

Ca(+) loading during reperfusion after myocardial ischemia is linked to reduced cardiac function. Like ischemic preconditioning (IPC), a volatile anesthetic given briefly before ischemia can reduce reperfusion injury. We determined whether IPC and sevoflurane preconditioning (SPC) before ischemia equivalently improve mechanical and metabolic function, reduce cytosolic Ca(2+) loading, and improve myocardial Ca(2+) responsiveness. Four groups of guinea pig isolated hearts were perfused: no ischemia, no treatment before 30-min global ischemia and 60-min reperfusion (control), IPC (two 2-min occlusions) before ischemia, and SPC (3.5 vol%, two 2-min exposures) before ischemia. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured at the left ventricular (LV) free wall with the fluorescent probe indo 1. Ca(2+) responsiveness was assessed by changing extracellular [Ca(2+)]. In control hearts, initial reperfusion increased diastolic [Ca(2+)] and diastolic LV pressure (LVP), and the maximal and minimal derivatives of LVP (dLVP/dt(max) and dLVP/dt(min), respectively), O(2) consumption, and cardiac efficiency (CE). Throughout reperfusion, IPC and SPC similarly reduced ischemic contracture, ventricular fibrillation, and enzyme release, attenuated rises in systolic and diastolic [Ca(2+)], improved contractile and relaxation indexes, O(2) consumption, and CE, and reduced infarct size. Diastolic [Ca(2+)] at 50% dLVP/dt(min) was right shifted by 32-53 +/- 8 nM after 30-min reperfusion for all groups. Phasic [Ca(2+)] at 50% dLVP/dt(max) was not altered in control but was left shifted by -235 +/- 40 nM [Ca(2+)] after IPC and by -135 +/- 20 nM [Ca(2+)] after SPC. Both SPC and IPC similarly reduce Ca(2+) loading, while augmenting contractile responsiveness to Ca(2+), improving postischemia cardiac function and attenuating permanent damage.     

Negative inotropic drugs alter indexes of cytosolic [Ca(2+)]-left ventricular pressure relationships after ischemia

Negative inotropic agents may differentially modulate indexes of cytosolic [Ca(2+)]-left ventricular (LV) pressure (LVP) relationships when given before and after ischemia. We measured and calculated [Ca(2+)], LVP, velocity ratios [[(d[Ca(2+)]/dt(max))/(dLVP/dt(max)); VR(max)] and [(d[Ca(2+)]/dt(min))/(dLVP/dt(min)); VR(min)]], and area ratio (AR; area [Ca(2+)]/area LVP per beat) before and after global ischemia in guinea pig isolated hearts. Ca(2+) transients were recorded by indo 1-AM fluorescence via a fiberoptic probe placed at the LV free wall. [Ca(2+)]-LVP loops were acquired by plotting LVP as a function of [Ca(2+)] at multiple time points during the cardiac cycle. Hearts were perfused with bimakalim, 2,3-butanedione monoxime (BDM), nifedipine, or lidocaine before and after 30 min of ischemia. Before ischemia, each drug depressed LVP, but only nifedipine decreased both LVP and [Ca(2+)] with a downward and leftward shift of the [Ca(2+)]-LVP loop. After ischemia, each drug depressed LVP and [Ca(2+)] with a downward and leftward shift of the [Ca(2+)]-LVP loop. Each drug except BDM decreased d[Ca(2+)]/dt(max); nifedipine decreased d[Ca(2+)]/dt(min), whereas lidocaine increased it, and bimakalim and BDM had no effect on d[Ca(2+)]/dt(min). Each drug except bimakalim increased VR(max) and VR(min) before ischemia; after ischemia, only BDM and nifedipine increased VR(max) and VR(min). Before and after ischemia, BDM and nifedipine increased AR, whereas lidocaine and bimakalim had no effect. At 30 min of reperfusion, control hearts exhibited marked Ca(2+) overload and depressed LVP. In each drug-pretreated group Ca(2+) overload was reduced on reperfusion, but only the group pretreated with nifedipine exhibited both higher LVP and lower [Ca(2+)]. These results show that negative inotropic drugs are less capable of reducing [Ca(2+)] after ischemia so that there is a relatively larger Ca(2+) expenditure for contraction/relaxation after ischemia than before ischemia. Moreover, the differential effects of pretreatment with negative inotropic drugs on [Ca(2+)]-LVP relationships after ischemia suggest that these drugs, especially nifedipine, can elicit cardiac preconditioning.     

Reduced mitochondrial Ca2+ loading and improved functional recovery after ischemia-reperfusion injury in old vs. young guinea pig hearts

Oxidative damage and impaired cytosolic Ca(2+) concentration ([Ca(2+)](cyto)) handling are associated with mitochondrial [Ca(2+)] ([Ca(2+)](mito)) overload and depressed functional recovery after cardiac ischemia-reperfusion (I/R) injury. We hypothesized that hearts from old guinea pigs would demonstrate impaired [Ca(2+)](mito) handling, poor functional recovery, and a more oxidized state after I/R injury compared with hearts from young guinea pigs. Hearts from young (～4 wk) and old (>52 wk) guinea pigs were isolated and perfused with Krebs-Ringer solution (2.1 mM Ca(2+) concentration at 37°C). Left ventricular pressure (LVP, mmHg) was measured with a balloon, and NADH, [Ca(2+)](mito) (nM), and [Ca(2+)](cyto) (nM) were measured by fluorescence with a fiber optic probe placed against the left ventricular free wall. After baseline (BL) measurements, hearts were subjected to 30 min global ischemia and 120 min reperfusion (REP). In old vs. young hearts we found: 1) percent infarct size was lower (27 ± 9 vs. 57 ± 2); 2) developed LVP (systolic-diastolic) was higher at 10 min (57 ± 11 vs. 29 ± 2) and 60 min (55 ± 10 vs. 32 ± 2) REP; 3) diastolic LVP was lower at 10 and 60 min REP (6 ± 3 vs. 29 ± 4 and 3 ± 3 vs. 21 ± 4 mmHg); 4) mean [Ca(2+)](cyto) was higher during ischemia (837 ± 39 vs. 541 ± 39), but [Ca(2+)](mito) was lower (545 ± 62 vs. 975 ± 38); 5) [Ca(2+)](mito) was lower at 10 and 60 min REP (129 ± 2 vs. 293 ± 23 and 122 ± 2 vs. 234 ± 15); 6) reduced inotropic responses to dopamine and digoxin; and 7) NADH was elevated during ischemia in both groups and lower than BL during REP. Contrary to our stated hypotheses, old hearts showed reduced [Ca(2+)](mito), decreased infarction, and improved basal mechanical function after I/R injury compared with young hearts; no differences were noted in redox state due to age. In this model, aging-associated protection may be linked to limited [Ca(2+)](mito) loading after I/R injury despite higher [Ca(2+)](cyto) load during ischemia in old vs. young hearts.     

Amiloride和Ni2+抑制缺血/复灌引起的大鼠心肌细胞内钙的增加

本文旨在研究Na+/H+交换以及Na+/Ca2 +交换对模拟缺血 /复灌引起的大鼠心肌细胞内游离钙水平变化的调节作用。分别利用模拟缺血液和正常台氏液对大鼠心肌细胞进行缺血 /复灌处理 ,在缺血期间分别应用Na+/H+交换抑制剂阿米洛利 (amiloride)、Na+/Ca2 +交换抑制剂NiCl2 以及无钙液 ,观察它们对细胞内游离Ca2 +浓度变化的影响。利用Zeiss LSM 5 10激光共聚焦显微镜检测、采集细胞内游离Ca2 +的指示剂Fluo 3 AM的荧光信号 ,计算出相对于正常(缺血前 )的相对荧光强度 ,以表示胞内游离Ca2 +浓度的变化。结果显示 ,模拟缺血引起大鼠心肌细胞内游离Ca2 +持续上升 ,缺血前的相对荧光强度值为 10 0 % ,模拟缺血 5min后为 140 3± 13 0 % (P <0 0 5 ) ,复灌 15min后为 142 8±15 5 % (P <0 0 5 )。经 10 0 μmol/Lamiloride、5mmol/LNiCl2 和无钙液分别预处理 ,模拟缺血 5min后的相对荧光强度分别为 10 1 4± 16 3 % (P <0 0 5 )、110 4± 11 1% (P <0 0 5 )和 10 7 1± 10 8(P <0 0 5 ) ;复灌 15min后则分别为 97 8±14 3 % (P <0 0 5 )、10 6 2± 14 5 % (P <0 0 5 )和 10 6 6± 15 7(P <0 0 5 )。另外 ,与对照组细胞相比 ,再灌注期间NiCl2和无钙液处理的细胞钙振荡的产生幅度明显减弱 ,amilorid     

Na(+)/Ca(2+) exchanger 2 is neuroprotective by exporting Ca(2+) during a transient focal cerebral ischemia in the mouse

Na(+)/Ca(2+) exchanger (NCX), by mediating Na(+) and Ca(2+) fluxes bi-directionally, assumes a role in controlling the Ca(2+) homeostasis in the ischemic brain. It has been suggested that the three isoforms of NCX (NCX1, 2 and 3) may be differentially involved in permanent cerebral ischemia. However, the role of NCX2 has not been defined in ischemic reperfusion injury after a transient focal cerebral ischemia. Furthermore, it is not known whether NCX2 imports or exports intracellular Ca(2+) ([Ca(2+)](i)) following ischemia and reperfusion. To define the role of NCX2 in ischemia and reperfusion, we examined mice lacking NCX2, in vivo and in vitro. After an in vitro ischemia, a significantly slower recovery in population spike amplitudes, a sustained elevation of [Ca(2+)](i) and an increased membrane depolarization were developed in the NCX2-deficient hippocampus. Moreover, a transient focal cerebral ischemia in vivo produced a larger infarction and more cell death in the NCX2-deficient mouse brain. In particular, in the wild type brain, NCX2-expressing neurons were largely spared from cell death after ischemia. Our results suggest that NCX2 exports Ca(2+) in ischemia and thus protects neuronal cells from death by reducing [Ca(2+)](i) in the adult mouse brain.     

哌替啶对心室肌收缩的抑制作用及其机制

为明确哌替啶对心脏收缩的直接效应 ,并探讨其相关机制。采用Langendorff灌流心脏模型 ,观察了哌替啶对大鼠心室收缩功能的影响 ,并用荧光测钙技术和膜片钳技术探讨了哌替啶作用的钙离子机制。结果显示 ,哌替啶剂量依赖性地降低离体灌流心脏的LVDP×HR、 +dP/dt和 -dP/dt,而升高LVEDP。在酶解分离的心室肌细胞上 ,哌替啶剂量依赖性地降低细胞收缩时的钙瞬变幅度 ,并升高舒张末期的钙水平。哌替啶不影响高浓度咖啡因诱导的内钙释放。哌替啶使L 型钙电流强度降低到给药前的 67 4± 10 1% ,而不改变钙通道的激活和失活电位。哌替啶减弱钙电流的作用并不能被阿片受体阻断剂纳洛酮所阻断。以上结果表明 ,哌替啶能通过非阿片受体介导的途径阻断细胞外钙离子的内流 ,对心室收缩产生直接的抑制作用     

Inflammation alters regional mitochondrial Ca²⁺ in human airway smooth muscle cells

Regulation of cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in airway smooth muscle (ASM) is a key aspect of airway contractility and can be modulated by inflammation. Mitochondria have tremendous potential for buffering [Ca(2+)](cyt), helping prevent Ca(2+) overload, and modulating other intracellular events. Here, compartmentalization of mitochondria to different cellular regions may subserve different roles. In the present study, we examined the role of Ca(2+) buffering by mitochondria and mitochondrial Ca(2+) transport mechanisms in the regulation of [Ca(2+)](cyt) in enzymatically dissociated human ASM cells upon exposure to the proinflammatory cytokines TNF-α and IL-13. Cells were loaded simultaneously with fluo-3 AM and rhod-2 AM, and [Ca(2+)](cyt) and mitochondrial Ca(2+) concentration ([Ca(2+)](mito)) were measured, respectively, using real-time two-color fluorescence microscopy in both the perinuclear and distal, perimembranous regions of cells. Histamine induced a rapid increase in both [Ca(2+)](cyt) and [Ca(2+)](mito), with a significant delay in the mitochondrial response. Inhibition of the mitochondrial Na(+)/Ca(2+) exchanger (1 μM CGP-37157) increased [Ca(2+)](mito) responses in perinuclear mitochondria but not distal mitochondria. Inhibition of the mitochondrial uniporter (1 μM Ru360) decreased [Ca(2+)](mito) responses in perinuclear and distal mitochondria. CGP-37157 and Ru360 significantly enhanced histamine-induced [Ca(2+)](cyt). TNF-α and IL-13 both increased [Ca(2+)](cyt), which was associated with decreased [Ca(2+)](mito) in the case of TNF-α but not IL-13. The effects of TNF-α on both [Ca(2+)](cyt) and [Ca(2+)](mito) were affected by CGP-37157 but not by Ru360. Overall, these data demonstrate that in human ASM cells, mitochondria buffer [Ca(2+)](cyt) after agonist stimulation and its enhancement by inflammation. The differential regulation of [Ca(2+)](mito) in different parts of ASM cells may serve to locally regulate Ca(2+) fluxes from intracellular sources versus the plasma membrane as well as respond to differential energy demands at these sites. We propose that such differential mitochondrial regulation, and its disruption, may play a role in airway hyperreactivity in diseases such as asthma, where [Ca(2+)](cyt) is increased.     

Cross-bridge kinetics modeled from myoplasmic [Ca2+] and LV pressure at 17 degrees C and after 37 degrees C and 17 degrees C ischemia

We modeled changes in contractile element kinetics derived from the cyclic relationship between myoplasmic [Ca(2+)], measured by indo 1 fluorescence, and left ventricular pressure (LVP). We estimated model rate constants of the Ca(2+) affinity for troponin C (TnC) on actin (A) filament (TnCA) and actin and myosin (M) cross-bridge (A x M) cycling in intact guinea pig hearts during baseline 37 degrees C perfusion and evaluated changes at 1) 20 min 17 degrees C pressure, 2) 30-min reperfusion (RP) after 30-min 37 degrees C global ischemia during 37 degrees C RP, and 3) 30-min RP after 240-min 17 degrees C global ischemia during 37 degrees C RP. At 17 degrees C perfusion versus 37 degrees C perfusion, the model predicted: A x M binding was less sensitive; A x M dissociation was slower; Ca(2+) was less likely to bind to TnCA with A x M present; and Ca(2+) and TnCA binding was less sensitive in the absence of A x M. Model results were consistent with a cold-induced fall in heart rate from 260 beats/min (37 degrees C) to 33 beats/min (17 degrees C), increased diastolic LVP, and increased phasic Ca(2+). On RP after 37 degrees C ischemia vs. 37 degrees C perfusion, the model predicted the following: A x M binding was less sensitive; A x M dissociation was slower; and Ca(2+) was less likely to bind to TnCA in the absence of A. M. Model results were consistent with reduced myofilament responsiveness to [Ca(2+)] and diastolic contracture on 37 degrees C RP. In contrast, after cold ischemia versus 37 degrees C perfusion, A x M association and dissociation rates, and Ca(2+) and TnCA association rates, returned to preischemic values, whereas the dissociation rate of Ca(2+) from A x M was ninefold faster. This cardiac muscle kinetic model predicted a better-restored relationship between Ca(2+) and cross-bridge function on RP after an eightfold longer period of 17 degrees C than 37 degrees C ischemia.     

Dynamics of matrix-free Ca2+ in cardiac mitochondria: two components of Ca2+ uptake and role of phosphate buffering

Mitochondrial Ca(2+) uptake is thought to provide an important signal to increase energy production to meet demand but, in excess, can also trigger cell death. The mechanisms defining the relationship between total Ca(2+) uptake, changes in mitochondrial matrix free Ca(2+), and the activation of the mitochondrial permeability transition pore (PTP) are not well understood. We quantitatively measure changes in [Ca(2+)](out) and [Ca(2+)](mito) during Ca(2+) uptake in isolated cardiac mitochondria and identify two components of Ca(2+) influx. [Ca(2+)](mito) recordings revealed that the first, MCU(mode1), required at least 1 μM Ru360 to be completely inhibited, and responded to small Ca(2+) additions in the range of 0.1 to 2 μM with rapid and large changes in [Ca(2+)](mito). The second component, MCU(mode2), was blocked by 100 nM Ru360 and was responsible for the bulk of total Ca(2+) uptake for large Ca(2+) additions in the range of 2 to 10 μM; however, it had little effect on steady-state [Ca(2+)](mito). MCU(mode1) mediates changes in [Ca(2+)](mito) of 10s of μM, even in the presence of 100 nM Ru360, indicating that there is a finite degree of Ca(2+) buffering in the matrix associated with this pathway. In contrast, the much higher Ca(2+) loads evoked by MCU(mode2) activate a secondary dynamic Ca(2+) buffering system consistent with calcium-phosphate complex formation. Increasing P(i) potentiated [Ca(2+)](mito) increases via MCU(mode1) but suppressed [Ca(2+)](mito) changes via MCU(mode2). The results suggest that the role of MCU(mode1) might be to modulate oxidative phosphorylation in response to intracellular Ca(2+) signaling, whereas MCU(mode2) and the dynamic high-capacity Ca(2+) buffering system constitute a Ca(2+) sink function. Interestingly, the trigger for PTP activation is unlikely to be [Ca(2+)](mito) itself but rather a downstream byproduct of total mitochondrial Ca(2+) loading.     

Separation of fluorescence signals from Ca2+ and NADH during cardioplegic arrest and cardiac ischemia

Determinations of intracellular [Ca(2+)](i) during ischemia using fluorescent indicators are hampered by overlapping cellular autofluorescence (AF), which largely depends on NADH. If Ca(2+) is to be determined under different kinds of ischemia, signal separation merits special attention. We used triple wavelength excitation fluorescence to separate autofluorescence from [Ca(2+)]-dependent fura-2 fluorescence. Excitation at 360 nm served as third, Ca(2+)-insensitive wavelength. Using an appropriate evaluation procedure, we separated Ca(2+)-dependent signals from autofluorescence which is semiquantitatively associated with NADH, an indicator of the cellular redox state. We compared changes of [Ca(2+)](i) in isolated hearts during ischemia following cardioplegic arrest with those after transient stop of nutritive perfusion. We observed [Ca(2+)] transients in spontaneously beating hearts, persisting during ischemic episodes, and an increase of mean [Ca(2+)](i). In contrast, cardioplegic arrest stopped periodical [Ca(2+)](i) transients and heart beats simultaneously. [Ca(2+)](i) remained at diastolic values, tended to decrease during the first minutes of cardioplegic arrest and then increased slowly. Autofluorescence increased under both conditions. During ischemia, this increase was faster than in cardioplegia experiments. It started after the last heart beat despite persisting perfusion. Our measurements demonstrate that rhythmical heart beat is essential for sufficient perfusion. Reduced [Ca(2+)](i) under cardioplegic arrest may influence metabolism.     

Sex-related effects on diabetes-induced alterations in calcium release in the rat heart

The present study was designed to determine whether the properties of local Ca(2+) release and its related regulatory mechanisms might provide insight into the role of sex differences in heart functions of control and streptozotocin-induced diabetic adult rats. Left ventricular developed pressure, the rates of pressure development and decay (+/-dP/dt), basal intracellular Ca(2+) level ([Ca(2+)](i)), and spatiotemporal parameters of [Ca(2+)](i) transients were found to be similar in male and female control rats. However, spatiotemporal parameters of Ca(2+) sparks in cardiomyocytes isolated from control females were significantly larger and slower than those in control males. Diabetes reduced left ventricular developed pressure to a lower extent in females than in males, and the diabetes-induced depressions in both +dP/dt and -dP/dt were less in females than in males. Diabetes elicited a smaller reduction in the amplitude of [Ca(2+)](i) transients in females than in males, a smaller reduction in sarcoplasmic reticulum-Ca(2+) load, and less increase in basal [Ca(2+)](i). Similarly, the elementary Ca(2+) events and their control proteins were clearly different in both sexes, and these differences were more marked in diabetes. Diabetes-induced depression of the Ca(2+) spark amplitude was significantly less in females than in matched males. Levels of cardiac ryanodine receptors (RyR2) and FK506-binding protein 12.6 in control females were significantly higher than those shown in control males. Diabetes induced less RyR2 phosphorylation and FK506-binding protein 12.6 unbinding in females. Moreover, total and free sulfhydryl groups were significantly less reduced, and PKC levels were less increased, in diabetic females than in diabetic males. The present data related to local Ca(2+) release and its related proteins describe some of the mechanisms that may underlie sex-related differences accounting for females to have less frequent development of cardiac diseases.     

Overexpression of Na+/Ca2+ exchanger gene attenuates postinfarction myocardial dysfunction

We monitored myocardial function in postinfarcted wild-type (WT) and transgenic (TG) mouse hearts with overexpression of the cardiac Na(+)/Ca(2+) exchanger. Five weeks after infarction, cardiac function was better maintained in TG than WT mice [left ventricular (LV) systolic pressure: WT, 41 +/- 2; TG, 58 +/- 3 mmHg; P < 0.05; maximum rising rate of LV pressure (+dP/dt(max)): WT, 3,750 +/- 346; TG, 5,075 +/- 334 mmHg/s; P < 0.05]. The isometric contractile response to beta-adrenergic stimulation was greater in papillary muscles from TG than WT mice (WT, 13.2 +/- 0.9; TG, 16.3 +/- 1.0 mN/mm(2) at 10(-4) M isoproterenol). The sarcoplasmic reticulum (SR) Ca(2+) content investigated by rapid cooling contractures in papillary muscles was greater in TG than WT mouse hearts. We conclude that myocardial function is better preserved in TG mice 5 wk after infarction, which results from enhanced SR Ca(2+) content via overexpression of the Na(+)/Ca(2+) exchanger.     

Critical role of sodium in cytosolic [Ca2+] elevations in cultured hippocampal CA1 neurons during anoxic depolarization

Although the extent of ischemic brain damage is directly proportional to the duration of anoxic depolarization (AD), the mechanism of cytosolic [Ca(2+)] ([Ca(2+)](c)) elevation during AD is poorly understood. To address the mechanism in this study, [Ca(2+)](c) was monitored in cultured rat hippocampal CA1 neurons loaded with a Ca-sensitive dye, fura-2FF, and exposed to an AD-simulating medium containing (in mmol/L): K(+) 65, Na(+) 50, Ca(2+) 0.13, glutamate 0.1, and pH reduced to 6.6. Application of this medium promptly elevated [Ca(2+)](c) to about 30 micromol/L, but only if oxygen was removed, the respiratory chain was inhibited, or if the mitochondria were uncoupled. These high [Ca(2+)](c) elevations depended on external Ca(2+) and could not be prevented by inhibiting NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors, or gadolinium-sensitive channels. However, they could be prevented by removing external Na(+) or simultaneously inhibiting NMDA and AMPA/kainate receptors; 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), an inhibitor of plasmalemmal Na(+)/Ca(2+) exchanger, partly suppressed them. The data indicate that the [Ca(2+)](c) elevations to 30 micromol/L during AD result from Na(+) influx. Activation of either NMDA or AMPA/kainate channels provides adequate Na(+) influx to induce these [Ca(2+)](c) elevations, which are mediated by KB-R7943-sensitive and KB-R7943-resistant mechanisms.     

Increase of [Ca(2+)](i) via activation of ATP receptors in PC-Cl3 rat thyroid cell line.

In PC-Cl3 rat thyroid cell line, ATP and UTP provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Thapsigargin (TG) caused a rapid rise in [Ca(2+)](i) and subsequent addition of ATP was without effect. The transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with the specific inhibitor of phospholipase C (PLC), U73122. These data suggest that the ATP-stimulated increment of [Ca(2+)](i) required InsP(3) formation and binding to its specific receptors in Ca(2+) stores. Desensitisation was demonstrated with respect to the calcium response to ATP and UTP in Fura 2-loaded cells. Further studies were performed to investigate whether the effect of ATP on Ca(2+) entry into PC-Cl3 cells was via L-type voltage-dependent Ca(2+) channels (L-VDCC) and/or by the capacitative pathway. Nifedipine decreased ATP-induced increase on [Ca(2+)](i). Addition of 2 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment of the cells with TG or with 100 microM ATP in Ca(2+)-free medium. These data indicate that Ca(2+) entry into PC-Cl3 stimulated with ATP occurs through both an L-VDCC and through a capacitative pathway. Using buffers with differing Na(+) concentrations, we found that the effects of ATP were dependent of extracellular Na(+), suggesting that a Na(+)/Ca(2+) exchange mechanism is also operative. These data suggest the existence, in PC-Cl3 cell line, of a P2Y purinergic receptor able to increase the [Ca(2+)](i) via PLC activation, Ca(2+) store depletion, capacitative Ca(2+) entry and L-VDCC activation.     

Enhanced charge-independent mitochondrial free Ca(2+) and attenuated ADP-induced NADH oxidation by isoflurane: Implications for cardioprotection

Modulation of mitochondrial free Ca(2+) ([Ca(2+)](m)) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia-reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca(2+)](m) and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2mM) on the magnitudes and time-courses of [Ca(2+)](m) and mitochondrial redox state (NADH), membrane potential (ΔΨ(m)), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10mM Na(+)- or K(+)-pyruvate/malate (NaPM or KPM) or Na(+)-succinate (NaSuc) followed by additions of isoflurane, 0.5mM CaCl(2) (≈200nM free Ca(2+) with 1mM EGTA buffer), and 250μM ADP. Isoflurane stepwise: (a) increased [Ca(2+)](m) in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨ(m) and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨ(m), and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨ(m), and matrix contraction with PM substrates. These findings suggest that isoflurane's effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca(2+) uptake by inhibiting the Na(+)/Ca(2+) exchanger (NCE), independent of changes in ΔΨ(m) and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca(2+)](m), while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level.     

Mechanisms of the lysophosphatidic acid-induced increase in [Ca(2+)](i) in skeletal muscle cells.

Although lysophosphatidic acid (LPA) is known to increase intracellularfree calcium concentration ([Ca(2+)](i)) in different cell types, the effect of LPA on the skeletal muscle cells is not known. The present study was therefore undertaken to examine the effect of LPA on the [Ca(2+)](i) in C2C12 cells. LPA induced a concentration and time dependent increase in [Ca(2+)](i), which was inhibited by VPC12249, VPC 32183 and dioctanoyl glycerol pyrophosphate, LPA1/3 receptor antagonists. Pertussis toxin, a G(i) protein inhibitor, also inhibited the LPA-induced increase in [Ca(2+)](i). Inhibition of tyrosine kinase activities with tyrphostin A9 and genistein also prevented the increase in [Ca(2+)](i) due to LPA. Likewise, wortmannin and LY 294002, phosphatidylinositol 3-kinase (PI3-K) inhibitors, inhibited [Ca(2+)](i) response to LPA. The LPA effect was also attenuated by ethylene glycolbis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), an extracellular Ca(2+) chelator, Ni(2+) and KB-R7943, inhibitors of the Na(+)-Ca(2+) exchanger; the receptor operated Ca(2+) channel (ROC) blockers, 2-aminoethoxydiphenyl borate and SK&F 96365. However, the L-type Ca(2+) channel blockers, verapamil and diltiazem; the store operated Ca(2+) channel blockers, La(3+) and Gd(3+); a sarcoplasmic reticulum calcium pump inhibitor, thapsigargin; an inositol trisphosphate receptor antagonist, xestospongin and a phospholipase C inhibitor, U73122, did not prevent the increase [Ca(2+)](i) due to LPA. Our data suggest that the LPA-induced increase in [Ca(2+)](i) might occur through G(i)-protein coupled LPA(1/3) receptors that may be linked to tyrosine kinase and PI3-K, and may also involve the Na(+)-Ca(2+) exchanger as well as the ROC. In addition, LPA stimulated C2C12 cell proliferation via PI3-K. Thus, LPA may be an important phospholipid in the regulation of [Ca(2+)](i) and growth of skeletal muscle cells.     

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.     

Feedback inhibition of sodium/calcium exchange by mitochondrial calcium accumulation

Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger were subjected to two periods of 5 and 3 min, respectively, during which the extracellular Na(+) concentration ([Na(+)](o)) was reduced to 20 mm; these intervals were separated by a 5-min recovery period at 140 mm Na(+)(o). The cytosolic Ca(2+) concentration ([Ca(2+)](i)) increased during both intervals due to Na(+)-dependent Ca(2+) influx by the exchanger. However, the peak rise in [Ca(2+)](i) during the second interval was only 26% of the first. The reduced rise in [Ca(2+)](i) was due to an inhibition of Na(+)/Ca(2+) exchange activity rather than increased Ca(2+) sequestration since the influx of Ba(2+), which is not sequestered by internal organelles, was also inhibited by a prior interval of Ca(2+) influx. Mitochondria accumulated Ca(2+) during the first interval of reduced [Na(+)](o), as determined by an increase in fluorescence of the Ca(2+)-indicating dye rhod-2, which preferentially labels mitochondria. Agents that blocked mitochondrial Ca(2+) accumulation (uncouplers, nocodazole) eliminated the observed inhibition of exchange activity during the second period of low [Na(+)](o). Conversely, diltiazem, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger, increased mitochondrial Ca(2+) accumulation and also increased the inhibition of exchange activity. We conclude that Na(+)/Ca(2+) exchange activity is regulated by a feedback inhibition process linked to mitochondrial Ca(2+) accumulation.     

Altered spatial calcium regulation enhances electrical heterogeneity in the failing canine left ventricle: implications for electrical instability

Myocytes across the left ventricular (LV) wall of the mammalian heart are known to exhibit heterogeneity of electrophysiological properties; however, the transmural variation of cellular electrophysiology and Ca(2+) homeostasis in the failing LV is incompletely understood. We studied action potentials (APs), the L-type calcium (Ca(2+)) current (I(Ca,L)), and intracellular Ca(2+) transients ([Ca(2+)](i)) of subendocardial (Endo), midmyocardial (Mid), and subepicardial (Epi) tissue layers in the canine normal and tachycardia pacing-induced failing left ventricles. Heart failure (HF) was associated with significant prolongation of the AP duration in Mid myocytes. There were no differences in I(Ca,L) density in normal Endo, Mid, and Epi myocytes, whereas in the failing heart, I(Ca,L) density was downregulated by 45% and 26% (at +10 mV) in Endo and Mid myocytes, respectively. The rates of sarcoplasmic reticulum (SR) Ca(2+) release and decay of the [Ca(2+)](i) were slowed, and the amplitude of the [Ca(2+)](i) was depressed in Endo and Epi myocytes isolated from failing, compared with normal, hearts. Experiments in sodium (Na(+))-free solutions showed that Epi and Mid myocytes of the failing ventricle exhibit a greater reliance on the Na(+)-Ca(2+) exchanger to remove cytosolic Ca(2+) than myocytes isolated from normal hearts. Simulation studies in Endo, Mid, and Epi canine myocytes demonstrate the importance of L-type current density and SR Ca(2+) uptake in modulating the potentially arrhythmogenic repolarization in HF. In conclusion, these results demonstrate that spatially heterogeneous decreases in I(Ca,L) and defective cytosolic Ca(2+) removal contribute to the altered [Ca(2+)](i) and AP profiles across the canine failing LV. These distinct electrophysiological features in myocytes from a failing heart contribute to a characteristic electrogram arising from increased dispersion of refractoriness across the LV, which may result in significant arrhythmogenic sequellae.     

Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger

Although the Na(+)/H(+) exchanger (NHE) is considered to be involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the Na(+)/Ca(2+) exchanger, the exact mechanisms of its participation in Ca(2+) handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca(2+) movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N-isobutyl)amiloride (MIA). [Ca(2+)](i) in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca(2+)](i) and augmented the KCl-induced increase in [Ca(2+)](i) in a concentration-dependent manner. The MIA-induced increase in basal [Ca(2+)](i) was unaffected by extracellular Ca(2+), antagonists of the sarcolemmal (SL) L-type Ca(2+) channel, and inhibitors of the SL Na(+)/Ca(2+) exchanger, SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. However, the MIA-induced increase in basal [Ca(2+)](i) was attenuated by inhibitors of SL Na(+)-K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+) transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca(2+) concentration and attenuated by agents that inhibit SL L-type Ca(2+) channels, the SL Na(+)/Ca(2+) exchanger, SL Na(+)-K(+)-ATPase, and SR Ca(2+) release channels and the SR Ca(2+) pump. However, the effect of MIA on the KCl-induced increase in [Ca(2+)](i) remained unaffected by treatment with inhibitors of SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca(2+)](i), whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca(2+)](i) in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca(2+)](i) and that MIA-induced increases in basal [Ca(2+)](i), as well as augmentation of the KCl-induced increase in [Ca(2+)](i), in cardiomyocytes are regulated differentially.