Effect of exercise training on Ca2+ release units of left ventricular myocytes of spontaneously hypertensive rats

In cardiomyocytes, calcium (Ca²⁺) release units comprise clusters of intracellular Ca²⁺ release channels located on the sarcoplasmic reticulum, and hypertension is well established as a cause of defects in calcium release unit function. Our objective was to determine whether endurance exercise training could attenuate the deleterious effects of hypertension on calcium release unit components and Ca²⁺ sparks in left ventricular myocytes of spontaneously hypertensive rats. Male Wistar and spontaneously hypertensive rats (4 months of age) were divided into 4 groups: normotensive (NC) and hypertensive control (HC), and normotensive (NT) and hypertensive trained (HT) animals (7 rats per group). NC and HC rats were submitted to a low-intensity treadmill running protocol (5 days/week, 1 h/day, 0% grade, and 50-60% of maximal running speed) for 8 weeks. Gene expression of the ryanodine receptor type 2 (RyR2) and FK506 binding protein (FKBP12.6) increased (270%) and decreased (88%), respectively, in HC compared to NC rats. Endurance exercise training reversed these changes by reducing RyR2 (230%) and normalizing FKBP12.6 gene expression (112%). Hypertension also increased the frequency of Ca²⁺ sparks (HC=7.61±0.26 vs NC=4.79±0.19 per 100 µm/s) and decreased its amplitude (HC=0.260±0.08 vs NC=0.324±0.10 ΔF/F₀), full width at half-maximum amplitude (HC=1.05±0.08 vs NC=1.26±0.01 µm), total duration (HC=11.51±0.12 vs NC=14.97±0.24 ms), time to peak (HC=4.84±0.06 vs NC=6.31±0.14 ms), and time constant of decay (HC=8.68±0.12 vs NC=10.21±0.22 ms). These changes were partially reversed in HT rats (frequency of Ca²⁺ sparks=6.26±0.19 µm/s, amplitude=0.282±0.10 ΔF/F₀, full width at half-maximum amplitude=1.14±0.01 µm, total duration=13.34±0.17 ms, time to peak=5.43±0.08 ms, and time constant of decay=9.43±0.15 ms). Endurance exercise training attenuated the deleterious effects of hypertension on calcium release units of left ventricular myocytes.     

A measurable reduction of s.r. Ca content follows spontaneous Ca release in rat ventricular myocytes

 The Ca content of the sarcoplasmic reticulum (s.r.) was measured in voltage-clamped rat ventricular myocytes from the integral of the Na-Ca exchange current evoked by applying caffeine to release the s.r. Ca content. Following spontaneous release of Ca from the s.r., the s.r. Ca content was decreased. The magnitude of this decrease was equal to that of the amount of calcium directly measured to have been pumped out of the cell during the spontaneous release. Following a spontaneous release, the s.r. Ca content recovered linearly. These results are shown to be consistent with the hypothesis that the frequency of spontaneous release is determined by the time taken for the cell and s.r. to reaccumulate the Ca²⁺ ions pumped out of the cell during spontaneous release. Received: 9 July 1997 / Accepted: 21 July 1997     

Inhibition of SERCA2 Ca2+-ATPases by Cs+

Replacement of K⁺ with Cs⁺ on the cytoplasmic side of the sarcoplasmic reticulum (SR) membrane reduces the maximum velocity (V_max) of Ca²⁺ uptake into the SR of saponin-permeabilized rat ventricular myocytes. To compare the sensitivity of the cardiac and smooth muscle/non-muscle forms of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a and -2b respectively) to replacement of K⁺ with Cs⁺, SERCA2a and SERCA2b were expressed in HEK-293 cells. Ca²⁺ uptake into HEK cell microsomes was inhibited by replacement of extravesicular K⁺ with Cs⁺ (V_max of SERCA2a-mediated Ca²⁺ uptake in CsCl was 80% of that in KCl; V_max of SERCA2b-mediated uptake was 70% of that in KCl). The Ca²⁺ sensitivity of uptake was decreased for both SERCA2a- and SERCA2b-mediated uptake and the Hill coefficients were increased in the presence of CsCl. The effects of Cs⁺ on uptake were associated with direct inhibition of the ATPase activity of SERCA2a and SERCA2b. Our results indicate that cation binding sites are present in both SERCA2 isoforms, although the extent to which SERCA2b is inhibited by K⁺ replacement is greater than that of SERCA2a or SERCA1. Consideration of these results and the recent molecular modeling work of others suggests that monovalent cations could interact with the Ca²⁺ binding region of SERCA.     

A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes

 This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine (10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca²⁺]_i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the measurements of [Ca²⁺]_i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114±18 μmol l^–1 and K _d of 0.59±0.17 μM (n=8). During the systolic rise of [Ca²⁺]_i, the measured changes of [Ca²⁺]_i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering and the time-course of both total cytoplasmic and s.r. Ca. Received: 14 October 1998 / Accepted: 6 November 1998     

Functional significance of Ca2+ in long-lasting fatigue of skeletal muscle

Repeated activation of skeletal muscle causes fatigue, which involves a reduced ability to produce force and slowed contraction regarding both the speed of shortening and relaxation. One important component in skeletal muscle fatigue is a reduced sarcoplasmic reticulum (SR) Ca²⁺ release. In the present review we will describe different types of fatigue-induced inhibition of SR Ca²⁺ release. We will focus on a type of long-lasting failure of SR Ca²⁺ release which is called low-frequency fatigue, because this type of fatigue may be involved in the muscle dysfunction and chronic pain experienced by computer workers. Paradoxically it appears that the Ca²⁺ released from the SR, which is required for contraction, may actually be responsible for the failure of SR Ca²⁺ release during low-frequency fatigue. We will also discuss the relationship between gross morphological changes in muscle fibres and long-lasting failure of SR Ca²⁺ release. Finally, a model linking muscle cell dysfunction and muscle pain is proposed. Accepted: 6 June 2000     

Estimation of myofibrillar responsiveness to Ca2+ in isolated rat ventricular myocytes

 To estimate myofibrillar responsiveness to Ca²⁺, we used the relation between cell length and intracellular [Ca²⁺] ([Ca²⁺]_i) during tetanic contractions of isolated ventricular myocytes. Enzymatically isolated rat ventricular myocytes were loaded with fura-2 AM (4 μM for 10 min) and excited alternately at 340 nm and 380 nm. The ratio (R) of fura-2 fluorescence at these wavelengths [F(340)/F(380), an index of [Ca²⁺]_i] and cell length (L) were measured simultaneously. Following treatment with thapsigargin (0.2 μM), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction every min and an instantaneous plot of R vs L (R-L trajectory) was constructed. The R-L trajectory followed the same path during cell shortening and re-lengthening, suggesting that dynamic equilibrium between R and L was achieved during tetanus. Increasing the extracellular [Ca²⁺] from 1 to 8 mM extended the R-L trajectory without a substantial shift of the relation. The Ca²⁺-sensitizing thiadiazinone derivative, EMD57033 (1 μM), shifted the R-L trajectory to the left (sensitization of the myofibrils to Ca²⁺), whereas the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (200 μM), shifted the R-L trajectory to the right (desensitization of the myofibrils to Ca²⁺), in agreement with previous results obtained using skinned preparations. We conclude that the R-L trajectory is useful for estimating the myofibrillar responsiveness to Ca²⁺ in isolated myocytes and may be beneficial for the evaluation of inotropic agents. Received: 13 March 1998 / Received after revision: 4 May 1998 / Accepted: 2 June 1998     

Multiple effects of caffeine on calcium current in rat ventricular myocytes

Caffeine exerts a number of different effects on L-type calcium current in rat ventricular myocytes. These include: (1) a slowing of inactivation that is comparable to, but not additive to, that produced by prior treatment of the cells with ryanodine (a selective sarcoplasmic reticulum Ca²⁺ releaser) or high concentrations of intracellular 1,2-bis[2-aminophenoxy]ethane-N,N,N,N-tetraacetic acid (BAPTA) (a fast Ca²⁺ chelator), (2) a stimulation of peak I _Ca that is comparable to, but not additive to that produced by prior treatment with isobutylmethylxanthine (a selective phosphodiesterase inhibitor), and (3) a dose-dependent decrease of peak I _Ca that is not prevented by pretreatment with any of these agents. None of the caffeine actions could be mimicked or prevented by administration of 8-phenyltheophylline, a specific adenosine receptor antagonist. We conclude that only the slowing of I _Ca inactivation is due to caffeine's ability to deplete the sarcoplasmic reticulum of calcium. The stimulatory effect of caffeine on peak I _Ca is probably due to phosphodiesterase inhibition, while caffeine's inhibitory effect on I _Ca is independent of these processes and could be a direct effect on the channel. The multiplicity of caffeine actions independent of its effects on the sarcoplasmic reticulum lead to the conclusion that ryanodine, though slower acting and essentially irreversible, is a more selective agent than caffeine for probing sarcoplasmic reticulum function and its effects on other processes.The experimental part of this work was published during the postdoctoral stay of I. Zahradník in the Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, TX 77555, USA     

Ca2+ store determines gating of store operated calcium entry in mammalian skeletal muscle

This work describes the gating of the store operated calcium entry (SOCE) in adult mammalian skeletal muscle. Flexor digitorum brevis fibers (FDB) were isolated from adult mice and exposed to conditions to deplete the sarcoplasmic reticulum (SR). A transient SR depletion caused either by repetitive depolarizations, chlorocresol (CMC) or, cyclopiazonic acid (CPA) induced a bell shaped calcium entry that raised the [Ca²⁺]_i to a maximum of 27.09 ± 4.35 nM from the resting value. The activation time to reach 10–90% of the maximum amplitude was 112 ± 10 s (n = 22). On the other hand, any mechanism that caused a permanent SR depletion (like thapsigargin, continuous CPA, or continuous CMC) triggered a calcium entry pathway that lasted 325 ± 23 s and raised the [Ca²⁺]_i to 129.50 ± 13.05 nM from the resting level (n = 28). Then, a prolonged depletion triggered an increase in [Ca²⁺]_i to higher values and for a longer time than when the SR is transiently depleted (p < 0.001). Our results, in skeletal muscle, showed that calcium store depletion was the signal for SOCE activation and how the SR got depleted was not relevant. Also, we found that SOCE deactivation was not caused by [Ca²⁺]_i but by the SR content. Our results suggest that the SR calcium content plays an important role in SOCE gating in mammalian skeletal muscle and a calcium sensor is located inside the SR.     

急性心肌梗死后1周兔心室肌细胞钠离子通道活性的变化

目的:研究急性心肌梗死(AMI)后心室肌细胞钠离子通道活性的变化。方法:采用结扎兔冠状动脉左前降支方法建立AMI动物模型, 应用膜片钳全细胞记录方法, 观察AMI后1周心外膜梗死区心肌细胞钠通道电流(I_Na)的变化。结果:正常对照组I_Na电流密度峰值(-30mV)为(45.5±5.33)PA/PF(n=12), 心梗组为(22.48±4.62)PA/PF(n=14), 明显低于对照组, P＜0.01, I_Na电流-电压关系曲线在心梗组明显下移;心梗组I_Na失活曲线较对照组明显左移, 对照组V_0.5为(-76.2±5.3)mV(n=5), 心梗组为(-89.1±5.6)mV(n=6), P＜0.05;心梗组钠通道灭活后恢复时程明显慢于对照组, 恢复曲线下移。结论:AMI可导致梗死区心室肌细胞I_Na下降、钠通道动力学发生变化, 引起心肌传导速度下降和不应性延长, 可能是导致AMI后出现折返性室性心律失常的原因。     

Effects of cytosolic Ca2+ on the Ca2+ content of the sarcoplasmic reticulum in saponin-permeabilized rat ventricular trabeculae

 Rat ventricular trabeculae were mounted for isometric tension recording, and then permeabilized with saponin. The Ca²⁺ concentration ([Ca²⁺]) within the permeabilized preparation (cytosolic [Ca²⁺]) was monitored continuously using Indo-1 and the integrals of Ca²⁺ transients resulting from brief caffeine application used as an index of the sarcoplasmic reticulum (SR) Ca²⁺ content. The relationship between SR Ca²⁺ content and cytosolic [Ca²⁺] was studied within the reported physiological range (i.e. 50–250 nmol · l^–1 Ca²⁺). Increasing cytosolic [Ca²⁺] from 50 nmol · l^–1 to 250 nmol · l^–1 increased the steady-state SR Ca²⁺ content about threefold. However, increasing [Ca²⁺] above 250 nmol · l^–1 typically resulted in spontaneous SR Ca²⁺ release, with no further increase in SR Ca²⁺ content. The SR Ca²⁺ content increased only slowly when cytosolic [Ca²⁺] was increased; it was unchanged 20 s after a rapid increase in cytosolic [Ca²⁺], but increased progressively to a new steady-state level during the following 1–2 min. In a parallel series of experiments using intact papillary muscles, increasing extracellular [Ca²⁺] (from 0.5 to 5 mmol · l^–1) significantly increased twitch tension within 20 s of the solution change. These results support previous suggestions that the SR Ca²⁺ content may increase when diastolic cytosolic [Ca²⁺] rises during inotropic interventions such as increased stimulus rate or extracellular [Ca²⁺]. However, the rate at which SR Ca²⁺ responds to changes in cytoplasmic [Ca²⁺] within the diastolic range does not appear rapid enough to explain the early potentiation of twitch tension in intact preparations after an increase in extracellular [Ca²⁺]. Received: 26 August 1997 / Accepted: 28 October 1997     

Effects of membrane potential on Na+ -dependent Mg2+ extrusion from rat ventricular myocytes

To study Mg2+ transport across the cell membrane, the cytoplasmic concentration of Mg2+ ([Mg2+](i)) in rat ventricular myocytes was measured with the fluorescent indicator furaptra (mag-fura-2) under Ca2+ -free conditions (0.1 mM EGTA) at 25 degrees C. The fluorescence ratio signal of furaptra was converted to [Mg2+](i) using calibration parameters previously estimated in myocytes (Watanabe and Konishi, Pflügers Arch 442: 35-40, 2001). After [Mg2+](i) was raised by loading the cells with Mg2+ in a solution containing 93 mM Mg(2+), the cells were voltage-clamped at a holding potential of -80 mV using the perforated patch-clamp technique with amphotericin B. At the holding potential of -80 mV, the reduction of extracellular Mg2+ to 1.0 mM caused a rapid decrease in [Mg2+](i) only in the presence of extracellular Na(+). The rate of the net Mg2+ efflux appeared to be dependent on the initial level of [Mg2+](i); the decrease in [Mg2+](i) was significantly faster in the myocytes markedly loaded with Mg2+. The rate of decrease in [Mg2+](i) was influenced little by membrane depolarization from -80 to -40 mV, but the [Mg2+](i) decrease accelerated significantly at 0 mV by, on average, approximately 40%. Hyperpolarization from -80 to -120 mV slightly but significantly slowed the decrease in [Mg2+](i) by approximately 20%. The results clearly demonstrate an extracellular Na(+)- and intracellular Mg2+ -dependent Mg2+ efflux activity, which is consistent with the Na(+)-Mg2+ exchange, in rat ventricular myocytes. We found that the apparent rate of Mg2+ transport depends slightly on the membrane potential: facilitation by depolarization and inhibition by hyperpolarization with no sign of reversal between -120 and 0 mV.     

Ca2+- and H+-dependent effects of crude bacterial phospholipase C on the hydroosmotic response of toad urinary bladder to serosal hypertonicity

Phospholipase C (EC 3.1.4.3.) from Clostridium perfringens (crude extracts) was used to study the role of phospholipids in the osmotic permeability of the urinary bladder of the toad. When added to the serosal bath (430 mU/ml) it inhibited the effects of antidiurectic hormone (ADH) and exogenous cyclic AMP. Under the same conditions the increase in osmotic flow produced by serosal hypertonicity (SH) was slightly enhanced by the lipase. The hydroosmotic effect of SH was greatly potentiated by the lipase by decreasing 10-fold the Ca²⁺ concentration. The SH-induced flow was inhibited by the lipase if the Ca²⁺ or the H⁺ concentration was increased 10-fold, but not if the increase in positive charges was produced by a concentration of Mg²⁺. Phospholipase C had no effect on the action of either ADH or SH if added to the mucosal bath. Serosal neuraminidase or phospholipase A₂ could not mimic the effect of phospholipase C on SH. The effect of phospholipase C on the response to SH was not modified if fatty acid-free bovine serum albumin was added to the bath. Therefore, the release of products of lipolysis into the bath do not seem to be responsible for the effects of phospholipase C on SH-induced water flow. The results suggest that the effects of the enzyme on the composition and rearrangement of lipids at the basolateral membrane produce modifications of the water flow. Ca²⁺ and H⁺ may modify the enzyme-substrate interaction, suggesting that different phospholipids may be differentially involved in the control of water permeability of the basolateral membrane. Changes in Ca²⁺ and H⁺ concentration may also activate or suppress different lipolytic enzymes of the nonhomogenous enzymatic complex employed. These results support the idea that ADH and SH converge in a final common site of action in order to increase the water permeability of the apical membrane, but they do so by activating different steps, SH acting mainly at a post-cyclic AMP site.     

1,6-二磷酸果糖对阿霉素引起大鼠心肌细胞内游离钙和肌浆网Ca2+-ATP酶活性改变的影响

目的:探讨1,6-二磷酸果糖(FDP)对阿霉素(ADR)所引起的大鼠心肌细胞内游离钙及心肌肌浆网钙-ATP酶(SRCa²⁺-ATPase)活性变化的影响。方法: 给大鼠腹腔注射ADR(2.5 mg·kg^-1,隔日1次,共6次),给ADR处理的大鼠腹腔注射不同剂量的FDP(隔日1次,共21次)进行干预。采用双抗体双夹心ELISA法定量测定血清肌钙蛋白I(CTnI);采用抗肌酸激酶同工酶(CK-MB)单克隆抗体包被小球测定血清CK-MB;用荧光分光光密度计测定心肌细胞内游离钙(MyoCa²⁺)浓度;用无机磷酸根法测定心肌肌浆网钙-ATP酶(SRCa²⁺-ATPase)活性。结果:FDP(300、600、1 200 mg·kg^-1)干预ADR处理的大鼠后,可显著降低血清CtnI、CK-MB及心肌细胞内MyoCa²⁺值,显著增高SRCa²⁺-ATPase活性(P＜0.01)。结论: FDP能通过降低心肌细胞内游离钙和对SRCa²⁺-ATPase活性的改变,起到减轻ADR对心肌的毒性作用。     

Ruthenium red affects the contractile apparatus but not sarcoplasmic reticulum Ca2+ release of skinned papillary muscle

Ruthenium red has been shown to have a positive inotropic effect on isolated perfused hearts. The cellular mechanism of this action is not clear. Ruthenium red is able to block the Ca²⁺ release channel in isolated sarcoplasmic reticulum (SR) vesicle and reconstituted channel preparations. However, the effect of ruthenium red on SR Ca²⁺ release has not been studied in skinned cardiac muscle preparations. In the present study we investigated the actions of ruthenium red on both the characteristics of force generation by the contractile apparatus and Ca²⁺ release from the SR in chemically skinned rat papillary muscle. Ruthenium red (2 and 10 M) significantly increased the Ca²⁺ sensitivity of the contractile apparatus (decreasing Ca²⁺ required for the half-maximal response from 1.56±0.04 M to 1.46±0.05 M) but had no effect on the maximal Ca²⁺-activated force in triton X-100 treated fibers. This result may suggest one explanation for the positive inotropic effect of ruthenium red on the heart. On the other hand, ruthenium red had no significant effect on either caffeine-induced Ca²⁺ release or Ca²⁺-induced Ca²⁺ release from the SR in saponin-skinned muscle fibers. Lack of a blocking effect on SR Ca²⁺ release by ruthenium red in skinned fibers suggests that the SR Ca²⁺ channels in intact preparations have characteristics that are different from those of either vesicular or reconstituted channel preparations.     

Modal gating transitions in cardiac ryanodine receptors during increases of Ca2+ concentration produced by photolysis of caged Ca2+

Channel adaptation is a basic property of the sarcoplasmic reticulum Ca²⁺-release channels/ryanodine receptors (RyRs). It allows channel activity to decay during sustained increases in the concentration of activating Ca²⁺. Despite the potential physiological importance of this self-confining process, its molecular mechanism is not well understood. To define the mechanism of adaptation we studied the dynamics of cardiac Ca²⁺-release channel (RyR) gating using the planar lipid bilayer technique in combination with photolysis of caged Ca²⁺ (DM-nitrophen). Channels activated by rapid and sustained increases in Ca²⁺ concentration (from 0.1 to 0.5 μmol/l) displayed three distinct gating modes, manifested as current records with frequent and long openings (H-mode), with rare and short openings (L-mode), and with no openings (I-mode). H-mode channel activity occurred primarily at early times while L- and I-modes predominated at late times after the rapid Ca²⁺ concentration increase. The decrease in probability of H-mode, mirrored by an increase in the probability of the I-mode, proceeded with a time constant similar to that observed for spontaneous decay in channel activity (i.e., adaptation) in ensemble average records. These results indicate that RyR adaptation transpires by a shift of channel gating from a high open probability mode to low open probability and inactivated modes of the channel. Received: 1 March 1999 / Received after revision: 16 April 1999 / Accepted: 19 April 1999     

Reduced sarcoplasmic reticulum content of releasable Ca2+ in rat soleus muscle fibres after eccentric contractions

AIM: The purpose was to evaluate the effects of fatiguing eccentric contractions (EC) on calcium (Ca2+) handling properties in mammalian type I muscles. We hypothesized that EC reduces both endogenous sarcoplasmic reticulum (SR) content of releasable Ca2+ (eSRCa2+) and myofibrillar Ca2+ sensitivity. METHODS: Isolated rat soleus muscles performed 30 EC bouts. Single fibres were isolated from the muscle and after mechanical removal of sarcolemma used to measure eSRCa2+, rate of SR Ca2+ loading and myofibrillar Ca2+ sensitivity. RESULTS: Following EC maximal force in whole muscle was reduced by 30% and 16/100 Hz force ratio by 33%. The eSRCa2+ in fibres from non-stimulated muscles was 45 +/- 5% of the maximal loading capacity. After EC, eSRCa2+ per fibre CSA decreased by 38% (P = 0.05), and the maximal capacity of SR Ca2+ loading was depressed by 32%. There were no effects of EC on either myofibrillar Ca2+ sensitivity, maximal Ca2+ activated force per cross-sectional area and rate of SR Ca2+ loading, or in SR vesicle Ca2+ uptake and release. CONCLUSIONS: We conclude that EC reduces endogenous SR content of releasable Ca2+ but that myofibrillar Ca2+ sensitivity and SR vesicle Ca2+ kinetics remain unchanged. The present data suggest that the long-lasting fatigue induced by EC, which was more pronounced at low frequencies (low frequency fatigue), is caused by reduced Ca2+ release occurring secondary to reduced SR content of releasable Ca2+.     

Activation of Ca2+-sensitive Cl– current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

 We investigated how Ca²⁺-sensitive transient outward current, I _to(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na⁺ (Na⁺ _i) using the whole-cell patch-clamp technique at 36°C. In cells dialysed with Na⁺-free solutions,the application of nicardipine (5 μM) to block L-type Ca²⁺ current (I _Ca) completely inhibited I _to(Ca). In cells dialysed with a [Na⁺]_i≥5 mM, however, I _to(Ca) could be observed after blockade of I _Ca, indicating the activity of an I _Ca-independent component. The amplitude of I _Ca-independent I _to(Ca) increased with voltage in a [Na⁺]_i-dependent manner. The block of Ca²⁺ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked I _Ca-independent I _to(Ca). In Ca²⁺-free bath solution I _to(Ca) was completely abolished. The application of 2 mM Ni²⁺ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na⁺/Ca²⁺ exchange, or perfusion with pipette solution containing XIP (10 μM), a selective blocker of the exchanger, blocked I _Ca-independent I _to(Ca). From these results we conclude that, in the presence of Na⁺ _i, I _to(Ca) can be activated via Ca²⁺-induced Ca²⁺ release triggered by Na⁺/Ca²⁺ exchange operating in the reverse mode after blockade of I _Ca. Received: 20 January 1998 / Received after revision: 6 July 1998 / Accepted: 25 July 1998     

Na+/Ca2+ exchange during Ca2+ repletion is not a prerequisite for the Ca2+ paradox in isolated rat hearts

 Ca²⁺ paradox damage has been suggested to be determined by Na⁺ entry during Ca²⁺ depletion and exchange of Na⁺ for Ca²⁺ during Ca²⁺ repletion. Since previously a Ca²⁺ paradox without prior increase of total intracellular [Na⁺] ([Na⁺]_i) has been observed, we investigated whether local accumulation of Na⁺ close to the inner side of the sarcolemma during Ca²⁺ depletion plays a role in the Ca²⁺ paradox by replacing all extracellular Na⁺ by Li⁺ 5 min before and during 10 min Ca²⁺-free perfusion (37°C) in isolated rat hearts (group I). Subsequently, hearts were perfused with a standard, Na⁺- and Ca²⁺-containing solution. Verapamil was used to prevent contracture due to the absence of Na⁺/Ca²⁺ exchange during Na⁺-free perfusion in the presence of Ca²⁺. In group II, the Ca²⁺-free period was omitted, and in group III normal extracellular [Na⁺] was used throughout. ²³Na-NMR was used to monitor intra- and extracellular Na⁺ signals. Total creatine kinase release was 2,977±413, 36±24 and 3170±297 IU/g dry weight in groups I, II and III respectively, indicating a full Ca²⁺ paradox in groups I and III. [Na⁺]_i decreased from 11.3±0.6 mM during control perfusion to 1.2±0.4 mM after 10 min Ca²⁺ depletion in group I, whereas in group III [Na⁺]_i was 10.9±2.2 mM during control perfusion and did not change significantly after 10 min Ca²⁺-free perfusion. It is concluded that accumulation of Na⁺ close to the inner side of the sarcolemma during Ca²⁺ depletion is not a prerequisite for the Ca²⁺ paradox. Received: 2 February 1998 / Received after revision: 31 March 1998 / Accepted: 9 April 1998     

Membrane Ca2+ interactions and contraction in denervated rat soleus muscle

Under voltage clamp conditions contractile responses and ionic currents of single fibres isolated from rat soleus, denervated for more than 20 days, were recorded in Na-free TEA containing solutions. The relationship between membrane potential and contraction has been analysed under various conditions. The addition of trivalent cations (La³⁺, Gd³⁺) resulted in a dose dependent reduction of the contractile response and similar effects were produced by polymyxin B (0.05–0.5 mM). By contrast in the presence of phospholipase D (1–5 U/ml) contractions were significantly increased for all values of depolarization. The time course of the change of tension amplitude after the application of Ca-free medium, was dependent on the amplitude, the duration and the frequency of the depolarization. Upon depolarization glycerol-treated fibres generated contractile responses which were similar to those recorded in normal muscle and were also dependent on [Ca]_o. It is proposed that in denervated soleus muscle the negatively charged phospholipids at the outside of the membrane were involved in the depolarization-contraction coupling by means of their Ca binding properties. The quantity of Ca binding sites would be dependent on [Ca]_o and membrane potential and their binding properties modified during and/or following variation in membrane potential.     

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake,spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca²⁺ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca²⁺ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca²⁺ content loaded into the SR, (2) the spontaneous Ca²⁺ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca²⁺ sensitivity in Triton X-100-treated preparations. Inhibition of Ca²⁺ loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca²⁺ loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca²⁺ sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca²⁺ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. Received: 28 July 1995/Received after revision: 11 December 1995/Accepted: 18 December 1995