Hypoxia delays the intracellular Ca2+ clearance by Na+-Ca2+ exchanger in human adult cardiac myocytes.

Transient myocardial ischemia during cardiac surgery causes a loss of energy sources, contractile depression, and accumulation of metabolites and H+ ion resulting in intracellular acidosis. The reperfusion following ischemic cardioplegia recovers intracellular pH, activates Na+-H+ exchange and Na+-Ca2+ exchange transports and consequently produces Ca2+ overload, which yields cell death. Among the various Ca2+ entry pathways, the Na+-Ca2+ exchanger is known to play one of the major roles during the ischemia/reperfusion of cardioplegia. Consequently, information on the changes in intracellular Ca2+ activities of human cardiac myocytes via the Na+-Ca2+ exchanger is imperative despite previous measurements of Ca2+ current of human single myocytes. In this study, human single myocytes were isolated from the cardiac tissues obtained during open-heart surgery and intracellular Ca2+ activity was measured with cellular imaging techniques employing fluorescent dyes. We report that the Na+-Ca2+ exchanger of adult cardiac myocytes is more susceptible to hypoxic insult than that of young patients.     

KB-R7943 reveals possible involvement of Na(+)-Ca2+ exchanger in elevation of intracellular Ca2+ in rat carotid arterial myocytes.

A Na(+)/Ca(2+) exchanger (NCX) is one of the major regulators of intracellular Ca(2+) concentration ([Ca(2+)](i)) in cardiac muscle cells. Although vascular smooth muscle myocytes also express NCX proteins, their functional role has not been clear, mainly due to the lack of specific inhibitors of NCX and relatively low levels of expression of NCX. In the present study, we have examined the involvement of NCX in the Na(+) deficient (0 Na(+)) elevation of [Ca(2+)](i) in rat carotid arterial myocytes using KB-R7943, an inhibitor of NCX. Perfusion with a Na(+)-free bathing solution, prepared by replacement of Na(+) with N-methyl-D-glucamine, induced an elevation of [Ca(2+)](i), which was effectively inhibited by KB-R7943 (IC(50)=3.5 microM). This inhibition was reversed by washout of KB-R7943. In contrast, D600, a blocker of voltage dependent L-type Ca(2+) channels (VDCC), did not affect the 0 Na(+)-induced elevation of [Ca(2+)](i). Treatment of myocytes with ryanodine abolished the elevation of [Ca(2+)](i) caused by caffeine but not that caused by 0 Na(+). Application of Cd(2+), which is known to block NCX as well as VDCC, also significantly inhibited the 0 Na(+) induced elevation. These results suggest that KB-R7943 inhibits the extracellular Na(+) dependent ([Na(+)](o)) change in [Ca(2+)](i) in rat carotid arterial myocytes, which is presumably activated by the reverse mode of NCX.     

Modulation of intracellular Na+ activity and cardiac force by norepinephrine and Ca2+

The actions of norepinephrine and high calcium on the electrical, mechanical, and intracellular sodium ion activities were studied in electrically driven canine cardiac Purkinje fibers under different conditions. It was found that norepinephrine and high calcium decrease intracellular sodium ion activity (aiNa). The exposure to either agent is followed by a transient decline of force that correlates with the lower aiNa. Inhibition of the Na+ -K+ pump by strophanthidin reduces or abolishes the decrease in aiNa by norepinephrine but not that by high calcium. It is concluded that norepinephrine and high calcium both decrease aiNa and thereby the contractile force but (unlike high calcium) norepinephrine acts through the stimulation of the Na+ -K+ pump.     

Modulation of Ca2+ channels by intracellular Mg2+ ions and GTP in frog ventricular myocytes

Under conditions of low intracellular [Mg²⁺] ([Mg²⁺]_i), achieved by dialysis with pipette solutions containing ethylenediamine tetraacetic acid (EDTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and adenosine triphosphate (ATP) as chelator, calcium currents through the L-type calcium channels (I _Ca) were increased in frog ventricular myocytes. Total suppression of phosphorylation by depleting the cell of ATP with a cocktail of β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP) 2-deoxyglucose and carboxylcyanide-M-chlorophenylhydrazone (CCCP) did not inhibit the increase in I _Ca in the Mg²⁺-deficient medium. Thus, the involvement of phosphorylation process in the increase in I _Ca was not likely. Effective suppression of this enhancement of I _Ca was achieved by the application of guanosine triphosphate (GTP). From the dose-response curve for GTP, the GTP concentration required for half-maximal inhibition (IC₅₀) was estimated to be 4.0 μM at pMg 6. This GTP-induced suppression of I _Ca is not due to the guanine nucleotide binding protein (G-protein) cascade, because both activators and inhibitors of G-protein, which are structural analogues of GTP, suppressed I _Ca similarly. Treatment with pertussis toxin (PTX) did not affect the inhibitory action of Mg²⁺ and GTP on I _Ca. GTP is therefore assumed to bind directly to the Ca²⁺ channel. Interaction of Mg²⁺ and GTP with the Ca²⁺ channel activated in the Mg²⁺-deficient medium was examined by comparing the dose/response curves for GTP at two different [Mg²⁺]. The IC₅₀ for GTP suppression was estimated to be 5.7 μM at pMg 6 and 6.9 μM at pMg 5. The results suggest strongly that Mg²⁺ and GTP independently bind and control Ca²⁺ channels. Received: 22 December 1995/Received after revision and accepted: 11 March 1996     

Mechanism of hyperthyroidism-induced modulation of the L-type Ca2+ current in guinea pig ventricular myocytes

The positive inotropic effects of thyroid hormone in the heart, increased force and velocity of contraction have been mostly attributed to modulation of myosin ATPase isoenzymes (V₁, V₂ and V₃), and sarcoplasmic reticulum Ca²⁺ pumping activity. In addition, we have suggested that the effects on ventricular contraction result from a thyroid hormone-induced increase in L-type Ca²⁺ current (I _{Ca, L}). Due to the central role of I _{Ca, L} in excitation-contraction coupling, we studied mechanisms whereby thyroid hormone augments this current. Since thyroid hormone modulates adenylate cyclase activity in various tissues, we tested the hypothesis that the hormone activates adenylate cyclase, leading to increased cyclic adenosine monophosphate (cAMP) levels, protein kinase A activation, Ca²⁺ channel phosphorylation and increased I _{Ca, L}. We therefore stimulated or inhibited different sites along the adenylate cyclase cascade, and measured I _{Ca, L} and isometric twitch in ventricular myocytes and papillary muscles from euthyroid and hyperthyroid guinea pigs. Our major findings were as follows. In euthyroid myocytes, 0.1 M isoproterenol (Iso) increased I _{Ca, L} (at V _M=0 mV) from –7.04±0.72 to –22.26±1.88 pA/pF, P<0.05, while in hyperthyroid myocytes (I _{Ca, L}=-21.48±2.94 pA/pF), Iso was ineffective. In euthyroid myocytes, intracellular application of cAMP (50 M) was as potent as Iso, but ineffective in hyperthyroid myocytes. In hyperthyroid myocytes, a protein kinase A inhibitor (2 M) lowered I _{Ca, L} from –26.82±1.54 to -10.17±1.70 pApF (P<0.05), but had no effect in euthyroid myocytes. In hyperthyroid myocytes, acetylcholine (ACh) (1 M) decreased I _{Ca, L} from –26.86±1.49 to –18.33±1.25 pA/pF (P<0.05), while in euthyroid myocytes ACh decreased I_{Ca, L} from –6.80±0.61 to –6.00±0.39 pA/pF (NS). Accordingly, in hyperthyroid papillary muscles, ACh decreased twitch tension by 36.4±2.8%, but in euthyroid preparations only by 9.4±5.1% (P<0.05). These findings suggest that thyroid-hormone-induced increase in I _{Ca, L} contributing to positive inotropy, is mediated by activation of the adenylate cyclase cascade.     

抗钠钙交换体特异位点抗体对大鼠单个心室肌细胞钙瞬变的影响

目的:观察抗钠钙交换体(Sodium calcium exchanger,NCX)特异位点124HNFTAGDLGPSTIVGSAAFNMF145 抗体对正常成年大鼠心室肌细胞钙瞬变的影响。方法:利用Langendorff 灌流方法,分离得到单个大鼠心室肌细胞;负载荧光染料Fura-2/ AM 和2%牛血清白蛋白后,利用离子影像分析系统记录激发光为340 nm 和380 nm 时心室肌细胞成对系列图像,计算钙瞬变峰值(F340/ F380)、细胞钙恢复90%时程(TR90 )以及钙敏感性(F340/ F380 与细胞缩短数值的比值)。结果:抗NCX 特异位点抗体可以增加正常成年大鼠单个心室肌细胞F340/ F380,缩短TR90 ,对钙敏感性无显著影响;尼卡地平和KB-R7943 分别预处理心室肌细胞可以抵消大部分抗体对F340/ F380 和TR90 的增加或缩短效应,联合使用二者预处理心室肌细胞后,该抗体对钙瞬变不再有显著影响。结论:抗NCX 特异位点124 HNFTAGDLGPSTIVGSAAFNMF145 抗体可以增加心室肌细胞钙瞬变峰值,同时缩短细胞钙恢复时程,这种效应主要与其激动L-型Ca2+通道和NCX 的作用有关。     

Mechanisms underlying the modulation of L-type Ca2+ channel by hydrogen peroxide in guinea pig ventricular myocytes

Although Cav1.2 Ca²⁺ channels are modulated by reactive oxygen species (ROS), the underlying mechanisms are not fully understood. In this study, we investigated effects of hydrogen peroxide (H₂O₂) on the Ca²⁺ channel using a patch-clamp technique in guinea pig ventricular myocytes. Externally applied H₂O₂ (1 mM) increased Ca²⁺ channel activity in the cell-attached mode. A specific inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) KN-93 (10 μM) partially attenuated the H₂O₂-mediated facilitation of the channel, suggesting both CaMKII-dependent and -independent pathways. However, in the inside-out mode, 1 mM H₂O₂ increased channel activity in a KN-93-resistant manner. Since H₂O₂-pretreated calmodulin did not reproduce the H₂O₂ effect, the target of H₂O₂ was presumably assigned to the Ca²⁺ channel itself. A thiol-specific oxidizing agent mimicked and occluded the H₂O₂ effect. These results suggest that H₂O₂ facilitates the Ca²⁺ channel through oxidation of cysteine residue(s) in the channel as well as the CaMKII-dependent pathway.     

Inactivation of cardiac Na+ channel simply through open states as revealed by single-channel analysis in guinea pig ventricular myocytes

Inactivation of the cardiac Na(+) channel was analyzed by recording channel currents from a cell-attached patch containing only one functional Na(+) channel in guinea-pig ventricular myocytes. A two-step test pulse, first to variable levels (Pulse 1) and then to -30 mV (Pulse 2) was applied from a holding potential of -140 mV. When a cumulative histogram was determined for the latency of first opening, the histogram was well fitted with a single exponential function at -70 to -30 mV of Pulse 1. The activation time course of ensemble average was virtually single exponential. Although the ensemble average of 500 sweeps showed various extents of inactivation during Pulse 1, the saturation level of the cumulative first-latency histogram at the end of the two-step pulse was almost constant (0.7-0.8), irrespective of Pulse 1. Even when the interval between successive test pulses was prolonged from 70 to 970 ms, the saturation level of the histogram was not modified. These findings are consistent with inactivation only through the open state. Thus, the apparent "blank sweep inactivation" does not necessarily indicate direct inactivation from closed states. These findings support the hypothesis that the inactivation of cardiac Na(+) channel occurs exclusively through the open state.     

Inactivation of outward Na(+)-Ca2+ exchange current in guinea-pig ventricular myocytes.

1. Outward Na(+)-Ca2+ exchange currents were measured in freshly dissociated guinea-pig myocytes to probe in intact cells the functional status of exchanger inactivation reactions, described previously in giant excised cardiac membranes patches. 2. When the cytoplasmic (pipette) solution contained 40 mM Na+ and 0.1 microM free Ca2+ (50 mM EGTA), the outward exchange current activated by extracellular Ca2+ decayed with time (time constant, 13.1 +/- 2.6 s; n = 6), and an inward current transient was observed upon removal of extracellular Ca2+. Both the current decay and the subsequent inward current transient were remarkably diminished with a saturating (100 mM) pipette Na+ concentration. 3. With 100 mM cytoplasmic Na+ and 140 mM extracellular Na+, a significant fraction of the exchanger population is predicted to be in an inactive state. Intracellular application of 2 mg ml-1 chymotrypsin and 5 microM sodium tetradecylsulphate, both of which decrease Na(+)-dependent inactivation in giant membrane patches, increased the outward exchange current by about 160-170%, suggesting that about 60-70% of exchangers might be inactivated. 4. With 100 mM cytoplasmic Na+ and no extracellular Na+ (replaced with 140 mM Li+), application of extracellular Ca+ was predicted to reorient exchanger binding sites from the extracellular side to the cytoplasmic side and thereby favour inactivation. During such protocols, the outward exchange current decayed by 60-80% when activated by extracellular Ca2+. The current decayed similarly when extracellular Ca2+ and Na+ were applied together, whereby current magnitudes were about 3-fold smaller. 5. The decay of outward exchange current usually followed a biexponential time course (5.8 +/- 3.5 and 27.3 +/- 16.3 s, means +/- S.D., n = 11). Intracellular application of 0.5-2 mg ml-1 trypsin attenuated the fast component more than the slow component, suggesting that the fast component reflects an inactivation process. 6. Current-voltage (I-V) relations of the outward exchange current became less steep during the inactivation protocols, but this flattening could not be correlated with inactivation. 7. Replacement of extracellular Li+ with N-methyl-D-glucamine (NMG), tetraethylammonium (TEA), sucrose or Cs+ resulted in a flattening of I-V relations and a decrease of the outward exchange current amplitude by approximately 3-fold, but the kinetics and extent of inactivation were not remarkably changed. Thus, the mechanism of inactivation appears to be independent of the mechanism(s) of activation by extracellular monovalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of PKC increases Na+-K+ pump current in ventricular myocytes from guinea pig heart

 We have previously shown activation of α₁-adrenergic receptors increases Na⁺-K⁺ pump current (I _p) in guinea pig ventricular myocytes, and the increase is eliminated by blockers of phosphokinase C (PKC). In this study we examined the effect of activators of PKC on I _p. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased I _P at each test potential without shifting its voltage dependence. The concentration required for a half-maximal response (K _0.5) was 6 μM at 15 nM cytosolic [Ca²⁺] ([Ca²⁺]_i) and13 nM at 314 nM [Ca²⁺]_i. The maximal increase at either [Ca²⁺]_i was about 30%. Another activator of PKC, 1,2-dioctanoyl-sn-glycerol (diC₈), increased I _p similarly. The effect of PMA on I _P was eliminated by the PKC inhibitor staurosporine, but not by the peptide PKI, an inhibitor of protein kinase A (PKA). PMA and α₁-adrenergic agonist effects both were sensitive to [Ca²⁺]_i, blocked by PKC inhibitors, unaffected by PKA inhibition, and increased I _p uniformly at all voltages. However, they differed in that α₁-activation caused a maximum increase of 15% vs 30% via PMA, and α₁-effects were less sensitive to [Ca²⁺]_i than PMA effects. These results demonstrate that activation of PKC causes an increase in I _p in guinea pig ventricular myocytes. Moreover, they suggest that the coupling of α₁-adrenergic activation to I _p is entirely through PKC, however α₁-activation may be coupled to a specific population of PKC whereas PMA is a more global agonist. Received: 21 August 1998 / Received after revision: 7 December 1998 / Accepted: 11 December 1998     

钙拮抗剂对高血压大鼠心房肌L-型钙通道和钠-钙离子交换器mRNA的影响

目的:观察钙拮抗剂地尔硫卓对Goldblatt高血压大鼠模型心房肌L-型钙通道α_1C亚单位(CaL-α_1C)和Na^＋-Ca²⁺交换器(NCX)mRNA的变化影响,在基因水平探讨其潜在的意义。方法:Sprague-Dawley大鼠,高血压组用U型银夹夹住左肾动脉,右肾保留;假手术组(S组)只分离左肾动脉,不夹银夹,套尾法监测尾动脉血压。高血压组术后1周开始治疗,分为高剂量地尔硫艹卓组(HD组)、低剂量地尔硫艹卓组(LD组)和对照组(C组),分别于治疗后4周与S组同时处死动物(各6只),RT-PCR半定量分析CaL-α_1C和NCXmRNA的表达量(以GAPDH为内参照)。结果:C组心房肌CaL-α_1C的mRNA水平分别是S组、HD组、LD组的2.5倍、2.4倍、2.1倍(P＜0.01),S组、HD组、LD组之间无显著差别。C组心房肌NCX的mRNA水平分别是S组、HD组、LD组的1.9倍、1.6倍、2.1倍(P＜0.01),S组、HD组、LD组之间无显著差别。结论:肾性高血压大鼠心房肌CaL-α_1C、NCX的mRNA水平均表现为上调,应用钙拮抗剂能够抑制其上调,抑制作用不依赖于血压水平的降低。     

Comparison of Na+-Ca2+ exchange current elicited from isolated rabbit ventricular myocytes by voltage ramp and step protocols

 In this study, the effects of three different voltage protocols on the Na⁺-Ca²⁺ exchange current (I _Na-Ca) of rabbit right ventricular myocytes were studied. Whole-cell patch-clamp recordings were made using a Cs⁺-based internal dialysis solution and external solutions designed to block major interfering currents. I _Na-Ca was measured at 35–37°C as (5 mM) Ni-sensitive current elicited by: a 2 s descending ramp (DR: +80 to –120 mV); a 2 s ascending ramp (AR: –120 to +80 mV) and 500 ms voltage steps (VS) between –120 and +80 mV. DR and AR were applied from –40 mV and elicited I _Na-Ca with reversal potentials (E _rev) of –17.6±2.5 mV (mean±SEM; n=16) and –46.2±4.1 mV (n=10; P=0.0001) respectively. This difference was maintained when the holding potential was –80 mV (–44.0±2.1 mV, n=24 and –86.3±4.8 mV, n=10; P=0.0001), when the internal Ca chelator (EGTA) was replaced with BAPTA (–19.5±1.8 mV and –46.3±1.6 mV, n=6; P=0.0003) and when DR and AR were applied alternately to the same cell. Experiments using modified ramp waveforms suggested a possible mechanism for these differences. Increases in subsarcolemmal Ca caused by Ca entry (coupled to Na extrusion) during the initial positive potential phase of the DR might have induced I _Na-Ca reversal at less negative potentials than observed with AR, during the initial phase of which subsarcolemmal Ca would not have accumulated. These data suggest that I _Na-Ca during voltage-clamp experiments can be significantly influenced by the type of voltage protocol chosen, as the protocol appears to induce subsarcolemmal changes in Ca and Na concentration that are independent of Ca buffering in the bulk cytosol and can occur on a pulse-to-pulse basis. Received: 23 October 1998 / Received after revision: 8 January 1999 / Accepted: 11 January 1999     

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     

Analysis of cardiac mitochondrial Na+-Ca2+ exchanger kinetics with a biophysical model of mitochondrial Ca2+ handling suggests a 3:1 stoichiometry

Calcium is a key ion and is known to mediate signalling pathways between cytosol and mitochondria and modulate mitochondrial energy metabolism. To gain a quantitative, biophysical understanding of mitochondrial Ca(2+) regulation, we developed a thermodynamically balanced model of mitochondrial Ca(2+) handling and bioenergetics by integrating kinetic models of mitochondrial Ca(2+) uniporter (CU), Na(+)-Ca(2+) exchanger (NCE), and Na(+)-H(+) exchanger (NHE) into an existing computational model of mitochondrial oxidative phosphorylation. Kinetic flux expressions for the CU, NCE and NHE were developed and individually parameterized based on independent data sets on flux rates measured in purified mitochondria. While available data support a wide range of possible values for the overall activity of the CU in cardiac and liver mitochondria, even at the highest estimated values, the Ca(2+) current through the CU does not have a significant effect on mitochondrial membrane potential. This integrated model was then used to analyse additional data on the dynamics and steady-states of mitochondrial Ca(2+) governed by mitochondrial CU and NCE. Our analysis of the data on the time course of matrix free [Ca(2+)] in respiring mitochondria purified from rabbit heart with addition of different levels of Na(+) to the external buffer medium (with the CU blocked) with two separate models--one with a 2:1 stoichiometry and the other with a 3:1 stoichiometry for the NCE--supports the hypothesis that the NCE is electrogenic with a stoichiometry of 3:1. This hypothesis was further tested by simulating an additional independent data set on the steady-state variations of matrix free [Ca(2+)] with respect to the variations in external free [Ca(2+)] in purified respiring mitochondria from rat heart to show that only the 3:1 stoichiometry model predictions are consistent with the data. Based on these analyses, it is concluded that the mitochondrial NCE is electrogenic with a stoichiometry of 3:1.     

氨甲酰胆碱与去甲肾上腺素对心室肌细胞钠钙交换电流的联合作用

交感和副交感神经在心脏活动的调节中起重要作用。交感神经末梢释放的去甲肾上腺素(NE)对心脏起正性肌力作用,而副交感神经末梢释放的乙酰胆碱(Ach)则对心脏起负性肌力作用。两者的作用相互拮抗。氨甲酰胆碱(CCh)是一种胆碱受体激动药,与胆碱受体结合后,产生与递质Ach相似的作用,而且它性质稳定,作用持久,常用作工具药代替Ach来研究胆碱受体激动后的效应。新近的研究表明高浓度的CCh可以起正性肌力作用。其机制还不清楚。那么当CCh与NE联合作用时,CCh是发挥激动作用还是抑制作用还有待观察。一般来说,心肌收缩的加强是细胞内游离钙离…     

Involvement of Ca2+ buffering and Na+/Ca2+ exchange in the positive staircase of contraction in guinea-pig ventricular myocytes

The mean sarcomere length (SL) of guinea-pig cardiac myocytes was recorded simultaneously with the whole-cell current under voltage-clamp conditions. After blocking both sarcoplasmic reticulum (SR) and L-type Ca(2+) channels with ryanodine, cyclopiazonic acid and nicardipine, strong depolarizing pulses induced only the tonic component of SL shortening through the reverse mode of Na(+)/Ca(2+) exchange (NCX). A positive staircase of SL shortening was observed on applying a train pulses to +60~+100 mV at 2 Hz and trans-membrane Ca(2+) flux was calculated from the time integral of the Na(+)/Ca(2+) exchange current ( I(NCX)). Changes in cytosolic [Ca(2+)] ([Ca(2+)](i)) were determined indirectly using the experimental [Ca(2+)](i)/SL relationship. Cellular Ca(2+) buffering was characterized by a lumped single-component system with a maximum binding capacity of 200 micro M and a dissociation constant of 613 nM. Despite the decrease in driving force, the amplitude of the outwards I(NCX) at +60 mV gradually increased along with the positive staircase. The model simulation suggested that this increase of outwards I(NCX) is caused by a dramatic increase in Ca(2+)-mediated activation of NCX.     

Ca2+ entry through cardiac L-type Ca2+ channels modulates β-adrenergic stimulation in mouse ventricular myocytes

 β-adrenergic receptor (β-AR) stimulation increases cardiac L-type Ca²⁺ channel (CaCh) currents via cAMP-dependent phosphorylation. We report here that the affinity and maximum response of CaCh to isoproterenol (Iso), in mouse ventricular myocytes were significantly higher when Ba²⁺ was used as the charge carrier (I _Ba) instead of Ca²⁺ (I _Ca). The EC₅₀ and maximum increase of peak currents were 43.7 ± 7.9 nM and 1.8 ± 0.1-fold for I _Ca and 23.3 ± 4.7 nM and 2.4 ± 0.1-fold for I _Ba. When cells were dialyzed with the faster Ca²⁺ chelator, BAPTA, both sensitivity and maximum response of I _Ca to Iso were significantly augmented compared to cells with EGTA (EC₅₀ of 23.1 ± 5.2 nM and maximal increase of 2.2 ± 0.1-fold). Response of I _Ca to forskolin was also significantly increased when cells were dialyzed with BAPTA or when currents were measured in Ba²⁺. In contrast, depletion of the sarcoplasmic reticulum (SR) Ca²⁺ stores by ryanodine did not alter sensitivity of I _Ca to Iso or forskolin. These results suggest that the Ca²⁺ entering through CaCh regulates cAMP-dependent phosphorylation, and such negative feedback may play a significant role in cellular Ca²⁺ homeostasis and contraction in cardiac cells during β-AR stimulation. Received: 10 December 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

Stimulation of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by proteinase pretreatment

The Na+-Ca2+ exchange activity of purified canine cardiac sarcolemmal vesicles can be strikingly stimulated if the vesicles are pretreated with a serine or thiol proteinase. The Km (Ca2+) for Na+i-dependent Ca2+ influx is reduced from 22.2 +/- 2.3 to 8.1 +/- 0.3 microM while Vmax is increased from 15.1 +/- 3.6 to 18.9 +/- 5.2 nmol Ca2+ . mg protein-1 . s-1. Na+o-dependent Ca2+ efflux is also stimulated by proteinase pretreatment although passive (Na+-independent) Ca2+ efflux from the sarcolemmal vesicles is unaffected. Proteinase treatment reduces the sensitivity of Na+-Ca2+ exchange to the inhibitors chlorpromazine and polymyxin B, but not to the inhibitor, palmitylcarnitine. Using a newly developed technique we are able to demonstrate that the Na+-Ca2+ exchange of inside-out sarcolemmal vesicles is being stimulated by proteinase treatment (Philipson, K. D., and A. Y. Nishimoto, J. Biol. Chem: 257, 5111-5117, 1982; this technique uses the ATP-dependent Na+ pump to preload only inside-out vesicles with Na+ prior to Na+-Ca2+ exchange). Right-side-out vesicles may also be stimulated.