Effects of sodium and potassium channel blockers on hyperinflation-induced slowly adapting pulmonary stretch receptor stimulation in the rat

The excitatory responses of slowly adapting pulmonary stretch receptor (SAR) activity to hyperinflation (inflation volume = 3 tidal volumes) for approximately 10 respiratory cycles were examined before and after administration of flecainide, a Na+ channel blocker, and 4-aminoprydine (4-AP), a K+ channel blocker. The experiments were performed in anesthetized, artificially ventilated rats after unilateral vagotomy. During hyperinflation the SARs increased their activity during inflation and decreased their discharge during deflation. The magnitude of increased SAR activity during inflation became more prominent as compared to that of decreased receptor activity during deflation. Flecainide treatment (6 mg/kg) that was sufficient to block veratridine (50 microg/kg)-induced SAR stimulation did not significantly alter the excitatory response of SAR activity to hyperinflation. Subsequent administration of 3 mg/kg flecainide (a total dose, 9 mg/kg) resulted in a greater inhibition of hyperinflation-induced SAR stimulation. Although administration of 4-AP (2 mg/kg) usually stimulated SAR activity, particularly in the deflation phase, in the control ventilation, 4-AP treatment had no significant effect on hyperinflation-induced SAR stimulation. These results suggest that the excitatory effect of hyperinflation on SAR activity may not be involved in the activation of either flecainide-sensitive Na+ channels or 4-AP-sensitive K+ channels.     

Effects of ouabain and flecainide on CO(2)-induced slowly adapting pulmonary stretch receptor inhibition in the rabbit

The inhibitory effect of CO2 on slowly adapting pulmonary stretch receptors (SARs) was examined before and after administration of ouabain, a Na+-K+ ATPase inhibitor, and flecainide, a Na+ channel blocker. The experiments were performed in anesthetized, artificially ventilated rabbits after vagus nerve section. CO2 inhalation (maximal tracheal CO2 concentration ranging from 9.2 % to 10.4%) for about 60 sec decreased the receptor activity during both inflation and deflation. The magnitude of decreased SAR activity during deflation was greater than that seen during inflation. Administration of ouabain (25 microg/kg) initially stimulated SAR activities during inflation and deflation, and after 20 min, the SAR response was still kept excitatory in both inflation and deflation phases. Under these conditions, CO2 inhalation inhibited SAR activities during inflation and deflation. Flecainide treatment (3 mg/kg) that abolished veratridine (30 microg/kg)-induced SAR excitation had no significant effect on the inhibitory responses of SAR activity to CO2. These results suggest that the inhibitory effect of CO2 occurs when ouabain results in intracellular Na+ concentration ([Na+]i) increases in the SAR endings, and that CO2-induced SAR inhibition may not be related to the reduction of influx of Na+ through voltage-gated Na+ channels.     

Inhibitory mechanism of slowly adapting pulmonary stretch receptors after release from hyperinflation in anesthetized rabbits

In anesthetized, artificially ventilated rabbits with vagus nerve section, release from 10 consecutive hyperinflations (inflation volume = 3 tidal volume) caused an inhibition of the slowly adapting pulmonary stretch receptor (SAR) activity for 16-22 sec. Intravenous administration of tetraethylammonium (TEA, 10 and 20 mg/kg), a K+ channel blocker, did not significantly alter either basal SAR discharge or tracheal pressure (PT). Although TEA treatment at 10.0 mg/kg had no significant effect on the magnitude and duration of inhibited SAR activity seen after release from hyperinflation, the increasing dose of this K+ channel blocker up to 20 mg/kg inhibited these effects of the receptor activity but this inhibition was small. The Na+ -K+ ATPase inhibitor ouabain (5 and 10 microg/kg) that had no significant effect on SAR activity and P(T) in the control abolished or attenuated the inhibitory action of SARs in a dose-dependent manner. Furthermore, the changes in dynamic lung compliance (Cdyn) and P(T) in response to post-hyperinflation were not significantly influenced by pretreatment with either TEA or ouabain. These results suggest that the inhibitory action of receptors seen during post-hyperinflation corresponded with the induction of slow afterhyperpolarization (sAHP), and that the mechanism of generating the sAHP of SARs is mainly mediated by the activation of Na+ -K+ pump activity.     

Ionic mechanisms of cardiac cell swelling induced by blocking Na+/K+ pump as revealed by experiments and simulation

Although the Na(+)/K(+) pump is one of the key mechanisms responsible for maintaining cell volume, we have observed experimentally that cell volume remained almost constant during 90 min exposure of guinea pig ventricular myocytes to ouabain. Simulation of this finding using a comprehensive cardiac cell model (Kyoto model incorporating Cl(-) and water fluxes) predicted roles for the plasma membrane Ca(2+)-ATPase (PMCA) and Na(+)/Ca(2+) exchanger, in addition to low membrane permeabilities for Na(+) and Cl(-), in maintaining cell volume. PMCA might help maintain the [Ca(2+)] gradient across the membrane though compromised, and thereby promote reverse Na(+)/Ca(2+) exchange stimulated by the increased [Na(+)](i) as well as the membrane depolarization. Na(+) extrusion via Na(+)/Ca(2+) exchange delayed cell swelling during Na(+)/K(+) pump block. Supporting these model predictions, we observed ventricular cell swelling after blocking Na(+)/Ca(2+) exchange with KB-R7943 or SEA0400 in the presence of ouabain. When Cl(-) conductance via the cystic fibrosis transmembrane conductance regulator (CFTR) was activated with isoproterenol during the ouabain treatment, cells showed an initial shrinkage to 94.2 +/- 0.5%, followed by a marked swelling 52.0 +/- 4.9 min after drug application. Concomitantly with the onset of swelling, a rapid jump of membrane potential was observed. These experimental observations could be reproduced well by the model simulations. Namely, the Cl(-) efflux via CFTR accompanied by a concomitant cation efflux caused the initial volume decrease. Then, the gradual membrane depolarization induced by the Na(+)/K(+) pump block activated the window current of the L-type Ca(2+) current, which increased [Ca(2+)](i). Finally, the activation of Ca(2+)-dependent cation conductance induced the jump of membrane potential, and the rapid accumulation of intracellular Na(+) accompanied by the Cl(-) influx via CFTR, resulting in the cell swelling. The pivotal role of L-type Ca(2+) channels predicted in the simulation was demonstrated in experiments, where blocking Ca(2+) channels resulted in a much delayed cell swelling.     

Systems analysis of digoxin kinetics and inotropic response in the rat heart: effects of calcium and KB-R7943

To gain more insight into the mechanistic processes controlling the kinetics of inotropic response of digoxin in the perfused whole heart, an integrated kinetic model was developed incorporating digoxin uptake, receptor binding (Na(+)-K(+)-ATPase inhibition), and cellular events linking receptor occupation and response. The model was applied to data obtained in the single-pass Langendorff-perfused rat heart for external [Ca(2+)] of 0.5 and 1.5 mM under control conditions and in the presence of the reverse-mode Na(+)/Ca(2+) exchange inhibitor KB-R7943 (0.1 microM) in perfusate. Outflow concentration and left ventricular developed pressure data measured for three consecutive doses (15, 30, and 45 microg) in each heart were analyzed simultaneously. While disposition kinetics of digoxin was determined by interaction with a heterogeneous receptor population consisting of a high-affinity/low-capacity and a low-affinity/high- capacity binding site, response generation was >80% mediated by binding to the high-affinity receptor. Digoxin sensitivity increased at lower external [Ca(2+)] due to higher stimulus amplification. Coadministration of KB-R7943 significantly reduced the positive inotropic effect of digoxin at higher doses (30 and 45 microg) and led to a saturated and delayed receptor occupancy-response relationship in the cellular effectuation model. The results provide further evidence for the functional heterogeneity of the Na(+)-K(+)-ATPase and suggest that in the presence of KB-R7943 a reduction of the Ca(2+) influx rate via the reverse mode Na(+)/Ca(2+) exchanger might become the limiting factor in digoxin response generation.     

alpha-Adrenoceptor stimulation-mediated negative inotropism and enhanced Na(+)/Ca(2+) exchange in mouse ventricle

Mechanisms underlying the negative inotropic response to alpha-adrenoceptor stimulation in adult mouse ventricular myocardium were studied. In isolated ventricular tissue, phenylephrine (PE), in the presence of propranolol, decreased contractile force by approximately 40% of basal value. The negative inotropic response was similarly observed under low extracellular Ca(2+) concentration ([Ca(2+)](o)) conditions but was significantly smaller under high-[Ca(2+)](o) conditions and was not observed under low-[Na(+)](o) conditions. The negative inotropic response was not affected by nicardipine, ryanodine, ouabain, or dimethylamiloride (DMA), inhibitors of L-type Ca(2+) channel, Ca(2+) release channel, Na(+)-K(+) pump, or Na(+)/H(+) exchanger, respectively. KB-R7943, an inhibitor of Na(+)/Ca(2+) exchanger, suppressed the negative inotropic response mediated by PE. PE reduced the magnitude of postrest contractions. PE caused a decrease in duration of the late plateau phase of action potential and a slight increase in resting membrane potential; time courses of these effects were similar to that of the negative inotropic effect. In whole cell voltage-clamped myocytes, PE increased the L-type Ca(2+) and Na(+)/Ca(2+) exchanger currents but had no effect on the inwardly rectifying K(+), transient outward K(+), or Na(+)-K(+)-pump currents. These results suggest that the sustained negative inotropic response to alpha-adrenoceptor stimulation of adult mouse ventricular myocardium is mediated by enhancement of Ca(2+) efflux through the Na(+)/Ca(2+) exchanger.     

Na+/Ca2+ exchanger activity modulates connective tissue growth factor mRNA expression in transforming growth factor beta1- and Des-Arg10-kallidin-stimulated myofibroblasts

Transforming growth factor (TGF)-beta and des-Arg(10)-kallidin stimulate the expression of connective tissue growth factor (CTGF), a matrix signaling molecule that is frequently overexpressed in fibrotic disorders. Because the early signal transduction events regulating CTGF expression are unclear, we investigated the role of Ca(2+) homeostasis in CTGF mRNA expression in TGF-beta1- and des-Arg(10)-kallidin-stimulated human lung myofibroblasts. Activation of the kinin B1 receptor with des-Arg(10)-kallidin stimulated a rise in cytosolic Ca(2+) that was extracellular Na(+)-dependent and extracellular Ca(2+)-dependent. The des-Arg(10)-kallidin-stimulated increase of cytosolic Ca(2+) was blocked by KB-R7943, a specific inhibitor of Ca(2+) entry mode operation of the plasma membrane Na(+)/Ca(2+) exchanger. TGF-beta1 similarly stimulated a KB-R7943-sensitive increase of cytosolic Ca(2+) with kinetics distinct from the des-Arg(10)-kallidin-stimulated Ca(2+) response. We also found that KB-R7943 or 2',4'-dichlorobenzamil, an amiloride analog that inhibits the Na(+)/Ca(2+) exchanger activity, blocked the TGF-beta1- and des-Arg(10)-kallidin-stimulated increases of CTGF mRNA. Pretreatment with KB-R7943 also reduced the basal and TGF-beta1-stimulated levels of alpha1(I) collagen and alpha smooth muscle actin mRNAs. These data suggest that, in addition to regulating ion homeostasis, Na(+)/Ca(2+) exchanger acts as a signal transducer regulating CTGF, alpha1(I) collagen, and alpha smooth muscle actin expression. Consistent with a more widespread role for Na(+)/Ca(2+) exchanger in fibrogenesis, we also observed that KB-R7943 likewise blocked TGF-beta1-stimulated levels of CTGF mRNA in human microvascular endothelial and human osteoblast-like cells. We conclude that Ca(2+) entry mode operation of the Na(+)/Ca(2+) exchanger is required for des-Arg(10)-kallidin- and TGF-beta1-stimulated fibrogenesis and participates in the maintenance of the myofibroblast phenotype.     

Involvement of Na(+)/Ca(2+) exchanger in endothelial NO production and endothelium-dependent relaxation

Endothelial nitric oxide (NO) synthase (eNOS) is controlled by Ca(2+)/calmodulin and caveolin-1 in caveolae. It has been recently suggested that Na(+)/Ca(2+) exchanger (NCX), also expressed in endothelial caveolae, is involved in eNOS activation. To investigate the role played by NCX in NO synthesis, we assessed the effects of Na(+) loading (induced by monensin) on rat aortic rings and cultured porcine aortic endothelial cells. Effect of monensin was evaluated by endothelium-dependent relaxation of rat aortic rings in response to acetylcholine and by real-time measurement of NO release from cultured endothelial cells stimulated by A-23187 and bradykinin. Na(+) loading shifted the acetylcholine concentration-response curve to the left. These effects were prevented by pretreatment with the NCX inhibitors benzamil and KB-R7943. Monensin potentiated Ca(2+)-dependent NO release in cultured cells, whereas benzamil and KB-R7943 totally blocked Na(+) loading-induced NO release. These findings confirm the key role of NCX in reverse mode on Ca(2+)-dependent NO production and endothelium-dependent relaxation.     

The Na+/Ca2+ exchanger is active and working in the reverse mode in human umbilical artery smooth muscle cells

The data presented in this work suggest that in human umbilical artery (HUA) smooth muscle cells, the Na(+)/Ca(2+) exchanger (NCX) is active and working in the reverse mode. This supposition is based on the following results: (i) microfluorimetry in HUA smooth muscle cells in situ showed that a Ca(2+)-free extracellular solution diminished intracellular Ca(2+) ([Ca(2+)](i)), and KB-R7943 (5microM), a specific inhibitor of the Ca(2+) entry mode of the exchanger, also decreased [Ca(2+)](i) (40.6+/-4.5% of Ca(2+)-free effect); (ii) KB-R7943 produced the relaxation of HUA rings (-24.7+/-7.3gF/gW, n=8, p<0.05); (iii) stimulation of the NCX by lowering extracellular Na(+) increases basal [Ca(2+)](i) proportionally to Na(+) reduction (Delta fluorescence ratio=0.593+/-0.141 for Na(+)-free solution, n=8) and HUA rings' contraction (peak force=181.5+/-39.7 for 130mM reduction, n=8), both inhibited by KB-R7943 and a Ca(2+)-free extracellular solution. In conclusion, the NCX represents an important Ca(2+) entry route in HUA smooth muscle cells.     

Functional coupling between the Na+/Ca2+ exchanger and nonselective cation channels during histamine stimulation in guinea pig tracheal smooth muscle

Airway smooth muscle (ASM) contracts partly due to an increase in cytosolic Ca(2+). In this work, we found that the contraction caused by histamine depends on external Na(+), possibly involving nonselective cationic channels (NSCC) and the Na(+)/Ca(2+) exchanger (NCX). We performed various protocols using isometric force measurement of guinea pig tracheal rings stimulated by histamine. We observed that force reached 53 +/- 1% of control during external Na(+) substitution by N-methyl-D-glucamine(+), whereas substitution by Li(+) led to no significant change (91 +/- 1%). Preincubation with KB-R7943 decreased the maximal force developed (52.3 +/- 5.6%), whereas preincubation with nifedipine did not (89.7 +/- 1.8%). Also, application of the nonspecific NCX blocker KB-R7943 and nifedipine on histamine-precontracted tracheal rings reduced force to 1 +/- 3%, significantly different from nifedipine alone (49 +/- 6%). Moreover, nonspecific NSCC inhibitors SKF-96365 and 2-aminoethyldiphenyl borate reduced force to 1 +/- 1% and 19 +/- 7%, respectively. Intracellular Ca(2+) measurements in isolated ASM cells showed that KB-R7943 and SKF-96365 reduced the peak and sustained response to histamine (0.20 +/- 0.1 and 0.19 +/- 0.09 for KB-R, 0.43 +/- 0.16 and 0.47 +/- 0.18 for SKF, expressed as mean of differences). Moreover, Na(+)-free solution only inhibited the sustained response (0.54 +/- 0.25). These data support an important role for NSCC and NCX during histamine stimulation. We speculate that histamine induces Na(+) influx through NSCC that promotes the Ca(2+) entry mode of NCX and Ca(V)1.2 channel activation, thereby causing contraction.     

Reverse Na(+)/Ca(2+)-exchange mediated Ca(2+)-entry and noradrenaline release in Na(+)-loaded peripheral sympathetic nerves

[(3)H]noradrenaline ([(3)H]NA) released from sympathetic nerves in the isolated main pulmonary artery of the rabbit was measured in response to field stimulation (2Hz, 1ms, 60V for 3min) in the presence of uptake blockers (cocaine, 3 x10(-5)M and corticosterone, 5 x10(-5)M). The [(3)H]NA-release was fully blocked by the combined application of the selective and irreversible 'N-type' voltage-sensitive Ca(2+)-channel (VSCC)-blocker omega-conotoxin (omega-CgTx) GVIA (10(-8)M) and the 'non-selective' VSCC-blocker aminoglycoside antibiotic neomycin (3x10(-3)M). Na(+)-loading (Na(+)-pump inhibition by K(+)-free perfusion) was required to elicit further NA-release after blockade of VSCCs (omega-CgTx GVIA+neomycin). In K(+)-free solution, in the absence of functioning VSCCs (omega-CgTx GVIA+neomycin), the fast Na(+)-channel activator veratridine (10(-5)M) further potentiated the nerve-evoked release of [(3)H]NA. This NA-release was significantly inhibited by KB-R7943, and fully blocked by Ca(o)(2+)-removal. However, Li(+)-substitution was surprisingly ineffective. The non-selective K(+)-channel blocker 4-aminopyridine (4-AP, 10(-4)M) also further potentiated the nerve-evoked release of NA in K(+)-free solution. This potentiated release was concentration-dependently inhibited by KB-R7943, significantly inhibited by Li(+)-substitution and abolished by Ca(o)(2+)-removal. It is concluded that in Na(+)-loaded sympathetic nerves, in which the VSCCs are blocked, the reverse Na(+)/Ca(2+)-exchange-mediated Ca(2+)-entry is responsible for transmitter release on nerve-stimulation. Theoretically we suppose that the fast Na(+)-channel and the exchanger proteins are close to the vesicle docking sites.     

Instrumental role of Na+ in NMDA excitotoxicity in glucose-deprived and depolarized cerebellar granule cells

In glucose-deprived cerebellar granule cells, substitution of extracellular Na+ with Li+ or Cs+ prevented N-methyl-D-aspartate (NMDA)-induced excitotoxicity. NMDA stimulated 45Ca2+ accumulation and ATP depletion in a Na-dependent manner, and caused neuronal death, even if applied while Na,K-ATPase was inhibited by 1 mM ouabain. The cells treated with NMDA in the presence of ouabain accumulated sizable 45Ca2+ load but most of them failed to elevate cytosolic [Ca2+] upon mitochondrial depolarization. Na/Ca exchange inhibitor, KB-R7943, inhibited Na-dependent and NMDA-induced 45Ca2+ accumulation but only if Na,K-ATPase activity was compromised by ouabain. In cells energized by glucose and exposed to NMDA without ouabain, KB-R7943 reduced NMDA-elicited ionic currents by 19% but failed to inhibit 45Ca2+ accumulation. It appears that a large part of NMDA-induced Ca2+ influx in depolarized and glucose-deprived cells is mediated by reverse Na/Ca exchange. A high level of reverse Na/Ca exchange operation is maintained by a sustained Na+ influx via NMDA channels and depolarization of the plasma membrane. In cells energized by glucose, however, most Ca2+ enters directly via NMDA channels because Na,K-ATPase regenerating Na+ and K+ concentration gradients prevents Na/Ca exchange reversal. Since under these conditions Na/Ca exchange extrudes Ca2+, its inhibition destabilizes Ca2+ homeostasis.     

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 microM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 microM) or Ni2+ (>100 microM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 microM but did at 30 microM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 microM), Ni2+ (300 microM), or KB-R7943 (30 microM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.     

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.     

KB-R7943 inhibits store-operated Ca(2+) entry in cultured neurons and astrocytes

We have studied cyclopiazonic acid (CPA)-sensitive store-operated Ca(2+) entry (SOCE) in cultured neurons and astrocytes and examined the effect of 2-[2-[4-(4-nitrobenzyloxy)phenyl]]isothiourea (KB-R7943), which is often used as a selective inhibitor of the Na(+)-Ca(2+) exchanger (NCX), on the SOCE. CPA increased transiently intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a sustained increase in [Ca(2+)](i) in neurons and astrocytes. The sustained increase in [Ca(2+)](i) depended on the presence of extracellular Ca(2+) and inhibited by SOCE inhibitors, but not by a Ca(2+) channel inhibitor. CPA also caused quenching of fura-2 fluorescence when the cells were incubated in Mn(2+)-containing medium. KB-R7943 at 10 microM inhibited significantly CPA-induced sustained increase in [Ca(2+)](i) in neurons and astrocytes. KB-R7943 also inhibited CPA-induced quenching of fura-2 fluorescence in the presence of extracellular Mn(2+). These results indicate that cultured neurons and astrocytes possess SOCE and that KB-R7943 inhibits not only NCX but also SOCE.     

增强Na+-Ca2+交换对大鼠心脏的正性变力作用及对哇巴因效应的加强

本文采用大鼠乳头肌张力测定及离体心脏灌流技术,研究大鼠心肌Na -Ca2 交换对乳头肌及离体灌流心肌变力性的影响。采用大鼠特异性Na -Ca2 交换激动剂E-4031能剂量依赖性地增加大鼠乳头肌的发展张力(P<0.05,n=6)及离体心脏的心泵功能(P<0.05,n=4);特异性Na -Ca2 交换抑制剂KB-R7943具有相反的效应,并可完全消除E-4031引起的正性变力作用。哇巴因(ouabain,0.5μmol/L)与E-4031(3μmol/L)联合使用,可使乳头肌发展张力由单独使用哇巴因时的0.25±0.03 g升高至0.29±0.04g(P<0.05,n=6);联合用药对大鼠离体心脏心泵功能的影响也强于哇巴因单独作用的效果。本研究结果证实,E-4031通过增强心肌Na -Ca2 交换,对大鼠乳头肌和离体心脏产生正性变力作用;与哇巴因合用时,它们的正性变力作用有相加作用。     

Mechanistic distinction between activation and inhibition of (Na(+)+K(+))-ATPase-mediated Ca2+ influx in cardiomyocytes

(Na(+)+K(+))-ATPase (NKA) mediates positive inotropy in the heart. Extensive studies have demonstrated that the reverse-mode Na(+)/Ca(2+)-exchanger (NCX) plays a critical role in increasing intracellular Ca(2+) concentration through the inhibition of NKA-induced positive inotropy by cardiac glycosides. Little is known about the nature of the NCX functional mode in the activation of NKA-induced positive inotropy. Here, we examined the effect of an NKA activator SSA412 antibody on (45)Ca influx in isolated rat myocytes and found that KB-R7943, a NCX reverse-mode inhibitor, fails to inhibit the activation of NKA-induced (45)Ca influx, suggesting that the Ca(2+) influx via the reverse-mode NCX does not mediate this process. Nifedipine, an L-type Ca(2+) channel (LTCC) inhibitor, completely blocks the activation of NKA-induced (45)Ca influx, suggesting that the LTCC is responsible for the moderate increase in intracellular Ca(2+). In contrast, the inhibition of NKA by ouabain induces 4.7-fold (45)Ca influx compared with the condition of activation of NKA. Moreover, approximately 70% of ouabain-induced (45)Ca influx was obstructed by KB-R7943 and only 30% was impeded by nifedipine, indicating that both the LTCC and the NCX contribute to the rise in intracellular Ca(2+) and that the NCX reverse-mode is the major source for the (45)Ca influx induced by the inhibition of NKA. This study provides direct evidence to demonstrate that the activation of NKA-induced Ca(2+) increase is independent of the reverse-mode NCX and pinpoints a mechanistic distinction between the activation and inhibition of the NKA-mediated Ca(2+) influx path ways in cardiomyocytes.     

The reverse mode of the Na(+)/Ca(2+) exchanger provides a source of Ca(2+) for store refilling following agonist-induced Ca(2+) mobilization

Agonist-induced contraction of airway smooth muscle (ASM) can be triggered by an elevation in the intracellular Ca(2+) concentration, primarily through the release of Ca(2+) from the sarcoplasmic reticulum (SR). The refilling of the SR is integral for subsequent contractions. It has been suggested that Ca(2+) entry via store-operated cation (SOC) and receptor-operated cation channels may facilitate refilling of the SR. Indeed, depletion of the SR activates substantial inward SOC currents in ASM that are composed of both Ca(2+) and Na(+). Accumulation of Na(+) within the cell may regulate Ca(2+) handling in ASM by forcing the Na(+)/Ca(2+) exchanger (NCX) into the reverse mode, leading to the influx of Ca(2+) from the extracellular domain. Since depletion of the SR activates substantial inward Na(+) current, it is conceivable that the reverse mode of the NCX may contribute to the intracellular Ca(2+) pool from which the SR is refilled. Indeed, successive contractions of bovine ASM, evoked by various agonists (ACh, histamine, 5-HT, caffeine) were significantly reduced upon removal of extracellular Na(+); whereas contractions evoked by KCl were unchanged by Na(+) depletion. Ouabain, a selective inhibitor of the Na(+)/K(+) pump, had no effect on the reductions observed under normal and zero-Na(+) conditions. KB-R7943, a selective inhibitor of the reverse mode of the NCX, significantly reduced successive contractions induced by all agonists without altering KCl responses. Furthermore, KB-R7943 abolished successive caffeine-induced Ca(2+) transients in single ASM cells. Together, these data suggest a role for the reverse mode of the NCX in refilling the SR in ASM following Ca(2+) mobilization.