Influence of a Na+-H+ exchange inhibitor ethylisopropylamiloride, a Na+-Ca2+ exchange inhibitor KB-R7943 and their combination on the increases in contractility and Ca2+ transient induced by angiotensin II in isolated adult rabbit ventricular myocytes

In rabbit, ventricular myocytes loaded with indo-1/AM, angiotensin II (0.1 nM-0.1 microM) exerted a positive inotropic effect with a significant increase in the amplitude of Ca2+ transients. For a given increase in cell shortening, the increase in Ca2+ transients induced by angiotensin II was less than that induced by elevation of extracellular Ca2+ concentration ([Ca2+]0) or isoprenaline, an indication that both the increase in mobilization of intracellular Ca2+ ions and myofibrillar sensitivity to Ca2+ ions contribute to the positive inotropic effect of angiotensin II. The effects of angiotensin II on Ca2+ transients and cell shortening were inhibited by the AT1 receptor antagonist losartan. A Na+ -H+ exchange inhibitor EIPA [5-(N-ethyl-N-isopropyl)amiloride] at 1 and 3 microM did not affect the Ca2+ transients and cell shortening, but it inhibited the angiotensin-II-induced responses in a concentration-dependent manner more effectively than the responses to elevation of [Ca2+]0, indicating that EIPA elicited a selective inhibitory action on the effects of angiotensin II. The observation that EIPA at 10 microM abolished the positive inotropic effect of angiotensin II without a significant depression of the inotropic response to elevation of [Ca2+]0 supports the selective action of EIPA at the high concentration on the response to angiotensin II. A novel selective Na+ -Ca2+ exchange (reverse mode) inhibitor KB-R7943, 2-[2-[4-(-nitrobenzyloxy)phenyl]ethyl] isothiourea methanesulphonate, at 0.3 and 1 microM inhibited also the responses to angiotensin II more effectively than the response to elevation of [Ca2+]0; however, over the same concentration range it suppressed significantly the amplitude of Ca2+ transients and cell shortening. Combination of EIPA (3 microM) and KB-R7943 (0.3 microM), each of which attenuated partially the angiotensin-II-induced responses, abolished the positive inotropic effect and the increase in Ca2+ transients induced by angiotensin II with much less depressant effect on the responses to elevation of [Ca2+]0. Thus, these ion exchange inhibitors exerted selective actions on the respective targets. The results with these selective inhibitors indicate that the activation of Na+ -H+ exchanger and subsequent modulation of the activity of Na+ -Ca2+ exchanger may be responsible for the increase in [Ca2+]i and the myofilament Ca2+ sensitization induced by stimulation of AT1 receptors by angiotensin II in rabbit ventricular myocytes.     

Role of Na+/Ca2+ exchange in endothelin-1-induced increases in Ca2+ transient and contractility in rabbit ventricular myocytes: pharmacological analysis with KB-R7943.

1. The effects of endothelin-1 (ET-1) on intracellular Ca2+ ion level and cell contraction were simultaneously investigated in rabbit ventricular cardiac myocytes loaded with indo-1/A1. The role of Na+/Ca2+ exchange in ET-1-induced positive inotropic effect (PIE) was examined by using KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate), a selective inhibitor of reverse mode Na+/Ca2+ exchange. 2. ET-1 at 0.3 pM - 1 nM increased cell contraction and Ca2+ transient (CaT) with EC50 values of 2.9 pM and 1.2 pM, respectively, and the increase in amplitude of CaT was much smaller relative to the PIE: ET-1 at 1 nM increased peak cell shortening by 237%, while it increased peak CaT by 167%. For a given level of PIE, ET-1-induced increase in CaT was much smaller than that induced by elevation of [Ca2+]o and by isoprenaline. Therefore, ET-1 shifted the relationship between peak CaT and cell shortening to the left relative to the relationship for increase in [Ca2+]o, an indication that ET-1 increased myofibrillar Ca2+ sensitivity. 3. KB-R7943 at 0.1 microM and higher inhibited contraction and CaT induced by 0.1 nM ET-1 and at 0.3 microM it abolished the increase in CaT while inhibiting the PIE by 48.1%. Over concentration range of 0.1-0.3 microM, KB-R7943 neither inhibited baseline contraction and CaT nor the isoprenaline-induced response, although at 1 microM and higher it had a significant inhibitory action on these responses. 4. These results indicate that in rabbit ventricular myocytes both increases in CaT and myofibrillar Ca2+ sensitivity contribute to the ET-induced PIE, and the activation of reverse mode Na+/Ca2+ exchange may play a crucial role in increase in CaT induced by ET-1 in rabbit ventricular cardiac myocytes.     

Endothelin-1 decreases [Ca2+]i via Na+/Ca2+ exchanger in CHO cells stably expressing endothelin ETA receptor

Endothelin ET(A) receptor couples to Gq/11 protein that transduces a variety of receptor signals to modulate diverse cellular responses including Ca2+ mobilization. Stimulation of endothelin ETA receptor with endothelin-1 is generally believed to induce an increase in intracellular Ca2+ concentration ([Ca2+]i) via Gq/11 protein. Here we provide the first convincing evidence that endothelin-1 elicited Gq/11 protein-dependent and -independent 'decrease' in [Ca2+]i via Na+/Ca2+ exchanger (NCX) in Chinese hamster ovary (CHO) cells stably expressing human endothelin ETA receptor. In the cells treated with 1 microM thapsigargin, an inhibitor of endoplasmic Ca2+ pump, that induces an increase in [Ca2+]i via capacitative Ca2+ entry, endothelin-1 induced a decrease in [Ca2+]i which was partially inhibited by YM-254890, a specific inhibitor of Gq/11, indicating that Gq/11-dependent and independent pathways are involved in the decrease. The endothelin-1-induced decrease in [Ca2+]i was markedly suppressed by 3',4'-dichlorobenzamil hydrochloride, a potent NCX inhibitor, and also by a replacement of extracellular Na+ with Li+, which was not transported by NCX, indicating a major role of NCX operating in the forward mode in the endothelin-1-induced decrease in [Ca2+]i. Molecular approach with RT-PCR demonstrated the expression of mRNA for NCX1, NCX2 and NCX3. These results suggest that stimulation of endothelin ETA receptor with endothelin-1 activates the forward mode NCX through Gq/11-dependent and -independent mechanisms: the NCX exports Ca2+ out of the cell depending on Na+ gradient across the cell membrane, resulting in the decrease in [Ca2+]i.     

Protection of ischemic rat spinal cord white matter: Dual action of KB-R7943 on Na+/Ca2+ exchange and L-type Ca2+ channels

The effect of the Na+/Ca(2+)-exchange inhibitor KB-R7943 was investigated in spinal cord dorsal column ischemia in vitro. Oxygen/glucose deprivation at 37 degrees C for 1 h causes severe injury even in the absence of external Ca2+. KB-R7943 was very protective in the presence and absence of external Ca2+ implicating mechanisms in addition to extracellular Ca2+ influx through Na+/Ca(2+)-exchange, such as activation of ryanodine receptors by L-type Ca2+ channels. Indeed, blockade of L-type Ca2+ by nimodipine confers a certain degree of protection of dorsal column against ischemia; combined application of nimodipine and KB-R7943 was not additive suggesting that KB-R7943 may also act on Ca2+ channels. KB-R7943 reduced inward Ba2+ current with IC50 = 7 microM in tsA-201 cells expressing Ca(v)1.2. Moreover, nifedipine and KB-R7943 both reduced depolarization-induced [Ca2+]i increases in forebrain neurons and effects were not additive. Nimodipine or KB-R7943 also reduced ischemic axoplasmic Ca2+ increase, which persisted in 0Ca2+/EGTA perfusate in dorsal column during ischemia. While KB-R7943 cannot be considered to be a specific Na+/Ca2+ exchange inhibitor, its profile makes it a very useful neuroprotectant in dorsal columns by: reducing Ca2+ import through reverse Na+/Ca2+ exchange; reducing influx through L-type Ca2+ channels, and indirectly inhibiting Ca2+ release from the ER through activation of ryanodine receptors.     

Endothelial nitric oxide attenuates Na+/Ca2+ exchanger-mediated vasoconstriction in rat aorta

BACKGROUND AND PURPOSE: The Na+/Ca2+ exchanger (NCX) may be an important modulator of Ca2+ entry and exit. The present study investigated whether NCX was affected by prostacyclin and nitric oxide (NO) released from the vascular endothelium, as NCX contains phosphorylation sites for PKA and PKG. EXPERIMENTAL APPROACH: Rat aortic rings were set up in organ baths. Tension was measured across the ring with a force transducer. KEY RESULTS: Lowering extracellular [Na+] ([Na+]o) to 1.18 mM induced vasoconstriction in rat endothelium-denuded aortic rings. This effect was blocked by the NCX inhibitor KB-R7943 (2-2-[4-(4-nitrobenzyloxy)phenyl] ethyl isothiourea methanesulphonate; 1 microM). In endothelium-intact aortic rings, decreasing [Na+]o did not constrict the aortic rings significantly, but after treatment with the guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 1 microM) or the NOS inhibitor L-NAME (N(omega)-nitro-L-arginine methyl ester; 50 microM), a vasoconstriction that was similar in size to that in endothelium-denuded preparations was evident. The vasorelaxation induced by the NO donor sodium nitroprusside sodium nitroprusside dihydrate (30 nM) was the same in the endothelium-denuded aortic rings preconstricted with either low Na+ (1.18 mM), the thromboxane A2 agonist U46619 (9,11-dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F(2alpha); 0.1 microM) or high K+ (80 mM). CONCLUSIONS AND IMPLICATIONS: The results suggest that the endothelium inhibits NCX operation via guanylate cyclase/NO. This is stronger than for other constrictors such as phenylephrine and may relate to concomitant NCX-stimulated NO release from the endothelium. This finding may be important where NCX operates in reverse mode, such as during ischaemia, and highlights a new mechanism whereby the endothelium modulates Ca2+ homoeostasis in vascular smooth muscle.     

Quantitative fluorescence measurement of cardiac Na+/Ca2+ exchanger inhibition by kinetic analysis in stably transfected HEK293 cells

We developed a method to quantitatively evaluate the potency of Na+/Ca2+ exchanger (NCX) inhibitors with fluorescence microscopy in NCX1-transfected HEK 293 cells. The reverse mode and forward mode NCX activities were measured as the ascending slope of the early phase increase in cytoplasmic Ca2+ concentration after change to low Na+ extracellular solution and the descending rate (inverse of the exponential time constant) on return to normal solution, respectively. Both modes of NCX were inhibited by SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline) and KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), and the concentration-inhibition relationships for both inhibitors were in good agreement with those previously reported in voltage clamped cardiomyocytes.     

Characterization of noradrenaline-induced increases in intracellular Ca2+ levels in Chinese hamster ovary cells stably expressing human alpha1A-adrenoceptor

The mechanism for noradrenaline (NA)-induced increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and physiological significance of Na(+) influx through receptor-operated channels (ROCs) and store-operated channels (SOCs) were studied in Chinese hamster ovary (CHO) cells stably expressing human alpha(1A)-adrenoceptor (alpha(1A)-AR). [Ca(2+)](i) was measured using the Ca(2+) indicator fura-2. NA (1 microM) elicited transient and subsequent sustained [Ca(2+)](i) increases, which were inhibited by YM-254890 (G(alphaq/11) inhibitor), U-73122 (phospholipase C (PLC) inhibitor), and bisindolylmaleimide I (protein kinase C (PKC) inhibitor), suggesting their dependence on G(alphaq/11)/PLC/PKC. Both phases were suppressed by extracellular Ca(2+) removal, SK&F 96365 (inhibitor of SOC and nonselective cation channel type-2 (NSCC-2)), LOE 908 (inhibitor of NSCC-1 and NSCC-2), and La(3+) (inhibitor of transient receptor potential canonical (TRPC) channel). Reduction of extracellular Na(+) and pretreatment with KB-R7943, a Na(+)/Ca(2+) exchanger (NCX) inhibitor, inhibited both phases of [Ca(2+)](i) increases. These results suggest that 1) stimulation of alpha(1A)-AR with NA elicits the transient and sustained increases in [Ca(2+)](i) mediated through NSCC-2 that belongs to a TRPC family; 2) Na(+) influx through these channels drives NCX in the reverse mode, causing Ca(2+) influx in exchange for Na(+) efflux; and 3) the G(alphaq/11)/PLC/PKC-dependent pathway plays an important role in the increases in [Ca(2+)](i).     

Effect of Na+/Ca2+ exchange inhibitor, KB-R7943 on ouabain-induced arrhythmias in guinea-pigs.

1. We investigated protective effects of KB-R7943, a Na+/Ca2+ exchange (NCX) inhibitor, on ouabain-induced tonotropy and arrhythmias in isolated whole atria and ouabain-induced changes in electrocardiogram (ECG) in the guinea-pig. 2. KB-R7943 (10 and 30 microM) suppressed the tonotropic effect of ouabain, and prolonged the onset time of extra-systole induced by ouabain in isolated atria. 3. The intravenous injection of KB-R7943 (1 and 3 mg kg-1) significantly increased the doses of ouabain required to induce ventricular premature beats (VPB), ventricular tachycardia (VT), ventricular fibrillation (VF) and cardiac arrest (CA) in anaesthetized guinea-pigs. 4. Lidocaine (Na+channel inhibitor) and R56865 (Na+ and Ca2+ overload inhibitor) also suppressed the ouabain-induced tonotropic effect and extra-systole in isolated atria, but Hoe-694 (Na+/H+ exchange inhibitor) or diltiazem (Ca2+ channel inhibitor) did not affect them. 5. Lidocaine also increased the doses of ouabain required to induce VPB, VT, VF and CA in anaesthetized guinea-pigs. 6. From these results, we conclude that KB-R7943 suppresses ouabain-induced arrhythmias through inhibition of the reverse-mode NCX.     

PPADS, a P2X receptor antagonist, as a novel inhibitor of the reverse mode of the Na⁺/Ca²⁺ exchanger in guinea pig airway smooth muscle

The Na(+)/Ca(2+)exchanger (NCX) principal function is taking 1 Ca(2+) out of the cytoplasm and introducing 3 Na(+). The increase of cytoplasmic Na(+) concentration induces the NCX reverse mode (NCX(REV)), favoring Ca(2+) influx. NCX(REV) can be inhibited by: KB-R7943 a non-specific compound that blocks voltage-dependent and store-operated Ca(2+) channels; SEA0400 that appears to be selective for NCX(REV), but difficult to obtain and SN-6, which efficacy has been shown only in cardiomyocytes. We found that PPADS, a P2X receptor antagonist, acts as a NCX(REV) inhibitor in guinea pig tracheal myocytes. In these cells, we characterized the NCX(REV) by substituting NaCl and NaHCO(3) with LiCl, resulting in the increase of the intracellular Ca(2+) concentration ([Ca(2+)]i) using fura 2-AM. We analyzed 5 consecutive responses of the NCX(REV) every 10 min, finding no differences among them. To evaluate the effect of different NCX(REV) blockers, concentration response curves to KB-R7943 (1, 3.2 and 10 μM), and SN-6 (3.2, 10 and 30 μM) were constructed, whereas PPADS effect was characterized as time- and concentration-dependent (1, 3.2, 10 and 30 μM). PPADS had similar potency and efficacy as KB-R7943, whereas SN-6 was the least effective. Furthermore, KCl-induced contraction, sensitive to D600 and nifedipine, was blocked by KB-R7943, but not by PPADS. KCl-induced [Ca(2+)]i increment in myocytes was also significantly decreased by KBR-7943 (10 μM). Our results demonstrate that PPADS can be used as a reliable pharmacological tool to inhibit NCX(REV), with the advantage that it is more specific than KB-R7943 because it does not affect L-type Ca(2+) channels.     

Positive inotropic effect of endothelin-1 in the neonatal mouse right ventricle

In neonatal mouse right ventricles, endothelin-1 (ET-1, 1-300 nM) induced a dose-dependent increase in twitch contractions and the dose-response curve was shifted to the right by BQ-123 (10 microM), an endothelin ET(A) receptor antagonist. The ET-1 (100 nM)-induced positive inotropy was accompanied by an increase in [Ca(2+)](i) transients without any change in the [Ca(2+)](i)-force relationship. Ryanodine (1 microM) partially decreased the [Ca(2+)](i) transients and contractile force, but did not affect the ET-1 (100 nM)-induced positive inotropy. Reduction of [Na(+)](o) elicited an increase in contractile force, and this effect was significantly inhibited by KB-R7943 (30 microM), an inhibitor of the Na(+)-Ca(2+) exchanger. KB-R7943 (30 microM) almost completely suppressed the positive inotropic effect of ET-1. Activation of protein kinase C (PKC) by phorbol 12,13-dibutylate (100 nM) decreased the contractile force, an effect which was suppressed by bisindolylmaleimide I (3 microM). On the other hand, the ET-1-induced positive inotropic effect was unaffected by bisindolylmaleimide I (3 microM). These results suggest that the positive inotropic effect of ET-1 in neonatal mouse right ventricles is caused by the increase in [Ca(2+)](i) transients through activation of the endothelin ET(A) receptor and the increase in Ca(2+) influx via the Na(+)-Ca(2+) exchanger during an action potential. Furthermore, the ET-1-induced positive inotropy is independent of the effects of PKC, which makes it distinct from the ET-1-mediated pathways reported for cardiac tissues in other species.     

Inhibition by SEA0400, a selective inhibitor of Na+/Ca2+ exchanger, of Na+ -dependent Ca2+ uptake and catecholamine release in bovine adrenal chromaffin cells

The effects of SEA0400, a selective inhibitor of the Na(+)/Ca(2+) exchanger (NCX), on Na(+)-dependent Ca(2+) uptake and catecholamine (CA) release were examined in bovine adrenal chromaffin cells that were loaded with Na(+) by treatment with ouabain and veratridine. SEA0400 inhibited Na(+)-dependent (45)Ca(2+) uptake and CA release, with the IC(50) values of 40 and 100 nM, respectively. The IC(50) values of another NCX inhibitor KB-R7943 were 1.8 and 3.7 microM, respectively. These results indicate that SEA0400 is about 40 times more potent than KB-R7943 in inhibiting NCX working in the reverse mode. In intact cells, SEA0400 and KB-R7943 inhibited CA release induced by acetylcholine and DMPP. The IC(50) values of SEA0400 were 5.1 and 4.5 microM and the values of KB-R7943 were 2.6 and 2.1 microM against the release induced by acetylcholine and DMPP, respectively, indicating that the potency of SEA0400 is about a half of that of KB-R7943 in inhibiting the nicotinic receptor-mediated CA release. The binding of [(3)H]nicotine with nicotinic receptors was inhibited by SEA0400 (IC(50) = 90 microM) and KB-R7943 (IC(50) = 12 microM). From these results, it is concluded that unlike KB-R7943, SEA0400 has a potent and selective action on NCX in bovine adrenal chromaffin cells.     

The Na(+)/Ca(2+) exchanger inhibitor KB-R7943 activates large-conductance Ca(2+)-activated K(+) channels in endothelial and vascular smooth muscle cells

The effect of the selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), KB-R7943, on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels was examined in cultured human umbilical vein endothelial cells (HUVECs) and freshly isolated mouse aortic smooth muscle cells (MASMCs). In voltage-clamped cells, KB-R7943 reversibly activated BK(Ca) currents in HUVECs and MASMCs. The EC(50) of KB-R7943 for BK(Ca) current activation in HUVECs was determined to be 6.78+/-0.7 microM. In inside-out and outside-out patches, KB-R7943 markedly increased BK(Ca) channel activity and slightly decreased single channel current amplitudes. In inside-out patches, KB-R7943 shifted the relationship between [Ca(2+)](i) and open probability (P(o)) to the left; the [Ca(2+)](i) required to evoke half-maximal activation changed from 1220+/-68 nM (in the absence of KB-R7943) to 620+/-199 nM (in the presence of 10 microM KB-R7943). In addition, KB-R7943 shifted the relationship between membrane potential and P(o) to the left; the membrane potential to evoke half-maximal activation changed from 76.86+/-1.09 mV (in the absence of KB-R7943) to 49.62+/-2.55 mV (in the presence of 10 microM KB-R7943). In conclusion, KB-R7943 was found to act as a potent BK(Ca) channel activator, which increases the sensitivity of BK(Ca) channels to cytosolic free Ca(2+) and membrane potential, and thereby BK(Ca) channel activity. These results should be considered when KB-R7943 is used as NCX blocker.     

A Na+/Ca2+ exchanger inhibitor, KB-R7943, caused negative inotropic responses and negative followed by positive chronotropic responses in isolated, blood-perfused dog heart preparations

Effects of a Na+/Ca2+ exchanger inhibitor, KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl] isothiourea methanesulfonate), on the sinoatrial nodal pacemaker activity, atrial contractility and ventricular contractility were investigated in the isolated and blood-perfused right atrium and left ventricle of the dog. KB-R7943 (0.03- 3 micromol) induced negative inotropic effects and negative followed by positive chronotropic effects in the right atrium and negative inotropic effects in the left ventricle. Neither atropine nor hexamethonium affected the cardiac responses to KB-R7943. Propranolol attenuated the positive chronotropic response to KB-R7943 but imipramine did not. Tetrodotoxin potentiated the positive chronotropic response to KB-R7943 in 6 of 11 isolated atria. When NaCl infusion increased atrial contractile force and atrial rate, KB-R7943-induced negative inotropic and chronotropic responses were attenuated in a dose-dependent manner. CaCl2 infusion potentiated the negative chronotropic response to KB-R7943 but did not affect the inotropic response significantly. On the other hand, ouabain (17 nmol) attenuated the negative inotropic response, but not chronotropic response, to KB-R7943. These results suggest that KB-R7943-induced cardiac effects relate to the Na+ activity, probably mediated through the Na+/Ca2+ exchanger, and the Na+/Ca2+ exchanger modifies the pacemaker activity and myocardial contractility in the dog heart.     

KB-R7943. Kanebo

Kanebo is investigating KB-R7943, a Na+/Ca2+ ion exchange inhibitor, for the potential treatment of ischemia and reperfusion injury. It inhibited the outward Na+/Ca2+ exchange current (iNCX) more potently than the inward current under unidirectional flow conditions; however, inward and outward current were inhibited equally under bidirectional conditions. The drug was a competitive inhibitor to external calcium, and the inhibition was reversible with a recovery t1/2 of about 30 s. The mammalian Na+/Ca2+ exchanger forms a multigene family of homologous proteins comprising three isoforms, NCX1, NCX2 and NCX3. By examining chimeric constructs between NCX1 and NCX3 expressed in CCL39 cells, it has been demonstrated that it is the conserved internal repeat regions (alpha-1 and alpha-2) of the exchanger that are critical for the drug's action.     

Molecular and functional characterization of the human platelet Na(+) /Ca(2+) exchangers

BACKGROUND AND PURPOSE The Na(+) /Ca(2+) exchanger is a bi-directional transporter that plays an important role in maintaining the concentration of cytosolic Ca(2+) ([Ca(2+) ](i) ) of quiescent platelets and increasing it during activation with some, but not all, agonists. There are two classes of Na(+) /Ca(2+) exchangers: K(+) -independent Na(+) /Ca(2+) exchanger (NCX) and K(+) -dependent Na(+) /Ca(2+) exchanger (NCKX). Platelets have previously been shown to express NCKX1. However, initial studies from our laboratory suggest that NCX may also play a role in platelet activation. The objective of this study was to determine if the human platelet expresses functional NCXs. EXPERIMENTAL APPROACH RT-PCR, DNA sequencing and Western blot analysis were utilized to characterize the human platelet Na(+) /Ca(2+) exchangers. Their function during quiescence and collagen-induced activation was determined by measuring [Ca(2+) ](i) with calcium-green/fura-red in response to: changes in the Na(+) and K(+) gradient, NCX pharmacological inhibitors (CBDMB, KB-R7943 and SEA0400) and antibodies specific to extracellular epitopes of the exchangers. KEY RESULTS Human platelets express NCX1.3, NCX3.2 and NCX3.4. The NCXs operate in the Ca(2+) efflux mode in resting platelets and also during their activation with thrombin but not collagen. Collagen-induced increase in [Ca(2+) ](i) was reduced with the pharmacological inhibitors of NCX (CBDMB, KB-R7943 or SEA0400), anti-NCX1 and anti-NCX3. In contrast, anti-NCKX1 enhanced the collagen-induced increase in [Ca(2+) ](i) . CONCLUSIONS AND IMPLICATIONS Human platelets express K(+) -independent Na(+) /Ca(2+) exchangers NCX1.3, NCX3.2 and NCX3.4. During collagen activation, NCX1 and NCX3 transiently reverse to promote Ca(2+) influx, whereas NCKX1 continues to operate in the Ca(2+) efflux mode to reduce [Ca(2+) ](i) .     

Na+/Ca2+ exchanger inhibitors inhibit neurite outgrowth in PC12 cells

To elucidate the role of Na(+)/Ca(2+) exchanger (NCX) in neurite outgrowth, we investigated the effects of NCX inhibitors on neurite outgrowth in PC12 cells. KB-R7943 and 3',4'-dichlorobenzamil, NCX inhibitors, inhibited the neurite outgrowth caused by nerve growth factor (NGF). NCX inhibitors inhibited the neurite outgrowth caused by dibutylyl cAMP, which rapidly reorganizes the cytoskeleton. KB-R7943 inhibited the neurite outgrowth caused by Y-27632, an inhibitor of Rho kinase (ROCK) that regulates actin. However, NCX inhibitors did not inhibit NGF-induced phosphorylation of extracellular signal-regulated kinase. These results suggest that NCX inhibitor affects downstream of the Rho-ROCK signal transduction pathways in neurite outgrowth.     

The novel Na+/Ca2+ exchange inhibitor KB-R7943 protects CA1 neurons in rat hippocampal slices against hypoxic/hypoglycemic injury

The selective Na+/Ca2+ exchange inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), significantly improves recovery of population spike amplitudes in rat hippocampal slices after hypoxia/hypoglycemia. Our data suggest that the Na+/Ca2+ exchanger, operating in reverse mode, contributes to hypoxia/hypoglycemia-induced injury in CA1 neurons.     

Calcium dynamics and vasomotion in rat mesenteric arteries

Smooth muscle cell calcium dynamics and diameter were measured in intact pressurized rat mesenteric artery segments during vasoconstriction and vasomotion. Arteries showed a certain norepinephrine (NE) threshold (0.3-0.4 microM) for the onset of vasomotion, during a cumulative NE concentration-response curve. This was due to a necessary [Ca2+]i threshold (increase over basal level of 22.2 +/- 2.6%) to elicit oscillations. The calcium oscillations obtained were synchronous over the entire vessel length and phase-shifted (in advance by 1.7 +/- 0.3 seconds) with respect to the diameter oscillations. A similar result was obtained using a KCl depolarization to contract the arteries, even though the [Ca2+]i threshold was much smaller in this case (increase over basal level of 9.9 +/- 4.3%), as compared with the NE-elicited vasomotion. Blockade of the Na+/K+-ATPase with 1 microM ouabain, or of the Na+/Ca2+ exchanger (NCX) with 1 microM KB-R 7943, did not abolish the calcium oscillations, thus showing that these two pumps are only modulatory elements, while on the other hand, voltage-gated calcium channels have been found to be important in the vasomotion mechanism.     

Protective effect of KB-R7943, a novel Na+/Ca2+ exchange inhibitor, on ischemic acute renal failure in rats

The effects of KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), a novel Na+/Ca2+ exchange inhibitor, on ischemic acute renal failure (ARF) in rats were examined. ARF was induced by clamping the left renal pedicle for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function was markedly diminished in ARF rats. Pretreatment with KB-R7943 (10 mg/kg, i.v.) markedly attenuated the ARF-induced renal dysfunction. Histopathological examination of the kidney of ARF rats revealed severe renal damage, which was suppressed by KB-R7943. Activation of the reverse mode of Na+/Ca2+ exchange seems to play an important role in the pathogenesis of ARF. A selective Na+/Ca2+ exchange inhibitor may be useful in cases of ARF.     

KB-R7943, a selective Na+/Ca2+ exchange inhibitor, protects against ischemic acute renal failure in mice by inhibiting renal endothelin-1 overproduction

We investigated whether the preischemic or postischemic treatment with KB-R7943, a novel and selective Na+/Ca2+ exchange inhibitor, has renal protective effects in mice with ischemic acute renal failure (ARF). Ischemic ARF was induced by clamping the left renal pedicle for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function was markedly diminished 24 h after reperfusion. Preischemic treatment with KB-R7943 attenuated the ARF-induced renal dysfunction. The ischemia/reperfusion-induced renal dysfunction was also overcome by postischemic treatment with KB-R7943. Histopathologic examination of the kidneys of ARF mice revealed severe renal damage such as tubular necrosis, proteinaceous casts in tubuli, and medullary congestion. Histologically evident damage and Ca2+ deposition in necrotic tubular epithelium were improved by preischemic treatment with KB-R7943. In addition, preischemic treatment with KB-R7943 significantly suppressed the increment of endothelin-1 (ET-1) content in the kidney at 2, 6, and 24 h after reperfusion. These findings suggest that Ca2+ overload via the reverse mode of Na+/Ca2+ exchange, followed by renal ET-1 overproduction, plays an important role in the pathogenesis of the ischemia/reperfusion-induced ARF. KB-R7943 may prove to be an effective therapeutic agent for cases of ischemic ARF in humans.