Selective Inhibitor Of Na Research Articles

Empagliflozin inhibits autophagy and mitigates airway inflammation and remodelling in mice with ovalbumin-induced allergic asthma

Empagliflozin, a selective inhibitor of Na+-glucose cotransporter-2, has been reported to exert anti-inflammatory and anti-fibrotic effects in addition to autophagy modulation. Addressing the role of autophagy in allergic asthma revealed controversial results. The potential effect of empagliflozin treatment on airway inflammation and remodelling as well as autophagy modulation in a murine model of allergic asthma was investigated. Over a 7-week period, male BALB/c mice were sensitized and challenged by intraperitoneal injection and inhalation of ovalbumin, respectively. Animals were treated with empagliflozin (10 mg/kg; orally) and/or rapamycin (an autophagy inducer; 4 mg/kg; intraperitoneally) before every challenge. Methacholine-induced airway hyperresponsiveness was evaluated one day after the last challenge. After euthanasia, serum, bronchoalveolar lavage fluid, and lung tissues were collected for biochemical, histopathological, and immunohistochemical assessment. Results revealed that empagliflozin decreased airway hyperresponsiveness, serum ovalbumin-specific immunoglobulin E, and bronchoalveolar lavage total and differential leukocytic counts. Levels of inflammatory and profibrotic cytokines (IL-4, IL-5, IL-13, IL-17, and transforming growth factor-β1) were all inhibited. Moreover, empagliflozin preserved pulmonary microscopic architecture and alleviated bronchiolar epithelial thickening, goblet cell hyperplasia, fibrosis and smooth muscle hypertrophy. These effects were associated with inhibition of ovalbumin-activated autophagic flux, as demonstrated by decreased LC3B expression and LC3BII/I ratio, as well as increased P62 expression. However, the therapeutic potential of empagliflozin was inhibited when rapamycin was co-administered. In conclusion, this study demonstrates that empagliflozin has immunomodulatory, anti-inflammatory, and anti-remodelling properties in ovalbumin-induced allergic asthma and suggests that autophagic flux inhibition may play a role in empagliflozin's anti-asthmatic effects.

Empagliflozin attenuates diabetic tubulopathy by improving mitochondrial fragmentation and autophagy.

We examined the effects of empagliflozin, a selective inhibitor of Na+-glucose cotransporter 2, on mitochondrial quality control and autophagy in renal tubular cells in a diabetic environment in vivo and in vitro. Human renal proximal tubular cells (hRPTCs) were incubated under high-glucose conditions. Diabetes was induced with streptozotocin in male C57BL/6J mice. Improvements in mitochondrial biogenesis and balanced fusion-fission protein expression were noted in hRPTCs after treatment with empagliflozin in high-glucose media. Empagliflozin also increased autophagic activities in renal tubular cells in the high-glucose environment, which was accompanied with mammalian target of rapamycin inhibition. Moreover, reduced mitochondrial reactive oxygen species production and decreased apoptotic and fibrotic protein expression were observed in hRPTCs after treatment with empagliflozin, even in the hyperglycemic circumstance. Importantly, empagliflozin restored AMP-activated protein kinase-α phosphorylation and normalized levels of AMP-to-ATP ratios in hRPTCs subjected to a high-glucose environment, which suggests the way that empagliflozin is involved in mitochondrial quality control. Empagliflozin effectively suppressed Na+-glucose cotransporter 2 expression and ameliorated renal morphological changes in the kidneys of streptozotocin-induced diabetic mice. Electron microscopy analysis showed that mitochondrial fragmentation was decreased and 8-hydroxy-2'-deoxyguanosine content was low in renal tubular cells of empagliflozin treatment groups compared with those of the diabetic control group. We suggest one mechanism related to the renoprotective actions of empagliflozin, which reverse mitochondrial dynamics and autophagy.

THE EFFECT OF HYPOXIA ON ELECTRICAL AND CONTRACTILE PROPERTIES OF SMOOTH MUSCLES OF THE GUINEA PIG URETER

Aim. The effect of hypoxia on the electrical and contractile activities of smooth muscles cells (SMCs) of the guinea pig ureter was studied by the method of the double sucrose bridge. Materials and methods. This method allows registering simultaneously parameters of the action potential (AP) and the contraction of SMCs, caused by an electrical stimulus. Results. It was found that lowering the oxygen content in the perfusion solution for 10 min resulted to an increase of electrical and contractile activity of ureteral SMCs. Addition of tetraethylammonium chloride (TEA, 5 mM) – nonselective blocker of potassium membrane conductance – in hypoxic conditions causing an additional increase in the amplitude of the AP, duration of the AP plateau and the contractile responses of smooth muscles. Thus, the hypoxia decreased the potassium membrane conductance of ureteral SMCs. Inhibition of the effect of the α1 -adrenergic receptors agonist phenylephrine (PE, 10 mM) on the electrical and contractile properties of SMCs in hypoxic condition indicate the involvement of the protein kinase C-dependent signaling system in effects of hypoxia. Pretreatment of ureteral smooth muscles with bumetanide (100 mM) – selective inhibitor of Na+,K+,2Cl- - cotransporter (NKCC) – caused a decrease of the activating effect of hypoxia on the SMCs of guinea pig ureter. Conclusion.Thus, the impact of hypoxia on the regulation of electrical activity and contractions of smooth muscles of guinea pig ureter may be due to changes in ion permeability of membranes SMCs and operation of ion-transporting systems.

Interaction of sodium bicarbonate and Na+/H+ exchanger inhibition in the treatment of acute metabolic acidosis in pigs.

Administration of NaHCO3 does not improve cellular function or reduce the mortality of acute lactic acidosis. This might be related to aggravation of intracellular acidosis, but it could also be due to activation of Na+/H+ exchanger with a deleterious increment in intracellular calcium ([Ca2+]i). This study examined the impact of coadministration of NaHCO3 and a selective inhibitor of Na+/H+ exchanger, sabiporide on cardiovascular function, changes in proinflammatory cytokines, and organ function in a model of acute lactic acidosis produced by hemorrhagic hypotension followed by infusion of lactic acid. Experimental, prospective study. Medical Center research laboratory. Male Yorkshire pigs. Anesthetized pigs were subjected to hypovolemia for 30 minutes and followed by DL-lactic acid infusion, and then either saline or sodium bicarbonate was infused. Hypovolemia followed by a DL-lactic acid infusion resulted in severe acidemia with a blood pH~6.8. Administration of NaHCO3 did not improve cardiovascular performance or decrease the levels of proinflammatory responses, whereas administration of sabiporide prior to acid or NaHCO3 infusion improved cardiopulmonary performance and blood oxygenation, reduced nuclear factor-κB activation, neutrophil accumulation, and proinflammatory cytokine production, and attenuated organ injury. Exposure of rat cardiac myocytes to a pH of 7.2 led to a marked increase of [Ca2+]i, and release of lactate dehydrogenase from cells which were further augmented after increase in external pH by addition of NaHCO3. Both the increase in [Ca2+]i and release of lactate dehydrogenase were attenuated in the presence of sabiporide. Coadministration of Na/H exchanger inhibitor with sodium bicarbonate improves cardiovascular performances, reduces proinflammatory responses, and attenuates organ injury. This improvement in these variables appears to be related to prevention of a rise in intracellular calcium occurring after both exposures to acid and bicarbonate.

Bumetanide, an Inhibitor of Cation-chloride Cotransporter Isoform 1, Inhibits γ-Aminobutyric Acidergic Excitatory Actions and Enhances Sedative Actions of Midazolam in Neonatal Rats

It has been shown that γ-aminobutyric acid exerts excitatory actions on the immature brain due to the increased expression of Na(+)-K(+)-2Cl(-) cotransporter isoform 1. The authors sought to clarify whether midazolam, a γ-aminobutyric acid-mimetic hypnotic agent, causes neuronal excitation that can be blocked by bumetanide, a selective inhibitor of Na(+)-K(+)-2Cl(-) cotransporter isoform 1. Furthermore, the authors examined whether bumetanide potentiates the sedative effects of midazolam in neonatal rats. The authors measured the effects of midazolam with or without bumetanide on the cytosolic Ca(2+) concentration ([Ca](2+)(i)) in hippocampal slices (n=3 in each condition) from rats at postnatal days 4, 7, and 28 (P4, P7, and P28) using fura-2 microfluorometry. Neuronal activity in the hippocampus and thalamus after intraperitoneal administration of midazolam with or without bumetanide was estimated by immunostaining of phosphorylated cyclic adenosine monophosphate-response element-binding protein (n=12 in each condition). Furthermore, the authors assessed effects of bumetanide on the sedative effect of midazolam by measuring righting reflex latency (n=6 in each condition). Midazolam significantly increased [Ca](2+)(i) in the CA3 area at P4 and P7 but not at P28. Bumetanide inhibited midazolam-induced increase in [Ca](2+)(i). Midazolam significantly up-regulated phosphorylated cyclic adenosine monophosphate-response element-binding protein expression in a bumetanide-sensitive manner in the hippocampus at P7 but not P28. Bumetanide enhanced the sedative effects of midazolam in P4 and P7 but not P28 rats. These results suggest that γ-aminobutyric acid A receptor-mediated excitation plays an important role in attenuated sedative effects of midazolam in immature rats.

Cardiometabolic Diseases and Related Complications: Current Status and Future Perspective

Cardiometabolic Diseases and Related Complications: Current Status and Future Perspective

Effects of MS-31-038, a novel Na(+)-H+ exchange inhibitor, on the myocardial infarct size in rats after postischemic administration.

The abilities of 2-(2-methylphenyl)-5,7-dimethoxy-4-quinolyl carbonylguanidine dihydrochloride (CAS 181048-29-3, MS-31-050) and 2-phenyl-8-(2-methoxyethoxy)-4-quinolyl carbonylguanidine bismethanesulfonate (CAS 181048-36-2, MS-31-038) in inhibiting Na(+)-H+ exchange, ischemia- and reperfusion-induced injury were determined and compared with those of 4-isopropyl-3-methylsulfonylbenzoyl guanidine methanesulfonate (CAS 159138-81-5, IMGM), a selective inhibitor of Na(+)-H+ exchange. MS-31-050 and IMGM exhibited comparable inhibitory effects on Na(+)-dependent pH recovery and antiarrhythmic effects during ischemia in anesthetized rats. In rats subjected to ischemia and reperfusion, MS-31-050 (10 mg/kg i.v.) significantly reduced the infarct size when given prior to the onset of ischemia. However, postischemic treatment with either MS-31-050 or IMGM failed to protect reperfused hearts. In contrast, MS-31-038 reduced the infarct size dramatically from 65.4 +/- 7.4% in control to 29.9 +/- 11.6% at 3 mg/kg and 9.8 +/- 3.4% at 10 mg/kg even when administered before the onset of reperfusion. These results suggest the beneficial effects of Na(+)-H+ exchange inhibitors on myocardial ischemia/reperfusion injury.

Ouabain-induced increase in dopamine release from mouse striatal slices is antagonized by riluzole.

We have examined the effects of riluzole, a neuroprotective drug which stabilizes voltage-dependent sodium channels in their inactivated state and inhibits the release of glutamate in-vivo and in-vitro, on the release of newly taken up [3H]dopamine induced by ouabain, a potent and selective inhibitor of Na+/K+-ATPase in mouse striatal slices in-vitro. Riluzole potently (IC50 (concentration resulting in 50% inhibition) = 0.9+/-0.3 microM) and dose-dependently antagonized ouabain-stimulated [3H]dopamine release, the effect being observed at low concentrations. Tetrodotoxin (1 microM) and nomifensine (10 microM) also abolished ouabain-induced [3H]dopamine release. Blockade of glutamate receptors with dizocilpine (1 microM) and 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione (YM-90K; 10 microM), alone or in combination, was without effect. Incubation of striatal slices with 50 microM La3+, which blocks voltage-dependent calcium channels, did not inhibit [3H]dopamine release induced by ouabain. The potent effects of riluzole observed in this model are probably related to its ability to block voltage-dependent sodium channels. The consequences of this activity are critically discussed in relation to the protective action of riluzole previously reported in various models of Parkinson's disease and other neurodegenerative disorders.

Regulatory volume increase in epithelial cells isolated from the mouse fourth ventricle choroid plexus involves Na +–H + exchange but not Na +–K +–2Cl − cotransport

The aim of this study was to determine the ability of choroid plexus epithelial cells to volume regulate when exposed to hypertonic solutions, and furthermore to identify the ion transporters involved in any volume regulation. Experiments were performed on cells freshly isolated, using the enzyme dispase, from the mouse fourth ventricle choroid plexus. Cell volume was measured using a video-imaging method. Cells used in this study were all of a similar morphology and had a mean volume of 0.71 pl. Cells shrank when superfused with hypertonic solutions to a minimum relative cell volume of 0.84 ± 0.01 ( n = 8) in 3 min. They then exhibited a regulatory volume increase (RVI) to reach a relative volume of 0.92 ± 0.02 over the following 12 min. The RVI was HCO 3 −-dependent, that is it was not observed in hepes-buffered solutions. A post-regulatory volume decrease RVI (post-RVD RVI) was also observed in cells following exposure to hypotonic solutions. The RVI and post-RVD RVI were inhibited by 10 µM 5-(N-ethyl-N-isopropyl) amiloride or 10 μM 5-(N-methyl-N-isobutyl) amiloride, both selective inhibitors of Na +–H + exchange (NHE). They were also inhibited by the anion transport inhibitor 100 µM 2,2′-(1,2-ethenediyl) bis (5-isothiocyanatobenzenesulfonic acid). The Na +–K +–2Cl − cotransporter inhibitor, 10 µM bumetanide, was without effect on either the RVI or the post-RVD RVI. The data indicate that NHE, probably in combination with Cl −–HCO 3 − exchangers, contributes to RVI in choroid plexus epithelial cells.

Effects of extracellular pH reductions on [3H]d-aspartate and [3H]noradrenaline release by presynaptic nerve terminals isolated from rat cerebral cortex

We analyzed the effects of extracellular pH reductions on the release of [(3)H]D: -aspartate ([(3)H]D: -ASP) and [(3)H]noradrenaline ([(3)H]NA) from cerebrocortical synaptosomes isolated from rats. Synaptosomes were superfused with standard medium at a physiologic pH of 7.4 and with acidified medium with a pH of 6.00, 5.50, or 5.0. Medium acidification produced pH-dependent stimulation of [(3)H]D: -ASP release. The increase amounted to 202 +/- 12.6% when the pH was reduced to 5.5. The [(3)H]D: -ASP release evoked by low pH (5.50) was still observed in the absence of Ca(2+) ions, but it was abolished by DL: -threo-beta-benzyloxyaspartate (DL: -TBOA) (100 microM), which inhibits neuronal glutamate/aspartate transport. Exposure to 5-(N,N-hexamethylene)-amiloride (EIPA) (30-100 microM), a selective inhibitor of Na(+)/H(+) exchange, caused concentration-dependent stimulation of [(3)H]D: -ASP release; the increase observed with EIPA 30 microM was 160 +/- 12%. The EIPA-induced release was not dependent on the presence of Ca(2+) ions in the medium, but it was abolished when synaptosomes were pretreated with 100 microM DL: -TBOA. Reduction of the extracellular pH (5.50-5.0) also stimulated the release of [(3)H]NA from rat cortical synaptosomes. Exposure to medium with a pH of 5.50 increased basal release by 136 +/- 9.5%. The release-stimulating effect of this medium was calcium-independent and abolished by 3 muM desipramine (DMI). [(3)H]NA release was also stimulated by EIPA. The increase induced by a concentration of 30 muM amounted to 136 +/- 9.50%, and this effect was calcium-independent and abolished by pretreatment with DMI (3 muM). These findings suggest that reduction of the extracellular pH stimulates release of [(3)H]D-ASP and [(3)H]NA by reversing neurotransmitter transport in the nerve terminal. This reversal might be activated by increased cytosolic concentrations of the transmitters caused by reduction of the pH gradient between the cytoplasm and the synaptic vesicles that take up the transmitters. This hypothesis is confirmed by the results of experiments conducted with EIPA. Selective blockade of Na(+)/H(+) exchange with this compound induces accumulation of H(+) in the nerve terminals and intracellular acidification, which leads to calcium-independent, transporter-mediated release of [(3)H]D: -ASP and [(3)H]NA.

Calpain activation contributes to hyperglycaemia-induced apoptosis in cardiomyocytes

Cardiomyocyte apoptosis contributes to cardiac complications of diabetes. The aim of this study was to investigate the role of calpain in cardiomyocyte apoptosis induced by hyperglycaemia. In cultured adult rat ventricular cardiomyocytes, high glucose (33 mM) increased calpain activity and induced apoptosis, concomitant with the impairment of Na+/K+ ATPase activity. These effects of high glucose on cardiomyocytes were abolished by various pharmacological calpain inhibitors, knockdown of calpain-1 but not calpain-2 using siRNA, or over-expression of calpastatin, a specific endogenous calpain inhibitor. The effect of calpain inhibition on cardiomyocyte apoptosis was abrogated by ouabain, a selective inhibitor of Na+/K+ ATPase. Furthermore, blocking gp91(phox)-NADPH oxidase activation, L-type calcium channels, or ryanodine receptors prevented calpain activation and apoptosis in high glucose-stimulated cardiomyocytes. In a mouse model of streptozotocin-induced diabetes, administration of different calpain inhibitors blocked calpain activation, increased the Na+/K+ ATPase activity, and decreased apoptosis in the heart. Calpain-1 activation induces apoptosis through down-regulation of the Na+/K+ ATPase activity in high glucose-stimulated cardiomyocytes and in vivo hyperglycaemic hearts. High glucose-induced calpain-1 activation is mediated through the NADPH oxidase-dependent pathway and associated with activation of L-type calcium channels and ryanodine receptors. Our data suggest that calpain activation may be important in the development of diabetic cardiomyopathy and thus may represent a potential therapeutic target for diabetic heart diseases.

Effects of the Noradrenergic System in Rat White Matter Exposed to Oxygen–Glucose DeprivationIn Vitro

Norepinephrine (NE) is released in excess into the extracellular space during oxygen-glucose deprivation (OGD) in brain, increasing neuronal metabolism and aggravating glutamate excitoxicity. We used isolated rat optic nerve and spinal cord dorsal columns to determine whether the noradrenergic system influences axonal damage in white matter. Tissue was studied electrophysiologically by recording the compound action potential (CAP) before and after exposure to 60 min of OGD at 36 degrees C. Depleting catecholamine stores with reserpine was protective and improved CAP recovery after 1 h of reperfusion from 17% (control) to 35%. Adding NE during OGD decreased CAP recovery to 8%, and adding NE to reserpine during OGD eliminated the protective effect of the latter. Selective inhibitors of Na(+)-dependent norepinephrine transport desipramine and nisoxetine improved recovery to 58% and 44%, respectively. alpha2 adrenergic receptor agonists UK14,304 and medetomidine improved CAP recovery to 41% and 46% after 1 h of OGD. Curiously, alpha2 antagonists alone were also highly protective (e.g., atipamezole: 86% CAP recovery), at concentrations that did not affect baseline excitability. The protective effect of alpha2 receptor modulation was corroborated by imaging fluorescent Ca(2+) and Na(+) indicators within axons during OGD. Both agonists and antagonists significantly reduced axonal Ca(2+) and Na(+) accumulation in injured axons. These data suggest that the noradrenergic system plays an active role in the pathophysiology of axonal ischemia and that alpha2 receptor modulation may be useful against white matter injury.

Acute effects of digoxin on plasma aldosterone and cortisol in monkeys

Digoxin, a cardiac glycoside, is used to increase cardiac contractility via inhibition of Na +/K +-adenosinetriphosphatase (ATPase) and increase intracellular calcium in congestive heart failure. Inhibitory effects of digoxin have been demonstrated on the biosynthesis of gonadal hormones and adrenal glucocorticoids in rats. However, acute effects of digoxin on levels of adrenal corticosteroid hormones in the primates in vivo are uncertain. Therefore, we test the hypothesis that a single injection of digoxin decreases the secretion of aldosterone and cortisol in monkeys. An intravenous injection of digoxin (1 μg/kg) inhibited basal and adrenocorticotropin (ACTH)- or KCl-stimulated aldosterone release in monkeys. Furthermore, digoxin induced a decrease in ACTH- and KCl-stimulated cortisol release. Administration of digoxin did not alter plasma concentrations of Na + and K +. Ouabain, a selective inhibitor of Na +/K +-ATPase, did not affect ACTH- or KCl-stimulated aldosterone and cortisol release. These results revealed that injection of digoxin induced an inhibitory effect on aldosterone and cortisol secretion in monkeys. Because ouabain did not affect levels of plasma aldosterone or cortisol, we suggest that (1) the Na +/K +-ATPase pathway may not be involved in the mechanism of action of digoxin on aldosterone or cortisol secretion in monkeys and/or (2) the Na +/K +-ATPase is more sensitive to digoxin than to ouabain in monkeys.

ProTx-II, a Selective Inhibitor of NaV1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors

Voltage-gated sodium (Na(V)1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to Na(V)1.7 as an essential contributor to pain signaling. Human loss-of-function mutations in SCN9A, the gene encoding Na(V)1.7, cause channelopathy-associated indifference to pain (CIP), whereas gain-of-function mutations are associated with two inherited painful neuropathies. Although the human genetic data make Na(V)1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires Na(V)1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with Na(V)1.7 channels, inhibiting Na(V)1.7 with an IC(50) value of 0.3 nM, compared with IC(50) values of 30 to 150 nM for other heterologously expressed Na(V)1 subtypes. This subtype selectivity was abolished by a point mutation in DIIS3. It is interesting that application of ProTx-II to desheathed cutaneous nerves completely blocked the C-fiber compound action potential at concentrations that had little effect on Abeta-fiber conduction. ProTx-II application had little effect on action potential propagation of the intact nerve, which may explain why ProTx-II was not efficacious in rodent models of acute and inflammatory pain. Mono-iodo-ProTx-II ((125)I-ProTx-II) binds with high affinity (K(d) = 0.3 nM) to recombinant hNa(V)1.7 channels. Binding of (125)I-ProTx-II is insensitive to the presence of other well characterized Na(V)1 channel modulators, suggesting that ProTx-II binds to a novel site, which may be more conducive to conferring subtype selectivity than the site occupied by traditional local anesthetics and anticonvulsants. Thus, the (125)I-ProTx-II binding assay, described here, offers a new tool in the search for novel Na(V)1.7-selective blockers.

Na +/H + exchanger inhibition at the onset of reperfusion decreases myocardial infarct size: role of reactive oxygen species

Background A burst of reactive oxygen species and activation of Na +/H + exchanger take place at the beginning of reperfusion. The aim of this study was to assess the possible interrelation of the inhibition of Na +/H + exchanger and reactive oxygen species about the determination of myocardial infarct size. Methods Isolated rat hearts were submitted to 40 min of coronary occlusion and 2 h of reperfusion. Infarct size was determined through triphenyltetrazolium chloride staining technique and was expressed as a percentage of risk area. Lipid peroxidation, as a marker of oxidative stress, was estimated by the concentration of thiobarbituric reactive substances. Results Treatment during the first 20 min of reperfusion with a selective inhibitor of Na +/H + exchanger 1 isoform, HOE 642 (cariporide; 10 μM), significantly diminished infarct size (15.1±2.4% vs. 31±2% in untreated hearts). The administration of a “scavenger” of hydroxyl radical, N-(2-mercaptopropionyl)-glycine (2 mM), decreased infarct size in an extent similar to that of cariporide (18±3%). The combination cariporide+ N-(2-mercaptopropionyl)-glycine did not produce additional protection (17±1.7%). Each intervention [HOE 642 or N-(2-mercaptopropionyl)-glycine] and its combination improved the postischemic recovery of myocardial systolic and diastolic functions in a similar extent. The content of the thiobarbituric reactive substances of untreated hearts (1012±144 nmol/g) decreased to 431±81, 390±82, and 433±41 after cariporide, N-(2-mercaptopropionyl)-glycine, and cariporide+ N-(2-mercaptopropionyl)-glycine treatments, respectively. Conclusions The present data support the conclusion that the cardioprotective effect of cariporide is associated with diminution of oxidative stress.

Na+/Ca2+exchanger contributes to asterosap-induced elevation of intracellular Ca2+concentration in starfish spermatozoa

Asterosap, a group of equally active isoforms of sperm-activating peptides from the egg jelly of the starfish Asterias amurensis, functions as a chemotactic factor for sperm. It transiently increases the intracellular cGMP level of sperm, which in turn induces a transient elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)). Using a fluorescent Ca(2+)-sensitive dye, Fluo-4 AM, we measured the changes in sperm [Ca(2+)](i) in response to asterosap. KB-R7943 (KB), a selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), significantly inhibited the asterosap-induced transient elevation of [Ca(2+)](i), suggesting that asterosap influences [Ca(2+)](i) through activation of a K+-dependent NCX (NCKX). An NCKX activity of starfish sperm also shows K(+) dependency like other NCKXs. Therefore, we cloned an NCKX from the starfish testes and predicted that it codes for a 616 amino acid protein that is a member of the NCKX family. Pharmacological evidence suggests that this exchanger participates in the asterosap-induced Ca(2+) entry into sperm.

A Selective Inhibitor of Na+/Ca2+ Exchanger, SEA0400, Preserves Cardiac Function and High-Energy Phosphates against Ischemia/Reperfusion Injury

The Ca2+ overload by Ca2+ influx via Na+/Ca2+ exchanger (NCX) is a critical mechanism in myocardial ischemia/reperfusion injury. We investigated protective effects of a novel selective inhibitor of NCX, SEA0400, on cardiac function and energy metabolism during ischemia and reperfusion. Langendorff-perfused rat hearts were exposed to 35 minutes global ischemia and 40 minutes reperfusion. Using 31P nuclear magnetic resonance spectroscopy, cardiac phosphocreatine (PCr), ATP, and pHi were monitored. SEA0400 did not change the basic cardiac function, but improved the recovery of left ventricular developed pressure (LVDP) after reperfusion (27.6 +/- 4.9 mm Hg in control, 101.2 +/- 19.3 mm Hg in 0.1 microM, and 115.5 +/- 13.3 mm Hg in 1 microM SEA0400, means +/- SE, n = 6, P < 0.05). SEA0400 reduced left ventricular end-diastolic pressure and increased coronary flow after reperfusion. SEA0400 improved the recoveries of cardiac phosphocreatine and ATP after reperfusion, but did not affect pHi. There were significant linear correlations between left ventricular developed pressure and cardiac phosphocreatine (r = 0.79, P < 0.05), and left ventricular developed pressure and ATP (r = 0.80, P < 0.05). However, SEA0400 increased the incidence and duration of reperfusion ventricular arrhythmias. SEA0400 added only after reperfusion also improved both the contractile function and energy metabolism. It is concluded that the selective inhibition of NCX may be effective to preserve high-energy phosphates and to improve cardiac function after reperfusion, but may not be able to prevent fatal arrhythmias.

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+/Ca2+ exchanger (NCX), on Na+-dependent Ca2+ 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 45Ca2+ uptake and CA release, with the IC50 values of 40 and 100 nM, respectively. The IC50 values of another NCX inhibitor KB-R7943 were 1.8 and 3.7 µM, 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 IC50 values of SEA0400 were 5.1 and 4.5 µM and the values of KB-R7943 were 2.6 and 2.1 µM 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 [3H]nicotine with nicotinic receptors was inhibited by SEA0400 (IC50 = 90 µM) and KB-R7943 (IC50 = 12 µM). From these results, it is concluded that unlike KB-R7943, SEA0400 has a potent and selective action on NCX in bovine adrenal chromaffin cells.

Effects of SEA0400, a Na +/Ca 2+ exchange inhibitor, on ventricular arrhythmias in the in vivo dogs

SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline), a novel and selective inhibitor of Na +/Ca 2+ exchanger, was investigated for its possible antiarrhythmic effects on arrhythmias of Ca 2+ overload induced by coronary ligation/reperfusion and by digitalis in the dog. SEA0400 (1.0 mg/kg) did not change the hemodynamics but slightly prolonged the QRS duration ( P<0.05). Pre-ischemic administration (10 min before coronary occlusion) of SEA0400 (1.0 mg/kg) and post-ischemic administration (1 min before reperfusion) of SEA0400 (0.3, 1.0 and 3.0 mg/kg) had no effects on the incidence of ventricular fibrillation induced by coronary ligation/reperfusion. On the other hand, SEA0400 (3.0 mg/kg) decreased the arrhythmic ratio in the digitalis arrhythmias ( P<0.01). However, atrioventricular block and cardiac standstill were induced in two digitalized dogs. In conclusion, SEA0400 has no significant antiarrhythmic effect on arrhythmias induced by coronary ligation/reperfusion, but has an obvious suppressing effect on tachyarrhythmias induced by digitalis in in vivo canine models.

Effects of SEA0400, a novel inhibitor of the Na +/Ca 2+ exchanger, on myocardial stunning in anesthetized dogs

Activation of the Na +/Ca 2+ exchanger may contribute to Ca 2+ overload during reperfusion after transient ischemia. We examined the effects of 2-[4-[(2,5-difluorophenyl) methoxy]phenoxy]-5-ethoxyaniline (SEA0400), a selective inhibitor of Na +/Ca 2+ exchange, on a canine model of ischemia/reperfusion injury (myocardial stunning). Myocardial stunning was induced by a 15-min occlusion of the left anterior descending coronary artery followed by a 4-h reperfusion in anesthetized open-chest dogs. Reperfusion gradually restored myocardial percent segment shortening but remained depressed during a 4-h reperfusion period. A bolus intravenous injection of SEA0400 (0.3 or 1.0 mg/kg), given 1 min before reperfusion, improved significantly the recovery of percent segment shortening in the ischemic/reperfused myocardium. SEA0400 did not affect the hemodynamics and electrocardiogram parameters. In addition, SEA0400 did not affect reperfusion-induced change in coronary blood flow. These results suggest that the Na +/Ca 2+ exchanger is involved in the stunned myocardium of dogs after reperfusion, and that SEA0400 has a protective effect against myocardial stunning in dogs.