Inhibition of synaptosomal membrane Na+-Ca2+ exchange transport by amiloride and amiloride analogues

Na+-Ca2+ exchange in rat brain synaptosomal plasmalemma vesicles is reversibly inhibited by amiloride (3,5-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide++ +). This drug (pKa = 8.7) inhibits Na+-dependent Ca2+ uptake more effectively at basic pH values than at neutral pH values, indicating that the positively charged form of amiloride is the active moiety. Twenty amiloride analogues were examined for ability to inhibit Na+-Ca2+ exchange. These studies demonstrate that the 6-chloro group, the 5-amino substituent, and the carbonyl guanidinium moiety are essential for drug inhibition of Na+-Ca2+ exchange. N-Benzyl amiloride derivatives such as 3,5-diamino-6-chloro-N-(benzylamino-aminomethylene)pyrazinecarb oxamide (benzamil) and 3,5-diamino-6-chloro-N-(2-phenethylamino-aminomethylene)p yrazinecarboxamide are more potent inhibitors of Na+-dependent Ca2+ uptake than is amiloride. The amiloride analogue pattern of interaction with the Na+-Ca2+ exchange system is distinct from the inhibition patterns of the epithelial Na+ channel and the Na+-H+ exchange transport system.     

Structure-activity relationships of amiloride and certain of its analogues in relation to the blockade of the Na+/H+ exchange system

Amiloride and 38 amiloride analogues were tested for their inhibitory action on the Na+/H+ exchanger of chick skeletal muscle cells. The unsubstituted guanidino group of amiloride is essential for the activity of the molecule, since substitution of its results in almost inactive molecules. Selected modification of position 3 and 5 substituents of amiloride have a less dramatic effect on its potency. Substitution of the 5-amino group of amiloride with alkyl or alkenyl groups produced compounds that were up to 140 times more potent than amiloride in inhibiting the Na+/H+ exchanger. Such molecules would appear to be preferable to use in place of amiloride in biochemical and physiological studies of the Na+/H+ exchanger.     

Inhibition of Na+/Ca2+ exchange by amiloride acting from opposite sides of cardiac sarcolemma

Amiloride inhibited the Na+Ca2+ exchange activity of cardiac sarcolemmal vesicles with similar affinities at the cis and trans sides of the membrane, estimated apparent Ki on both sides of the sarcolemma being similar. The extent of amiloride inhibition on Na+/Ca2+ exchange activity was decreased by alkaline pH only when the drug was acting from the external side of the vesicle sarcolemma, whereas when vesicles were preincubated with the drug at different pH values, amiloride appeared to act as a weak permeant base, being a more effective inhibitor at alkaline pH values. In fact, a rise in the pH of the preincubation medium may favour the entry and consequently the effect of the drug on the exchanger. The pH dependence of the inhibition of Na+/Ca2+ exchange activity by either extravesicular or intravesicular amiloride was consistent with the hypothesis that in both cases the protonated drug was the active form. Evidence is presented that the pattern of interaction of amiloride on the Na+/Ca2+ exchange system strictly depended on the sidedness of drug action. In fact, while Na+ protected against inhibition by amiloride when it was acting on the same side of the vesicle membrane as the drug, it synergically interacted with amiloride to inhibit exchange activity when it was acting on the opposite side of the sarcolemma as the drug. Furthermore, only extravesicular amiloride removed the stimulation of Na+/Ca2+ exchange activity in Ca2+-treated vesicles.     

Inhibition by amiloride of both adenylate cyclase activity and the Na+/H+ antiporter in fish erythrocytes

In fish erythrocytes isoproterenol stimulates cellular accumulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and produces a large increase in sodium permeability which corresponds to the activation of Na+/H+ exchanges and chloride-dependent sodium uptake. The stimulation of sodium transport by isoproterenol was reproduced by adding cyclic AMP or forskolin to the medium and was blocked by propranolol. This increase in sodium permeability was completely inhibited by amiloride at the relatively high levels (0.1-1 mM) of the diuretic required to inhibit the activity of the Na+/H+ exchanger under physiological conditions in various biological systems. It was shown that amiloride inhibited cyclic AMP accumulation. This effect, which was reversible and dose-dependent (ED50 6 X 10(-6) M-maximal effect 0.5 mM), resulted from the inhibition of the catalytic unit of adenylate cyclase. Amiloride also directly inhibited the sodium entry system but the Na transporter was less sensitive than adenylate cyclase to amiloride (ED50 6 X 10(-5) M). It appears from the data presented in this report that the inhibition of sodium permeability observed in fish erythrocytes in the presence of amiloride can result either from the effect of the diuretic on the adenylate cyclase system or from the effect on the sodium transport system, depending on the conditions in which amiloride is used. Thus, caution is required when interpreting amiloride action in terms of inhibition of specific transport processes.     

Novel amiloride analog allosterically modulates the alpha 2-adrenergic receptor but does not inhibit Na+/H+ exchange.

Two novel amiloride analogs have been synthesized during the course of efforts to develop a photoaffinity label for the amiloride allosteric domain on alpha 2-adrenergic receptors. One of these, 5-[N-2'-aminoethyl-N'-isopropyl]amiloride-N-[4"-azidosalicylamide] (A-EIA-AS), markedly accelerates the rate of dissociation of [3H]yohimbine from affinity-purified alpha 2-adrenergic receptors, an assay for allosteric modulation of receptor-adrenergic ligand interactions. In contrast, this agent does not appreciably inhibit Na+/H+ exchange, measured as 5-(N-ethyl-N-isopropyl)amiloride (EIA)-inhibitable 22Na+ uptake into cultured renal epithelial cells. A second analog, 5-[N-2'-(4"-azidosalicylamidino)ethyl-N'- isopropyl]amiloride (ASA-EIA), does not foster an accelerated rate of dissociation of [3H]yohimbine binding from the alpha 2 receptor but does block the ability of A-EIA-AS to do so, suggesting that ASA-EIA and A-EIA-AS interact at a common binding site. Interestingly, the ability of EIA to accelerate [3H]yohimbine dissociation is not blocked by ASA-EIA, a finding that may indicate that EIA and A-EIA-AS allosterically modulate alpha 2 receptor-ligand interactions via distinct or nonoverlapping binding sites.     

六肽FRCRSFa对大鼠心室肌Na^＋／Ca^＋交换的抑制

AIM: To study the effect of Phe-Arg-Cys-Arg-Ser-Phe-CONH2 (FRCRSFa) on Na+/Ca2+ exchange and its specificity in rat ventricular myocytes. METHODS: Na+/Ca2+ exchange current (INa+/Ca2+) and other currents were measured using whole-cell voltage clamp technique. RESULTS: A concentration-dependent inhibition of hexapeptide FRCRSFa on Na +/Ca2+ exchange was observed in rat ventricular myocytes. IC50 of inward and outward INa+/Ca2+ were 2 and 4 micromol/L, respectively. FRCRSFa 5 micromol/L did not affect L-type Ca2+ current, voltage-gated Na+ current, transient outward K+ current, and inward rectifier K+ current. CONCLUSION: These data indicate that FRCRSFa is an available inhibitor of Na+/Ca2+ exchange with relative selectivity and m ay be valuable for studies of the Na+/Ca2+ exchange in cardiac myocytes.     

Na~+/H~+交换泵1激活与心肌肥大

钠氢交换泵 1介导缺血及再灌流引起的心肌损伤。近期的研究提示钠氢交换泵 1也介导长期不良刺激引起的心肌肥大和心衰。钠氢交换泵 1可能是引起心肌肥大的多种因素信息传导下游区的共同媒介 ,比如血管紧张素Ⅱ ,肾上腺α1、β1受体兴奋等。抑制钠氢交换泵 1可能会成为防治心衰的一种新方法。     

Regulation of G protein-coupled receptor function by Na+/H+ exchange regulatory factors

Many G protein-coupled receptors (GPCR) exert patterns of cell-specific signaling and function. Mounting evidence now supports the view that cytoplasmic adapter proteins contribute critically to this behavior. Adapter proteins recognize highly conserved motifs such as those for Src homology 3 (SH3), phosphotyrosine-binding (PTB), and postsynaptic density 95/discs-large/zona occludens (PDZ) docking sequences in candidate GPCRs. Here we review the behavior of the Na+/H+ exchange regulatory factor (NHERF) family of PDZ adapter proteins on GPCR signalling, trafficking, and function. Structural determinants of NHERF proteins that allow them to recognize targeted GPCRs are considered. NHERF1 and NHERF2 are capable also of modifying the assembled complex of accessory proteins such as β-arrestins, which have been implicated in regulating GPCR signaling. In addition, NHERF1 and NHERF2 modulate GPCR signaling by altering the G protein to which the receptor binds or affect other regulatory proteins that affect GTPase activity, protein kinase A, phospholipase C, or modify downstream signaling events. Small molecules targeting the site of NHERF1-GPCR interaction are being developed and may become important and selective drug candidates.     

Role of nitric oxide and free radicals in cardioprotection by blocking Na+/H+ and Na+/Ca2+ exchange in rat heart

Inhibition of Na(+)/H(+) (NHE) and Na(+)/Ca(2+) (NCE) exchangers prevents myocardial ischemia/reperfusion injury by preventing cardiomyocyte Ca(2+) overload. We hypothesized that it may influence ischemic/reperfused myocardium also indirectly by preventing endothelial Ca(2+) accumulation, and thereby by attenuating reperfusion-induced formation of nitric oxide (NO) and/or oxygen free radicals. Langendorff-perfused rat hearts were subjected to 30-min ischemia and 30-min reperfusion. Myocardial outflow of NO (nitrite+nitrate) and hydroxyl radical (*OH, salicylate method), and functional recoveries were followed during reperfusion. In all groups, there was a transient rise in NO and *OH outflow upon reperfusion. An inhibitor of NHE, cariporide (10 microM) [(4-Isopropyl-3-methylsulfonyl-benzoyl)-quanidine methanesulfonate], and an inhibitor of the reverse mode of NCE, KB-R7943 (5 microM) (2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate), decreased NO and *OH formation, reduced contracture, and improved the recovery of mechanical function during reperfusion, compared to the untreated hearts. The formation of NO was reduced by 40% by 100 microM N(G)-methyl-L-arginine acetate salt (L-NMMA, NO synthase inhibitor), and not affected by 50 microM L-NMMA. *OH formation, contracture, and the functional recoveries were affected neither by 50 nor by 100 microM L-NMMA. Also, the effects of cariporide and KB-R7943 were unaffected by 100 microM L-NMMA. This study shows for the first time that the inhibition of NHE and NCE attenuates post-ischemic myocardial formation of NO and *OH, suggesting that prevention of Ca(2+) overload is cardioprotective via these mechanisms. The results indicate, however, that NO synthase pathway did not interfere with the protection afforded by NHE or NCE in our model.     

Inhibition of superoxide dismutase by 2-methoxyoestradiol analogues and oestrogen derivatives: structure-activity relationships

Superoxide dismutases catalyse the dismutation of highly reactive superoxide ions to produce hydrogen peroxide and several lines of evidence suggest that these enzymes play important roles in the development and response to treatment of human cancers. For example, Mn-containing superoxide dismutase is frequently overexpressed in various cancer types and can contribute to resistance to apoptosis. 2-Methoxyoestradiol is a naturally occurring metabolic product of 17beta-oestradiol that inhibits tubulin polymerization and possesses growth inhibitory and cytotoxic activity in vitro and in vivo. More recently 2-methoxyoestradiol has also been shown to inhibit superoxide dismutase (SOD) in a tetrazolium salt based enzyme assay, suggesting that oestrogen derivatives might be useful starting points for the development of effective, non-toxic enzyme inhibitors. Here we have tested the SOD inhibiting activity of a range of oestrogen derivatives to determine structural features important for enzyme inhibition.     

Na+/H+ exchange inhibitor SM-20220 improves endothelial dysfunction induced by ischemia-reperfusion

Endothelial cells play an important role in the physiologic homeostasis of the cerebral circulation. Previously, we showed that the Na+/H+ exchanger (NHE) inhibitor SM-20220 (N-(aminoimino-methyl)-1-methyl-1H-indole-2-carboxamide methanesulfonate) improved ischemic brain injury. In this study, we investigated the effect of SM-20220 on cerebrovascular dysfunction after ischemia-reperfusion, focusing on the kinds of dysfunction that involved endothelial function. In cultured bovine brain microvascular endothelial cells (BBMCs), the IC50 value for the NHE activity of SM-20220 was 4 x 10(-8) M. SM-20220 also reduced the cell injury induced by hypoxia/aglycemia-reoxygenation in BBMCs, with statistical significance at 10(-7) M (P<0.05). Next, the effect of SM-20220 on disruption of the blood-brain barrier and cerebral blood flow were evaluated using transient middle cerebral artery (MCA) occlusion models. Intravenous infusion of SM-20220 (0.4 mg/kg per hour for 1 h) attenuated the extravasation of Evans blue, a blood-brain barrier disruption indicator, into cerebral tissue on the day after transient ischemia (P<0.05). The occlusion of the MCA decreased the cerebral blood flow in the MCA territory by about 20%, and only about 45% of the preischemic value was recovered at 1-h reperfusion. A bolus injection of SM-20220 (1 mg/kg, i.v.) improved the postischemic hypoperfusion by about 75%, without causing changes in the systemic blood pressure. These results indicate that the protective effect of NHE inhibitor on ischemic brain injury may be at least partially mediated by the prevention of endothelial dysfunction.     

Na+/H+ exchange inhibitor SM-20220 attenuates leukocyte adhesion induced by ischemia-reperfusion

Leukocytes play a key role in ischemia-reperfusion-induced tissue injuries. It has been suggested that blocking the Na+/H+ exchanger improves ischemic injuries such as stroke. In this study, we investigated the effect of the Na+/H+ exchanger inhibitor SM-20220 (N-[aminoiminomethyl]- 1-methyl-1H-indole-2-carboxamide methanesulfonate) on leukocyte-endothelial cell interactions during ischemia-reperfusion. SM-20220 (0.3-1.0 mg/kg i.v.) given after ischemia significantly attenuated the leukocyte adhesion in the mesenteric postcapillary venules that was induced by transient superior mesenteric artery occlusion. At 60 min after reperfusion, the numbers of adherent leukocytes in groups treated with vehicle or SM-20220 (0.3 mg/kg) were 15.1+/-2.9 cells/100 microm/3 min and 3.0+/-0.7 cells/100 microm/3 min (p < 0.01), respectively. In a transient middle cerebral artery occlusion model, i.v. infusion of SM-20220 (0.4 mg/kg per hour) for 1 h, beginning 1 h after the start of occlusion, significantly reduced both the infarct size and the increase in brain myeloperoxidase activity, compared with the vehicle group (p < 0.01 and p < 0.05, respectively). In summary, this is the first evidence that the leukocyte adhesion to the endothelium that is induced by ischemia-reperfusion is attenuated by the inhibition of Na+/H+ exchanger activity in vivo. Our results suggest that Na+/H+ exchanger inhibitors may prevent ischemia-reperfusion injuries such as stroke partly through the attenuation of leukocyte-endothelial cell interactions.     

Comparison of the protective actions of Na+/H+ and Na+/Ca2+ exchange inhibitors in ischemic/reperfused rat hearts

The protective effects of the Na⁺/H⁺ exchange inhibitors amiloride, EIPA (5‐(N‐ethyl‐N‐isopropyl)‐amiloride), and HOE 694 (3‐methylsulfonyl‐4‐(1‐piperidino) benzoyl‐guanidine) and the Na⁺/Ca²⁺ exchange inhibitor, DCB (3,4‐Dichlorobenzamil) on ischemia (30 min) / reperfusion (30 min) injury were studied using Langendorff perfused rat hearts. EIPA and HOE 694 given before ischemia protected the heart during reperfusion from mechanical and metabolic disturbances. A weak protective effect was observed with amiloride, but not with DCB. The cardioprotective efficacies of these compounds correlated with their potencies as Na⁺/H⁺ exchange inhibitors as assessed by the NH₄Cl prepulse method. None of the inhibitors was effective when given at reperfusion. EIPA and HOE 694 decreased myocardial rigidity as assessed by the resting tension (RT) which elevated during reperfusion. EIPA led to a more marked attenuation of RT elevation during reperfusion rather than ischemia, whereas diltiazem, a Ca²⁺ channel blocker, suppressed RT elevation during ischemia but did not cause a further attenuation of RT during reperfusion. Treatment with EIPA as well as diltiazem before ischemia showed a direct negative chronotropic effect. Cardioprotective effects were also observed with diltiazem. These results suggest that Na⁺/H⁺ exchange plays a more important role in ischemia‐reperfusion‐induced myocardial injury than does Na⁺/Ca²⁺ exchange. The cardioprotective effects of EIPA appear to be produced by Ca²⁺ channel blockade during ischemia and by Na⁺/H⁺ exchange inhibition during reperfusion. Drug Dev. Res. 48:160–170, 1999. © 1999 Wiley‐Liss, Inc.     

Amiloride analogues induce responses in isolated rat cardiovascular tissues by inhibition of Na+/Ca2+ exchange

Summary The role of inhibition of Na⁺/Ca²⁺ exchange in the positive inotropic, negative chronotropic and vasorelaxant responses to amiloride and some of its analogues was investigated in isolated cardiovascular tissues from female Wistar rats. The compounds tested were amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA, a potent inhibitor of Na⁺/H⁺ exchange), phenamil and 2,4-dimethylbenzamil (DMB), both potent Na⁺ channel inhibitors with activity against Na⁺/Ca²⁺ exchange, and 5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil (CBDMB), a potent inhibitor of Na⁺/Ca²⁺ exchange with reduced activity against Na⁺ channels compared with its parent compound DMB.Phenamil, DMB and CBDMB increased the force of contraction of right ventricular papillary muscles with similar potencies (-log EC₅₀ values: 4.77 ± 0.06, 5.09 ± 0.09, 4.97 ± 0.17 respectively), while amiloride and EIPA gave small negative inotropic responses. All compounds gave negative chronotropic responses at similar concentrations to those which exerted inotropic effects. Inhibition of KCl contraction of endothelium-free aortic rings was observed with all compounds tested. Phenamil, DMB and CBDMB but not amiloride or EIPA showed a shift to the left of the concentration-response curves in the presence of intact endothelium.These results provide further evidence for positive inotropic and endothelium-dependent vasorelaxant effects of amiloride analogues mediated by inhibition of Na⁺/Ca²⁺ exchange. Send offprint requests to J. R. Bourke at the above address     

Alpha 1-adrenoceptor stimulation increases intracellular pH and Ca2+ in cardiomyocytes through Na+/H+ and Na+/Ca2+ exchange

The effects of alpha 1-adrenergic stimulation on intracellular pH (pHi) and Ca2+ concentration ([Ca2+]i) were investigated in isolated rat cardiomyocytes with fluorescence dyes, BCECF and fura-2, respectively. In the presence of 5 or 25 mM HCO3- norepinephrine (NE) increased pHi in a dose-dependent manner. Intracellular alkalinization was inhibited by prazosin and phentolamine but not by yohimbine. NE-induced alkalinization was inhibited in the presence of a Na+/H+ exchange inhibitor (5-(N,N-hexamethylene) amiloride (HMA)), a C kinase inhibitor (H-7) or a calmodulin inhibitor (W-7), or in the absence of extracellular Na+. NE also increased [Ca2+]i following the pHi increase, which was abolished in the absence of extracellular Na+ or Ca2+. This Ca2+ influx was inhibited by HMA but not by diltiazem (10(-5) M). Thus, we conclude that alpha 1-adrenergic stimulation enhances Na+/H+ exchange by activation of C kinase, thereby allowing intracellular alkalinization, and that subsequent activation of Na+/Ca2+ exchange increases Ca2+ influx.     

Na+/H+ exchange inhibitors reverse lactate-induced depression in postischaemic ventricular recovery.

1. By use of pharmacological approaches, the present study examined the hypothesis that the deleterious effect of lactate on postischaemic ventricular recovery may be mediated, at least in part, by enhanced activation of the Na+/H+ exchanger at the time of reperfusion. 2. Spontaneously beating isolated hearts of the rat were subjected to 15 min zero-flow global ischaemia followed by 30 min reperfusion. The effects of lactate (10, 20 or 40 mM) were studied by adding it 20 min before ischaemia whereas reperfusion was carried out with lactate-free buffer. 3. Pretreatment with 20 or 40 mM lactate significantly reduced postischaemic recovery of developed force to 17 +/- 3% and 16 +/- 4% of preischaemic values (P < 0.05) compared to a 78 +/- 4% recovery in control hearts. Similarly, recovery in ventricular rate was significantly reduced to 34 +/- 7.6% and 38 +/- 12% with 20 and 40 mM lactate, respectively compared to 97.5 +/- 6.4% recovery in control hearts. At a concentration of 10 mM, lactate was without effect on either force or ventricular rate recovery. 4. Coadministration of either of two Na+/H+ exchange inhibitors, amiloride (174 microM) or 5-N,N-hexamethylene amiloride (HMA, 1 microM) with lactate and inclusion of the two drugs during the first 5 min of reperfusion resulted in reversal of lactate-induced inhibition of force recovery with observed recoveries of 69 +/- 6.7% and 64 +/- 5% with amiloride and HMA, respectively. Similarly, recovery in ventricular rate was significantly enhanced to 92 +/- 10% and 89 +/- 6% with amiloride and HMA, respectively compared to 38 +/- 12% recovery in control hearts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Na+ channel or Na+/H+ exchanger attenuates the hydrogen peroxide-induced derangements in isolated perfused rat heart.

The effect of tetrodotoxin, a specific inhibitor of the Na+ channel, and 5-(N,N-dimethyl)-amiloride, a specific inhibitor of the Na+/H+ exchanger, on the mechanical and metabolic derangements induced by hydrogen peroxide (H2O2) was studied in the isolated perfused rat heart. The isolated rat heart was perfused aerobically at a constant flow rate and driven electrically. H2O2 (600 microM) decreased the left ventricular developed pressure and increased the left ventricular end-diastolic pressure (i.e. mechanical dysfunction), decreased the tissue levels of adenosine triphosphate and adenosine diphosphate (i.e. metabolic derangement), and increased the tissue level of malondialdehyde (i.e. lipid peroxidation). These mechanical and metabolic derangements induced by H2O2 were significantly attenuated by tetrodotoxin (3 microM) or 5-(N,N-dimethyl)-amiloride (15 microM). Neither tetrodotoxin nor 5-(N,N-dimethyl)-amiloride modified the tissue malondialdehyde level, which was increased by H2O2. In the normal (H2O2-untreated) heart, neither tetrodotoxin nor 5-(N,N-dimethyl)-amiloride affected the mechanical function and energy metabolism. These results suggested that inhibition of the Na+ channel or Na+/H+ exchanger was effective in attenuating the H2O2-induced mechanical dysfunction and metabolic derangements in the isolated perfused rat heart.     

Na~+/H~+交换泵在人肺癌组织中的表达

目的 为了探讨肺癌细胞NHE-1基因的表达情况。方法 采用了mRNA原位杂交和图像分析技术对27例人肺癌手术切除的新鲜标本进行有关NHE-1基因表达水平方面的研究,并对其pH调节的分子机制和特点进行了初步探讨。结果 肺癌组织细胞的NHE-1 mRNA表达水平显著的高于正常肺组织(P<0.001)。结论 人肺癌组织细胞通过上调NHE-1基因表达以增加其细胞膜NHE-1的数量分布,这可能是在整体条件下pH调节的主要分子机制和特点。NHE-1基因的超表达对维持人肺癌细胞在机体内的抗凋亡生长起着重要作用,因此在以pH调节机制为靶点的肿瘤治疗研究领域中此项研究具有积极意义。