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Biomedical subjects

Takahiro Iwamoto

Publications and source records attributed to Takahiro Iwamoto.

At least 19 recordsLinked to original sources
PubMed2006

YM-244769, a novel Na+/Ca2+ exchange inhibitor that preferentially inhibits NCX3, efficiently protects against hypoxia/reoxygenation-induced SH-SY5Y neuronal cell damage.

We investigated the pharmacological properties and interaction domains of N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide (YM-244769), a novel potent Na(+)/Ca(2+) exchange (NCX) inhibitor, using various NCX-transfectants and neuronal and renal cell lines. YM-244769 preferentially inhibited intracellular Na(+)-dependent (45)Ca(2+) uptake via NCX3 (IC(50) = 18 nM); the inhibition was 3.8- to 5.3-fold greater than for the uptake via NCX1 or NCX2, but it did not significantly affect extracellular Na(+)-dependent (45)Ca(2+) efflux via NCX isoforms. We searched for interaction domains with YM-244769 by NCX1/NCX3-chimeric analysis and determined that the alpha-2 region in NCX1 is mostly responsible for the differential drug response between NCX1 and NCX3. Further cysteine scanning mutagenesis in the alpha-2 region identified that the mutation at Gly833 markedly reduced sensitivity to YM-244769. Mutant exchangers that display either undetectable or accelerated Na(+)-dependent inactivation, had markedly reduced sensitivity or hypersensitivity to YM-244769, respectively. YM-244769, like 2-[2-[4-(4-nitrobenzyloxyl)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), protected against hypoxia/reoxygenation-induced cell damage in neuronal SH-SY5Y cells, which express NCX1 and NCX3, more efficiently than that in renal LLC-PK(1) cells, which exclusively express NCX1, whereas 2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester (SN-6) suppressed renal cell damage to a greater degree than neuronal cell damage. These protective potencies consistently correlated well with their inhibitory efficacies for the Ca(2+) uptake via NCX isoforms existing in the corresponding cell lines. Antisense knockdown of NCX1 and NCX3 in SH-SY5Y cells confirmed that NCX3 contributes to the neuronal cell damage more than NCX1. Thus, YM-244769 is not only experimentally useful as a NCX inhibitor that preferentially inhibits NCX3, but also has therapeutic potential as a new neuroprotective drug.

Animals↗
PubMed2006

Topics on the Na+/Ca2+ exchanger: role of vascular NCX1 in salt-dependent hypertension.

Excess salt intake is a major risk factor for hypertension. However, the molecular mechanisms underlying salt-dependent hypertension remain obscure. Our recent studies using selective Na(+)/Ca(2+) exchange inhibitors and genetically engineered mice provide compelling evidence that salt-dependent hypertension is triggered by Ca(2+) entry through Na(+)/Ca(2+) exchanger type 1 (NCX1) in arterial smooth muscle. Endogenous cardiac glycosides, which may contribute to salt-dependent hypertension, seem to be necessary for NCX1-mediated hypertension. Intriguingly, recent studies by Dostanic-Larson et al. using knock-in mice with modified cardiac glycoside binding affinity of Na(+),K(+)-ATPases demonstrate that this binding site plays an important physiological role in blood pressure control. Thus, when cardiac glycosides inhibit Na(+),K(+)-ATPase in arterial smooth muscle cells, the elevation of local Na(+) on the submembrane area is believed to facilitate Ca(2+) entry through NCX1, resulting in vasoconstriction. This proposed pathway may have enabled us to explain how to link dietary salt to hypertension.

Animals↗
PubMed2006

Intravascular two-dimensional tissue strain imaging.

Our goal is to achieve the precise quantitative imaging of tissue elasticity in clinical settings. In the present study, we measured basic ultrasonic characteristics of atherosclerosis by two-dimensional (2D) intravascular tissue velocity imaging. Radio-frequency (RF) signal from a clinically used IVUS apparatus was digitized at 500 MSa/s and stored in a workstation. First, the correlation coefficient between two consecutive frames was calculated in the rotational direction and the rotational disuniformity was corrected to obtain the maximum correlation coefficient. Then, the polar coordinate images were converted into rectangular coordinate images and the images were divided into 64 by 64 square shaped regions of interest (ROIs). The correlation and displacement of the ROIs between the consecutive two frames were calculated by template matching method. Two-dimensional tissue velocity was defined as the vectors of displacement of ROI with 0.7 and more correlation. IVUS studies were performed in directional coronary atherectomy (DCA) procedures. The specimens excised by DCA were stained with Elastica-Masson's trichrome staining and CD68 immunochemical staining. Eleven cases (including two no re-flow cases and one perforation case) were intraoperatively observed by IVUS and the specimens obtained by DCA were observed by optical microscopy. The specimen from homogeneous 2D strain was collagen dominant fibrosis and the specimen from a lesion with complex vectors contained CD68 positive cells and degenerated collagen fibers, which indicated the plaque was vulnerable.

Algorithms↗
PubMed2006

Vascular Na+/Ca2+ exchanger: implications for the pathogenesis and therapy of salt-dependent hypertension.

The Na+/Ca2+ exchanger is an ion transporter that exchanges Na+ and Ca2+ in either Ca2+ efflux or Ca2+ influx mode, depending on membrane potential and transmembrane ion gradients. In arterial smooth muscle cells, the Na+/Ca2+ exchanger is thought to participate in the maintenance of vascular tone by regulating cytosolic Ca2+ concentration. Recent pharmacological and genetic engineering studies have revealed that the Ca2+ influx mode of vascular Na+/Ca2+ exchanger type-1 (NCX1) is involved in the pathogenesis of salt-dependent hypertension. SEA0400, a specific Na+/Ca2+ exchange inhibitor that preferentially blocks the Ca2+ influx mode, lowers arterial blood pressure in salt-dependent hypertensive models, but not in normotensive rats or other types of hypertensive rats. Furthermore, heterozygous mice with reduced expression of NCX1 are resistant to development of salt-dependent hypertension, whereas transgenic mice with vascular smooth muscle-specific overexpression of NCX1 readily develop hypertension after high-salt loading. SEA0400 reverses the cytosolic Ca2+ elevation and vasoconstriction induced by nanomolar ouabain, as well as humoral factors in salt-loaded animals. One possibility is that circulating endogenous cardiotonic steroids may be necessary for NCX1-mediated hypertension. These findings help to explain how arterial smooth muscle cells in blood vessels contribute to salt-elicited blood pressure elevation and suggest that NCX1 inhibitors might be therapeutically useful for salt-dependent hypertension.

Animals↗
PubMed2006

[Na+/Ca2+ exchanger(NCX1) and salt-sensitive hypertension].

Hypertension is the most common chronic disease, and is the leading risk factor for death caused by stroke, myocardial infarction, and end-stage renal failure. The critical importance of excess salt intake in the pathogenesis of hypertension is widely recognized. However, the molecular mechanisms underlying salt-sensitive hypertension remain obscure. Recent studies using selective inhibitors and genetically engineered mice provide compelling evidence that salt-sensitive hypertension is triggered by Ca2+ entry through Na+/Ca2+ exchanger type-1 (NCX1) in vascular smooth muscle. Intriguingly, endogenous Na+ pump inhibitors seem to be necessary for NCX1-mediated hypertension. These findings have enabled us to explain how high salt intake leads to hypertension, and further to describe the potential of vascular NCX1 as a new therapeutic or diagnostic target for salt-sensitive hypertension.

Aniline Compounds↗
PubMed2005

Sodium pump alpha2 subunits control myogenic tone and blood pressure in mice.

A key question in hypertension is: How is long-term blood pressure controlled? A clue is that chronic salt retention elevates an endogenous ouabain-like compound (EOLC) and induces salt-dependent hypertension mediated by Na(+)/Ca(2)(+) exchange (NCX). The precise mechanism, however, is unresolved. Here we study blood pressure and isolated small arteries of mice with reduced expression of Na(+) pump alpha1 (alpha1(+/-)) or alpha2 (alpha2(+/-)) catalytic subunits. Both low-dose ouabain (1-100 nm; inhibits only alpha2) and high-dose ouabain (> or =1 microm; inhibits alpha1) elevate myocyte Ca(2)(+) and constrict arteries from alpha1(+/-), as well as alpha2(+/-) and wild-type mice. Nevertheless, only mice with reduced alpha2 Na(+) pump activity (alpha2(+/-)), and not alpha1 (alpha1(+/-)), have elevated blood pressure. Also, isolated, pressurized arteries from alpha2(+/-), but not alpha1(+/-), have increased myogenic tone. Ouabain antagonists (PST 2238 and canrenone) and NCX blockers (SEA0400 and KB-R7943) normalize myogenic tone in ouabain-treated arteries. Only the NCX blockers normalize the elevated myogenic tone in alpha2(+/-) arteries because this tone is ouabain independent. All four agents are known to lower blood pressure in salt-dependent and ouabain-induced hypertension. Thus, chronically reduced alpha2 activity (alpha2(+/-) or chronic ouabain) apparently regulates myogenic tone and long-term blood pressure whereas reduced alpha1 activity (alpha1(+/-)) plays no persistent role: the in vivo changes in blood pressure reflect the in vitro changes in myogenic tone. Accordingly, in salt-dependent hypertension, EOLC probably increases vascular resistance and blood pressure by reducing alpha2 Na(+) pump activity and promoting Ca(2)(+) entry via NCX in myocytes.

Animals↗
PubMed2005

Down-regulation of Na+/Ca2+ exchanger by fluvastatin in rat cardiomyoblast H9c2 cells: involvement of RhoB in Na+/Ca2+ exchanger mRNA stability.

We investigated the effect of fluvastatin (Flv), an HMG-CoA reductase inhibitor, on Na(+)/Ca(2+) exchanger 1 (NCX1) expression in H9c2 cardiomyoblasts. Reverse transcriptase-polymerase chain reaction analyses revealed that Flv decreased NCX1 mRNA in a concentration- and time-dependent manner and NCX1 protein. This effect of Flv was caused by the inhibition of HMG-CoA reductase, because Flv did not affect the NCX1 mRNA in the presence of mevalonate. Flv-induced down-regulation of NCX1 mRNA was also cancelled by farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), suggesting an involvement of small G-proteins. However, overexpression of neither constitutive active RhoA nor Ras affected NCX1 mRNA. In contrast, intracellular expression of C3 toxin, a specific inhibitor of Rho family proteins, decreased NCX1 mRNA, suggesting that Flv decreases NCX1 mRNA by inhibiting a signaling pathway of Rho family proteins other than RhoA. On the other hand, lysophosphatidylcholine (LPC), an activator of Rho signaling, increased both NCX1 mRNA and protein in a C3 toxin-sensitive manner. Western blot analyses revealed that membrane-associated RhoB, which is isoprenylated by either FPP or GGPP, was decreased by Flv but was increased by LPC. Selective inhibition of gene expression by short interfering RNA duplex showed that RhoB but not RhoA is involved in the regulation of NCX1 mRNA and protein. When transcription was blocked by 5,6-dichlorobenzimidazole riboside, the NCX1 mRNA stability was decreased by Flv. Long-term treatment of rat with Flv in vivo also down-regulated the cardiac NCX1 mRNA. These results suggest that a RhoB-mediated signaling pathway regulates cardiac NCX1 levels by controlling the NCX1 mRNA stability.

Animals↗
PubMed2005

Adenosine induces ATP release via an inositol 1,4,5-trisphosphate signaling pathway in MDCK cells.

ATP is released into extracellular space as an autocrine/paracrine molecule by mechanical stress and pharmacological-receptor activation. Released ATP is partly metabolized by ectoenzymes to adenosine. In the present study, we found that adenosine causes ATP release in Madin-Darby canine kidney cells. This release was completely inhibited by CPT (an A1 receptor antagonist), U-73122 (a phospholipase C inhibitor), 2-APB (an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor blocker), thapsigargin (a Ca2+-ATPase inhibitor), and BAPTA/AM (an intracellular Ca2+ chelator), but not by DMPX (an A2 receptor antagonist). However, forskolin, epinephrine, and isoproterenol, inducers of cAMP accumulation, failed to release ATP. Adenosine increased intracellular Ca2+ concentrations that were strongly blocked by CPT, U-73122, 2-APB, and thapsigargin. Moreover, adenosine enhanced accumulations of Ins(1,4,5)P3 that were significantly reduced by U-73122 and CPT. These data suggest that adenosine induces the release of ATP by activating an Ins(1,4,5)P3 sensitive-Ca2+ pathway through the stimulation of A1 receptors.

Adenosine↗
PubMed2005

Different cation sensitivities and binding site domains of Na+-Ca2+-K+ and Na+-Ca2+ exchangers.

We examined inhibitory effects of external multivalent cations Ni(2+), Co(2+), Cd(2+), La(3+), Mg(2+), and Mn(2+) on reverse-mode exchange of the K(+)-dependent Na(+)/Ca(2+) exchanger NCKX2 and the K(+)-independent exchanger NCX1 expressed in CCL-39 cells by measuring the rate of Ca(2+) uptake with radioisotope tracer and electrophysiological techniques. The apparent affinities for block of Ca(2+) uptake by multivalent cations was higher in NCKX2 than NCX1, and the rank order of inhibitory potencies among these cations was different. Additional experiments also showed that external Li(+) stimulated reverse-mode exchange by NCX1, but not NCKX2 in the presence of 5 mM K(+). Thus, both exchangers exhibited differential sensitivities to not only K(+) but also many other external cations. We attempted to locate the putative binding sites within the alpha motifs for multivalent cations by site-directed mutagenesis experiments. The cation affinities of NCKX2 were altered by mutations of amino acid residues in the alpha-1 motif, but not by mutations in the alpha-2 motif. These results contrast with those for NCX1 where mutations in both alpha-1 and alpha-2 motifs have been shown previously to affect cation affinities. Susceptibility tests with sulfhydryl alkylating agents suggested that the alpha-1 and alpha-2 motifs are situated extracellularly and intracellularly, respectively, in both exchangers. A topological model is proposed in which the extracellular-facing alpha-1 motif forms an external cation binding site that includes key residues N203, G207C, and I209 in NCKX2, while both alpha-1 and alpha-2 motifs together form the binding sites in NCX1.

Amino Acid Motifs↗
PubMed2005

Salt-sensitive hypertension, Na+/Ca2+ exchanger, and vascular smooth muscle.

Hypertension is the most common chronic disease and is the leading risk factor for death caused by stroke, myocardial infarction, and end-stage renal failure. The critical importance of excess salt intake in the pathogenesis of hypertension is widely recognized. However, the molecular mechanisms underlying salt-sensitive hypertension remain obscure. Recent studies using selective Na(+)/Ca(2+) exchanger (NCX) inhibitors and genetically engineered mice provide compelling evidence that salt-sensitive hypertension is triggered by Ca(2+) entry through NCX type 1 (NCX1) in arterial smooth muscle. Cardiotonic steroids, such as endogenous ouabain, which may contribute to the pathogenesis of salt-sensitive hypertension, seem to be necessary for NCX1-mediated hypertension. These findings have enabled us to explain how high salt intake leads to hypertension and further to describe the potential of vascular NCX1 as a new therapeutic or diagnostic target for salt-sensitive hypertension.

Aniline Compounds↗
PubMed2004

Salt-sensitive hypertension is triggered by Ca2+ entry via Na+/Ca2+ exchanger type-1 in vascular smooth muscle.

Excessive salt intake is a major risk factor for hypertension. Here we identify the role of Na(+)/Ca(2+) exchanger type 1 (NCX1) in salt-sensitive hypertension using SEA0400, a specific inhibitor of Ca(2+) entry through NCX1, and genetically engineered mice. SEA0400 lowers arterial blood pressure in salt-dependent hypertensive rat models, but not in other types of hypertensive rats or in normotensive rats. Infusion of SEA0400 into the femoral artery in salt-dependent hypertensive rats increases arterial blood flow, indicating peripheral vasodilation. SEA0400 reverses ouabain-induced cytosolic Ca(2+) elevation and vasoconstriction in arteries. Furthermore, heterozygous NCX1-deficient mice have low salt sensitivity, whereas transgenic mice that specifically express NCX1.3 in smooth muscle are hypersensitive to salt. SEA0400 lowers the blood pressure in salt-dependent hypertensive mice expressing NCX1.3, but not in SEA0400-insensitive NCX1.3 mutants. These findings indicate that salt-sensitive hypertension is triggered by Ca(2+) entry through NCX1 in arterial smooth muscle and suggest that NCX1 inhibitors might be useful therapeutically.

Alternative Splicing↗
PubMed2004

Forefront of Na+/Ca2+ exchanger studies: physiology and molecular biology of monovalent cation sensitivities in Na+/Ca2+ exchangers.

Sensitivities of the reverse-mode Na+/Ca2+ exchange activity measured as the Na+i-dependent Ca2+ uptake to extracellular monovalent cations K+, Li+, and Na+ were compared between the K+ -dependent (NCKX2) and the K+ -independent Na+/Ca2+ exchanger (NCX1) overexpressed in a fibroblast cell. Interestingly, the exchange activity of NCKX2 was not influenced by Li+ while it was increased by K+. On the contrary, the activity of NCX1 was increased by Li+. Thus, the cation sensitivities to K+ and Li+ markedly differed between NCKX2 and NCX1. In addition, Na+ exerted a significantly smaller inhibitory effect on the activity in NCKX2 than in NCX1. The Na+/Ca2+ exchange activities of NCKX2 and NCX1 are considered to be regulated differentially via the respective binding site domains that have distinct sensitivities to the external monovalent cations.

Amino Acid Sequence↗
PubMed2004

Forefront of Na+/Ca2+ exchanger studies: molecular pharmacology of Na+/Ca2+ exchange inhibitors.

The Na+/Ca2+ exchanger (NCX) is an ion transporter that exchanges Na+ and Ca2+ in either Ca2+ efflux or Ca2+ influx mode, depending on membrane potential and transmembrane ion gradients. In myocytes, neurons, and nephron cells, NCX is thought to play an important role in the regulation of intracellular Ca2+ concentration. Recently, the benzyloxyphenyl derivatives KB-R7943, SEA0400, and SN-6 have been developed as selective NCX inhibitors. Currently, SEA0400 is the most potent and selective inhibitor. These inhibitors possess different isoform-selectivities, although they have similar properties, such as Ca2+ influx mode-selectivity and I1 inactivation-dependence. Recent site-directed mutagenesis has revealed that these inhibitors possess some molecular determinants (Phe-213, Val-227, Tyr-228, Gly-833, and Asn-839) for interaction with NCX1. These benzyloxyphenyl derivatives are expected to be useful tools to study the physiological roles of NCX. Moreover, such inhibitors may have therapeutic potential as a new remedy for ischemic disease, arrhythmias, heart failure, and hypertension.

Aniline Compounds↗
PubMed2004

Effects of amiodarone on mutant Na+/Ca2+ exchangers expressed in CCL 39 cells.

Using the whole cell voltage clamp, we reported previously that amiodarone acutely inhibits Na+/Ca2+ exchange current (INCX) in guinea pig cardiac ventricular myocytes. Intracellular application of trypsin via the patch pipette attenuated the blocking effect of amiodarone, suggesting that amiodarone affects the Na+/Ca2+ exchanger (NCX) from the cytoplasmic side. Here, we attempted to detect the site of amiodarone inhibition using wild type NCX1, mutants, and NCX3 expressed in CCL39 fibroblasts. INCX was recorded by ramp pulses. Amiodarone at 30 microM inhibited INCX by 80% in cells expressing wild type NCX1. However, 30 microM amiodarone inhibited INCX by about 55% in cells expressing mutant NCX1 with amino acids 217-671 (DeltaXIP) or 247-671 (Delta247-671) deleted in the long intracellular loop between the transmembrane segments (TM) 5 and 6. INCXs from NCX mutants deleted of cytoplasmic TM1-2, TM3-4 or the C-terminus were inhibited by amiodarone to a similar extent as the wild type. Amiodarone also inhibited INCX of NCX3 by 76%. These results suggest that a long intracellular loop may be involved in the inhibition of NCX1 by amiodarone, but that other intracellular loops, XIP region or C terminus are not involved in the amiodarone inhibition of NCX1.

Amiodarone↗
PubMed2004

Development and application of Na+/Ca2+ exchange inhibitors.

The Na+/Ca2+ exchanger (NCX) is an ion transporter that exchanges Na+ and Ca2+ in either Ca2+ efflux or Ca2+ influx mode, depending on the ion gradients across the plasma membrane and the membrane potential. In heart, smooth muscle cells, neurons, and nephron cells, the NCX is thought to play an important role in the regulation of intracellular Ca2+ concentration. Recently, a novel selective inhibitor (KB-R7943 and SEA0400) of the Ca2+ influx mode of the NCX has been developed. NCX inhibitor is expected to be a pharmaceutical agent that offers effective protection against ischemia/reperfusion injury in several organs such as heart and kidney. Here, we summarize pharmacological profiles of KB-R7943 and SEA0400, the molecular mechanism of its action, and its future prospect as a novel pharmaceutical agent.

Aniline Compounds↗
PubMed2004

Endothelin-1 enhances the activity of Na+/Ca2+ exchanger type 1 in renal epithelial cells.

The Na+/Ca2+ exchanger, an ion transporter across the plasma membrane, is considered to play a role in calcium reabsorption in the renal nephron. We found that endothelin-1 enhanced Na+/Ca2+ exchange activity in renal epithelial LLC-PK1 cells. Treatment with endothelin-1 increased concomitantly the phosphorylation of Na+/Ca2+ exchanger type 1 (NCX1). Chelerythrine and prolonged exposure to phorbol 12-myristate 13-acetate abolished the activation of NCX1 induced by endothelin-1. To further analyze the activation mechanism of NCX1 by endothelin-1, we examined the effects of endothelin-1 in LLC-PK1 cells expressing NCX1 with mutated exchanger inhibitory peptide regions, which have either no Na+-dependent inactivation (XIP-4YW) or accelerated inactivation (F223E). The exchange activities in LLC-PK1 cells expressing wild-type NCX1 or F223E were stimulated by endothelin-1, but not in cells expressing XIP- 4YW. These results suggest that endothelin-1 activates NCX1 in renal epithelial cells through the pathway of protein kinase C and the process related to Na-dependent inactivation.

Animals↗