Multidrug resistance protein transporter and Ins(1,4,5)P3-sensitive Ca²+-signaling involved in adenosine triphosphate export via Gq protein-coupled NK2-receptor stimulation with neurokinin A.

The purpose of this study is to identify the membrane transport machinery and cell signaling involved in the neurokinin A-inducible release of adenosine triphosphate (ATP) as an autocrine/paracrine signal from cultured guinea-pig taenia coli (T. coli) smooth muscle cells (SMCs). ATP release evoked by neurokinin A was inhibited by L-659877, a NK(2)-receptor antagonist; by modulators for Ins(1,4,5)P(3)-sensitive Ca(2+)-signaling, U-73122, thapsigargin, and 2-APB; and by W-7, a calmodulin inhibitor, and staurosporine, a protein kinase C (PKC) inhibitor, but not by wortmannin, a phosphoinositide 3-kinase inhibitor. The evoked release was suppressed by a multidrug resistance protein (MRP)-transporter inhibitors, MK-571, indomethacin, and benzbromarone, but not by CFTR-inh 172, a CFTR-Cl(-) channel blocker, and α-glycyrrhetinic acid, a gap junction hemichannel blocker. Neurokinin A caused a marked accumulation of Ins(1,4,5)P(3) and an increase in [Ca(2+)](i) in the cultured cells. These findings suggest that stimulation of Gq/(11) protein-coupled NK(2) receptor with neurokinin A caused a substantial release of ATP from cultured T. coli SMCs and that the evoked release may be mediated by Ins(1,4,5)P(3)-sensitive Ca(2+)-signaling, further by PKC and Ca(2+)/calmodulin signals, and finally by an activation of MRP transporters as the membrane device.

Ins(1,4,5)P(3) facilitates ATP accumulation via phosphocreatine/creatine kinase in the endoplasmic reticulum extracted from MDCK cells.

So far, the content and accumulation of ATP in isolated endoplasmic reticulum (ER) are little understood. First, we confirmed using electron microscopic and Western blotting techniques that the samples extracted from MDCK cells are endoplasmic reticulum (ER). The amounts of ATP in the extracted ER were measured from the filtrate after a spinning down of ultrafiltration spin column packed with ER. When the ER sample (5mug) after 3days freezing was suspended in intracellular medium (ICM), 0.1% Triton X and ultrapure water (UPW), ATP amounts from the ER with UPW were the highest and over 10 times compared with that from the control with ICM, indicating that UPW is the most effective tool in destroying the ER membrane. After a 10-min-incubation with ICM containing phosphocreatine (PCr)/creatine kinase (CK) of the fresh ER. ATP amounts in the filtrate obtained by spinning down were not changed from that in the control (no PCr/CK). However, ATP amounts in the filtrate from the second spinning down of the ER (treated with PCr/CK) suspended in UPW became over 10-fold compared with the control. When 1muM inositol(1,4,5)trisphosphate (Ins(1,4,5)P(3)) was added in the incubation medium (ICM with PCr/CK), ATP amounts from the filtrate after the second spinning down were further enhanced around three times. This enhancement was almost canceled by Ca(2+)-removal from ICM and by adding thapsigargin, a Ca(2+)-ATPase inhibitor, but not by 2-APB and heparin, Ins(1,4,5)P(3) receptor antagonists. Administration of 500muM adenosine to the incubation medium (with PCr/CK) failed to enhance the accumulation of ATP in the ER. These findings suggest that the ER originally contains ATP and ATP accumulation in the ER is promoted by PCr/CK and Ins(1,4,5)P(3).

MRP transporters as membrane machinery in the bradykinin-inducible export of ATP.

Adenosine triphosphate (ATP) plays the role of an autocrine/paracrine signal molecule in a variety of cells. So far, however, the membrane machinery in the export of intracellular ATP remains poorly understood. Activation of B2-receptor with bradykinin-induced massive release of ATP from cultured taenia coli smooth muscle cells. The evoked release of ATP was unaffected by gap junction hemichannel blockers, such as 18alpha-glycyrrhetinic acid and Gap 26. Furthermore, the cystic fibrosis transmembrane regulator (CFTR) coupled Cl(-) channel blockers, CFTR(inh)172, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, Gd3(+) and glibenclamide, failed to suppress the export of ATP by bradykinin. On the other, the evoked release of ATP was greatly reduced by multidrug resistance protein (MRP) transporter inhibitors, MK-571, indomethacin, and benzbromarone. From western blotting analysis, blots of MRP 1 protein only, but not MRP 2 and MRP 3 protein, appeared at 190 kD. However, the MRP 1 protein expression was not enhanced after loading with 1 muM bradykinin for 5 min. Likewise, niflumic acid and fulfenamic acid, Ca2(+)-activated Cl(-) channel blockers, largely abated the evoked release of ATP. The possibility that the MRP transporter system couples with Ca2(+)-activated Cl(-) channel activities is discussed here. These findings suggest that MRP transporters, probably MRP 1, unlike CFTR-Cl(-) channels and gap junction hemichannels, may contribute as membrane machinery to the export of ATP induced by G-protein-coupled receptor stimulation.

Modulation of P2X receptors in dorsal root ganglion neurons of streptozotocin-induced diabetic neuropathy.

Painful diabetic neuropathy causes hyperalgesia and does not respond to commonly used analgesics such as non-steroidal anti-inflammatory drugs or opioids at doses below those producing disruptive side effects. In the present study, we examined the effect of P2X receptor antagonists, which are known to modulate the pain pathway, on mechanical hyperalgesia in streptozotocin (STZ)-induced diabetic mice. The paw withdrawal frequency measured by von Frey filaments, began to significantly increase 5 days after STZ injection and was maintained for more than 14 days. Intrathecal administration of P2X receptor antagonists (PPADS and TNP-ATP) inhibited the mechanical allodynia in diabetic mice. The levels of P2X(2) and P2X(3) receptors mRNA were significantly increased in diabetic mice at 14 days after the intravenous injection of STZ. These results suggest that the upregulation of P2X(2), P2X(3) and/or P2X(2/3) receptor in DRG neurons is associated with mechanical allodynia in STZ-induced diabetic mice.

Caffeine-inducible ATP release is mediated by Ca2+-signal transducing system from the endoplasmic reticulum to mitochondria.

Adenosine triphosphate (ATP) is released as an autocrine/paracrine signal from a variety of cells. The present study was undertaken to clarify the Ca2+-signal pathway involved in the caffeine-inducible release of ATP from cultured smooth muscle cells (SMC). The release of ATP induced by caffeine (3 mM) was almost completely inhibited by ryanodine and tetracaine, but not by 2-APB, thus being mediated by ryanodine receptors (RyR). The expression of messenger RNA from only RyR-2 was detected in the cells. Furthermore, the induced release was attenuated by mitochondrial inhibitors, rotenone and oligomycin and by Cl- channel blockers, niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Increase in Ca2+-signals with fluo 4 and rhod-2 caused by caffeine were reduced by tetracaine and oligomycin plus carbonyl cyanide m-chlorophenylhydrazone, respectively. A close spatial relation between the endoplasmic reticulum (ER) and mitochondria was electromicroscopically observed in the SMC, supporting the existence of a Ca2+-signaling bridge on both the organelli. These results suggest that caffeine stimulates ryanodine receptor (RyR-2) and facilitates a Ca2+-signal transducing system from ER to mitochondria, and then, the signal appears to accelerate the ATP synthesis in mitochondria. In addition, the mitochondrial event may lead further cell signaling to the cell membrane and activates Cl- channels, resulting in the extracellular release of cytosolic ATP.

Endoplasmic reticulum is a key organella in bradykinin-triggered ATP release from cultured smooth muscle cells.

ATP has broad functions as an autocrine/paracrine molecule. The mode of ATP release and its intracellular source, however, are little understood. Here we show that bradykinin via B(2)-receptor stimulation induces the extracellular release of ATP via the inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]-signaling pathway in cultured taenia coli smooth muscle cells. It was found that bradykinin also increased the production of Ins(1,4,5)P(3) and 2-APB-inhibitable [Ca(2+)](i). The evoked release of ATP was suppressed by the Ca(2+)-channel blockers, nifedipine, and verapamil. Moreover, the extracellular release of ATP was elicited by photoliberation of Ins(1,4,5)P(3). Bradykinin caused a quick and transient accumulation of intracellular ATP from cells treated with 1% perchloric acid solution (PCA), but not with the cell lysis buffer. Peak accumulation was prevented by 2-APB and thapsigargin, but not by nifedipine or verapamil, inhibitors of extracellular release of ATP. These findings suggest that bradykinin elicits the extracellular release of ATP that is mediated by the Ins(1,4,5)P(3)-induced Ca(2+) signaling and, finally, leads to a Ca(2+)-dependent export of ATP from the cells. Furthermore, the bradykinin-induced transient accumulation of ATP in the cells treated with PCA may imply a possible release of ATP from the endoplasmic reticulum.

Mitochondria play an important role in adenosine-induced ATP release from Madin-Darby canine kidney cells.

We previously found that adenosine stimulates ATP release from Madin-Darby canine kidney (MDCK) cells, by activating an Ins(1,4,5)P(3) sensitive-calcium (Ca(2+)) pathway through the stimulation of A(1) receptors. Thus, we investigated the intracellular pathway of ATP efflux after the rise in intracellular Ca(2+) in MDCK cells. Adenosine evoked an increase in mitochondrial Ca(2+) using Rhod-2/AM, a mitochondrial Ca(2+) indicator. Adenosine-induced ATP release was inhibited by mitochondrial modulators, such as oxidative phosphorylation modulators (carbonyl cyanide 3-chlorophenylhydrazone and oligomycin), mitochondrial ADP/ATP carrier inhibitors (N-ethylmaleimide, carboxyatractyloside and bongkrekic acid), a mitochondrial Na(+)-Ca(2+) exchange inhibitor (CGP-37157). In addition, mitochondrial modulators significantly reduced intracellular ATP content. On the other hand, 2-deoxy-glucose (2-DG) induced a greater decrease in intracellular ATP content than mitochondrial modulators. ATP release was still induced by adenosine in the presence of 5mM 2-DG. These results suggest that mitochondria play an important role in the signaling pathway of adenosine-triggered ATP release in MDCK cells.

Involvement of Na+/Ca2+ exchanger type-1 in ischemia-induced neovascularization in the mouse hindlimb.

The Na+/Ca2+ exchanger (NCX) is considered to be involved in endothelial nitric oxide (NO) production and endothelium-dependent vasorelaxation, but little is known about the physiological and pathological roles of endothelial NCX in these processes. We examined the role of NCX1 in neovascularization in mice with hindlimb ischemia. Unilateral hindlimb ischemia was induced surgically in wild-type and heterozygous NCX1 knockout mice (NCX1+/-) mice. We found that in NCX1+/- mice, blood flow recovery was significantly augmented compared with that in wild-type mice. N(G)-nitro-L-arginine methyl ester treatment eliminated enhanced angiogenesis observed in NCX1+/- mice. These results suggest that NCX1 is involved in eNOS-dependent angiogenesis.

A novel method for preserving human lungs using a super-cooling system.

PURPOSE: To ensure the suitable preservation of isolated lungs, a super-cooling system was used to cool water to temperatures as low as -5 degrees C without freezing. DESCRIPTION: After lung tissues were obtained from patients with lung cancer, they were kept at -5 degrees C or 4 degrees C for as many as 5 days, and then they were histologically and biochemically examined. To evaluate biochemical stability, tissues after storage were passively sensitized with immunoglobulin E and then incubated with anti-immunoglobulin-E antibody. EVALUATION: Although tissues preserved at -5 degrees C for 5 days had an almost normal appearance with intact cilia on bronchial epithelium and normal endothelium, tissues stored at 4 degrees C showed degradation of these structures. Single-stranded DNA, a sign of DNA cleavage, was frequently noted in tissues stored at 4 degrees C, but only rarely observed at -5 degrees C. A significant amount of cysteinyl-leukotrienes was generated from tissues stored at -5 degrees C for 3 days, but there was no response to antibody stimulation from tissues stored at 4 degrees C. CONCLUSIONS: Super-cooling systems may provide useful applications as a novel preserving method.

Effects of inducible nitric oxide synthase inhibitors on asthma depending on administration schedule.

The effectiveness of two inducible nitric oxide synthase (iNOS) inhibitors on allergic airway inflammation was investigated under different administration schedules. Rats sensitized to ovalbumin (OVA) were exposed to OVA for 3 consecutive days. Both iNOS inhibitors showed markedly different effects between two pretreatment schedules: pretreatment before each of three OVA exposures S1 and before the third exposure alone S2. S1 pretreatment resulted in higher pulmonary resistance than triple OVA alone. This potentiation was associated with increased eosinophil infiltration and malondialdehyde levels in the lungs, which were suppressed by superoxide dismutases (SODs) but not by methylprednisolone. However, the S2 administration of both iNOS inhibitors completely suppressed the airway response. Administration by schedule S1 completely suppressed plasma nitrite and nitrate levels, but that by S2 caused only a slight suppression. The triple OVA exposures resulted in the upregulation of iNOS in alveolar macrophages and arginase activity, Mn- and Cu/Zn-SOD expression, and nitrotyrosine and lipid peroxide deposition in the airway. However, inhibitors administered by schedule S1 suppressed this upregulation, but further potentiated nitrotyrosine, which in turn was inhibited by SOD. Although iNOS inhibitors may be beneficial for asthma, repeated administration may be detrimental because of extensive reduction of NO and downregulation of SOD.

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.

Contribution of anaphylatoxins to allergic inflammation in human lungs.

The contribution of complement activation to allergic asthma remains controversial. In order to elucidate the role played by the complement split products, anaphylatoxins C3a and C5a, we evaluated their effects on production of cysteinyl-leukotrienes (cysLTs) by human lung fragments following an anaphylactic reaction. The lung tissues obtained from two patients with lung cancer showed C5aR-, C5L2R-, and C3aR-mRNA expression. When the chopped lung fragments passively sensitized with human IgE were incubated with anti-human IgE antibody, a significant amount of cysLTs was generated in comparison with the control (without anti-IgE antibody). The co-addition of human C5a at doses of 0.1 to 10 ng/ml to the anti-IgE antibody potentiated cysLT production. The response was bell-shaped in distribution, significant, and peaked at a C5a concentration of 1 ng/ml. The co-addition of human C3a up to 1,000 ng/ml seemed to increase cysLT production, but not to any significant extent. A novel C5a receptor complementary peptide, acetylated peptide A, dose-dependently inhibited cysLT production by the human lung fragments following the anaphylactic reaction in the presence of 1 ng/ml C5a. However, this peptide did not inhibit cysLT production in the presence of 100 ng/ml C3a. It is suggested that the anaphylatoxin C5a potentiates cysLT production in human lung tissues and contributes to allergic inflammation in disorders such as asthma, thus acetylated peptide A may be useful for suppressing allergic inflammation in the lungs.

A new approach to finding specific dopamine D4 receptor agonists.

This paper describes a new approach to finding specific dopamine D4 receptor agonists based on pharmacological analysis of the contractile response to ATP in guinea pig vas deferens. A partial cDNA of the dopamine D4 receptor of the vas deferens was identified. In the vas deferens, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis revealed the existence of dopamine D4 receptor mRNA and D4 receptor protein, respectively. ATP (10(-7) M) induced a transient phasic contraction in the presence of prazosin (10(-7) M), an alpha1-adrenoceptor antagonist. This contraction was potentiated by dopamine receptor agonists in a concentration-dependent manner; and was antagonized by 8-Methyl-6-(4-methyl-1-piperazinyl)-11H-pyrido[2,3-b][1,4]benzodiazepine (JL-18), a dopamine D4 receptor antagonist, but not by raclopride, a dopamine D2 and D3 receptor antagonist. Assay methods utilizing contractile responses to ATP may be available for identifying novel dopamine D4 receptor agonists.

Comparison of effects of nitric oxide synthase (NOS) inhibitors on plasma nitrite/nitrate levels and tissue NOS activity in septic organs.

An excessive production of nitric oxide (NO) by NO synthase (NOS) is considered to contribute to circulatory disturbance, tissue damage, and refractory hypotention, which are often observed in septic disorders. It is anticipated that a selective inducible NOS (iNOS) inhibitor with excellent pharmacokinetics may be potentially effective as a novel and potent therapeutic intervention in sepsis. We examined whether or not a selective iNOS inhibitor shows iNOS selectivity at the tissue level, when administered systemically. The effects of four NOS inhibitors on plasma nitrite/nitrate (NOx) and tissue NOS levels were compared in major organs (lungs, liver, heart, kidneys, and brain) 6 hr after the injection of E. coli lipopolysaccharide (LPS) into male Wistar-King rats. The rats treated with the three iNOS inhibitors (N-(3-(aminomethyl)benzyl)acetamidine (1400W), (1 S, 5 S, 6 R, 7 R )-2-aza-7-chloro-3-imino-5-methylbicyclo [4.1.0] heptane hydrochloride (ONO-1714), and aminoguanidine) administered 1 hr after LPS injection, showed dose-dependent decreases in plasma NOx levels and NOS activity in the lungs. The non-selective NOS inhibitor (N(G)-methyl-L-arginine (L-NMMA)) had an effect only at the maximum dose. The differences in in vitro iNOS selectivity among these drugs did not correlate with iNOS selectivity at the tissue level. The relationship between plasma NOx levels and NOS activity in the lungs showed a linear relationship with or without the NOS inhibitors. In conclusion, the iNOS selectivity of these drugs does not seem to differ at the tissue level. Plasma NOx levels may be a useful indicator of lung NOS activity.

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.

Comparison study between the mechanisms of allergic asthma amelioration by a cysteinyl-leukotriene type 1 receptor antagonist montelukast and methylprednisolone.

We investigated the effects of cysteinyl-leukotriene (cysLT) type 1 receptor antagonist montelukast (MK) and compared them with those of methylprednisolone (MP) in an allergic asthma model. Rats sensitized to ovalbumin (OVA) received repeated intratracheal exposure to OVA for up to 3 consecutive days. Pretreatment with MK or MP before OVA exposure inhibited late airway response (LAR) and reduced cellular infiltration into the bronchial submucosa after the triple OVA. The amount of N-acetyl-leukotriene E(4) in the bile was significantly reduced by pretreatment with MK or MP, suggesting that both drugs reduced the production of cysLTs in the lungs. In the in vitro study, when the fragments of lungs that had been repeatedly pretreated with MK or MP and exposed to OVA were removed and incubated with OVA, the coaddition of either drug significantly reduced cysLT production. In contrast, the cysLT production following the addition of OVA to the lung fragments that had not received in vivo pretreatment with either drug was inhibited by MK but not by MP. These results indicate that MK and MP inhibit LAR by suppressing the infiltration of inflammatory cells into the bronchial submucosa, thereby inhibiting the production of cysLTs in the lungs, and that MK but not MP may inhibit cysLT production directly. The different effects on cysLT production between the two drugs may provide a rationale for the use of combination therapy with cysLT(1) receptor antagonists and steroids for the treatment of asthma.

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.

[The mechanism of ATP release as an autocrine/paracrine molecule].

Many studies have been performed to clarify the underlying mechanisms of the release of ATP as an autocrine / paracrine signaling molecule. So far, there is a variety of findings on the mode of release of this nucleotide. This review focused on the possible mechanisms of ATP release. The ATP binding cassette, especially CFTR (cystic fibrosis transmembrane conductance regulator), is a strong candidate for a channel or a transporter for outward movement of ATP. CFTR, which is activated via phosphorylation by protein kinase A, causes an opening of channels for Cl(-) and ATP(4-), releasing ATP. However, the possible involvement of CFTR in ATP release is still under dispute. As another candidate of the membrane machinery, the hemichannel of gap junction has been raised. Mechanical stress and photoliberation of caged InsP(3) induce the release of ATP as a paracrine through the hemichannel accompanied with the increase of [Ca(2+)]i. These events result in the Ca(2+)wave as cell-to-cell communications. In conclusion, an authoritative view of the mechanism of ATP release remains to be made clear in future studies.

The exchanger inhibitory peptide region-dependent inhibition of Na+/Ca2+ exchange by SN-6 [2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester], a novel benzyloxyphenyl derivative.

We investigated the properties and interaction domains of SN-6 [2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester], a newly synthesized and selective Na(+)/Ca(2+) exchange (NCX) inhibitor. SN-6 (0.3-30 microM) inhibited preferentially intracellular Na(+)-dependent (45)Ca(2+) uptake (i.e., the reverse mode) compared with extracellular Na(+)-dependent (45)Ca(2+) efflux (i.e., the forward mode) in NCX1-transfected fibroblasts. SN-6 was 3- to 5-fold more inhibitory to (45)Ca(2+) uptake in NCX1 (IC(50) = 2.9 microM) than to that in NCX2 or NCX3 but not to that in NCKX2. We searched for regions that may form the SN-6 receptor by NCX1/NCX3-chimeric analyses and determined that amino acid regions 73 to 108 and 193 to 230 in NCX1 are mostly responsible for the differential drug response between NCX1 and NCX3. Further site-directed mutagenesis revealed that double substitutions of Val227 and Tyr228 in NCX1, which exist within the exchanger inhibitory peptide (XIP) region, mimicked the different drug response. In addition, F213R, G833C, and N839A mutations in NCX1 resulted in loss of drug sensitivity. Exchangers with mutated XIP regions, which display either undetectable or accelerated Na(+)-dependent inactivation, had markedly reduced sensitivity or hypersensitivity to SN-6, respectively. Cell ATP depletion enhanced the inhibitory potency of SN-6. Therefore, SN-6 at lower doses (IC(50) = 0.63 microM) potently protected against hypoxia/reoxygenation-induced cell damage in renal tubular cells overexpressing NCX1, suggesting that this drug predominantly works under hypoxic/ischemic conditions. These properties of SN-6, which may be derived from its interaction with the XIP region, are advantageous to developing it as a new anti-ischemic drug.

Molecular determinants of Na+/Ca2+ exchange (NCX1) inhibition by SEA0400.

SEA0400 is a potent and selective Na(+)/Ca(2+) exchanger (NCX) inhibitor. We evaluated the inhibitory effects of SEA0400 on Na(+)(i)-dependent (45)Ca(2+) uptake and whole-cell Na(+)/Ca(2+) exchange currents in NCX-transfected fibroblasts. SEA0400 preferentially inhibited (45)Ca(2+) uptake by NCX1 compared with inhibitions by NCX2, NCX3, and NCKX2. SEA0400 also selectively blocked outward exchange currents from NCX1 transfectants. We searched for regions that may form the SEA0400 receptor in the NCX1 molecule by NCX1/NCX3 chimeric analysis. The results suggest that the first intracellular loop and the fifth transmembrane segment are mostly responsible for the differential drug responses between NCX1 and NCX3. Further site-directed mutagenesis revealed that multiple mutations at Phe-213 markedly reduced sensitivity to SEA0400 without affecting that to KB-R7943. We also found that Gly-833-to-Cys mutation (within the alpha-2 repeat) greatly reduced the inhibition by SEA0400, but unexpectedly the NCX1 chimera with an alpha-2 repeat from NCKX2 possessed normal drug sensitivity. In addition, exchangers with mutated exchanger inhibitory peptide regions, which display either undetectable or accelerated Na(+)-dependent inactivation, had a markedly reduced sensitivity or hypersensitivity to SEA0400, respectively. To verify the efficacy of the NCX inhibitor, we examined the renoprotective effect of SEA0400 in a hypoxic injury model using porcine renal tubular cells. SEA0400 protected against hypoxia/reoxygenation-induced cell damage in tubular cells expressing wild-type NCX1 but not in cells expressing SEA0400-insensitive mutants. These results suggest that Phe-213, Gly-833, and residues that eliminate Na(+)-dependent inactivation are critical determinants for the inhibition by SEA0400, and their mutants are very useful for checking the pharmacological importance of NCX inhibition by SEA0400.