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We investigated the effects of water-soluble low-molecular-weight β-(1,3-1,6) D-glucan isolated from Aureobasidium pullulans1A1 strain black yeast (LMW-β-glucan) on mast cell-mediated anaphylactic reactions. Although it is known that LMW-β-glucan has anti-tumor, anti-metastatic and anti-stress effects, the roles of LMW-β-glucan in immediate-type allergic reactions have not been fully investigated. We examined whether LMW-β-glucan could inhibit mast cell degranulation and passive cutaneous anaphylaxis (PCA). LMW-β-glucan dose-dependently inhibited the degranulation of both rat basophilic leukemia (RBL-2H3) and cultured mast cells (CMCs) activated by calcium ionophore A23187 or IgE. However, LMW-β-glucan had no cytotoxicity towards RBL-2H3 cells and CMCs. Furthermore, orally administered LMW-β-glucan inhibited the IgE-induced PCA reaction in mice. These results show LMW-β-glucan to be a possible compound for the effective therapeutic treatment of allergic diseases.

Syntrophin is known to be a component of the dystrophin-glycoprotein complex (DGC), a membrane/cytoskeleton-anchoring structure that is essential for the maintenance of viability of sarcolemma. We purified DGC from hearts of human and several animal species, and compared their protein composition. While almost all components of DGC were present in various species, proteins with the apparent molecular mass of 50–65 kDa corresponding to syntrophin isoforms were very different among them. Three isoforms of syntrophin (a1, ß1, ß2) were expressed in hamster, rat and canine ventricles, whereas only a1-isoform was mainly expressed in human and rabbit ventricles. Immunohistochemical analysis revealed that a1-and ß2-syntrophins were co-localized in sarcolemma and in T-tubules of canine ventricles. However, despite membrane localization of most syntrophins, subcellular fractionation revealed that part of syntrophins were recovered in the cytosolic fraction devoid of other components of DGC, raising the possibility that syntrophins may play multiple roles in various intracellular sites of cardiac muscle cells. Species-dependent expression and unique subcellular localization of syntrophins in cardiac muscle may contribute to the variable severity of muscle dysgenesis caused by the same primary defect in components of DGC of human and other animal species. (Mol Cell Biochem 268: 59–66, 2005)

Using Na+/Ca2+exchanger (NCX1)-deficient mice, the pathophysiological role of Ca2+overload via the reverse mode of NCX1 in ischemia/reperfusion-induced renal injury was investigated. Because NCX1−/−homozygous mice die of heart failure before birth, we used NCX1+/−heterozygous mice. NCX1 protein in the kidney of heterozygous mice decreased to about half of that of wild-type mice. Expression of NCX1 protein in the tubular epithelial cells and Ca2+influx via NCX1 in renal tubules were markedly attenuated in the heterozygous mice. Ischemia/reperfusion-induced renal dysfunction in heterozygous mice was significantly attenuated compared with cases in wild-type mice. Histological renal damage such as tubular necrosis and proteinaceous casts in tubuli in heterozygous mice were much less than that in wild-type mice. Ca2+deposition in necrotic tubular epithelium was observed more markedly in wild-type than in heterozygous mice. Increases in renal endothelin-1 content were greater in wild-type than in heterozygous mice, and this reflected the difference in immunohistochemical endothelin-1 localization in necrotic tubular epithelium. When the preischemic treatment with KB-R7943 was performed, the renal functional parameters of both NCX1+/+and NCX1+/−acute renal failure mice were improved to the same level. These findings strongly support the view that Ca2+overload via the reverse mode of Na+/Ca2+exchange, followed by renal endothelin-1 overproduction, plays an important role in the pathogenesis of ischemia/reperfusion-induced renal injury.

The membrane domain of the NaCa2exchanger (NCX) contains two conserved internal repeat sequences, designated the ?-1 and ?-2 repeats. We have studied the topological disposition of residues in the ? repeats and a neighboring region by substituted cysteine accessibility scanning as well as the functional importance of these residues by kinetically evaluating transport activities of cysteine-substituted or other site-directed NCX1 mutants. The results suggest that the ?-1 repeat contains a reentrant loop originating from extracellular side of the membrane, while the ?-2 repeat and its neighboring region contain a complex reentrant loop structure originating from the cytoplasmic side. We identified several residues in the ?-1 repeat loop whose mutation caused significant alterations in the interaction of NCX1 with a transport substrate Ca2oand inhibitory ions Ni2and Co2. On the other hand, we found residues in the ?-2 repeat loop region whose mutation altered the interaction with an activating ion Liand an inhibitory drug KB-R7943 in addition to the effect on Ca2oand Ni2. Collectively, our data suggest that these ?-repeat regions participate in the formation of ion translocation pathway of the exchanger and that the ?-1 repeat loop plays an important role in ion selection.

The calcineurin B homologous protein (designated CHP1) has been shown to be a common essential cofactor for the plasma membrane Na(+)/H(+) exchangers (NHEs) (Pang, T., Su, X., Wakabayashi, S., and Shigekawa, M. (2001) J. Biol. Chem. 276, 17367-17372). In this study, we characterized the function of another isoform of CHP (designated CHP2) that has a 61% amino acid identity with CHP1. CHP2, like CHP1, conferred the ability to NHEs 1-3 to express a high exchange activity by binding to the juxtamembrane region of the cytoplasmic domain of the exchanger, but it interacts more strongly (approximately 5-fold) with NHE1 than does CHP1. Although CHP1 is expressed ubiquitously at relatively high levels, CHP2 expression was extremely low in most human tissues but was higher in tumor cells. We produced stable cell clones overexpressing either CHP1 or CHP2 in which one of them is predominantly bound to NHE1. Serum (10%) induced a significant cytoplasmic alkalinization (0.1-0.2 pH unit) in cells co-expressing CHP1 and NHE1 but not in cells co-expressing CHP2 and NHE1. In the latter, pH(i) was high (7.4-7.5) even in the absence of serum, suggesting that NHE1 was already activated. Surprisingly, most (>80%) of CHP2/NHE1 cells unlike CHP1/NHE1 cells were viable even after long serum starvation (>7 days). Thus, the expression of CHP2 appears to protect cells from serum deprivation-induced death by increasing pH(i). These properties of CHP2/NHE1 cells are similar to those of malignantly transformed cells. We propose that serum-independent activation of NHE1 by bound CHP2 is one of the key mechanisms for the maintenance of high pH(i) and the resistance to serum deprivation-induced cell death in malignantly transformed cells.

Using Na+/Ca2+ exchanger (NCX1)-deficient mice, the pathophysiological role of Ca2+ overload via the reverse mode of the Na+/Ca2+ exchanger in ischaemia/reperfusion-induced renal injury was investigated. Since NCX1-/- homozygous mice die of heart failure before birth, we utilized NCX1+/- heterozygous mice. The ischaemia/reperfusion-induced renal dysfunction in heterozygous mice were significantly attenuated compared with cases in wild-type mice. Also, histological renal damage such as tubular necrosis and proteinaceous casts in tubuli in heterozygous mice were much less than that in wild-type mice. Ca2+ deposition in necrotic tubular epithelium was observed more markedly in wild-type than in heterozygous mice. The increase in renal endothelin-1 (ET-1) content was significantly greater in wild-type than in heterozygous mice, and this reflected the difference in immunohistochemical ET-1 localization in necrotic tubular epithelium. We conclude that Ca2+ overload via the reverse-mode of Na+/Ca2+ exchange, followed by renal ET-1 overproduction, plays an important role in the pathogenesis of ischaemia/reperfusion-induced acute renal failure.

The NHE6 protein is a unique Na+/H+exchanger isoform believed to localize in mitochondria. It possesses a hydrophilic N-terminal portion that is rich in positively charged residues and many hydrophobic segments. In the present study, signal sequences in the NHE6 molecule were examined for organelle localization and membrane topogenesis. When the full-length protein was expressed in COS7 cells, it localized in the endoplasmic reticulum and on the cell surface. Furthermore, the protein was fully N-glycosylated. When green fluorescent protein was fused after the second (H2) or third (H3) hydrophobic segment, the fusion proteins were targeted to the endoplasmic reticulum (ER) membrane. The localization pattern was the same as that of fusion proteins in which green fluorescent protein was fused after H2 of NHE1. In an in vitrosystem, H1 behaved as a signal peptide that directs the translocation of the following polypeptide chain and is then processed off. The next hydrophobic segment (H2) halted translocation and eventually became a transmembrane segment. The N-terminal hydrophobic segment (H1) of NHE1 also behaved as a signal peptide. Cell fractionation studies using antibodies against the 15 C-terminal residues indicated that NHE6 protein localized in the microsomal membranes of rat liver cells. All of the NHE6 molecules in liver tissue possess an endoglycosidase H-resistant sugar chain. These findings indicate that NHE6 protein is targeted to the ER membrane via the N-terminal signal peptide and is sorted to organelle membranes derived from the ER membrane.

The Na+/H+exchangers (NHEs) comprise a family of transporters that catalyze cell functions such as regulation of the pH and volume of a cell and epithelial absorption of Na+and bicarbonate. Ubiquitous calcineurin B homologous protein (CHP or p22) is co-localized and co-immunoprecipitated with expressed NHE1, NHE2, or NHE3 independently of its myristoylation and Ca2+binding, and its binding site was identified as the juxtamembrane region within the carboxyl-terminal cytoplasmic domain of exchangers. CHP binding-defective mutations of NHE1–3 or CHP depletion by injection of the competitive CHP-binding region of NHE1 into Xenopusoocytes resulted in a dramatic reduction (>90%) in the Na+/H+exchange activity. The data suggest that CHP serves as an essential cofactor, which supports the physiological activity of NHE family members.

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) is a potent and selective Na+/Ca2+exchange (NCX) inhibitor that is 3-fold more inhibitory to NCX3 than to NCX1 or NCX2. Here we searched for amino acid residues that may form the KB-R7943 receptor in the exchanger by analyzing the function of chimeras between NCX1 and NCX3 as well as of their site-directed mutants. We found that the highly conserved α-2 repeat of the exchanger is almost exclusively responsible for the difference in drug response of the isoforms. Such difference was mostly reproduced by single substitutions of residues in the α-2 repeat (V820G or Q826V in NCX1 and A809V or A809I in NCX3), suggesting their importance in drug sensitivity. Cysteine scanning mutagenesis of the α-2 repeat of NCX1 identified one residue (Gly833) that caused a large (≥ 30-fold) reduction in drug sensitivity. We found that the Gly-to-Thr substitution caused even larger reduction in drug sensitivity. Interestingly, extracellularly applied KB-R7943 at 0.8 μM markedly inhibited the whole-cell outward exchange current, whereas the drug applied intracellularly at 30 μM did not. These results suggest that KB-R7943 inhibits the exchanger from the external side in intact cells and that a region of the α-2 repeat of NCX1 containing Gly833 may participate in the formation of the drug receptor. Because we suggested previously that Gly833 is accessible from the inside of a cell, the results raised an interesting possibility that this residue may alter its position during Na+/Ca2+exchange in such a way that it becomes accessible to external drug.

The effect of 2,3‐butanedione monoxime (BDM), a ‘chemical phosphatase’, on Na+/Ca2+exchange current (INCX) was investigated using the whole‐cell voltage‐clamp technique in single guinea‐pig cardiac ventricular myocytes and in CCL39 fibroblast cells expressing canine NCX1.INCXwas identified as a current sensitive to KB‐R7943, a relatively selective NCX inhibitor, at 140 mMNa+and 2 mMCa2+in the external solution and 20 mMNa+and 433 nMfree Ca2+in the pipette solution.In guinea‐pig ventricular cells, BDM inhibited INCXin a concentration‐dependent manner. The IC50value was 2.4 mMwith a Hill coefficients of 1. The average time for 50% inhibition by 10 mMBDM was 124±31 s (n=5).The effect of BDM was not affected by 1 μMokadaic acid in the pipette solution, indicating that the inhibition was not viaactivation of okadaic acid‐sensitive protein phosphatases.Intracellular trypsin treatment viathe pipette solution significantly suppressed the inhibitory effect of BDM, implicating an intracellular site of action of BDM.PAM (pralidoxime), another oxime compound, also inhibited INCXin a manner similar to BDM.Isoprenaline at 50 μMand phorbol 12‐myristate 13‐acetate (PMA) at 8 μMdid not reverse the inhibition of INCXby BDM.BDM inhibited INCXin CCL39 cells expressing NCX1 and in its mutant in which its three major phosphorylatable serine residues were replaced with alanines.We conclude that BDM inhibits INCXbut the mechanism of inhibition is not by dephosphorylation of the Na+/Ca2+exchanger as a ‘chemical phosphatase’.

1 The effect of 2,3-butanedione monoxime (BDM), a ‘chemical phosphatase’, on Na+, a2+ exchange current (INCX) was investigated using the whole-cell voltage-clamp technique in single guinea-pig cardiac ventricular myocytes and in CCL39 fibroblast cells expressing canine NCX1. 2 INCX was identified as a current sensitive to KB-R7943, a relatively selective NCX inhibitor, at 140 mM Na+ and 2 mM Ca2+ in the external solution and 20 mM Na+ and 433 nM free Ca2+ in the pipette solution. 3 In guinea-pig ventricular cells, BDM inhibited INCX in a concentration-dependent manner. The IC50 value was 2.4 mM with a Hill coefficients of 1. The average time for 50% inhibition by 10 mM BDM was 124±31 s (n=5). 4 The effect of BDM was not affected by 1 μM okadaic acid in the pipette solution, indicating that the inhibition was not via activation of okadaic acid-sensitive protein phosphatases. 5 Intracellular trypsin treatment via the pipette solution significantly suppressed the inhibitory effect of BDM, implicating an intracellular site of action of BDM. 6 PAM (pralidoxime), another oxime compound, also inhibited INCX in a manner similar to BDM. 7 Isoprenaline at 50 μM and phorbol 12-myristate 13-acetate (PMA) at 8 μM did not reverse the inhibition of INCX by BDM. 8 BDM inhibited INCX in CCL39 cells expressing NCX1 and in its mutant in which its three major phosphorylatable serine residues were replaced with alanines. 9 We conclude that BDM inhibits INCX but the mechanism of inhibition is not by dephosphorylation of the Na+, a2+ exchanger as a ‘chemical phosphatase’. British Journal of Pharmacology (2001) 132, 1317 – 1325

We studied the effect of point mutation within the putative 11th transmembrane domain (TM11) of the Na+/H+exchanger NHE1 on the plasma membrane expression. Of the 19 mutants tested, two mutants (Tyr454 or Arg458 replaced by Cys) were retained in the endoplasmic reticulum. Interestingly, Y454C was expressed on the cell surface when one of the endogenous cysteine residues at position 8, 133, 421, or 477 was substituted with alanine. Random mutagenesis at Cys8 and its surrounding residues in the cytosolic N‐tail revealed that replacement of Cys8 with Ala was the only identified single residue mutation that rescued Y454C. These results suggest that the abnormal conformation of the region of TM11 containing the Y454C mutation is compensated by the second mutation within other domains such as the N‐tail. This approach may provide evidence for the interdomain interaction in NHE1.

Ca2+, which enters cardiac myocytes through voltage-dependent Ca2+channels during excitation, is extruded from myocytes primarily by the Na+/Ca2+exchanger (NCX1) during relaxation. The increase in intracellular Ca2+concentration in myocytes by digitalis treatment and after ischemia/reperfusion is also thought to result from the reverse mode of the Na+/Ca2+exchange mechanism. However, the precise roles of the NCX1 are still unclear because of the lack of its specific inhibitors. We generated Ncx1-deficient mice by gene targeting to determine the in vivofunction of the exchanger. Homozygous Ncx1-deficient mice died between embryonic days 9 and 10. Their hearts did not beat, and cardiac myocytes showed apoptosis. No forward mode or reverse mode of the Na+/Ca2+exchange activity was detected in null mutant hearts. The Na+-dependent Ca2+exchange activity as well as protein content of NCX1 were decreased by ∼50% in the heart, kidney, aorta, and smooth muscle cells of the heterozygous mice, and tension development of the aortic ring in Na+-free solution was markedly impaired in heterozygous mice. These findings suggest that NCX1 is required for heartbeats and survival of cardiac myocytes in embryos and plays critical roles in Na+-dependent Ca2+handling in the heart and aorta.

The membrane topology of the human Na+/H+exchanger isoform 1 (NHE1) was assessed by substituted cysteine accessibility analysis. Eighty-three cysteine residues were individually introduced into a functional cysteineless NHE1, and these mutants were expressed in the exchanger-deficient PS120 cells. The topological disposition of introduced cysteines was determined by labeling with a biotinylated maleimide in the presence or absence of preincubation with the membrane-impermeable sulfhydryl reagent, 2-trimethylammoniumethyl-methanethiosulfonate in streptolysin O-permeabilized or nonpermeabilized cells. We proposed a new model for the topology of NHE1 that is significantly different from the model derived from hydropathy analysis. In this model, NHE1 is composed of 12 transmembrane segments (TMs) with the N and C termini located in the cytosol. The large, last extracellular loop in the membrane domain of the original model was suggested to comprise an intracellular loop, a new transmembrane segment (TM11), and an extracellular loop in the new model. Interestingly, cysteines at 183 and 184 and at 324 and 325 mapped to intracellular loops connecting TMs 4 and 5 (IL2) and TMs 8 and 9 (IL4), respectively, were accessible to sulfhydryl reagents from the outside. Furthermore, exchange activities of two mutants, R180C and Q181C, within IL2 were markedly inhibited by external MTSET. These data suggest that part of IL2 or IL4 may be located in a pore-lining region that is accessible from either side of the membrane and involved in ion transport.

We studied the effect of point mutation within the putative 11th transmembrane domain (TM11) of the Na +/H +exchanger NHE1 on the plasma membrane expression. Of the 19 mutants tested, two mutants (Tyr454 or Arg458 replaced by Cys) were retained in the endoplasmic reticulum. Interestingly, Y454C was expressed on the cell surface when one of the endogenous cysteine residues at position 8, 133, 421, or 477 was substituted with alanine. Random mutagenesis at Cys8 and its surrounding residues in the cytosolic N-tail revealed that replacement of Cys8 with Ala was the only identified single residue mutation that rescued Y454C. These results suggest that the abnormal conformation of the region of TM11 containing the Y454C mutation is compensated by the second mutation within other domains such as the N-tail. This approach may provide evidence for the interdomain interaction in NHE1.

Externally applied Ni2+, which apparently competes with Ca2+in all three isoforms of Na+/Ca2+exchanger, inhibits exchange activity of NCX1 or NCX2 with a 10-fold higher affinity than that of NCX3, whereas stimulation of exchange by external Li+is significantly greater in NCX2 and NCX3 than in NCX1 (Iwamoto, T., and Shigekawa, M. (1998) Am. J. Physiol.275, C423–C430). Here we identified structural domains in the exchanger that confer differential sensitivity to Ni2+or Li+by measuring intracellular Na+-dependent45Ca2+uptake in CCL39 cells stably expressing NCX1/NCX3 chimeras or mutants. We found that two segments in the exchanger corresponding mostly to the internal α-1 and α-2 repeats are individually responsible for the alteration of Ni2+sensitivity, both together accounting for ∼80% of the difference between NCX1 and NCX3. In contrast, the segment corresponding to the α-2 repeat fully accounts for the differential Li+sensitivity between the isoforms. The Ni2+sensitivity was mimicked, respectively, by simultaneous substitution of two amino acids in the α-1 repeat (N125G/T127I in NCX1 and G159N/I161T in NCX3) and substitution of one amino acid in the α-2 repeat (V820A in NCX1 and A809V in NCX3). On the other hand, the Li+sensitivity was mimicked by double substitution mutation in the α-2 repeat (V820A/Q826V in NCX1 and A809V/V815Q in NCX3). Single substitution mutations at Asn125and Val820of NCX1 caused significant alterations in the interactions of the exchanger with Ca2+and Ni2+, and Ni2+and Li+, respectively, although the extent of alteration varied depending on the nature of side chains of substituted residues. Since the above four important residues are mostly in the putative loops of the α repeats, these regions might form an ion interaction domain in the exchanger.

Hydropathy analysis predicts 11 transmembrane helices in the cardiac Na+/Ca2+exchanger. Using cysteine susceptibility analysis and epitope tagging, we here studied the membrane topology of the exchanger, in particular of the highly conserved internal α‐1 and α‐2 repeats. Unexpectedly, we found that the connecting loop in the α‐1 repeat forms a re‐entrant membrane loop with both ends facing the extracellular side and one residue (Asn‐125) being accessible from the inside and that the region containing the α‐2 repeat is mostly accessible from the cytoplasm. Together with other data, we propose that the exchanger may consist of nine transmembrane helices.

The rat L6 skeletal muscle cell line was used to study expression of the dystrophin-containing glycoprotein complex and its interaction with the integrin system involved in the cell-matrix adhesion reaction. A complex of dystrophin and its associated proteins was fully expressed in L6 myotubes, from which anti-dystrophin or anti-α-sarcoglycan co-precipitated integrin α5β1and other focal adhesion-associated proteins vinculin, talin, paxillin, and focal adhesion kinase. Immunostaining and confocal microscopy revealed that dystrophin, α-sarcoglycan, integrin α5β1, and vinculin exhibited overlapping distribution in the sarcolemma, especially at focal adhesion-like, spotty structures. Adhesion of cells to fibronectin- or collagen type I-coated dishes resulted in induction of tyrosine phosphorylation of α- and γ-sarcoglycans but not β-sarcoglycan. The same proteins were also tyrosine-phosphorylated when L6 cells in suspension were exposed to Arg-Gly-Asp-Ser peptide. All of these tyrosine phosphorylations were inhibited by herbimycin A. On the other hand, treatment of L6 myotubes with α- and γ-sarcoglycan antisense oligodeoxynucleotides resulted in complete disappearance of α- and γ-sarcoglycans and in significant reduction of levels of the associated focal adhesion proteins, which caused about 50% reduction of cell adhesion. These results indicate the existence of bidirectional communication between the dystrophin-containing complex and the integrin adhesion system in cultured L6 myocytes.

By using a patch clamp technique, we examined the effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G kinase) on Ca2+-activated maxi K+ channels in canine coronary artery smooth muscle cells. Maxi K+ channels (274±4 pS in symmetrical 140 mM KCl at 24–26°C) were activated by cytoplasmic Ca2+ and were completely blocked by 100 nM charybdotoxin (CTX). G kinase (300 U/ml) added to the cytoplasmic face of the membrane patch shifted the voltage dependence of these channels by about 25 mV in the negative direction in the presence of 1 µM Ca2+, 50 µM cGMP and 1 mM magnesium adenosine triphosphate. At -50 mV and 1 µM Ca2+, G kinase treatment increased the mean number of open channels 4.5-fold compared with the control. a-Human atrial natriuretic peptide (ANP, 100 nM) reduced the isometric tension of coronary arterial rings elicited by 14 or 24 mM KCl, but failed to relax the artery contracted by 34 mM KCl. Addition of 100 nM CTX augmented tension development elicited by 24 mM KCl and totally prevented ANP from relaxing the arterial rings. These results indicate that G kinase-dependent protein phosphorylation activates maxi K+ channels in canine coronary smooth muscle, and further suggest that the G kinase-induced activation of maxi K+ channels may cause hyperpolarization and relaxation of coronary artery.

The dystrophin–glycoprotein complex was isolated from hamster ventricular muscle by a method involving homogenization of muscle directly in the presence of 1% digitonin, followed by chromatography on succinylated wheat germ agglutinin agarose, Diethyl aminoethyl (DEAE) cellulose, and/or immunoaffinity agarose. Protein yield of the DEAE cellulose-purified dystrophin–glycoprotein complex was 120±30 (n=3)μg per 5 g hamster ventricular muscle. The cardiac dystrophin–glycoprotein complex, unlike the skeletal muscle counterpart, could not be solubilized from a microsomal fraction with digitonin or some other detergents. By sodium dodecyl sulfate gel electrophoresis, protein composition of the dystrophin–glycoprotein complexes from hamster cardiac muscle was found to be significantly different from that of rabbit skeletal muscle which has been extensively studied. This difference mainly arises from the species difference, because in hamster the cardiac and skeletal muscle complexes exhibited essentially the same protein composition. In rabbit, on the other hand, there are differences between the cardiac and skeletal complexes in the relative abundance of 60 and 64 kDa proteins and in the apparent M_rofα-dystroglycan. We found that the content of the dystrophin–glycoprotein complex, estimated by quantitative immunoblot assay, is at least 5 times more abundant in cardiac than in skeletal muscle in hamster and rabbit.

This study investigated the selective effects of intracellular (pH_i) or extracellular change in pH on reoxygenation-induced Ca²⁺ overload in simulated myocardial hypoxia. Experiments were performed in cultured cardiomyocytes isolated from the ventricle of neonatal ICR mouse. A model of chemical hypoxia with 2 mM sodium cyanide was developed to mimic the ATP depletion of hypoxia. This chemical hypoxia was "reoxygenated" and the dynamics in intracellular Ca²⁺ concentration ([Ca²⁺]) and pH_i were monitored using the fluorescent dyes fura-2 and 2', 7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. During a 40-min chemical hypoxia, pH_i progressively fell from 7.2 to 6.6. Reoxygenation with control solution caused rapid recovery of pH_i and a marked increase in [Ca²⁺]_i (1884 ± 136 nM). Intracellular acidotic reoxygenation produced by lactate apparently prolonged the time course of pH_i recovery and significantly suppressed reoxygenation-induced Ca²⁺ overload (1170 ± 118 nM, P = 0.008). Extracellular acidotic reoxygenation with 2-(N-morpholino) ethanesulfonic acid (pK = 5.96) buffer somewhat suppressed the Ca²⁺ overload; however, the maximal value of [Ca²⁺]_i was not reduced significantly compared with the control (1790 ± 122 nM, P = 0.130). In addition, inhibition of Na'-H' exchange by amiloride potentiated prolongation of intracellular acidosis during reoxygenation and resulted in a minimal increase in [Ca²⁺]_i (985 ± 102 nM, P = 0.004). These results suggest that reoxygenation-induced Ca²⁺ overload is closely correlated with intracellular pH in the initial phase of reoxygenation, and the protective effects of extracellular acidosis is principally mediated by intracellular acidification of reoxygenated cardiomyocytes. Copyright 1995, 1999 Academic Press

The sequence of 4968 (or 4976 with an insertion) amino acids composing the ryanodine receptor from rabbit cardiac sarcoplasmic reticulum has been deduced by cloning and sequencing the cDNA. This protein is homologous in amino acid sequence and shares characteristic structural features with the skeletal muscle ryanodine receptor. Xenopusoocytes injected with mRNA derived from the cardiac ryanodine receptor cDNA exhibit Ca 2+-dependent Cl −current in response to caffeine, which indicates the formation of functional calcium release channels. RNA blot hybridization analysis with a probe specific for the cardiac ryanodine receptor mRNA shows that the stomach and brain contain a hybridizable RNA species with a size similar to that of the cardiac mRNA. This result, in conjunction with cloning and analysis of partial cDNA sequences, suggests that the brain contains a cardiac type of ryanodine receptor mRNA.

We have investigated the distribution of the sarcolemmal Ca 2+transporters in hamster and dog ventricular myocytes by immunocytochemical and membrane fractionation techniques. The data suggest that the DHP receptor α1 subunit and the Na +/Ca 2+exchanger are present in surface sarcolemma as well as T-tubule membranes located at the cardiac dyads. Compared with these Ca 2+transporters, the sarcolemmal Ca 2+-ATPase is much less abundant in the latter fraction. Thus the sarcolemmal Ca 2+-ATPase seems to be located predominantly in surface sarcolemma.

We examined the Ca2+handling property and cell function of CCL39 fibroblasts highly overexpressing the cardiac isoform (NCX1) of Na+/Ca2+exchanger. In NCX1 transfectants in 146 mM Na+, ionomycin, α-thrombin or thapsigargin only produced a small transient increase in [Ca2+]icompared to the large increase seen in control cells, although resting [Ca2+]iwas not significantly different between these cells. In Na+-free medium, in contrast, the [Ca2+]iresponses in NCX1 transfectants and control cells stimulated with these agents were not different, indicating that the Ca2+content of the intracellular store(s) does not decrease on NCX1 transfection. The expression levels of the endoplasmic reticulum and plasma membrane Ca2+-ATPases, and thrombin- or serum-stimulated cell growth were not altered in NCX1 transfectants. The latter finding suggests that Ca2+signaling in the nucleus is not impaired appreciably. On fluorescence imaging and confocal microscopy, we found that [Ca2+] did not increase in the peripheral cytoplasm of these cells treated with α-thrombin in Na+-containing medium. In these NCX1 transfectants, activation of the plasma membrane Ca2+-activated K+channels by thrombin or ionomycin was markedly suppressed, and the integrin-mediated adhesion to substrate was significantly delayed compared with control cells. NCX1-overexpressing CCL39 cells thus seem to be a good model with which we can study the Ca2+-regulated membrane processes under physiologically relevant conditions.

Chinese hamster ovary (CHO) cells stably coexpressing cardiac calcium ion channel [L-type calcium channel or ryanodine receptor (RyR)] and the calcium- sensitive bioluminescent protein aequorin were generated by transfecting aequorin cDNA. In a selected clone, C1-17, carrying the L-type calcium channel, depolarization induced by high concentration of K+produces aequorin luminescence. In another clone, R3-7, carrying RyR, caffeine produces aequorin luminescence. In the presence of selective calcium ion channel blockers, the aequorin luminescence was inhibited in a dose-dependent manner. These results indicate that functionally expressed calcium ion channels in these transformants can be monitored through the activation of endogenous aequorin luminescence following a physiological signal similar to that of native calcium channel. Moreover, the aequorin system compared very well with Fura-2 measurements. Thus, the recombinant cell models, which expressed cloned calcium channel and aequorin, will contribute to the elucidation of Ca2+movement through the cell surface and intracellular calcium ion channels.

The Na+/H+exchanger isoforms NHE1 and NHE3 are regulated differently by various stimuli. Calcium has been recognized as one of the major second messengers in such exchanger regulation. We previously proposed that Ca2+-induced activation of NHE1 occurs via displacement of its autoinhibitory domain from the H+modifier site due to direct binding of Ca2+/calmodulin. To further validate this hypothesis, the functional role of the cytoplasmic domain was studied in both wild-type and chimeric exchangers, i.e.NHE1, NHE3, NHE1 with the cytoplasmic domain of NHE3(N1N3), and NHE3 with the cytoplasmic domain of NHE1(N3N1). After expression in exchanger-deficient fibroblasts (PS120), early response (<80 s) to external stimuli was assessed as 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+uptake. Among stimuli tested (ionomycin, α-thrombin, phorbol ester, hyperosmotic stress, and platelet-derived growth factor) that are all known to activate NHE1, only ionomycin and thrombin induced a significant intracellular Ca2+mobilization and early activation of 22Na+uptake, implying that Ca2+is a main regulator of NHE1 in the early phase of the agonist response. However, all the stimuli did not activate NHE3 or N1N3. In contrast, a significant stimulation of 22Na+uptake in response to ionomycin and thrombin was observed in N3N1, accompanied by an alkaline shift of pHisensitivity (∼0.2 pH units). Deletion of the cytoplasmic calmodulin-binding domain within N3N1 resulted in a constitutive alkaline shift of pHisensitivity and abolished the activation by ionomycin and thrombin. Together, these data reinforce our concept of Ca2+-induced activation of NHE1. Furthermore, they provide evidence for a functional interaction of the autoinhibitory domain of NHE1 with the H+-modifier site of a different isoform, NHE3.

We have investigated the distribution of the sarcolemmal Ca2+transporters in hamster and dog ventricular myocytes by immunocytochemical and membrane fractionation techniques. The data suggest that the DHP receptor α1 subunit and the Na+/Ca2+exchanger are present in surface sarcolemma as well as T‐tubule membranes located at the cardiac dyads. Compared with these Ca2+transporters, the sarcolemmal Ca2+‐ATPase is much less abundant in the latter fraction. Thus the sarcolemmal Ca2+‐ATPase seems to be located predominantly in surface sarcolemma.

The cardiac Na+/Ca2+exchanger (NCX1) plays a major role in the extrusion of Ca2+from cardiomyocytes. We studied the role of protein phosphorylation in the regulation of cardiac NCX1 using CCL39 stably overexpressing the canine cardiac NCX1 and rat neonatal cardiomyocytes. In both cell types, the NCX1 protein immunoprecipitated with a chicken anti-NCX1 antibody exhibited a significant basal phosphorylation that was further enhanced by treatment with endothelin-1, acidic fibroblast growth factor, phorbol 12-myristate 13-acetate, or okadaic acid. In contrast, calphostin C, K252a, or EGTA inhibited the phosphorylation. The phosphorylation occurred on two major tryptic phosphopeptides (P1 and P2) exclusively on serine residues. Evidence is presented suggesting that P2 was derived from an N-terminal half (amino acids 240-475) of the central cytoplasmic domain of NCX1 and was phosphorylated directly by protein kinase C (PKC). The agents that increased NCX1 phosphorylation significantly enhanced both the forward and reverse modes of Na+/Ca2+exchange. This exchange activation exhibited a very good correlation with the NCX1 phosphorylation. In NCX1-transfected cells, PKC down-regulation following prolonged exposure to phorbol 12-myristate 13-acetate abolished the acidic fibroblast growth factor-induced activation of exchange activity. On the other hand, cell ATP depletion reduced the exchange activity and abolished the effects of the above agents on exchange activity. These results indicate that the cardiac NCX1 is up-regulated by PKC-catalyzed phosphorylation. The cardiac NCX1 thus could play an important role in the previously reported negative inotropic actions of phorbol esters and other PKC-activating agents.

Although the Na+/Ca2+exchanger is one of the major Ca2+extrusion systems in excitable tissues, little is known about its regulation via protein phosphorylation. We now present evidence that the Na+/Ca2+exchanger is phosphorylated in quiescent and growth factor-stimulated cultured aortic smooth muscle cells. The Na+/Ca2+exchanger was isolated from 32P-labeled cells by immunoprecipitation with a specific polyclonal antibody. Phosphorylation of the exchanger was increased by up to 1.7-fold in response to platelet-derived growth factor-BB (PDGF-BB), α-thrombin, or phorbol 12-myristate 13-acetate (PMA). However, angiotensin II did not enhance the phosphorylation significantly. The extent of phosphorylation appeared to correlate with the growth factor-induced increase in cell 1,2-diacylglycerol. At least four phosphopeptides (P1 to P4) were detected by tryptic phosphopeptide map analysis of the phosphorylated exchanger, suggesting that phosphorylation occurred at multiple sites. PDGF-BB and PMA increased phosphorylation of the same phosphopeptides (in particular P1). Phosphorylated amino acids were exclusively serine residues in both quiescent and stimulated cells. We found that growth factors enhanced Na+/Ca2+exchange activity and that there was a good correlation between the growth factor-induced stimulations of phosphorylation and exchange activity. PDGF-BB-induced activation of the exchanger was abolished by prior long treatment of cells with PMA. These results suggest that the Na+/Ca2+exchanger is activated by protein kinase C-dependent phosphorylation in response to growth factors in vascular smooth muscle cells.

In ventricular muscle from 30‐ to 60‐day‐old Bio 14.6 cardiomyopathic hamsters, dystrophin‐associated glycoproteins of 43, 50 and 150 kDa are markedly reduced in abundance. In particular, the 50‐kDa glycoprotein is totally deficient in the sareolemma of myopathic ventricular myocytes as revealed by immunofluorescence microscopy. The dystrophin‐glycoprotein complex formation is defective in the cardiomyopathic hamster heart, because dystrophin and the glycoproteins behave independently when digitonin‐solubilized ventricular homogenates are fractionated on wheat germ agglutinin beads or anti‐dystrophin immunoaffinity beads.

The sequence of 4968 (or 4976 with an insertion) amino acids composing the ryanodine receptor from rabbit cardiac sarcoplasmic reticulum has been deduced by cloning and sequencing the cDNA. This protein is homologous in amino acid sequence and shares characteristic structural features with the skeletal muscle ryanodine receptor. Xenopusoocytes injected with mRNA derived from the cardiac ryanodine receptor cDNA exhibit Ca2+‐dependent Cl−current in response to caffeine, which indicates the formation of functional calcium release channels. RNA blot hybridization analysis with a probe specific for the cardiac ryanodine receptor mRNA shows that the stomach and brain contain a hybridizable RNA species with a size similar to that of the cardiac mRNA. This result, in conjunction with cloning and analysis of partial cDNA sequences, suggests that the brain contains a cardiac type of ryanodine receptor mRNA.

Overlay and co‐sedimentation assays using recombinant α1‐syntrophin proteins revealed that two regions of α1‐syntrophin, i.e. aa 274–315 and 449–505, contain high‐affinity binding sites for F‐actin (Kd0.16–0.45 μM), although only a single high‐affinity site (Kd0.35 μM) was detected in the recombinant full‐length syntrophin. We also found that actomyosin fractions prepared from both cardiac and skeletal muscle contain proteins recognized by anti‐syntrophin antibody. These data suggest a novel role for syntrophin as an actin binding protein, which may be important for the function of the dystrophin‐glycoprotein complex or for other cell functions. We also found that α1‐syntrophin binds calmodulin at two distinct sites with high (Kd15 nM) and low (Kd0.3 μM) affinity.

Overlay and co-sedimentation assays using recombinant α1-syntrophin proteins revealed that two regions of α1-syntrophin, i.e. aa 274–315 and 449–505, contain high-affinity binding sites for F-actin ( Kd0.16–0.45 μM), although only a single high-affinity site ( Kd0.35 μM) was detected in the recombinant full-length syntrophin. We also found that actomyosin fractions prepared from both cardiac and skeletal muscle contain proteins recognized by anti-syntrophin antibody. These data suggest a novel role for syntrophin as an actin binding protein, which may be important for the function of the dystrophin-glycoprotein complex or for other cell functions. We also found that α1-syntrophin binds calmodulin at two distinct sites with high ( Kd15 nM) and low ( Kd0.3 μM) affinity.

This study investigated the effects of guarana seed extract (GSE) on an anti-allergic mechanism. GSE orally administered inhibited the anti-dinitrophenol IgE-induced passive cutaneous anaphylaxis reaction in mice. Furthermore, it inhibited the degranulation of rat basophilic leukemia RBL-2H3 cells. It had no cytotoxicity on RBL-2H3 cells. These results show that GSE is a candidate for effective therapeutic material for allergic diseases.

ATP hydrolysis with CaATP as a substrate was characterized at 0°C and pH 7.0 using purified ATPase preparations of sarcoplasmic reticulum and compared with that with MgATP as a substrate. The maximal rate of enzyme phosphorylation and the Kmvalue for the phosphorylation were 8 to 10 times less for CaATP than for MgATP. These substrates appeared to act as a competitive inhibitor with respect to each other in enzyme phosphorylation. The phosphoenzyme formed from CaATP turned over at a much slower rate than that formed from MgATP because conversion rate of the ADP-sensitive (E1P) to ADP-insensitive (E2P) phosphoenzyme was very slow. E2P’s formed from both CaATP and MgATP were similar in that T:hey decomposed spontaneously at comparable rates and that KCl accelerated their decomposition. These results suggest that the metal component of the substrate basically determines affinity of the substrate to the enzyme and the catalytic mechanism of subsequent reaction steps.