Detachment of cultured cells from the substratum induced by the neutrophil-derived oxidant NH2Cl: synergistic role of phosphotyrosine and intracellular Ca2+ concentration.

The neutrophil-derived, membrane-permeating oxidant, NH2Cl, (but not the non-membrane-permeating chloramine, taurine-NHCl) induced detachment of fetal mouse cardiac myocytes and other cell types (fibroblasts, epithelial cells, and endothelial cells) from the culture dish, concomitant with cell shrinkage ("peeling off"). Stimulated human neutrophils also induced peeling off of cultured mouse cardiac myocytes when the latter were pretreated with inhibitors of .OH and elastase. Immunofluorescence microscopy revealed that the NH2Cl-induced peeling off of WI-38 fibroblasts is accompanied by disorganization of integrin alpha 5 beta 1, vinculin, stress fibers, and phosphotyrosine (p-Tyr)-containing proteins. Decrease in the content of the p-Tyr-containing proteins of the NH2Cl-treated cells was analyzed by immunoblotting techniques. Coating of fibronectin on the culture dish prevented both NH2Cl-induced peeling off and a decrease in p-Tyr content. Preincubation with a protein-tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4), also prevented NH2Cl-induced peeling off, suggesting that dephosphorylation of p-Tyr is necessary for peeling off. NH2Cl-induced peeling off was accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiac myocytes and WI-38 fibroblasts. The absence of extracellular Ca2+ prevented both NH2Cl-induced peeling off and increased [Ca2+]i, both of which did occur on subsequent incubation of the cells in Ca2+-containing medium. These observations suggest that an increase in [Ca2+]i is also necessary for peeling off. Depletion of microsomal and cytosolic Ca2+ by incubation with the microsomal Ca2+-ATPase inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ) plus EGTA prevented both NH2Cl-induced increases in [Ca2+]i and peeling off. Direct inhibition of microsomal Ca2+ pump activity by NH2Cl may participate in the NH2Cl-induced [Ca2+]i increment. A combination of p-Tyr dephosphorylation by genistein (an inhibitor of tyrosine kinase) and an increase in [Ca2+]i by BHQ could also induce peeling off. All these observations suggest a synergism between p-Tyr dephosphorylation and increased [Ca2+]i in NH2Cl-induced peeling off.

Cardiac Na(+)-Ca(2+) exchange: molecular and pharmacological aspects.

The Na(+)-Ca(2+) exchanger (NCX) is one of the essential regulators of Ca(2+) homeostasis in cardiomyocytes and thus an important modulator of the cardiac contractile function. The purpose of this review is to survey recent advances in cardiac NCX research, with particular emphasis on molecular and pharmacological aspects. The NCX function is thought to be regulated by a variety of cellular factors. However, data obtained by use of different experimental systems often appear to be in conflict. Where possible, we endeavor to provide a rational interpretation of such data. We also provide a summary of current work relating to the structure and function of the cardiac NCX. Recent molecular studies of the NCX protein are beginning to shed light on structural features of the ion translocation pathway in the NCX membrane domain, which seems likely to be formed, at least partly, by the phylogenetically conserved alpha-1 and alpha-2 repeat structures and their neighboring membrane-spanning segments. Finally, we discuss new classes of NCX inhibitors with improved selectivity. One of these, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), appears to exhibit unique selectivity for Ca(2+)-influx-mode NCX activity. Data obtained with these inhibitors should provide a basis for designing more selective and clinically useful drugs targeting NCX.

Rapid reconstitution and characterization of highly-efficient sarcoplasmic reticulum Ca pump.

The Ca pump was reconstituted from the purified sarcoplasmic reticulum ATPase and excess soybean phospholipids by the freeze-thaw sonication procedure in the presence of cholate. In the absence of Ca precipitating agents, the reconstituted proteoliposomes accumulated Ca2+ at an initial rate of up to 0.7 mumol/mg per min at 25 degrees C, and a value of 1.54 was obtained for the coupling ratio between Ca uptake and Ca2+-dependent ATPase activities. The proteoliposomes were mainly unilamellar vesicles but were heterogeneous with respect to their size. When reconstituted at a lipid/protein ratio of 40, proteoliposomes had a buoyant density of about 1.04 and their average internal volume was 1.4-1.6 microliters/mg of phospholipids. More than 95% of the ATPase was incorporated randomly into these proteoliposomes and the fraction of proteoliposomes that represented about 50% of the total intravesicular isotope space contained right-side-out oriented enzyme. 86Rb efflux from the 86Rb-loaded proteoliposomes was found to be slow even at 25 degrees C. Therefore, the proteoliposomes prepared by the present simple method should be useful for the study of the side-specific interaction of ions such as alkali metal cations with the sarcoplasmic reticulum Ca pump.

Protein-protein interaction of detergent-solubilized Ca2(+)-ATPase during ATP hydrolysis analyzed by low-angle laser light scattering photometry coupled with high-performance gel chromatography.

Protein-protein interaction of detergent-solubilized Ca2(+)-ATPase was examined, employing low-angle laser light scattering photometry coupled with high-performance gel chromatography. When solubilized with octa(ethylene glycol) mono-n-dodecyl ether (C12E8) and chromatographed in the presence of 0.3 mg/ml C12E8, the Ca2(+)-ATPase emerged as a single peak with an intermediate molecular weight between the monomer and the dimer, showing a dissociation-association equilibrium of the two components. In the presence of 50 micrograms/ml phosphatidylcholine and 0.3 mg/ml C12E8 at 0 degrees C, the Ca2(+)-ATPase (0.8 mg) emerged as the two distinct components with molecular weights of 125,000 +/- 2100 (n = 3) and 211 300 +/- 7300 (n = 3), indicating that there was no rapid interconversion between the monomer and the dimer. Under the latter conditions, addition of ATP induced fusion of two components. The apparent molecular weight of the fused peak shifted from the monomer to the dimer as the amount of protein increased. Addition of ADP or adenosine 5'-(beta, gamma-methylene triphosphate), however, did not induce such fusion of the peaks. The ATP-induced fusion of the peaks was not observed either in 5 mM CaCl2, the conditions in which the rate of ATP hydrolysis was extremely slow. Thus, the solubilized Ca2(+)-ATPase underwent a rapid interconversion between the monomer and the dimer during ATP hydrolysis. These results suggest that the protein-protein interaction during ATP hydrolysis is an intrinsic nature of Ca2(+)-ATPase and that such interaction may be important for Ca2+ transport by Ca2(+)-ATPase in the sarcoplasmic reticulum membranes.

Stimulatory and inhibitory effects of dimethylsulfoxide, propranolol and chlorpromazine on the partial reactions of ATPase of sarcoplasmic reticulum.

The effects of dimethylsulfoxide, propranolol and chlorpromazine on the partial reactions of the ATPase of sarcoplasmic reticulum were investigated. When analyzed according to a reaction scheme in which the ADP-sensitive (E1P) and ADP-insensitive (E2P) phosphoenzymes occur sequentially and P1 is derived from the latter, dimethylsulfoxide decreased the rate of E2P hydrolysis whereas it stimulated the rate of the E1P to E2P conversion. Propranolol increased the rate of E2P hydrolysis while it decreased the rate of the E1P to E2P conversion. Propranolol exerted an additional effect, presumably inhibition of the phosphoenzyme formation. These effects of dimethylsulfoxide and propranolol can account for both the stimulatory and inhibitory effects of these drugs on the overall rate of ATP hydrolysis observed in the presence and absence of added alkali metal salts. Chlorpromazine accelerated E2P hydrolysis whereas it appeared to inhibit the E1P to E2P conversion. These effects of chlorpromazine appear able to account for its stimulatory and inhibitory effects on the overall rate of ATP hydrolysis in the presence and absence of alkali metal salts. In the presence of chlorpromazine, however, the rate of Pi liberation during the steady state ATP hydrolysis was found to be greater than the hydrolysis rate of E2P. This finding suggests that under these conditions Pi is derived not only from E2P but also from source(s) other than E2P.

Stimulation of adenosine triphosphatase activity of sarcoplasmic reticulum by adenylyl methylene diphosphate.

The effects of adenylyl methylene diphosphate (AMD), a non-hydrolyzable ATP analogue, were examined in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. The Ca2+-dependent APTase activity measured at 5 degrees C and pH 7.0 in 5.2 micrometer [gamma-32P]ATP and in the absence of added alkali metal salts was stimulated by added AMD. The steady state level of phosphoenzyme, however, was not decreased greatly by added AMP under these conditions. The hydrolysis of the phosphoenzyme formed at the steady state in the absence of added alkali metal salts was accelerated by added AMD to an extent that can account for the stimulation of the ATPase activity. At 5 degrees C and pH 7.0 the maximum stimulation of phosphoenzyme hydrolysis by AMD and the Km value for this ATP analogue were 4.3-fold and 40 micrometer, respectively. These results provide further support for our previous conclusion (Shigekawa, M., Dougherty, J.P. and Katz, A.M. (1978) J.Biol. Chem. 253, 1442--1450) that 2 classes of ATP site exist in the calcium pump ATPase in the absence of added alkali metal salts, one being the catalytic site and the other being the regulation site which activates the activity of the catalytic site.

The modification of the unidirectional calcium fluxes of sarcoplasmic reticulum vesicles by monovlent cation ionophroes.

Calcium uptake by rabbit skeletal muscle sarcoplasmic reticulum vesicles in phosphate-containing media exhibits time-dependent changes that arise from changing rates of calcium influx and efflux. The monovalent cation ionophore gramicidin, added before the start of the calcium uptake reaction, delayed the spontaneous calcium release that normally occurred after approx. 6 min in such reactions; the rate of calcium efflux was inhibited while calcium influx was little affected. Under these conditions, Ca2+-activated ATPase activity could remain unaltered. Gramicidin stimulated calcium uptake irrespective of the presence of a K+ gradient across the vesicle membrane. Valinomycin stimulated calcium uptake in a manner similar to that for gramicidin even in an NaCl-containing medium lacking potassium. Thus, dissipation of a transmembrane K+ gradient is unlikely to account for the effects of these ionophores on the spontaneous changes in calcium flux rates. Addition of gramicidin to partially calcium-filled vesicles inhibited the phase of spontaneous calcium reuptake because both calcium influx and efflux wre inhibited. Addition of gramicidin to partially calcium-filled vesicles in the presence of a water-soluble protein, such as bovine serum albumin, creatine kinase or pyruvate kinase, markedly stimulated calcium uptake. This stimulatory effect was due primarily to inhibition of calcium efflux, calcium influx being minimally influenced by the ionophore. After cleavage of the 100,000 dalton ATPase to 50,000 dalton fragments, which was not associated with changes in Ca2+-activated ATPase activity or initial calcium uptake rate, gramicidin increased rather than decreased calcium content when added to vesicles after the initial maximum in calcium content. Thus, the ability of monovalent cation ionophores to block calcium efflux from calcium-filled vesicles may reflect their interaction with a portion of the Ca2+-activated ATPase protein.

Time-dependent changes of calcium influx and efflux rates in rabbit skeletal muscle sarcoplasmic reticulum.

Unfractionated and low buoyant density sarcoplasmic reticulum vesicles released calcium spontaneously after ATP- or acetyl phosphate-supported calcium uptake when internal Ca2+ was stabilized by the use of 50 mM phosphate as calcium-precipitating anion. This spontaneous calcium release could not be attributed to falling Ca2+ concentration outside the vesicles (Ca02+), substrate depletion, ADP accumulation, nonspecific membrane deterioration of the attainment of a high vesicular calcium content. Instead, spontaneous calcium release was directly proportional to Ca02+ at the time that calcium content was maximal. A causal relationship between high Ca02+ and spontaneous calcium release was suggested by the finding that elevation of Ca02+ from less than 1 microM to 3--5 microM increased the rate and extent of calcium release. The spontaneous calcium release was due both to acceleration of calcium efflux and slowing of calcium influx that was not accompanied by a significant change in the rate of ATP hydrolysis. Neither reversal of the transmembrane KCl gradient nor incubation with cation and proton ionophores abolished the spontaneous calcium release. The persistence of calcium release under conditions where the membrane was permeable to both anions and cations makes it unlikely that this phenomenon is due to a changing transmembrane potential. The similarity between the Ca2+ dependence of spontaneous calcium release and of calcium uptake, along with other similarities between these processes, suggest that calcium release is mediated by the calcium pump in these membranes.

Effects of cyclic tensile forces on the expression of vascular endothelial growth factor (VEGF) and macrophage-colony-stimulating factor (M-CSF) in murine osteoblastic MC3T3-E1 cells.

It has been reported that vascular endothelial growth factor (VEGF), expressed by osteoblasts, can induce osteoclast recruitment and thus affects bone remodeling. The purpose of this study was to investigate the effects of cyclic tensile forces on the expression of VEGF and macrophage-colony-stimulating factor (M-CSF) in osteoblastic MC3T3-E1 cells. VEGF and M-CSF gene expression and protein concentration were determined by real-time PCR and enzyme-linked immunoassay. The expression of VEGF and M-CSF mRNA in the experimental group was higher than in the control group. The increase in the concentration of VEGF and M-CSF protein in the experimental group was time-dependent. Moreover, gadolinium (an S-A channel inhibitor), but not nifedipine (L-Type Ca2+ channel blocker), treatment reduced the concentration of VEGF and M-CSF mRNA and protein in the experimental groups. These findings suggest that cyclic tensile forces increase the expression of VEGF and M-CSF in osteoblastic MC3T3-E1 cells via a stretch-activated channel (S-A channel).

Amyloid beta protein deposition in osteopetrotic (op/op) mice is reduced by injections of macrophage colony stimulating factor.

The deposition of amyloid beta (Abeta) protein is a neuropathological change that characterizes Alzheimer's disease. Animals with the osteopetrosis (op/op) mutation suffer from a general skeletal sclerosis, a significantly reduced number of macrophages and osteoclasts in various tissues, and have no systemic macrophage colony stimulating factor (M-CSF). This study examined the effect that M-CSF injections had on Abeta deposition and microglial cell distribution in the brains of normal and op/op mice. Abeta-positive plaques were detected in the cerebral cortex of op/op mice, but not in normal mice. M-CSF reduced the numbers of Abeta-positive plaques in op/op mice. The microglial cell population was reduced in op/op mice compared with normal mice, and M-CSF increased the numbers to 65.8% of that observed in normal mice. Our results suggest that a clearer understanding of the role that microglial cells play in Abeta deposition may help determine the mechanisms involved in the pathogenesis of Alzheimer's disease.

Na+/Ca2+ exchanger overexpression impairs calcium signaling in fibroblasts: inhibition of the [Ca2+] increase at the cell periphery and retardation of cell adhesion.

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, alpha-thrombin or thapsigargin only produced a small transient increase in [Ca2+]i compared to the large increase seen in control cells, although resting [Ca2+]i was not significantly different between these cells. In Na+-free medium, in contrast, the [Ca2+]i responses 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 alpha-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.

Second mutations rescue point mutant of the Na(+)/H(+) exchanger NHE1 showing defective surface expression.

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.

Ca(2+)-ATPase distributes differently in cardiac sarcolemma than dihydropyridine receptor alpha 1 subunit and Na+/Ca2+ exchanger.

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 alpha 1 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 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.

Alpha1-syntrophin has distinct binding sites for actin and calmodulin.

Overlay and co-sedimentation assays using recombinant alpha1-syntrophin proteins revealed that two regions of alpha1-syntrophin, i.e. aa 274-315 and 449-505, contain high-affinity binding sites for F-actin (Kd 0.16-0.45 microM), although only a single high-affinity site (Kd 0.35 microM) 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 alpha1-syntrophin binds calmodulin at two distinct sites with high (Kd 15 nM) and low (Kd 0.3 microM) affinity.

Primary structure and functional expression from cDNA of the cardiac ryanodine receptor/calcium release channel.

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. Xenopus oocytes 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.

Defective association of dystrophin with sarcolemmal glycoproteins in the cardiomyopathic hamster heart.

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 sarcolemma 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.

Unique topology of the internal repeats in the cardiac Na+/Ca2+ 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 alpha-1 and alpha-2 repeats. Unexpectedly, we found that the connecting loop in the alpha-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 alpha-2 repeat is mostly accessible from the cytoplasm. Together with other data, we propose that the exchanger may consist of nine transmembrane helices.

Inhibitory effect of 2,3-butanedione monoxime (BDM) on Na(+)/Ca(2+) exchange current in guinea-pig cardiac ventricular myocytes.

1. The effect of 2,3-butanedione monoxime (BDM), a 'chemical phosphatase', on Na(+)/Ca(2+) exchange current (I(NCX)) 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. I(NCX) was identified as a current sensitive to KB-R7943, a relatively selective NCX inhibitor, at 140 mM Na(+) and 2 mM Ca(2+) in the external solution and 20 mM Na(+) and 433 nM free Ca(2+) in the pipette solution. 3. In guinea-pig ventricular cells, BDM inhibited I(NCX) in a concentration-dependent manner. The IC(50) 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 microM 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 I(NCX) in a manner similar to BDM. 7. Isoprenaline at 50 microM and phorbol 12-myristate 13-acetate (PMA) at 8 microM did not reverse the inhibition of I(NCX) by BDM. 8. BDM inhibited I(NCX) 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 I(NCX) but the mechanism of inhibition is not by dephosphorylation of the Na(+)/Ca(2+) exchanger as a 'chemical phosphatase'.

Multiple mechanisms of arachidonic acid release in Chinese hamster ovary cells transfected with cDNA of substance P receptor.

We investigated the release of [3H]arachidonic acid ([3H]AA) and its relationship to the formation of [3H]inositol trisphosphate ([3H]IP3) elicited by substance P (SP) in prelabeled Chinese hamster ovary cells stably expressing the SP receptor. Activation of the SP receptor resulted in a concentration- and time-dependent stimulation of [3H]AA release. Half-maximal release was obtained at 10(-9) M, comparable to that for [3H]IP3 formation reported previously, and the maximal release effected by 0.1 microM SP was 8 to 10-fold above the basal value. Both the [3H]AA release and the [3H]-IP3 accumulation stimulated in the cells by 0.1 microM SP were concentration-dependently blocked with the specific SP receptor antagonist CP-96,345, with IC50 values of 2.5 and 0.4 microM, respectively. The time course of [3H]AA release showed a biphasic pattern: an initial rapid release essentially independent of Ca2+, followed by a sustained release markedly suppressed by removal of extracellular Ca2+ or chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxyethane)-N,N,N',N'-tetraacetic acid (BAPTA). While pretreatment with pertussis toxin (200 ng/mL, 6 hr) did not block [3H]IP3 formation, it did reduce [3H]AA release by 50% at 1 and 10 min after SP stimulation. Treatment of the cells with a phorbol ester, a protein kinase C activator, augmented the SP-stimulated [H]AA release, and sphingosine, a protein kinase C inhibitor, reversed the phorbol ester-potentiated [3H]AA release, but not the release stimulated by SP alone, suggesting a synergistic effect of protein kinase C on SP-stimulated AA release. These results demonstrate that SP, acting at the SP receptor, stimulates [3H]AA release via mechanisms that are (1) mediated by a pertussis toxin-sensitive G-protein, (2) dependent on extracellular Ca2+, and (3) enhanced by activation of protein kinase C.