Publications by authors named "Condrescu M"

Inhibitors of Na(+)/Ca(2+) exchange (NCX) such as KB-R7943 and SEA0400 are thought to act by promoting an inactive state of the exchanger induced by elevated cytosolic Na(+) concentrations (Na(+)-dependent inactivation). This conclusion is based mainly on experiments in excised patches from frog oocytes expressing NCX and has not been fully tested in intact cells. Here we characterize the inhibitory effects of KB-R7943 and other amphiphilic cations on NCX activity in transfected Chinese hamster ovary (CHO) cells expressing the cardiac isoform of the Na(+)/Ca(2+) exchanger (NCX1.

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The Na(+)-Ca(2+) exchanger (NCX) links transmembrane movements of Ca(2+) ions to the reciprocal movement of Na(+) ions. It normally functions primarily as a Ca(2+) efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca(2+) influx under certain conditions. Na(+) and Ca(2+) ions exert complex regulatory effects on NCX activity.

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High concentrations of cytosolic Na(+) ions induce the time-dependent formation of an inactive state of the Na(+)/Ca(2+) exchanger (NCX), a process known as Na(+)-dependent inactivation. NCX activity was measured as Ca(2+) uptake in fura 2-loaded Chinese hamster ovary (CHO) cells expressing the wild-type (WT) NCX or mutants that are hypersensitive (F223E) or resistant (K229Q) to Na(+)-dependent inactivation. As expected, 1) Na(+)-dependent inactivation was promoted by high cytosolic Na(+) concentration, 2) the F223E mutant was more susceptible than the WT exchanger to inactivation, whereas the K229Q mutant was resistant, and 3) inactivation was enhanced by cytosolic acidification.

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This report describes the influence of fluid flow and osmotically induced volume changes on Na(+)-Ca(2+) exchange (NCX) activity in transfected CHO cells. Exchange activity was measured as Na(+)-dependent Ca(2+) or Ba(2+) fluxes using the fluorescent probe fura-2. When exchange activity was initiated by superfusing Ba(2+)-containing solutions over the cells for a 20 s interval, a high rate of Ba(2+) uptake was observed while the solution was being applied but the rate of Ba(2+) uptake declined > 10-fold when the solution flow ceased.

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Exchange activity is regulated principally by cytosolic Na+, Ca2+, and PIP2. However, the properties of these modes of regulation that have emerged from excised patch studies appear to be poorly suited to regulating exchange activity on a beat-to-beat basis. Here we summarize recent findings from our lab indicating that (a) allosteric activation by Ca2+ exhibits hysteresis, (b) elevated concentrations of cytosolic Na+ induce a mode of activity that no longer requires regulatory Ca2+ activation, and (c) the requirement for PIP2 is reduced or eliminated after allosteric Ca2+ activation.

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The activity of the cardiac Na(+)-Ca(2+) exchanger (NCX1.1) is allosterically regulated by Ca(2+), which binds to two acidic regions in the cytosolically disposed central hydrophilic domain of the NCX protein. A mutation in one of the regulatory Ca(2+) binding regions (D447V) increases the half-activation constant (K(h)) for allosteric Ca(2+) activation from approximately 0.

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In the present study, the bovine cardiac Na(+)/Ca(2+) exchanger (NCX1.1) was expressed in Chinese hamster ovary cells. The surface distribution of the exchanger protein, externally tagged with the hemagglutinin (HA) epitope, was associated with underlying actin filaments in regions of cell-to-cell contact and also along stress fibers.

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Chinese hamster ovary cells expressing the bovine cardiac Na+-Ca2+ exchanger (NCX1.1) accumulated Cd2+ after a lag period of several tens of seconds. The lag period reflects the progressive allosteric activation of exchange activity by Cd2+ as it accumulates within the cytosol.

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Allosteric regulation by cytosolic Ca2+ of Na(+)/Ca2+ exchange activity in the Ca2+ efflux mode has received little attention because it has been technically difficult to distinguish between the roles of Ca2+ as allosteric activator and transport substrate. In this study, we used transfected Chinese hamster ovary cells to compare the Ca2+ efflux activities in nontransfected cells and in cells expressing either the wild-type exchanger or a mutant, Delta(241-680), that operates constitutively; i.e.

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The activity of the cardiac Na+/Ca2+ exchanger is stimulated allosterically by Ca2+, but estimates of the half-maximal activating concentration have varied over a wide range. In Chinese hamster ovary cells expressing the cardiac Na+/Ca2+ exchanger, the time course of exchange-mediated Ca2+ influx showed a pronounced lag period followed by an acceleration of Ca2+ uptake. Lag periods were absent in cells expressing an exchanger mutant that was not dependent on regulatory Ca2+ activation.

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La3+ uptake was measured in fura 2-loaded Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger (NCX1.1). La3+ was taken up by the cells after an initial lag phase of 50-60 s and achieved a steady state within 5-6 min.

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Na(+)/Ca(2+) exchange activity was studied in transfected Chinese hamster ovary (CHO) cells expressing the wild-type cardiac exchanger (NCX1.1) or mutants created by site-directed mutagenesis. The activity of the wild-type exchanger, but not exchanger mutants deficient in Ca(2+)-dependent activation, was inhibited by sphingolipids such as ceramide and sphingosine.

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Na(+)/Ca(2+) exchange activity in Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger was inhibited by the short chain ceramide analogs N-acetylsphingosine and N-hexanoylsphingosine (5-15 micrometer). The sphingolipids reduced exchange-mediated Ba(2+) influx by 50-70% and also inhibited the Ca(2+) efflux mode of exchange activity. The biologically inactive ceramide analog N-acetylsphinganine had only modest effects on exchange activity.

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The objective of this study was to assess the contribution of Na+-Ca2+ exchange activity to Ca2+ efflux at various cytosolic Ca2+ concentrations ([Ca2+]i) in transfected Chinese hamster cells expressing the bovine cardiac Na+-Ca2+ exchanger. Ionomycin was added to fura-2 loaded cells and the resulting [Ca2+]i transient was monitored in Ca2+-free media with or without extracellular Na+. The presence of Na+ reduced both the amplitude and duration of the [Ca2+]i transient.

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The effects of the protein phosphatase inhibitors calyculin A and okadaic acid on Na(+)/Ca(2+) exchange activity were examined in transfected Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger. Incubating the cells for 5-10 min with 100 nM calyculin A reduced exchange-mediated (45)Ca(2+) uptake or Ba(2+) influx by 50-75%. Half-maximal inhibition of (45)Ca(2+) uptake was observed at 15 nM calyculin A.

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Transfected Chinese hamster ovary cells stably expressing the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca2+ concentrations ([Ca2+]i) that activate Na+/Ca2+ exchange activity. Ba2+ influx was measured in fura 2-loaded, ionomycin-treated cells under conditions in which the intracellular Na+ concentration was clamped with gramicidin at approximately 20 mM.

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We examined Ba2+ influx using isotopic and fura-2 techniques in transfected Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells). Ba2+ competitively inhibited exchange-mediated 45Ca2+ uptake with a Ki approximately 3 mM.

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Our experiments with transfected cells provide new insights into the role of Na-Ca exchange activity in Ca homeostasis and emphasize the role of local interactions in determining exchanger function. Thus, the effects of ATP depletion and cytochalasin D highlight the influence of the actin cytoskeleton in regulating exchange activity. Cytoskeletal interactions could provide a mechanism for modulating exchange activity by mechanical stretch and might constitute a novel feedback mechanism for regulating contractile activity in the heart.

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Chinese hamster ovary cells expressing the bovine cardiac Na/Ca exchanger were treated with ouabain to increase [Na+]i and stimulate Ca2+ influx by Na/Ca exchange. Depletion of cellular ATP inhibited 45Ca uptake by 40% or more and reduced the half-maximal Na+ concentration for inhibition of 45Ca uptake from 90 to 55 mM. ATP depletion also reduced the rate of rise in [Ca2+]i when [Na+]o was reduced and inhibited the decline in [Ca2+]i when high [Na+]o was restored.

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The cardiac Na+/Ca2+ antiporter moves 3 Na+ across the plasma membrane in exchange for a single Ca2+ moving in the opposite direction. It is the principal Ca2+ efflux mechanism in myocardial cells; however, it also contributes to Ca2+ influx under certain conditions. It is particularly abundant in the heart, but is also expressed in other tissues such as smooth and skeletal muscle, the kidney and the brain.

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A line of Chinese hamster ovary (CHO) cells called CK1.4 was produced by transfection with the gene for the bovine cardiac Na(+)-Ca2+ exchanger. CK1.

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Two clones (p17 and p13), each containing the complete coding sequence for the bovine cardiac Na+/Ca2+ exchanger, were obtained from a lambda gt10 cDNA library by screening with cDNA probes from the canine exchanger. The coding sequence of clone p17 was 92 and 98% identical to the canine cDNA at the nucleotide and amino acid levels, respectively. Nine of the 21 amino acid differences between the two exchangers were found within the 32-amino acid signal sequence.

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Squid axons display a high activity of Na+/Ca2+ exchange which is largely increased by the presence of external K+, Li+, Rb+ and NH+4. In this work we have investigated whether this effect is associated with the cotransport of the monovalent cation along with Ca2+ ions. 86Rb+ influx and efflux have been measured in dialyzed squid axons during the activation (presence of Ca2+i) of Ca2+o/Na+i and Ca2+i/Ca2+o exchanges, while 86Rb+ uptake was determined in squid optic nerve membrane vesicles under equilibrium Ca2+/Ca2+ exchange conditions.

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A Ca-stimulated ATPase activity (pH 9.5) associated with the tegumental membrane enriched (TME) fraction of Schistosoma mansoni adults was partially inhibited by NAP-taurine or by increasing concentrations of chlorpromazine; endogenous calmodulin was found associated with the TME fraction. A similar activity (pH 8.

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