Modulation of Na/sup +/Ca/sup 2 +/ exchange in sarcolemmal vesicles by intravesicular Ca/sup 2 +

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When cardiac sarcolemmal vesicles were incubated at 37°C in 160 mM NaCl containing 0.5 mM CaClâ and subsequently assayed for Na-Ca exchange activity, they exhibited a threefold increase in the initial rate of ⁴⁵Ca/sup 2 +/ uptake compared with vesicles incubated without added CaClâ. Removal of endogenous Ca/sup 2 +/ by incubation of the vesicles with 0.1 mM ethylene-bis(..beta..-aminoethylether)-N,N'-tetraacetic acid (EGTA) resulted in a 35% inhibition in exchange activity. The pretreatment with CaClâ produced an acceleration of Na-Ca exchange activity rather than an increase in Ca/sup 2 +/ uptake due to Ca-Ca exchange. Pretreatment of the vesicles with CaClâ lowered the apparent K/sub m/ of the exchange system for Ca/sup 2 +/. The effects of the Ca treatment were reversed by subsequently incubating the vesicles with EGTA. In contrast to the effects of intravesicular Ca/sup 2 +/ on Na/sub i/-dependent Ca/sup 2 +/ uptake, external Ca/sup 2 +/ had no effect on Naâ-dependent Ca/sup 2 +/ efflux. The results suggest that an understanding of the kinetics of the Na-Ca exchange system may be hampered by the autoacceleration of exchange activity that occurs during initial rate measurements as Ca/sup 2 +/ accumulates within the vesicles. This phenomenon may contribute to the variability that exists among different vesicle preparations in their apparent K/sub m/ values for Ca/sup 2 +/.

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... On the basis of this criterion, a Ca 2ϩ /Ca 2ϩ exchange that appears to be mediated by the Na ϩ /Ca 2ϩ exchanger has been identified in various types of cells. A few examples are squid axons (35,88,107,249,254), barnacle muscle (686,754,757,800), mammalian neurons (98), and cardiac muscle (763,863). ...
... This modulatory effect of intracellular Ca 2ϩ could explain the inhibition of exchanger-mediated Ca 2ϩ entry by intracellular Ca 2ϩ chelators such as quin 2 (10) and EGTA (859). Also, removal of intravesicular Ca 2ϩ by preincubating cardiac sarcolemmal vesicles with EGTA also reduces Na i -dependent Ca 2ϩ uptake (763). This catalytic effect of internal Ca 2ϩ does not involve PKC because specific calmodulin inhibitors have little effect on the exchangermediated current in cardiac myocytes (512). ...
The Na+/Ca2+ exchanger, an ion transport protein, is expressed in the plasma membrane (PM) of virtually all animal cells. It extrudes Ca2+ in parallel with the PM ATP-driven Ca2+ pump. As a reversible transporter, it also mediates Ca2+ entry in parallel with various ion channels. The energy for net Ca2+ transport by the Na+/Ca2+ exchanger and its direction depend on the Na+, Ca2+, and K+ gradients across the PM, the membrane potential, and the transport stoichiometry. In most cells, three Na+ are exchanged for one Ca2+. In vertebrate photoreceptors, some neurons, and certain other cells, K+ is transported in the same direction as Ca2+, with a coupling ratio of four Na+ to one Ca2+ plus one K+. The exchanger kinetics are affected by nontransported Ca2+, Na+, protons, ATP, and diverse other modulators. Five genes that code for the exchangers have been identified in mammals: three in the Na+/Ca2+ exchanger family (NCX1, NCX2, and NCX3) and two in the Na+/Ca2+ plus K+ family (NCKX1 and NCKX2). Genes homologous to NCX1 have been identified in frog, squid, lobster, and Drosophila. In mammals, alternatively spliced variants of NCX1 have been identified; dominant expression of these variants is cell type specific, which suggests that the variations are involved in targeting and/or functional differences. In cardiac myocytes, and probably other cell types, the exchanger serves a housekeeping role by maintaining a low intracellular Ca2+ concentration; its possible role in cardiac excitation-contraction coupling is controversial. Cellular increases in Na+ concentration lead to increases in Ca2+ concentration mediated by the Na+/Ca2+ exchanger; this is important in the therapeutic action of cardiotonic steroids like digitalis. Similarly, alterations of Na+ and Ca2+ apparently modulate basolateral K+ conductance in some epithelia, signaling in some special sense organs (e.g., photoreceptors and olfactory receptors) and Ca2+- dependent secretion in neurons and in many secretory cells. The juxtaposition of PM and sarco(endo)plasmic reticulum membranes may permit the PM Na+/Ca2+ exchanger to regulate sarco(endo)plasmic reticulum Ca2+ stores and influence cellular Ca2+ signaling.
... As has been described in the Introduction, the inhibitory effect of Ca chelator on Na-Ca exchange has been reported qualitatively by various workers in squid axon (Baker, 1972;Baker and McNaughton, 1976;Allen and Baker, 1985), single cardiac ventricular cells (Kimura et al., 1986), and more recently in dog sarcolemma vesicles (Reeves and Poronnik, 1987). Systematic investigation, however, has been carried out only by DiPolo (1979) in squid axon, where he found the activation threshold was at 40 nM but that the Na~-dependent Ca influx did not saturate even at 0.8 #M. ...
... In cardiac vesicles, Philipson and Nishimoto (1982) measured 33 ~M. Reeves and Poronnik (1987) recently reported that variability of Km values in vesicle studies (15-40 ~M, see review by Philipson, 1985) may be attributed to the change in [Ca] at the activa-tor site (not transportedy. The overall values of Kin[Call, however, are considerably lower than Km [Ca]o. ...
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Na-Ca exchange current was measured at various concentrations of internal Na [( Na]i) and Ca [( Ca]i) using intracellular perfusion technique and whole-cell voltage clamp in single cardiac ventricular cells of guinea pig. Internal Ca has an activating effect on Nai-Cao exchange beginning at approximately 10 nM and saturating at approximately 50 nM with a half maximum [Ca]i (Km[Ca]i) of 22 nM (Hill coefficient, 3.7). Measurement of Nai-Cao exchange current at various concentration of [Na]i revealed an apparent Km[Na]i of 20.7 +/- 6.9 mM (n = 14) with imax of 3.5 +/- 1.2 microA/microF. For [Ca]i transported by the exchange, a Km[Ca]i of 0.60 +/- 0.24 microM (n = 8) with an imax of 3.0 +/- 0.54 microA/microF was obtained by measuring Nao-Cai exchange current. These values are apparently different from the values for the external binding site which have been reported previously. Whether Na and Ca compete for the external binding site, and if so, how it affects the binding constants was then investigated. Outward Nai-Cao exchange current became larger by reducing [Na]o. The double reciprocal plot of the current magnitude and [Ca]o at different [Na]o revealed a competitive interaction between Na and Ca. In the absence of competitor [Na]o, an apparent Km[Ca]o of 0.14 mM was obtained. When comparing internal and external Km values, the external value is markedly larger than the internal one and thus we conclude that binding sites of the Na-Ca exchange molecule are at least apparently asymmetrical between the inside and outside of the membrane.
... Both Ca 2+ influx and Ca 2+ efflux via NCX transport activate dynamically, dependent on [Ca 2+ ] i . In NCX expressed in Chinese hamster ovary cells, Ca 2+ influx activated from a low-[Ca 2+ ] i state on removal of external Na + with a delayed sigmoidal time course, persisting for some seconds after [Ca 2+ ] i was reduced (Reeves & Poronnik, 1987;Reeves & Condrescu, 2003). These features indicate positive feedback of inwardly transported Ca 2+ to augment and promote further activation. ...
Cardiac Na/Ca exchange (NCX) activity is regulated by cytosolic Ca ([Ca]i). The physiological role and dynamics of this process in intact cardiomyocytes are largely unknown. We examined NCX Ca activation in intact rabbit and mouse cardiomyocytes at 37°C. Sarcoplasmic reticulum (SR) function was blocked and cells were bathed in 2 mM Ca. We probed Ca activation without voltage clamp by applying Na-free solution for 5 sec bouts, repeated each 10 sec, which should evoke cytosolic Ca ([Ca]i) transients due to Ca influx via NCX. In rested rabbit myocytes, Ca influx was undetectable even after 0 Na applications were repeated for 2-5 min or more, suggesting NCX was inactive. After external electric field stimulation pulses were applied, to admit Ca via L-type Ca channels, 0 Na bouts activated Ca influx efficaciously, indicating NCX had become active. Ca activation increased with more field pulses, reaching maximum after typically 15-20 pulses (1 Hz). On rest, NCX deactivated with a time constant of typically 20-40 sec. Increased cytosolic [Na], either in rabbit as a result of inhibiting Na/K pumping, or in mouse where normal [Na]i is higher vs rabbit, sensitized NCX to self-activation by 0 Na bouts. In experiments with SR functional but initially empty, activation time course was slowed. Possibly, SR initially accumulated Ca that would otherwise cause activation. We modeled Ca activation as a fourth-order highly cooperative process (K0.5act = 375 nM), with dynamics severalfold slower than the cardiac cycle. We incorporated this NCX model in an established ventricular myocyte model, which allowed us to predict responses to twitch stimulation under physiological conditions with SR intact. Model NCX fractional activation increased from 0.1 to 1.0, as frequency was increased from 0.2 to 2 Hz. By adjusting Ca activation on a multibeat time scale, NCX might better maintain stable long-term Ca balance while contributing to the ability of myocytes to produce Ca transients over a wide range of intensity.
... First, we determined that rates of calcium uptake were linear to a good approximation under all conditions used (not shown). Second, since the rate of Na-Ca exchange in vesicles has been shown to be promoted by intravesicular calcium, 13 we investigated the effect of calcium loading on measured initial uptake rates. This was accomplished by adding 350 /AM Ca to the cells during the final 2 minutes of incubation of concentrated cells with ouabain to give a final free calcium of 100 /AM. ...
General anesthetics, typically octanol, were found to inhibit the influx of calcium in isolated sodium-loaded adult rat heart cells, using 45Ca, quin 2, or indo 1. Inhibition by octanol, like inhibition by sodium, was competitive with calcium. Octanol and sodium together inhibited calcium influx synergistically. At physiological levels of extracellular calcium and sodium, the EC50 was 177 +/- 37 microM for octanol and 48 +/- 5 microM for decanol. These values are threefold to fourfold larger than those reported to cause 50% loss of righting reflex in tadpoles, a measure of their anesthetic effectiveness. We conclude that general anesthetics inhibit Na-Ca exchange at the sarcolemma. We suggest that octanol inhibits like sodium, and the synergism stems from the cooperativity of sodium inhibition at the binding and regulatory sites of the exchanger. Insofar as Na-Ca exchange may regulate inotropy, the inhibition of Na-Ca exchange by general anesthetics could contribute to their negative inotropic effect.
... The apparent affinity of the Ca i 2ϩ regulatory site in the squid is close to 400 nM. This stimulatory effect of Ca i 2ϩ , also called "allosteric," has been demonstrated in several preparations including cardiac sarcolemmal vesicles (218), barnacle muscle fibers, intact myocytes, cardiac giant excised patches, and NCX1 clones expressed heterologously in alien cells (165,179,189,190,228). The values of the apparent Ca i 2ϩ affinities vary within preparations and are summarized in Table 2. ...
The Na(+)/Ca(2+) exchanger's family of membrane transporters is widely distributed in cells and tissues of the animal kingdom and constitutes one of the most important mechanisms for extruding Ca(2+) from the cell. Two basic properties characterize them. 1) Their activity is not predicted by thermodynamic parameters of classical electrogenic countertransporters (dependence on ionic gradients and membrane potential), but is markedly regulated by transported (Na(+) and Ca(2+)) and nontransported ionic species (protons and other monovalent cations). These modulations take place at specific sites in the exchanger protein located at extra-, intra-, and transmembrane protein domains. 2) Exchange activity is also regulated by the metabolic state of the cell. The mammalian and invertebrate preparations share MgATP in that role; the squid has an additional compound, phosphoarginine. This review emphasizes the interrelationships between ionic and metabolic modulations of Na(+)/Ca(2+) exchange, focusing mainly in two preparations where most of the studies have been carried out: the mammalian heart and the squid giant axon. A surprising fact that emerges when comparing the MgATP-related pathways in these two systems is that although they are different (phosphatidylinositol bisphosphate in the cardiac and a soluble cytosolic regulatory protein in the squid), their final target effects are essentially similar: Na(+)-Ca(2+)-H(+) interactions with the exchanger. A model integrating both ionic and metabolic interactions in the regulation of the exchanger is discussed in detail as well as its relevance in cellular Ca(i)(2+) homeostasis.
The ATP dependence of the Na-Ca exchanger was investigated in isolated adult rat heart cells to evaluate the extent to which ATP depletion after a period of ischemia plus reperfusion in whole hearts could limit calcium uptake by Na-Ca exchange. A standard state for measurement of Na-Ca exchange activity that could be used with cells depleted of ATP to different degrees was defined. This was a state of zero sarcolemmal gradient for sodium, potassium, and pH and was achieved by incubation of the cells for 5 minutes with EDTA, EGTA, ouabain, and nigericin. Heterogeneity of cell ATP levels was minimized by using a protocol of total ATP depletion by incubation under conditions similar to ischemia, followed by reoxygenation to give partial restoration of ATP levels. No ATP was regenerated when cells were reoxygenated in the presence of rotenone, and such cells showed a very low rate of calcium uptake. Without rotenone, cells showed an almost complete restoration of Na-Ca exchange activity, in spite of a restoration of ATP levels to only one third of control values. Thus, the dependence of calcium uptake on ATP was highly nonlinear under these conditions. The calculated Km for ATP was no more than 10% of normal ATP levels. We conclude that ATP depletion after ischemia plus reperfusion is unlikely to limit the rate of calcium uptake through Na-Ca exchange in the whole heart if at least one quarter of the ATP is restored. In addition, we measured the apparent ATP dependence of calcium uptake by Na-Ca exchange in cells under conditions in which we previously had concluded that cell ATP distributions were very heterogeneous: when cells undergo contracture during incubation with oligomycin and without glucose. A linear relation between calcium uptake rate and ATP was observed at all ATP levels. This can be understood if cells in contracture that are incubated with oligomycin cannot take up calcium because of low ATP, whereas rod-shaped cells are able to retain a full uptake capability. This result further supports our conclusion that the ATP level declines catastrophically to near zero in these oligomycin-incubated cells just before contracture.
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