Interaction of intracellular ion buffering with transmembrane-coupled ion transport

The role of the Na/Ca exchanger in the control of cellular excitability and tension development is a subject of current interest in cardiac physiology. It has been suggested that this coupled transporter is responsible for rapid changes in intracellular calcium activity during single beats, generation of plateau currents, which control action potential duration, and control of intracellular sodium during Na/K pump suppression, which may occur during terminal states of ischemia. The actual behavior of this exchanger is likely to be complex for several reasons. First, the exchanger transports two ionic species and thus its instantaneous flux rate depends on both intracellular sodium and calcium activity. Secondly, the alteration in intracellular calcium activity, which is caused by a given transmembrane calcium flux, and which controls the subsequent exchanger rate, is a complex function of available intracellular calcium buffering. The buffers convert the ongoing transmembrane calcium fluxes into changes in activity that are a small and variable fraction of the change in total calcium concentration. Using a number of simple assumptions, we model changes in intracellular calcium and sodium concentration under the influence of Na/Ca exchange, Na/K ATPase and Ca-ATPase pumps, and passive sodium and calcium currents during periods of suppression and reactivation of the Na/K ATPase pump. The goal is to see whether and to what extent general notions of the role of the Na/Ca exchanger used in planning and interpreting experimental studies are consistent with its function as derived from current mechanistic assumptions about the exchanger. We find, for example, that based on even very high estimates of intracellular calcium buffering, it is unlikely that Na/Ca exchange alone can control intracellular sodium during prolonged Na/K pump blockade. It is also shown that Na/Ca exchange can contaminate measurements of Na/K pump currents under a variety of experimental conditions. The way in which these and other functions are affected by the dissociation constants and total capacity of the intracellular calcium buffers are also explored in detail.     

Role of the Na(+)-Ca(2+) exchanger as an alternative trigger of CICR in mammalian cardiac myocytes

Ca(2+) influx through the L-type Ca(2+) channels is the primary pathway for triggering the Ca(2+) release from the sarcoplasmic reticulum (SR). However, several observations have shown that Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger current (I(Na-Ca)) could also trigger the Ca(2+) release. The aim of the present study was to quantitate the role of this alternative pathway of Ca(2+) influx using a mathematical model. In our model 20% of the fast sodium channels and the Na(+)-Ca(2+) exchanger molecules are located in the restricted subspace between the sarcolemma and the SR where triggering of the calcium-induced calcium release (CICR) takes place. After determining the strengths of the alternative triggers with simulated voltage-clamps in varied membrane voltages and resting [Na](i) values, we studied the CICR in simulated action potentials, where fast sodium channel current contributes [Na](i) of the subspace. In low initial [Na](i) the Ca(2+) influx via the L-type Ca(2+) channels is the major trigger for Ca(2+) release from the SR, and the Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger cannot trigger the CICR. However, depending on the initial [Na](i), the contribution of the Ca(2+) entry via the exchanger may account for 25% (at [Na](i) = 10 mM) to nearly 100% ([Na](i) = 30 mM) of the trigger Ca(2+). The shift of the main trigger from L-type calcium channels to the exchanger reduced the delay between the action potential upstroke and the intracellular calcium transient. This may contribute to the function of the myocyte in physiological situations where [Na](i) is elevated. These main results remain the same when using different estimates for the most crucial parameters in the modeling or different models for the exchanger.     

The contributions of plasma membrane Na+-Ca2+-exchange and the Ca2+-ATPase to the regulation of cytosolic calcium ([Ca2+]i) in a clonal pituitary cell line (AtT-20) of mouse corticotropes

Single cell calcium microfluorimetry was used to examine the regulation of [Ca2+]i homeostasis in a clonal cell line of corticotropes (AtT-20 cells). Single cells, loaded with fura-2/AM, were exposed briefly to elevated potassium chloride (KCI, 40 mM, 5 sec). The time constant of decay of the [Ca2+]i signal was used as an index of [Ca2+]i extrusion and/or sequestration. Substitution of extracellular sodium with lithium, N-methyl-D-glucamine (NMDG), or Tris, increased resting levels of [Ca2+]i and significantly increased the time constant of [Ca2+]i decay by 40% compared to control indicating the participation of Na+-Ca2+-exchange. Prior exposure of single cells to thapsigargin (1 microM) or BuBHQ (10 microM). inhibitors of the SERCA Ca2+-ATPases, and/or the mitochondrial uncoupler FCCP (1 microM) did not significantly change the time constant of [Ca2+]i decay following KCl. Lanthanum ions (La3+), applied during the decay of the KCI-induced increase in [Ca2+]i, significantly increased the time constant of the return of [Ca2+]i to resting levels by 70% compared to control. Brief exposure of cells to sodium orthovanadate, an inhibitor of ATP-dependent pump activity, slowed and longer exposures prevented, the return of [Ca2+]i to resting levels. We conclude that neither intracellular SERCA pumps nor mitochondrial uptake contribute significantly to [Ca2+]i sequestration following a [Ca2+]i load and that the plasma membrane Ca2+-ATPase contributes to a greater extent than the Na+-Ca2+-exchanger to the return of [Ca2+]i to resting levels following a [Ca2+]i load under these experimental conditions.     

Ca2+ mobilization can occur independent of acceleration of Na+/H+ exchange in thrombin-stimulated human platelets

Intracellular free Ca2+ [( Ca2+]i) and pH (pHi) were measured simultaneously by dual wavelength excitation in thrombin-stimulated human platelets double-labeled with the fluorescent probes fura2 and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein to determine the relationship between changes in [Ca2+]i and pHi, respectively. At 37 degrees C, thrombin (0.5 or 0.1 units/ml) increased [Ca2+]i with no detectable lag period to maximum levels within 13 s followed by a slow return to resting levels. There was a transient decrease in pHi within 9 s that was immediately followed by an alkalinization response, attributable to activation of Na+/H+ exchange, that raised pHi above resting levels within 22 s. At 10-15 degrees C, thrombin-induced changes in [Ca2+]i and pHi were delayed and therefore better resolved, although no differences in the magnitude of changes in [Ca2+]i and pHi were observed. However, the increase in [Ca2+]i had peaked or was declining before the alkalinization response was detected, suggesting that Ca2+ mobilization occurs before activation of Na+/H+ exchange. In platelets preincubated with 5-(N-ethyl-N-isopropyl)amiloride or gel-filtered in Na+-free buffer (Na+ replaced with N-methyl-D-glutamine) to inhibit Na+/H+ exchange, thrombin stimulation caused a rapid, sustained decrease in pHi. Under these conditions there was complete inhibition of the alkalinization response, whereas Ca2+ mobilization was only partially inhibited. Nigericin (a K+/H+ ionophore) caused a rapid acidification of more than 0.3 pH unit that was sustained in the presence of 5-(N-ethyl-N-isopropyl)amiloride. Subsequent stimulation with thrombin resulted in slight inhibition of Ca2+ mobilization. These data show that, in human platelets stimulated with high or low concentrations of thrombin, Ca2+ mobilization can occur without a functional Na+/H+ exchanger and in an acidified cytoplasm. We conclude that Ca2+ mobilization does not require activation of Na+/H+ exchange or preliminary cytoplasmic alkalinization.     

Stimulation of polymorphonuclear leukocytes by dicyclohexylcarbodiimide: calcium homeostasis

The involvement of calcium in N,N'-dicyclohexylcarbodiimide (DCCD)-mediated stimulation of guinea pig neutrophils was investigated. Exposure to DCCD resulted in a fast though moderate elevation of cytosolic calcium concentration. Exchange experiments indicated that DCCD enhanced 45Ca2+ efflux without affecting uptake of the radioisotope from the medium. Plasma membranes isolated from DCCD-stimulated cells failed to support ATP-dependent 45Ca2+ uptake indicating inhibition of their Ca-ATPase. The finding that the enhanced efflux of 45Ca2+ depended on the presence of Na+ ions in the medium implicated a Na+/Ca2+ exchanger in efflux of the ion observed in DCCD-stimulated neutrophils. This is the first indication for the participation of this carrier in calcium homeostasis in stimulated neutrophils. Experiments carried out with 14C-DCCD indicated covalent binding of the reagent to 20 and 150 Kd membrane proteins.     

Protein kinase C alpha enhances sodium-calcium exchange during store-operated calcium entry in mouse platelets

A rise in intracellular calcium concentration ([Ca(2+)](i)) is necessary for platelet activation. A major component of the [Ca(2+)](i) elevation occurs through store-operated Ca(2+) entry (SOCE). The aim of this study was to understand the contribution of the classical PKC isoform, PKCα to platelet SOCE, using platelets from PKCα-deficient mice. SOCE was reduced by approximately 50% in PKCα(-/-) platelets, or following treatment with bisindolylmaleimide I, a PKC inhibitor. However, TG-induced Mn(2+) entry was unaffected, which suggests that divalent cation entry through store-operated channels is not directly regulated. Blocking the autocrine action of secreted ADP or 5-HT on its receptors did not reproduce the effect of PKCα deficiency. In contrast, SN-6, a Na(+)/Ca(2+) exchanger inhibitor, did reduce SOCE to the same extent as loss of PKCα, as did replacing extracellular Na(+) with NMDG(+). These treatments had no further effect in PKCα(-/-) platelets. These data suggest that PKCα enhances the extent of SOCE in mouse platelets by regulating Ca(2+) entry through the Na(+)/Ca(2+) exchanger.     

Calcium adaptation to sodium pump inhibition in a human megakaryocytic cell line

The unique characteristics of the platelet Na/Ca exchanger, i.e., its dependence on both transmembrane Na and K gradients, render it highly sensitive to Na pump inhibition. In this project, we observed that the human megakaryocytic cell line CHRF-288 expresses both the ₁- and ₃-isoforms of the Na-K-ATPase. Inhibition of the Na pump increased the RNA and protein expressions of sarco(endo)plasmic reticulum Ca-ATPase 2b, cytosolic Na and Ca, and the freely exchangeable Ca in the endoplasmic reticulum. These changes occurred in concert with diminished store-operated Ca entry and an increase in the maximal activity of the Na/Ca exchanger. Inhibition of the Na pump by ouabain was more effective in inducing these changes than diminishing medium K. Collectively, these observations point to an integrative effort to counteract the impact of Na pump inhibition by Ca sequestration into the endoplasmic reticulum, diminished Ca entry, and increased activity of the Na/Ca exchanger. The implications of these findings in platelet biology are discussed. platelets; thrombosis; potassium; stroke     

Activation of a calcium entry pathway by sodium pyrithione in the bag cell neurons of Aplysia

The ability of sodium pyrithione (NaP), an agent that produces delayed neuropathy in some species, to alter neuronal physiology was accessed using ratiometric imaging of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in fura PE-filled cultured Aplysia bag cell neurons. Bath-application of NaP evoked a [Ca(2+)](i) elevation in both somata and neurites with an EC(50) of approximately 300 nM and a Hill coefficient of approximately 1. The response required the presence of external Ca(2+), had an onset of 3-5 min, and generally reached a maximum within 30 min. 2-Methyl-sulfonylpyridine, a metabolite and close structural analog of NaP, did not elevate [Ca(2+)](i). Under whole-cell current-clamp recording, NaP produced a approximately 14 mV depolarization of resting membrane potential that was dependent on external Ca(2+). These data suggested that NaP stimulates Ca(2+) entry across the plasma membrane. To minimize the possibility that a change in cytosolic pH was the basis for NaP-induced Ca(2+) entry, bag cell neuron intracellular pH was estimated with the dye 2',7'-bis(carboxyethyl-5(6)-carboxy-fluorescein acetoxy methylester. Exposure of the neurons to NaP did not alter intracellular pH. The slow onset and sustained nature of the NaP response suggested that a cation exchange mechanism coupled either directly or indirectly to Ca(2+) entry could underlie the phenomenon. However, neither ouabain, a Na(+)/K(+) ATPase inhibitor, nor removal of extracellular Na(+), which eliminates Na(+)/Ca(2+) exchanger activity, altered the NaP-induced [Ca(2+)](i) elevation. Finally, the possibility that NaP gates a Ca(2+)-permeable ion channel in the plasma membrane was examined. NaP did not appear to activate two major forms of bag cell neuron Ca(2+)-permeable ion channels, as Ca(2+) entry was unaffected by inhibition of voltage-gated Ca(2+) channels using nifedipine or by inhibition of a voltage-dependent, nonselective cation channel using a high concentration of tetrodotoxin. In contrast, two potential store-operated Ca(2+) entry current inhibitors, SKF-96365 and Ni(2+), attenuated NaP-induced Ca(2+) entry. We conclude that NaP activates a slow, persistent Ca(2+) influx in Aplysia bag cell neurons.     

Ionic ligand interactions with the intracellular loop of the sodium-calcium exchanger. Modulation by ATP

In the last decade, there has been a large increase in the study of the Na(+)/Ca(2+) exchanger due to its implications in physiological and pathophysiological processes at the cell and organ levels. Key areas of these studies have been molecular biology, regulation and physiology-pathophysiology of the exchanger. There are three main types of regulation that take place at the large intracellular loop of the Na(+)/Ca(2+) exchanger: (i) ionic (sodium inactivation, calcium regulation and proton inhibition), (ii) metabolic (ATP as phosphoryl group donor), and (iii) genetic (alternative splicing). This review analyzes the most recent data on the mutual interactions of regulatory ionic ligands (Ca(2+), Na(+), H(+)) and how they are secondarily modulated by MgATP, emphasizing the importance of the binding of Ca(2+) to its regulatory site as an essential requirement for the exchange function. Intracellular protons and sodium inhibit the Na(+)/Ca(2+) exchanger by reducing the apparent affinity of the Ca(i)-regulatory site for Ca(2+). Although the metabolic pathways are different in the mammalian heart (membrane lipids) and squid nerve cells (soluble cytosolic regulatory protein), the final mechanism for the protective effect of MgATP is the same: a reduction of Na(i)(+)-H(i)(+) binding affinities facilitating the attachment of Ca(2+) to its regulatory site. Kinetic models, which partially analyzed some of these ionic and metabolic interactions, can be integrated into a single scheme where the Ca(i)-regulatory site plays a central role.     

Rapid secretagogue-induced activation of Na+H+ exchange in rat parotid acinar cells. Possible interrelationship between volume regulation and stimulus-secretion coupling.

We demonstrate a rapid activation of the Na+/H+ exchanger in intact rat parotid acini in response to muscarinic (carbachol; K1/2 = 0.4 microM) and alpha-adrenergic (epinephrine; K1/2 = 0.1 microM) stimulation. This rapid activation is apparently distinct from the relatively "slow" activation of the exchanger (t1/2 greater than or equal to 5 min) reported previously (Manganel, M., and Turner, R. J. (1989) J. Membr. Biol. 111, 191-198). This rapid activation is not produced by treatment of acini with active diacylglycerol analogues nor prevented by protein kinase inhibitors, arguing against the involvement of protein kinase C-dependent processes. Stimulation of the exchanger is, however, produced by concentrations of ionomycin which yield intracellular calcium levels in the physiologic (secretagogue-induced) range. In addition, chelation of intracellular calcium with 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocks the effect of carbachol, but calmodulin antagonists are without effect. The possibility that the rapid activation of the Na+/H+ exchanger may be associated with cell shrinkage arising from carbachol-induced calcium mobilization is explored. In support of this suggestion we present evidence that: (i) the Na+/H+ exchanger is stimulated by shrinkage of these cells, (ii) the carbachol dose dependence of Na+/H+ exchange activation correlates well with that of shrinkage (but not with that of intracellular calcium levels), and (iii) maneuvers which blunt carbachol- or calcium-induced shrinkage also blunt activation of the exchanger. We suggest that this osmoregulatory response may play an important role in maintaining ionic homeostasis during the acinar fluid secretory process.     

Mechanism of rise and decay of thapsigargin-evoked calcium signals in MDCK cells

We studied the effect of thapsigargin on intracellular calcium levels ([Ca2+]i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. Thapsigargin elevated [Ca2+]i dose dependently with an EC50 of approximately 0.15 microM. The Ca2+ signal consisted of a slow rise, a gradual decay and a plateau. Depletion of the endoplasmic reticulum Ca2+ store with thapsigargin for 7 min abolished the [Ca2+]i increases evoked by bradykinin. Removal of extracellular Ca2+ reduced the thapsigargin response by approximately 50%. The Ca2+ signal was initiated by Ca2+ release from the internal store followed by capacitative Ca2+ entry (CCE). The thapsigargin-evoked CCE was abolished by La3 and Gd3+, and was partly inhibited by SKF 96365 and econazole. After depletion of the internal Ca2+ store for 30 min with another inhibitor of the internal Ca2+ pump, cyclopiazonic acid, thapsigargin failed to increase [Ca2+]i, thus suggesting that the thapsigargin-evoked Ca2+ influx was solely due to CCE. We investigated the mechanism of decay of the thapsigargin response. Pretreatment with La3+ (or Gd3+) or alkalization of extracellular medium to pH 8 significantly potentiated the Ca2+ signal; whereas pretreatment with carbonylcyanide m-chlorophynylhydrozone (CCCP) or removal of extracellular Na+ had no effect. Collectively, our results imply that thapsigargin increased [Ca2+]i in MDCK cells by depleting the internal Ca2+ store followed by CCE, with both pathways contributing equally. The decay of the thapsigargin response might be significantly governed by efflux via the plasmalemmal Ca2+ pump.     

Mitogen-stimulated release of inositol phosphates in human fibroblasts

Mitogenic stimulation of quiescent human fibroblasts (HSWP) with a growth factor mixture (consisting of epidermal growth factor (EGF), insulin, bradykinin, and vasopressin) rapidly induces an increase in Na influx via a Ca-mediated activation of an amiloride-sensitive Na/H exchanger. Inositol phosphates (specifically inositol-1',4',5'-phosphate) have been implicated in mediating the mobilization of intracellular Ca stores in other cell types and we have now completed a detailed analysis of the mitogen-induced release of inositol phosphates in HSWP cells. Stimulation of inositol trisphosphate release is rapid (within 5 s) and reaches a maximum level (416-485% basal) within 10-15 s after the addition of growth factor mixture. Inositol bisphosphate and inositol monophosphate reach maximum levels by 30 s (1257% basal) and 60 s (291% basal), respectively. Levels of all three compounds then decay toward basal levels but remain elevated (150-350% of basal levels) after 10 min of incubation with mitogens. The effects of different combinations of these growth factors and of the bee venom peptide, melittin, have also been determined. We have also found that 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate, which prevents the mitogen-induced rise in intracellular calcium activity and activation of Na influx, does not alter the mitogen-stimulated accumulation of inositol trisphosphate. In addition, the calcium ionophore A23187, which increases cytosolic Ca activity and induces a Na influx, does not stimulate the release of inositol trisphosphate. Assays performed in the presence of lithium, which inhibits inositol phosphate monophosphatase, promotes the prolonged and enhanced accumulation of inositol monophosphate. Treatment with the phospholipase inhibitor mepacrine or pretreatment with dexamethasone reduces the amount of inositol phosphates released upon mitogenic stimulation. Hence mitogenic stimulation of HSWP cells leads to the rapid stimulation of inositol phosphate release via a calcium-independent mechanism and suggests inositol trisphosphate as a candidate to mediate the release of intracellular calcium stores which is involved in the processes responsible for the activation of the Na/H exchanger.     

Changes in cytosolic free calcium concentration in isolated rat parotid cells by cholinergic and beta-adrenergic agonists

The alteration in the concentration of cytosolic free calcium ([Ca2+]i) in isolated rat parotid cells caused by autonomic agents was directly measured using the Ca-sensitive fluorescent probe, quin2. [Ca2+]i of unstimulated cells was estimated to be 162.7 +/- 3.2 nM in normal medium. Carbachol (CCh) and isoproterenol (ISP) caused a rapid rise in [Ca2+]i in a dose-dependent manner. Maximum increases in [Ca2+]i induced by CCh and ISP were approximately 100% and 25% of resting level, respectively. In Ca-free medium, CCh produced a small, rapid rise in [Ca2+]i, followed by a slow decay and a return to resting level within 3-4 min, while all doses of ISP tested failed to change [Ca2+]i. These results suggest that CCh mobilizes Ca2+ from both extracellular and intracellular pools and then results in a rise in [Ca2+]i, whereas ISP may slightly mobilize only the extracellular Ca pool.     

Overshooting cytosolic Ca2+ signals evoked by capacitative Ca2+ entry result from delayed stimulation of a plasma membrane Ca2+ pump

The effect of capacitative Ca2+ entry on cytosolic free Ca2+ concentration ([Ca2+]c) was examined in calf pulmonary artery endothelial cells treated with thapsigargin. Restoration of extracellular Ca2+ evoked an overshoot in [Ca2+]c: the initial rate of Ca2+ influx was 12.4 +/- 0.5 nM/s as [Ca2+]c rose monoexponentially (time constant, tau = 36 +/- 2 s) to a peak (322 +/- 16 nM) before declining to 109 +/- 14 nM after 2000 s. Rates of Ca2+ removal from the cytosol were measured throughout the overshoot by recording the monoexponential decrease in [Ca2+]c after rapid removal of extracellular Ca2+. The time constant for recovery (tau rec decreased from 54 +/- 4 s when Ca2+ was removed after 10 s to its limiting value of 8.8 +/- 1.0 s when it was removed after 2000 s. The time dependence of the changes in tau rec indicate that an increase in [Ca2+]c is followed by a delayed (tau = 408 s) stimulation of Ca2+ removal, which fully reverses (tau approximately 185 s) after Ca2+ entry ceases. Numerical simulation indicated that the changes in Ca2+ removal were largely responsible for the overshooting pattern of [Ca2+]c. Because prolonged (30 min) Ca2+ entry did not increase the total 45Ca2+ content of the cells, an increased rate of Ca2+ extrusion across the plasma membrane most likely mediates the Ca2+ removal, and since it persists in the absence of extracellular Na+, it probably results from stimulation of a plasma membrane Ca2+ pump. We conclude that delayed stimulation of a plasma membrane Ca2+ pump by capacitative Ca2+ entry may protect cells from excessive increases in [Ca2+]c and contribute to oscillatory changes in [Ca2+]c.     

Ethanol protects the heart against the calcium paradox injury

Rat hearts were depleted of Ca2+ (less than 10(-9) M) for 10 min, followed by 15 min of Ca2+-repletion. The calcium paradox injury occurs during Ca2+-repletion, after a period of calcium depletion. The calcium paradox injury was assessed by percent recovery (hemodynamics, [Ca2+]i, and energy levels) during Ca2+-repletion. A decrease in Na+ concentration during Ca2(+)-depletion did not allow for recovery during Ca2(+)-repletion, however 2.5% and 5% ethanol during Ca2(+)-depletion allowed for an approximate 50% recovery during Ca2(+)-repletion. A combination of ethanol (2.5% or 5%) with a low extracellular Na+ concentration (88 mM) allowed for complete recovery. Ethanol prevented a depletion of diastolic [Ca2+]i during Ca2(+)-depletion, and allowed for a return of normal diastolic [Ca2+]i during Ca2(+)-repletion. Ethanol modulates the activity of the Na+/Ca2+ exchanger and protects against the Ca2(+)-paradox injury.     

Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons

Proteolytic cleavage of the Na(+)/Ca(2+) exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca(2+) dynamics, calpain activation and cell viability, in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. Selective overactivation of AMPA receptors caused the reversal of the NCX, which accounted for approximately 30% of the rise in intracellular free calcium concentration ([Ca(2+)](i)). The NCX reverse-mode inhibitor, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943), partially inhibited the initial increase in [Ca(2+)](i), and prevented a delayed increase in [Ca(2+)](i). In parallel, overactivation of AMPA receptors strongly activated calpains and led to the proteolysis of NCX3. KB-R7943 prevented calpain activation, cleavage of NCX3 and was neuroprotective. Silencing of NCX3 reduced Ca(2+) uptake, calpain activation and was neuroprotective. Our data show for the first time that NCX reversal is an early event following AMPA receptor stimulation and is linked to the activation of calpains. Since calpain activation subsequently inactivates NCX, causing a secondary Ca(2+) entry, NCX may be viewed as a new suicide substrate operating in a Ca(2+)-dependent loop that triggers cell death and as a target for neuroprotection.     

Sulfur dioxide derivatives modulate Na/Ca exchange currents and cytosolic [Ca2+]i in rat myocytes

We have recently shown that sulfur dioxide (SO(2)) derivatives (bisulfite and sulfite, 1:3 M/M) modulated L-type calcium, sodium, and potassium channels in rat myocytes. The aim of this study was to investigate whether SO(2) derivatives could alter Na/Ca exchanger current and the intracellular free [Ca(2+)]. The nickel-sensitive Na/Ca exchanger current was measured in rat myocytes exposed to ramp pulses in Tyrode's solution containing ouabain, nifedipine, and +/-Ni (5 mmol/l). Myocytes were loaded with the fluorescent Ca(2+) indicator Fura-2/AM to estimate intracellular Ca(2+) concentration. SO(2) derivatives significantly inhibited both outward and inward Ni-sensitive Na/Ca exchanger currents without a shift in the reversal potential. The intracellular free [Ca(2+)] was raised by SO(2) derivatives in several concentrations. SO(2) derivatives increased [Ca(2+)](i) in rat myocytes and its mechanism might involve SO(2) derivatives significantly inhibiting Na/Ca exchanger current and enhancing L-type calcium channel.     

Allosteric activation of sodium-calcium exchange activity by calcium: persistence at low calcium concentrations

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. We assumed that the rate of Ca2+ uptake during the acceleration phase reflected the degree of allosteric activation of the exchanger and determined the value of cytosolic Ca2+ ([Ca2+]i) at which the rate of Ca2+ influx was half-maximal (Kh). After correcting for the effects of mitochondrial Ca2+ uptake and fura-2 buffering, Kh values of approximately 300 nM were obtained. After an increase in [Ca2+]i, the activated state of the exchanger persisted following a subsequent reduction in [Ca2+]i to values <100 nM. Thus, within 30 s after termination of a transient increase in [Ca2+]i, exchange-mediated Ca2+ entry began without a lag period and displayed a linear rate of Ca2+ uptake in most cells; a sigmoidal time course of Ca2+ uptake returned 60-90 s after the transient increase in [Ca2+]i was terminated. Relaxation of the activated state was accelerated by the activity of the endoplasmic reticulum Ca2+ pump, suggesting that local Ca2+ gradients contribute to maintaining exchanger activation after the return of global [Ca2+]i to low values.     

Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation

Sodium-calcium exchange current was isolated in inside-out patches excised from guinea pig ventricular cells using the giant patch method. The outward exchange current decayed exponentially upon activation by cytoplasmic sodium (sodium-dependent inactivation). The kinetics and mechanism of the inactivation were studied. (a) The rate of inactivation and the peak current amplitude were both strongly temperature dependent (Q10 = 2.2). (b) An increase in cytoplasmic pH from 6.8 to 7.8 attenuated the current decay and shifted the apparent dissociation constant (Kd) of cytoplasmic calcium for secondary activation of the exchange current from 9.6 microM to < 0.3 microM. (c) The amplitude of exchange current decreased synchronously over the membrane potential range from -120 to 60 mV during the inactivation, indicating that voltage dependence of the exchanger did not change during the inactivation process. The voltage dependence of exchange current also did not change during secondary modulation by cytoplasmic calcium and activation by chymotrypsin. (d) In the presence of 150 mM extracellular sodium and 2 mM extracellular calcium, outward exchange current decayed similarly upon application of cytoplasmic sodium. Upon removal of cytoplasmic sodium in the presence of 2-5 microM cytoplasmic free calcium, the inward exchange current developed in two phases, a fast phase within the time course of solution changes, and a slow phase (tau approximately 4 s) indicative of recovery from sodium-dependent inactivation. (e) Under zero-trans conditions, the inward current was fully activated within solution switch times upon application of cytoplasmic calcium and did not decay. (f) The slow recovery phase of inward current upon removal of cytoplasmic sodium was also present under the zero-trans condition. (g) Sodium-dependent inactivation shows little or no dependence on membrane potential in guinea pig myocyte sarcolemma. (h) Sodium-dependent inactivation of outward current is attenuated in rate and extent as extracellular calcium is decreased. (i) Kinetics of the sodium-dependent inactivation and its dependence on major experimental variables are well described by a simple two-state inactivation model assuming one fully active and one fully inactive exchanger state, whereby the transition to the inactive state takes place from a fully sodium-loaded exchanger conformation with cytoplasmic orientation of binding sites (E1.3Ni).     

Parathyroid hormone-activated calcium channels in an osteoblast-like clonal osteosarcoma cell line. cAMP-dependent and cAMP-independent calcium channels

Changes in free cytosolic calcium were measured in UMR-106 cells in response to parathyroid hormone (PTH) stimulation. Bovine PTH-(1-34) induced an increase in [Ca2+]i with the contour of the rise in [Ca2+]i occurring in three successive phases: a rapid increase in [Ca2+]i occurring within seconds, rapid decrement in [Ca2+]i to near-resting levels within 1 min, and slow increment in [Ca2+]i. Phase one and phase three increases in [Ca2+]i were dependent on medium calcium. The phase one rise in [Ca2+]i was inhibitable by the calcium channel blockers lanthanum and verapamil. Only the phase one rise in [Ca2+]i was blocked by preincubation of the cells with the phorbol ester, phorbol 12-myristate 13-acetate. This channel was also blocked when cellular cAMP levels were increased prior to PTH stimulation. The phase two decrement of [Ca2+]i was due to the rapid inactivation of the phase one calcium channel. The phase three rise in [Ca2+]i was mediated by cellular cAMP levels. This cAMP-dependent Ca2+ channel was insensitive to pretreatment of the cells with phorbol diesters and showed low sensitivity to Ca2+ channel blockers. It is concluded that UMR-106 cells respond to PTH stimulation by the activation of a cAMP-independent Ca2+ channel. This channel rapidly inactivates. The subsequent PTH-dependent increase in cellular cAMP is followed by activation of a cAMP-dependent Ca2+ channel resulting in a slow rise in [Ca2+]i.