Modulation of functional and optimal (Na+-K+)ATPase activity during the cell cycle of neuroblastoma cells

Functional and optimal activities of the (Na+-K+)ATPase, as determined by ouabain-sensitive K+ influx in intact cells and ATP hydrolysis in cell homogenates respectively, have been measured during the cell cycle of neuroblastoma (clone Neuro-2A) cells. The cells were synchronized by selective detachment of mitotic cells. The ouabain-sensitive K+ influx decreased more than fourfold from 1.62 +/- 0.11 nmoles/min/10(6) cells to 0.36 +/- 0.25 nmoles/min/10(6) cells on passing from mitosis to early G1 phase. On entry into S phase a transient sixfold increase to 2.07 +/- 0.30 nmoles/min/10(6) cells was observed, followed by a rapid decline, after which the active K+ influx rose again steadily from 1.03 +/- 0.25 nmoles/min/10(6) cells in early S phase to 2.10 +/- 0.92 nmoles/min/10(6) cells just prior to the next mitosis. The ouabain-insensitive component rose linearly through the cycle in the same manner as the protein content/cell. Combining total K+ influx values with efflux data obtained previously showed that net loss of K+ occurred with transition from mitosis to G1 phase while net accumulation occurred with entry into S. Throughout mid-S phase net K+ flux was virtually zero, but a large net influx occurred again just before the next mitosis. The (Na+-K+)ATPase activity measured in cell homogenates decreased rapidly from mitosis to G1 phase and increased steadily throughout S phase, but the transient activation on entry into S phase was not observed. Complete inhibition of the (Na+-K+)ATPase mediated K+ influx by ouabain (5 mM) prevents the cells from entering S phase, while partial inhibition by lower concentrations of ouabain (0.2 and 0.5 mM; km = 0.17 mM) causes partial blockage in G1 and, to a lesser extent, a reduced rate of progression through the rest of the cell cycle. We conclude that the transient increase in (Na+-K+)ATPase mediated K+ influx at the G1/S transition is a prerequisite for entry into S phase, while maintenance of adequate levels of K+ influx is necessary for normal rate of progression through the rest of the cell cycle.     

The effect of Na+ on Ca2+ homeostasis in unstimulated platelets

Platelets maintain a low cytosolic free Ca2+ concentration by limiting Ca2+ influx from plasma and promoting Ca2+ efflux. The present studies examine the role of the plasma membrane Na+ gradient in these processes. The Na+ gradient in intact unstimulated platelets was altered by incubating the platelets with ouabain or by replacing extracellular Na+ with N-methyl-D-glucamine or choline. Ca2+ flux across the plasma membrane and the amount of exchangeable Ca2+ in the platelet cytosol were measured by observing 45Ca2+ influx and efflux under steady-state conditions. The cytosolic free Ca2+ concentration was measured with the fluorescent probe quin2. At extracellular Na+ concentrations below 50 mM, the size of the cytosolic exchangeable Ca2+ pool increased by 48%. The size of the exchangeable Ca2+ pool sequestered in the dense tubular system increased by 356%. Ca2+ flux across the plasma membrane increased by 38%. There was, however, no change in total platelet Ca2+ and little, if any, change in the cytosolic free Ca2+ concentration. Similar effects were produced by incubating platelets with ouabain. These observations demonstrate a marked influence of the plasma membrane Na+ gradient on Ca2+ homeostasis in platelets. The nature of the changes, however, suggests that Na+/Ca2+ exchange cannot be sole basis for Ca2+ efflux from platelets.     

Sodium-potassium exchange in sea urchin egg. II. Ionic events stimulating the Na+-K+ pump activity at fertilization

The preceding paper (Ciapa et al., 1984) provided biochemical and kinetic characterization of the Na+-K+ exchange in Paracentrotus lividus eggs. The present work is a study of the ionic events involved in the stimulation of the Na+-K+ transporter after fertilization. Fertilization in low Na+-external medium containing amiloride (0.1 mM) suppresses the stimulation of the net efflux of H+ and 86Rb uptake. Activation of eggs with the ionophore A23187 leads to stimulation of both Na+-H+ exchange and ouabain-sensitive 86Rb influx. When eggs were activated with A23187 in artificial seawater, 86Rb uptake and 24Na influx showed similar saturable kinetics with respect to the external Na+. A23187 treatment of eggs in Na+-free artificial seawater did not stimulate the Na+-K+ exchange until 10 mEq Na+ was added. Activation of eggs by NH4Cl (5 mM) stimulated 86Rb influx and Na+ exit; both fluxes were ouabain sensitive. Monensin increased cell Na+ of unfertilized eggs without any significant increase in intracellular pH: a condition in which 86Rb influx was not markedly stimulated. Addition of 10 mEq Na+ to unfertilized eggs in Na+-free artificial seawater stimulated 86Rb uptake but to a lower extent that did 10 mEq Na+ plus sperm. It is concluded that (1) the stimulation of the Na+-K+ pump at fertilization has an absolute requirement for the Na+-H+ exchange; (2) the alkalinization of eggs resulting from the acid efflux is a prerequisite for the enhancement of the Na+-K+ pump; (3) the amount of Na+ entering eggs at fertilization determines the intensity of the Na+-K+ exchange; (4) early events of fertilization such as exocytosis and calcium release which may be involved in the stimulation of the Na+-K+ pump must necessarily be coupled to cell alkalinization.     

Clearance of store-released Ca2+ by the Na+-Ca2+ exchanger is diminished in aortic smooth muscle from Na+-K+-ATPase alpha 2-isoform gene-ablated mice

Two alpha-isoforms of the Na+-K+-ATPase are expressed in vascular smooth muscle cells (VSMCs). The alpha 1-isoform is proposed to serve a cytosolic housekeeping role, whereas the alpha 2-isoform modulates Ca2+ storage via coupling to the Na+-Ca2+ exchanger (NCX) in a subsarcolemmal compartment. To evaluate the ramifications of this proposed interaction, Ca2+-store load and the contributions of the primary Ca2+ transporters to Ca2+ clearance were studied in aortic VSMCs from embryonic wild-type (WT) and Na+-K+-ATPase alpha 2-isoform gene-ablated, homozygous null knockout (alpha 2-KO) mice. Ca2+ stores were unloaded by inhibiting the sarco(endo)plasmic reticulum Ca2+-ATPase with cyclopiazonic acid (CPA) in Ca2+-free media to limit Ca2+ influx. Ca2+ clearance by the plasma membrane Ca2+-ATPase (PMCA), NCX, or mitochondria was selectively inhibited. In WT VSMCs, NCX accounted for 90% of the Ca2+ efflux. In alpha 2-KO VSMCs, preferential clearance of store-released Ca2+ by NCX was lost, whereas PMCA activity was increased. Selective inhibition of the alpha 2-isoform (0.5 microM ouabain for 20 min), before treatment with CPA enhanced the store load in VSMCs from WT, but not alpha 2-KO mice. A subsequent analysis of capacitative Ca2+ entry (CCE) indicated that the magnitude of Ca2+ influx was significantly greater in alpha 2-KO cells. Our findings support the concept of a subsarcolemmal space where the alpha 2-isoform coupled with NCX modulates Ca2+-store function and, thereby, CCE.     

Epinephrine stimulates the Na+-K+ ATPase in isolated rat jejunal crypt cells

The effect of epinephrine on the activity of the Na+-K+ ATPase was studied in isolated rat jejunal cells. The activity of the pump was assessed by measuring the ouabain inhibitable K+ accumulation by the enterocytes using 86Rb as a tracer. Epinephrine stimulated significantly the Na+-K+ ATPase in crypt cells but not in villus cells. This effect was still apparent in presence of propranolol and prazocin but disappeared in presence of yohimbine. Amiloride did not affect the epinephrine-induced stimulation. Calcium channel blockers and dibutyryl cAMP enhanced the activity of the pump, and exerted respectively overlapping and additive effects with epinephrine, when added simultaneously. The calcium ionophore A23187 inhibited the basal activity of the ATPase and the stimulatory effect of epinephrine disappeared in its presence. These results suggest that epinephrine stimulates the Na+-K+ ATPase in jejunal crypt cells by activating alpha2 receptors and decreasing intracellular calcium, and not by altering cAMP levels.     

Distinct effects of various amino acids on 45Ca2+ fluxes in rat pancreatic islets.

The effects of three types of amino acids on 45Ca2+ fluxes in rat pancreatic islets have been compared. Alanine, a non-insulinotropic neutral amino acid, transported with Na+, increased 45Ca2+ efflux in the presence or in the absence of extracellular Ca2+, but not in the absence of Na+. Its effects in Na+-solutions were practically abolished by 7 mM-glucose. Alanine slightly stimulated 45Ca2+ influx (5 min uptake) only when Na+ was present. Two insulinotropic cationic amino acids (arginine and lysine) triggered similar changes in 45Ca2+ efflux. They accelerated the efflux in the presence of Ca2+ and inhibited the efflux in a Ca2+-free medium, whether glucose was present or not. In an Na+-free Ca2+-medium, arginine and lysine markedly accelerated 45Ca2+ efflux, but this effect was suppressed by 7 mM-glucose. Arginine stimulated 45Ca2+ influx irrespective of the presence or absence of glucose and Na+. Leucine, a neutral insulinotropic amino acid well metabolized by islet cells, inhibited 45Ca2+ efflux from the islets in a Ca2+-free medium; this effect was potentiated by glutamine. In the presence of Ca2+ and Na+, leucine was ineffective alone, but triggered a marked increase in 45Ca2+ efflux when combined with glutamine. In an Na+-free Ca2+-medium, leucine accelerated 45Ca2+ efflux to the same extent with or without glutamine. Leucine also stimulated 45Ca2+ influx in the presence or in the absence of Na+, but its effects were potentiated by glutamine only in the presence of Na+. The results show that amino acids of various types cause distinct changes in 45Ca2+ fluxes in pancreatic islets. Certain of these changes involve an Na+-mediated mobilization of cellular Ca2+ from sequestering sites where glucose appears to exert an opposite effect.     

Na,K-ATPase in excitation-contraction coupling of vascular smooth muscle from cattle.

Lowering the extracellular K+ content from 6 to 0.6 mM causes a rise, and elevation from 6 to 8.5 mM a fall of 45Ca++ efflux from the vascular smooth muscle cells of the arteria carotis communis of cattle. In contrast, a level of 17 mM K+ has no influence. Removal of extracellular calcium does not block these effects. 10(-4) M ouabain also induces a rise in Ca++ efflux, additional potassium reduction then being without effect; 10(-9) M ouabain is of no influence. The 45Ca++ efflux kinetics correlates with the activity of the isolated Na,K-ATPase. Tonus increases of the vascular strips by 10(-4) M ouabain and potassium deficiency cannot be blocked by 4 mM lanthanum or removal of extracellular calcium. Unlike sodium, potassium stimulates the active Ca++ binding and the activity of the Ca-ATPase of the microsomal fraction. The ative Ca++ binding of the mitochondria is stimulated by both ions. It is postulated that the activity of the plasma membrane Na,K-pump is able to regulate the tonus of big arteries through alteration of Ca++ storage processes.     

Ca2+ transport by intact synaptosomes: the voltage-dependent Ca2+ channel and a re-evaluation of the role of sodium/calcium exchange

The verapamil-sensitive Ca2+ channel in the synaptosomal plasma membrane is investigated. Verapamil is without effect on Ca2+ uptake or steady-state content in synaptosomes with a polarized plasma membrane, but completely inhibits the additional Ca2+ uptake following plasma-membrane depolarization by high [K+], by veratridine plus ouabain or by high concentrations of the permeant cation tetraphenylphosphonium. Verapamil-insensitive Ca2+ influx and steady-state content are identical in polarized and depolarized synaptosomes, even though the Na+ electrochemical potential is greatly decreased in the latter, indicating that Na+/Ca2+ exchange is not a significant mechanism for Ca2+ efflux under these conditions. A transient Na+-dependent Ca2+ efflux can only be observed on addition of Na+ to Na+-depleted depolarized synaptosomes. While 0.2 mM verapamil decreases the ate of 86Rb+ efflux and 22Na+ entry during depolarization induced by veratridine plus ouabain, the final steady-state Na+ accumulation is not inhibited. Ca2+ efflux from synaptosomes following mitochondrial depolarization does not occur by a verapamil-sensitive pathway.     

Exogeneous diacylglycerols downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes.

In this study, cell permeable diacylglycerols, sn-1,2-dioctanoglycerol (DiC8), and sn-1-oleoyl-2-acetylglycerol (OAG) were found to downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes. Both DiC8 and OAG decreased the binding of [3H]ouabain to intact oocytes while phorbol esters did not appreciably influence the same. These diacylglycerols inhibited the amiloride-sensitive 22Na+ influx and ouabain-sensitive 86Rb+ uptake in the oocytes. Furthermore, DiC8 prevented the 22Na+ efflux from the oocytes preloaded with 22Na+. Addition of H-7 to DiC8- and OAG-treated oocytes stimulated the pump activity curtailed by the two latters. The impairment of Na(+)-K+ pump activity by diacylglycerols suggests that protein kinase C activators may stimulate endocytosis of membrane-coupled Na(+)-K+ ATPase.     

Patulin-induced ion flux in cultured renal cells and reversal by dithiothreitol and glutathione: a scanning electron microscopy (SEM) X-ray microanalysis study

Patulin (PAT), a compound produced by certain species of Aspergillus, Penicillium, and Byssochlamys, is frequently found associated with agricultural commodities. PAT has many effects on membrane function, including the inhibition of the isolated Na+-K+ ATPase. In this study, a scanning electron microscope equipped with an energy dispersive spectroscopy X-ray microanalysis system was used to examine individual cultured renal epithelial cells (LLC-PK1) in order to determine the effects of PAT on the relative intracellular ion concentrations. The estimated EC50 (60 min) for both sodium influx and potassium efflux was between 10 and 50 microns for ouabain. For PAT, the EC50 (60 min) was 250 microns for sodium influx and 100 microns for potassium efflux. However, 1 mM patulin at 240 min caused complete reversal of the sodium and potassium content of cells, and 1 mM ouabain at 240 min did not. The effect of patulin on sodium and potassium flux was both concentration and time dependent and was reversed by dithiothreitol and glutathione. PAT (250 microM) but not ouabain (250 microM) induced massive blebbing of LLC-PK1 cells. Thus, the interaction of PAT with cellular membranes involves both alterations in the regulation of intracellular ion content and the cytoskeleton. We hypothesize that patulin alters intracellular ion content via Na+-K+ ATPase and non-Na+-K+ ATPase mechanisms.     

Instrumental role of Na+ in NMDA excitotoxicity in glucose-deprived and depolarized cerebellar granule cells

In glucose-deprived cerebellar granule cells, substitution of extracellular Na+ with Li+ or Cs+ prevented N-methyl-D-aspartate (NMDA)-induced excitotoxicity. NMDA stimulated 45Ca2+ accumulation and ATP depletion in a Na-dependent manner, and caused neuronal death, even if applied while Na,K-ATPase was inhibited by 1 mM ouabain. The cells treated with NMDA in the presence of ouabain accumulated sizable 45Ca2+ load but most of them failed to elevate cytosolic [Ca2+] upon mitochondrial depolarization. Na/Ca exchange inhibitor, KB-R7943, inhibited Na-dependent and NMDA-induced 45Ca2+ accumulation but only if Na,K-ATPase activity was compromised by ouabain. In cells energized by glucose and exposed to NMDA without ouabain, KB-R7943 reduced NMDA-elicited ionic currents by 19% but failed to inhibit 45Ca2+ accumulation. It appears that a large part of NMDA-induced Ca2+ influx in depolarized and glucose-deprived cells is mediated by reverse Na/Ca exchange. A high level of reverse Na/Ca exchange operation is maintained by a sustained Na+ influx via NMDA channels and depolarization of the plasma membrane. In cells energized by glucose, however, most Ca2+ enters directly via NMDA channels because Na,K-ATPase regenerating Na+ and K+ concentration gradients prevents Na/Ca exchange reversal. Since under these conditions Na/Ca exchange extrudes Ca2+, its inhibition destabilizes Ca2+ homeostasis.     

Environmental salinity and sodium and chloride exchanges across the gill of Tilapia mossambica.

10 Freshwater-(FW)-adapted, one-third seawater (1/3 SW)-adapted and seawater (SW) adapted Tilapia mossambica were compared for their branchial Na+ influx and efflux as well as Cl- efflux. Na+ and Cl- effluxes were identical. Rates of effluxes were in 1/3 SW- and in SW-adapted fish 10 times and 200 times higher respectively than in FW specimens. 20 Shock due to handling and transfer to small experimental chambers induced, within 20 to 45 min., a considerable increase in Na+ efflux and a more discrete augmentation of the Na+ influx. 30 Branchial Mg++-and Na+-K+ activated ATPase activities increased significantly upon adaptation from FW to 1/3 SW. No significant increase was apparent upon adaptation from 1/3 SW to SW. 40 The trans-branchial potential observed in SW Tilapia resembled the pattern previously described in other species of teleosts.     

Calcium mobilization by cadmium or decreasing extracellular Na+ or pH in coronary endothelial cells

Replacing extracellular Na+ with choline transiently increased cytoplasmic free Ca2+ ([Ca2+]i) more than 5-fold in coronary endothelial cells. Removing external Na+ stimulated 45Ca2+ efflux approximately 4-fold and influx approximately 1.7-fold. The stimulation of efflux was independent of extracellular Ca2+ and the osmotic Na+ substitute. The release of stored Ca2+, rather than Ca2+ influx via Na(+)-Ca2+ exchange, probably causes the increase in [Ca2+]i and 45Ca2+ efflux. Cadmium or decreasing external, not intracellular, pH transiently increased [Ca2+]i. Cd2+ and some other divalent metals also stimulated 45Ca2+ efflux. The potency order of the metals that stimulated efflux was Cd2+ greater than CO2+ greater than Ni2+ greater than Fe2+ greater than Mn2+. Incubating the cells with Zn2+ prior to assaying efflux in the absence of Zn2+ strongly inhibited the stimulation of 45Ca2+ efflux by Cd2+, pH 6, and the removal of external Na+ without affecting the stimulation of efflux by ATP. These findings support the hypothesis that certain trace metals or decreasing external Na+ or pH trigger the release of stored Ca2+ by stimulating a cell surface "receptor."     

Calcium efflux and cycling across the synaptosomal plasma membrane.

Ca2+ efflux from intact synaptosomes is investigated. Net efflux can be induced by returning synaptosomes from media with elevated Ca2+ or high pH to a normal medium. Net Ca2+ efflux is accelerated when the Na+ electrochemical potential gradient is collapsed by veratridine plus ouabain. Under steady-state conditions at 30 degrees C, Ca2+ cycles across the plasma membrane at 0.38 nmol . min-1 . mg-1 of protein. Exchange is increased by 145% by veratridine plus ouabain, both influx and efflux being increased. Increased influx is probably due to activation of voltage-dependent Ca2+ channels, since it is abolished by verapamil. The results indicate that, at least under conditions of low Na+ electrochemical gradient, some pathway other than a Na+/Ca2+ exchange must operate in the plasma membrane to expel Ca2+.     

Na+ channel and Na+-K+ ATPase involvement in norepinephrine- and veratridine-stimulated metabolism in perfused rat hind limb.

In the constant flow perfused rat hind limb, norepinephrine (NE) evoked increases in oxygen uptake (VO2) and lactate efflux (LE) were inhibited by the cardiac glycoside ouabain (1 mM), without interrupting the NE-mediated vasoconstriction. The membrane labilizer veratridine, previously shown to increase VO2 and LE, without increasing perfusion pressure, was also shown to be inhibited by the cardiac glycoside ouabain, as well as by the ouabain analogues digitoxin and digoxin. The stimulatory actions of veratridine on VO2 were inhibitable by low doses of the specific sodium channel blocker tetrodotoxin (TTX), while NE effects were unaffected, suggesting that NE may be acting via a TTX-insensitive sodium channel. It is concluded that agents such as NE (a vasoconstrictor) or veratridine (a membrane labilizer), which stimulate VO2 in the perfused rat hind limb, do so by increasing Na+ influx. The observed increases in oxygen consumption and LE are due to Na+-K+ ATPase activity to pump Na+ out of the cell at the expense of ATP turnover. Energy dissipation due to Na+ cycling may be a form of facultative thermogenesis attributable to NE that can be stimulated by membrane labilizers such as veratridine in the constant flow perfused rat hind limb.     

Synergism between ouabain and monensin in neurogenic responses of guinea-pig vas deferens

Interrelations between ouabain, a Na+-K+ ATPase inhibitor, and monensin, a Na+ ionophore, on noradrenaline liberation and contractile activity were evaluated in the guinea-pig vas deferens. Monensin (1 microM) per se elicited a small contraction of the tissue. However, amplitude and time to the peak of large and sustained contractions evoked by 10 microM ouabain were potentiated and markedly shortened, respectively, by monensin. Contractions elicited by ouabain with or without monensin were prevented by 3 microM phentolamine or by pretreatment with reserpine. Contractions evoked by K+-free solution were augmented by monensin. In an HPLC study, noradrenaline outflow from the vas deferens was moderately and considerably increased by monensin (10 microM) and ouabain (100 microM), respectively. The ouabain-evoked output of noradrenaline was enhanced in the presence of monensin and the time course for maximum noradrenaline release was shortened, as was the contractile activity. This enhanced outflow after ouabain plus monensin was reserpine sensitive but not tetrodotoxin sensitive. Furthermore, this noradrenaline outflow was roughly halved in Na+-deficient medium, but was unaltered in Ca2+-free medium. These findings suggest that the synergistic effect of ouabain and monensin on noradrenaline liberation from the guinea-pig vas deferens may be due to an elevation of cytoplasmic Ca2+ concentrations, presumably resulting from a stimulation of intracellular Na+-Ca2+ exchange system, but not enhanced Ca2+ entry.     

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells. Evidence for separate modes of operation of the transporter

(1) Unidirectional K+ (86Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K+ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 . 10(-3) M ouabain. (3) Measurements of net K+ loss and Na+ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K+ (Cl-) outward net flux uncoupled from net Na+ movement. Net K+ (Cl-) outward flux was half-maximally inhibited by 2 microM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K+ influx/efflux were essentially unchanged when compared to untreated controls.     

Vascular Na+/Ca2+ exchanger: implications for the pathogenesis and therapy of salt-dependent hypertension

The Na+/Ca2+ exchanger is an ion transporter that exchanges Na+ and Ca2+ in either Ca2+ efflux or Ca2+ influx mode, depending on membrane potential and transmembrane ion gradients. In arterial smooth muscle cells, the Na+/Ca2+ exchanger is thought to participate in the maintenance of vascular tone by regulating cytosolic Ca2+ concentration. Recent pharmacological and genetic engineering studies have revealed that the Ca2+ influx mode of vascular Na+/Ca2+ exchanger type-1 (NCX1) is involved in the pathogenesis of salt-dependent hypertension. SEA0400, a specific Na+/Ca2+ exchange inhibitor that preferentially blocks the Ca2+ influx mode, lowers arterial blood pressure in salt-dependent hypertensive models, but not in normotensive rats or other types of hypertensive rats. Furthermore, heterozygous mice with reduced expression of NCX1 are resistant to development of salt-dependent hypertension, whereas transgenic mice with vascular smooth muscle-specific overexpression of NCX1 readily develop hypertension after high-salt loading. SEA0400 reverses the cytosolic Ca2+ elevation and vasoconstriction induced by nanomolar ouabain, as well as humoral factors in salt-loaded animals. One possibility is that circulating endogenous cardiotonic steroids may be necessary for NCX1-mediated hypertension. These findings help to explain how arterial smooth muscle cells in blood vessels contribute to salt-elicited blood pressure elevation and suggest that NCX1 inhibitors might be therapeutically useful for salt-dependent hypertension.     

Effects of low extracellular sodium on cytosolic ionized calcium. Na+-Ca2+ exchange as a major calcium influx pathway in kidney cells

The effects of extracellular Na+ (Na+o) on cytosolic ionized calcium (Ca2+i) and on calcium and sodium fluxes were measured in monkey kidney cells (LLC-MK2). Ca2+i was measured with aequorin and the ion fluxes with 45Ca and 22Na. Na+-free media rapidly increased Ca2+i from 60 to a maximum of about 700 nM in 2-3 min. After the peak, Ca2+i declined and reached a plateau of about twice the resting Ca2+i. The peak Ca2+i was inversely proportional to Na+o and directly proportional to the extracellular calcium concentration (Ca2+o). On the other hand, a pH of 6.8 reduced and Ca2+o substitution with Sr2+ completely blocked the Ca2+i response to low Na+o. A Na+-free medium stimulated calcium efflux from the cells 4-5-fold, a response which was abolished in the absence of extracellular Ca2+. Na+-free media also stimulated calcium influx and sodium efflux. The cell calcium content, however, was not increased. These results indicate that removal of extracellular Na+ increases Ca2+i by stimulating calcium influx and not by inhibiting calcium efflux; the increased calcium influx takes place on the Na+-Ca2+ antiporter operating in the reverse mode in exchange for sodium efflux. The increased calcium efflux occurs as a consequence of the rise in Ca2+i and presumably takes place on the (Ca2+-Mg2+) ATPase-dependent calcium pump.     

Kinetics and stoichiometry of coupled Na efflux and Ca influx (Na/Ca exchange) in barnacle muscle cells

Coupled Na+ exit/Ca2+ entry (Na/Ca exchange operating in the Ca2+ influx mode) was studied in giant barnacle muscle cells by measuring 22Na+ efflux and 45Ca2+ influx in internally perfused, ATP-fueled cells in which the Na+ pump was poisoned by 0.1 mM ouabain. Internal free Ca2+, [Ca2+]i, was controlled with a Ca-EGTA buffering system containing 8 mM EGTA and varying amounts of Ca2+. Ca2+ sequestration in internal stores was inhibited with caffeine and a mitochondrial uncoupler (FCCP). To maximize conditions for Ca2+ influx mode Na/Ca exchange, and to eliminate tracer Na/Na exchange, all of the external Na+ in the standard Na+ sea water (NaSW) was replaced by Tris or Li+ (Tris-SW or LiSW, respectively). In both Na-free solutions an external Ca2+ (Cao)-dependent Na+ efflux was observed when [Ca2+]i was increased above 10(-8) M; this efflux was half-maximally activated by [Ca2+]i = 0.3 microM (LiSW) to 0.7 microM (Tris-SW). The Cao-dependent Na+ efflux was half-maximally activated by [Ca2+]o = 2.0 mM in LiSW and 7.2 mM in Tris-SW; at saturating [Ca2+]o, [Ca2+]i, and [Na+]i the maximal (calculated) Cao-dependent Na+ efflux was approximately 75 pmol#cm2.s. This efflux was inhibited by external Na+ and La3+ with IC50's of approximately 125 and 0.4 mM, respectively. A Nai-dependent Ca2+ influx was also observed in Tris-SW. This Ca2+ influx also required [Ca2+]i greater than 10(-8) M. Internal Ca2+ activated a Nai-independent Ca2+ influx from LiSW (tracer Ca/Ca exchange), but in Tris-SW virtually all of the Cai-activated Ca2+ influx was Nai-dependent (Na/Ca exchange). Half-maximal activation was observed with [Na+]i = 30 mM. The fact that internal Ca2+ activates both a Cao-dependent Na+ efflux and a Nai-dependent Ca2+ influx in Tris-SW implies that these two fluxes are coupled; the activating (intracellular) Ca2+ does not appear to be transported by the exchanger. The maximal (calculated) Nai-dependent Ca2+ influx was -25 pmol/cm2.s. At various [Na+]i between 6 and 106 mM, the ratio of the Cao-dependent Na+ efflux to the Nai-dependent Ca2+ influx was 2.8-3.2:1 (mean = 3.1:1); this directly demonstrates that the stoichiometry (coupling ratio) of the Na/Ca exchange is 3:1. These observations on the coupling ratio and kinetics of the Na/Ca exchanger imply that in resting cells the exchanger turns over at a low rate because of the low [Ca2+]i; much of the Ca2+ extrusion at rest (approximately 1 pmol/cm2.s) is thus mediated by an ATP-driven Ca2+ pump.(ABSTRACT TRUNCATED AT 400 WORDS)