Ca2+-activated Na+ fluxes in human red cells. Amiloride sensitivity

The effect of Ca2+ on the ouabain- and bumetanide-resistant Na+ fluxes in intact red cells was studied at relatively constant internal Ca2+, membrane potential, and cell volume. The red cell calcium concentration was modified using the ionophore A23187. In fresh red cells, the Na+ influx and efflux (1.2 +/- 0.13 and 0.26 +/- 0.07 mmol/liter cells x h, respectively) were not affected by amiloride (1 mM). When external Ca2+ was raised from 0 to 150 microM, in the presence of A23187, both the Na+ influx and efflux were stimulated (about 3.5-fold). The Ca2+-activated Na+ efflux and influx had an apparent Km for activation by Ca2+o of about 25 microM. The Ca2+-dependent Na+ transport was inhibited 30-60% by amiloride (ID50 = 17.3 +/- 8 microM). Amiloride, however, had no effect on the Ca2+-dependent K+ influx. The amiloride-sensitive (AS) transport pathway was a linear function of the Na+o concentration in the range from 0 to 75 mM. The Ca2+i activation seems to depend on the metabolic integrity of red cells. 1) It does not take place in ATP-depleted red cells; 2) ATP-repletion of ATP-depleted red cells fully restored AS Na influx; and 3) ATP-enrichment (ATP-red cells) enhanced the AS Na influx by about 100%. The Ca2+-activated AS Na+ influx was not affected by either DIDS or trifluoperazine. The present results indicate that in human erythrocytes an increase in internal Ca2+ activates on otherwise silent AS Na+-transport system, which is dependent on the metabolic integrity of the red cells.     

Influence of external K+ on potassium efflux in isolated chicken enterocytes.

1. Efflux of K+ was measured in pre-loaded (86Rb+) chicken enterocytes incubated in buffers with external K+ concentration ([K+]0) between 1 and 40 mM. 2. A decrease in [K+]0 from 6 to 1 mM reduced the rate constant of K+ efflux, whereas it was stimulated by increasing [K+]0 from 6 to 40 mM. 3. The inhibitory effect of low [K+]0 on K+ efflux was: (i) higher than that expected from a change in the electrical driving force, suggesting that membrane K+ permeability has been decreased, and (ii) attenuated by A23187 and Na(+)-free buffers. 4. The effect of A23187 on K(+)-induced K+ efflux was abolished by apamin and that of Na(+)-free buffers by apamin, quinine or verapamil, which suggests that the effect of low K+ on K+ efflux seems to be due to decreased intracellular Ca2+ concentration. 5. The stimulatory effect of 40 mM K0+ on K+ exit can be accounted for by an increase in the electrical driving force. 6. The efflux of K+ at 40 mM K0 appears to occur through Ca2(+)-activated K+ channels (KCa) since it was prevented by 500 microM quinine and unaffected by bumetanide or 3,4-diaminopyridine. 7. In addition, the current results show that an increase in external K+ concentration reduced the ability of quinine to inhibit KCa channels, and even abolished that of Ba2+ and apamin.     

Calcium transport mechanisms in dog red blood cells studied from measurements of initial flux rates

Ca2+ efflux from dog red blood cells loaded with Ca2+ using the A23187 ionophore could be separated into two main components: (1) Mg- and ATP-dependent (active transport) and (2) dependent on external Na (K1/2 around 15 mM); at 80 microM internal free Ca the relative magnitudes of these fluxes were 70% and 30% respectively. The Na-dependent Ca2+ efflux had the following additional properties: (i) it was partially inhibited by ATP depletion or preincubation with vanadate, but it was not affected by Mg2+ depletion; (ii) it failed to be stimulated by external monovalent cations other than Na: (iii) it was stimulated by reduction in the internal Na+ concentration. Both active and Na-dependent Ca2+ efflux remained unchanged in hypotonic solutions or in solutions with alkaline pH (8.5). In cells containing ATP and Mg2+, external Ca2+ inhibited Ca2+ efflux (K1/2 around 1 mM); on the other hand, in Mg-free dog red cells external Ca2+ stimulated Ca2+ efflux (K1/2 about 30 microM). In Mg-depleted red cells incubated in the absence of external Na2+, Ca2+ influx as a function of external Ca2+ followed a monotonically saturable function (K1/2 around 20 microM): addition of Na resulted in (i) inhibition of Ca2+ influx and (ii) a sigmoid relationship between flux and external Ca2+. Intracellular Ca2+ stimulated the external Na-dependent Ca2+ efflux along a sigmoid curve (K1/2 around 30 microM); on the other hand the Ca pump had a biphasic response to internal Ca2+: stimulation at low internal Ca2+ (K1/2 between 1 and 10 microM), followed by a decline at internal Ca2+ concentrations higher than 50 microM.     

Regulatory changes in the K+, Cl- and water contents of HeLa cells incubated in an isosmotic high K(+)-medium

HeLa cells had their normal medium replaced by an isosmotic medium containing 80 mM K+, 70 mM Na+ and 100 microM ouabain. The cellular contents of K+ first increased and then decreased to the original values, that is, the cells showed a regulatory decrease (RVD) in size. The initial increase was not inhibited by various agents except by substitution of medium Cl- with gluconate. In contrast, the regulatory decrease was inhibited strongly by addition of either 1 mM quinine, 10 microM BAPTA-AM without medium Ca2+, or 0.5 mM DIDS, and partly by either 1 mM EGTA without medium Ca2+, 10 microM trifluoperazine, or substitution of medium Cl- with NO3-. Addition of DIDS to the NO3(-)-substituted medium further suppressed the K+ loss but the effect was incomplete. Intracellular Ca2+ showed a transient increase after the medium replacement. These results suggest that the initial increase in cell K+ is a phenomenon related to osmotic water movement toward Donnan equilibrium, whereas the regulatory K+ decrease is caused by K+ efflux through Ca(2+)-dependent K+ channels. The K+ decrease induced a decrease in cellular water, i.e., RVD. The K+ efflux may be more selectively associated with Cl- efflux through DIDS-sensitive channels than the efflux of other anions.     

Effects of Lead Ions on Events Associated with Exocytosis in Isolated Bovine Adrenal Medullary Cells

Lead buffers (citrate and Tiron) were used to investigate the effects of low concentrations (0.1-6 microM) of Pb2+ on stimulus-secretion coupling in isolated bovine chromaffin cells. Nicotinic agonists and high K elicit secretion by enhancing Ca2+ influx into chromaffin cells. Pb2+ inhibited the catecholamine secretion in response to 500 microM carbachol and 77 mM K+ depolarization but was without significant effect on basal secretion. Pb2+ also inhibited the influx of 45Ca occurring in response to these agents. The K0.5 values for inhibition suggest that the carbachol-evoked flux is more sensitive to Pb2+ than influx in response to a direct depolarization. When extracellular calcium was lowered in the absence of Pb2+, both secretion and 45Ca entry were reduced. The effects of Pb2+ were comparable to those of lowered Ca2+. 22Na influx through nicotinic receptor-mediated channels, measured in the presence of tetrodotoxin (2 microM) and ouabain (50 microM), was inhibited by Pb2+. The results suggest that Pb2+ inhibits exocytotic catecholamine secretion by inhibiting Ca2+ influx. The differential sensitivity to Pb2+ of K- and carbachol-evoked 45Ca flux, coupled with the 22Na measurements, indicates that Pb2+ inhibits the movement of ions through acetylcholine-induced channels as well as through voltage-sensitive calcium channels.     

Calcium regulates cAMP-induced potassium ion efflux in Dictyostelium discoideum

The chemoattractant cAMP elicits a transient efflux of K+ in cell suspensions of Dictyostelium discoideum. This cellular response displayed half-maximal activity at about 1 microM cAMP and saturated at 100 microM cAMP, cAMP-stimulated K+ efflux, measured with a K+-sensitive electrode, depended on the extracellular free Ca2+ concentration ([Ca2+]0) and was maximal in the presence of EGTA. Usually more than 90% of the K+ release could be inhibited by the addition of Ca2+. Half-maximal reduction occurred at about 2 microM [Ca2+]0. Inhibition was also observed in the presence of caffeine or A23187, drugs known to elevate the intracellular free Ca2+ concentration ([Ca2+]i). Under conditions where [Ca2+]0 was maintained at a low level, half-maximal inhibition was 1 mM for caffeine and 3 microM for A23187. These results indicate that Cai2+ is involved in the regulation of K+ efflux. Simultaneous measurements of Ca2+ uptake and K+ efflux induced by cAMP as well as free running oscillations of both ions revealed that initiation and termination of Ca2+ uptake slightly preceded those of K+ efflux.     

The Suppression of Stimulus-Evoked Release of Amino Acid Neurotransmitters from Synaptosomes by Verapamil

Verapamil at 200 microM, prevented the respiratory stimulation, K+ loss, transmitter release, and 45Ca2+ entry into incubated synaptosomes evoked by veratrine (25 to 75 microM) or by high K+ (56 mM). Verapamil (100 microM) also blocked gamma-aminobutyric acid homoexchange, whilst tetrodotoxin was ineffective. Much lower concentrations of verapamil (less than 1 microM) blocked the 45Ca2+ entry caused by veratrine, but not its action in releasing neurotransmitter or K+. It is concluded that verapamil, at 30 to 200 microM, blocks active Na+ channels, thereby preventing depolarization. At greater than 1 microM, verapamil blocks Ca+ channels selectively.     

The effect of ferricyanide with iodoacetate in calcium-free solution on passive cation permeability in human red blood cells: comparison with the Gardos-effect and with the influence of PCMBS on passive cation permeability

Freshly prepared human red blood cells incubated with 5 mM ferricyanide, 0.2 mM iodoacetate and 2 mM adenosine in the presence of 5 mM EGTA demonstrate comparable increases in Na+ and K+ permeability (ferricyanide effect). This effect is unrelated to the Ca2+-activated K+ channel (Gardos effect) since influx of Ca2+ from outside the cell is excluded. Also this effect is different from the non-specific Na+ and K+ permeability change elicited by PCMBS. These differences become obvious by using various reagents. For example, A23187 and quinidine exert opposite effects in Gardos and ferricyanide experiments, where A23187 and atebrin react oppositely in the latter and in PCMBS experiments. The ferricyanide effect described here does not involve formation of nonspecific channels. The change in Na+ permeability separately from K+ permeability under certain circumstances suggests a more specific effect.     

Volume regulation by human lymphocytes. Role of calcium

Human peripheral blood lymphocytes regulate their volumes in hypotonic solutions. In hypotonic media in which Na+ is the predominant cation, an initial swelling phase is followed by a regulatory volume decrease (RVD) associated with a net loss of cellular K+. In media in which K+ is the predominant cation, the rapid initial swelling is followed by a slower second swelling phase. 86Rb+ fluxes increased during RVD and returned to normal when the original volume was approximately regained. Effects similar to those induced by hypotonic stress could also be produced by raising the intracellular Ca++ level. In isotonic, Ca++- containing media cells were found to shrink upon addition of the Ca++ ionophore A23187 in K+-free media, but to swell in K+-rich media. Exposure to Ca++ plus A23187 also increased 86Rb+ fluxes. Quinine (75 microM), an inhibitor of the Ca++-activated K+ pathway in other systems blocked RVD, the associated K+ loss, and the increase in 86Rb+ efflux. Quinine also inhibited the volume changes and the increased 86Rb fluxes induced by Ca++ plus ionophore. The calmodulin inhibitors trifluoperazine, pimozide and chlorpromazine blocked RVD as well as Ca++ plus A23187-induced volume changes. Trifluoperazine also prevented the increase in 86Rb+ fluxes and K+ loss induced by hypotonicity. Chlorpromazine sulfoxide, a relatively ineffective calmodulin antagonist, was considerably less potent as an inhibitor of RVD than chlorpromazine. It is suggested than an elevation in cytoplasmic [Ca++], triggered by cell swelling, increases the plasma membrane permeability to K+, the ensuing increased efflux of K+, associated anions, and osmotically obliged water, leading to cell shrinking (RVD).     

Distinct metal ion binding sites on Ca(2+)-activated K+ channels in inside-out patches of human erythrocytes.

Effects of Cd2+, Co2+, Pb2+, Fe2+ and Mg2+ (1-100 microM) on single-channel properties of the intermediate conductance Ca(2+)-activated K+ (CaK) channels were investigated in inside-out patches of human erythrocytes in a physiological K+ gradient. Cd2+, Co2+ and Pb2+, but not Fe2+ and Mg2+, were able to induce CaK channel openings. The potency of the metals to open CaK channels in human erythrocytes follows the sequence Pb2+, Cd2+ > Ca2+ > or = Co2+ > Mg2+, Fe2+. At higher concentrations Pb2+, Cd2+ and Co2+ block the CaK channel by reducing the opening frequency and the single-channel current amplitude. The potency of the metals to reduce CaK channel opening frequency follows the sequence Pb2+ > Cd2+, Co2+ > Ca2+, which differs from the potency sequence Cd2+ > Pb2+, Co2+ > Ca2+ to reduce the unitary single-channel current amplitude. Fe2+ reduced the channel opening frequency and enhanced the two open times of CaK channels activated by Ca2+, whereas up to 100 microM Mg2+ had no effect on any of the measured single-channel parameters. It is concluded that the activation of CaK channels of human erythrocytes by various metal ions occurs through an interaction with the same regulatory site at which Ca2+ activates these channels. The different potency orders for the activating and blocking effects suggest the presence of at least one activation and two blocking sites. A modulatory binding site for Fe2+ exists as well. In addition, the CaK channels in human erythrocytes are distinct from other subtypes of Ca(2+)-activated K+ channels in their sensitivity to the metal ions.     

Modulation of the Ca2+- or Pb2+-activated K+-selective channels in human red cells. I. Effects of propranolol

To study the effect of propranolol on the Ca2+- or Pb2+-activated K+ permeability in human erythrocytes, K+ effluxes were compared with single-channel currents. The results demonstrate that propranolol has a twofold effect: (1) it renders the channel protein more sensitive to Ca2+ or Pb2+; and (2) it simultaneously inhibits channel activity and slightly reduces single-channel conductance. The number of active channels is not affected.     

Calcium-activated potassium channels in chondrocytes.

The presence of calcium-activated potassium channels in chondrocytes of growing cartilage was tested. Results obtained with fura-2 on cultured resting chondrocytes indicate that the cells respond to an elevation of extracellular calcium concentration ([Ca2+]o) from 0.1 to 2 mM increasing the intracellular concentration of the ion ([Ca2+]i) from 117 to 187 nM. This increment may be blocked by 3 microM La3+. Patch clamp experiments in cell-attached configuration showed that, when [Ca2+]i rises, the open probability (Po) of the K+ channels increases. Increments in both Po and unitary currents of the K+ channels can be obtained after applying 2.5 microM A23187 with 2 mM [Ca2+]o. Hence, the results demonstrate that, in chondrocytes, a class of Ca(2+)-activated K+ channels is present and their activity is related to an increase of [Ca2+]i.     

Ca-induced K transport in human red blood cell ghosts containing arsenazo III. Transmembrane interactions of Na, K, and Ca and the relationship to the functioning Na-K pump

Increasing free intracellular Ca (Cai) from less than 0.1 microM to 10 microM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Cai. Ca-stimulated K transport was activated 50% at 2-3 microM free Cai and the Na-K pump was inhibited 50% by 5-10 microM free Cai. Free Cai from 1 to 8 microM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect of Ca on the two systems. 3 microM trifluoperazine inhibited Ca-stimulated K efflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 microM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 microM initiated a homogeneous loss of K. In ATP-deficient ghosts containing Cai equilibrated with A23187, K transport was activated at the same free Cai as in the ghosts containing 2 mM ATP. Neither Cao nor the presence of an inward Ca gradient altered the effect of free Cai on the permeability to K. In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na- and K-induced changes in free Cai or sensitivity to Cai. At constant free Cai, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Cai markedly decreased the rate of efflux at 2 mM Ko, but had no effect when Ko was greater than or equal to 20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Cai must be at a site that is independent of those responsible for the interaction of Na and K. In the presence of A23187, this activating site is half-maximally stimulated by approximately 2 microM free Ca and is not influenced by the concentration of ATP. The partial inhibition of Ca-stimulated K efflux by trifluoperazine in ghosts containing ATP suggests that calmodulin could be involved in the activation of K transport by Cai.(ABSTRACT TRUNCATED AT 400 WORDS)     

Extracellular Ca2+ ameliorates NaCl-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+ -permeable channels

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+ -induced K+ efflux through outwardly directed, K+ -permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+ -sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.     

K(Ca) channel blockers reveal hyperpolarization and relaxation to K+ in rat isolated mesenteric artery

Raising extracellular K+ concentration ([K+](o)) around mesenteric resistance arteries reverses depolarization and contraction to phenylephrine. As smooth muscle depolarizes and intracellular Ca(2+) and tension increase, this effect of K+ is suppressed, whereas efflux of cellular K+ through Ca(2+)-activated K+ (K(Ca)) channels is increased. We investigated whether K+ efflux through K(Ca) suppresses the action of exogenous K+ and whether it prestimulates smooth muscle Na(+)-K(+)-ATPase. Under isometric conditions, 10.8 mM [K+](o) had no effect on arteries contracted >10 mN, unless 100 nM iberiotoxin (IbTX), 100 nM charybdotoxin (ChTX), and/or 50 nM apamin were present. Simultaneous measurements of membrane potential and tension showed that phenylephrine depolarized and contracted arteries to -32.2 +/- 2.3 mV and 13.8 +/- 1.6 mN (n = 5) after blockade of K(Ca), but 10.8 mM K+ reversed fully the responses (107.6 +/- 8.6 and 98.8 +/- 0.6%, respectively). Under isobaric conditions and preconstriction with phenylephrine, 10.7 mM [K+](o) reversed contraction at both 50 mmHg (77.0 +/- 8.5%, n = 9) and 80 mmHg (83.7 +/- 5.5%, n = 5). However, in four additional vessels at 80 mmHg, raising K+ failed to reverse contraction unless ChTX was present. Increases in isometric and decreases in isobaric tension with phenylephrine were augmented by either ChTX or ouabain (100 microM), whereas neither inhibitor altered tension under resting conditions. Inhibition of cellular K+ efflux facilitates hyperpolarization and relaxation to exogenous K+, possibly by indirectly reducing the background activation of Na(+)-K(+)-ATPase.     

Effect of cyclic nucleotides on calcium dependent K+ channels, in human erythrocytes

K+ efflux has been analyzed in human erythrocytes incubated in a K+ free medium containing ouabain, bumetanide, CaCl2, and the Ca2+ ionophore A23187. In these conditions, a K+ efflux, which is exponentially dependent on the concentration of A23187 present in the medium, has been observed. This flux is almost completely abolished by either quinine or EGTA, so that, the above K+ efflux has been considered Ca2+ dependent. The effects of cAMP, and cGMP, have been tested on this flux. Ca2+ dependent K+ efflux decreases in presence of millimolar concentrations of cAMP in the medium. The addition of methyl-isobutyl-xanthine to the incubation medium containing cAMP enhances the inhibitory effect of this compound. cGMP also inhibits the Ca2+ dependent K+ efflux. Our results suggest that cyclic nucleotides may modulate the activation of Ca2+ dependent K+ channels in human erythrocytes.     

Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line

Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl- cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4-aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187-stimulated uptake and efflux were consistent with a channel-rather than a carrier-mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone-activated electrogenic Cl- secretion.     

Rat myometrial Na/K ATPase is increased by serum but not by isoproterenol and relaxin

1. Na/K ATPase activity in rat myometrial cells in culture exhibited a Kapp of 0.93 mM for Rb+ and a Ki of 31 microM for ouabain with respect to Rb+. 2. 86Rb+ uptake was stimulated by serum and monensin but was not affected by the uterine relaxants isoproterenol and relaxin in 0.5-7.5 mM Rb+. Nonetheless, these relaxants elicited significant increases in 45Ca2+ efflux under similar conditions. 3. These data suggest that increased Na/Ca exchange resulting from a stimulation of Na/K ATPase is not involved in the mechanism of action of relaxin and isoproterenol in the uterus.     

Na+-dependent regulation of extramitochondrial Ca2+ by rat-liver mitochondria

The presence and significance of Na+-induced Ca2+ release from rat liver mitochondria was investigated by the arsenazo technique. Under the experimental conditions used, the mitochondria, as expected, avidly extracted Ca2+ from the medium. However, when the uptake pathway was blocked with ruthenium red, only a small rate of 'basal' release of Ca2+ was seen (0.3 nmol Ca2+ X min-1 X mg-1), in marked contrast to earlier reports on a rapid loss of sequestered Ca2+ from rat liver mitochondria. The addition of Na+ in 'cytosolic' levels (20 mM) led to an increase in the release rate by about 1 nmol Ca2+ X min-1 X mg-1. This effect was specific for Na+. The significance of this Na+-induced Ca2+ release, in relation to the Ca2+ uptake mechanism, was investigated (in the absence of uptake inhibitors) by following the change in the extramitochondrial Ca2+ steady-state level (set point) induced by Na+. A five-fold increase in this level, from less than 0.2 microM to more than 1 microM, was induced by less than 20 mM Na+. The presence of K+ increased the sensitivity of the Ca2+ homeostat to Na+. The effect of Na+ on the extramitochondrial level was equally well observed in an K+/organic-anion buffer as in a sucrose buffer. Liver mitochondria incubated under these circumstances actively counteracted a Ca2+ or EGTA challenge by taking up or releasing Ca2+, so that the initial level, as well as the Na+-controlled level, was regained. It was concluded that liver mitochondria should be considered Na+-sensitive, that the capacity of the Na+-induced efflux pathway was of sufficient magnitude to enable it to influence the extramitochondrial Ca2+ level biochemically and probably also physiologically, and that the mitochondria have the potential to act as active, Na+-dependent regulators of extramitochondrial ('cytosolic') Ca2+. It is suggested that changes of cytosolic Na+ could be a mediator between certain hormonal signals (notably alpha 1-adrenergic) and changes in this extramitochondrial ('cytosolic') Ca2+ steady state level.     

Modulation of depolarization stimulated 45Ca2+ uptake in cultured neuronal cells by calcium channel activators and antagonists

The effect of dihydropyridine calcium agonists and antagonists on 45Ca2+ uptake into primary neuronal cell cultures was investigated. K+ stimulated neuronal 45Ca2+ accumulation in a concentration dependent manner. This effect was further enhanced by the calcium agonists Bay K 8644 and (+)-(S)-202-791 with EC50 values of 21 nM and 67 nM respectively. The calcium antagonists PN 200-110 and (-)-(R)-202-791 inhibited Bay K 8644 (1 microM) stimulated uptake with IC50 values of 20 nM and 130 nM respectively. 45Ca2+ efflux from neuronal cells was measured in the presence and absence of Na+. Efflux occurred at a much greater rate from cells incubated in the presence of Na+, indicating the existence of an active Na+/Ca2+ exchanger in these neurons. The data suggests that voltage sensitive calcium channels of these neurons are sensitive to dihydropyridines and thus that dihydropyridine binding sites have a functional role in these neuronal cultures.