Activation of Ca2+ influx by metabolic substrates in Saccharomyces cerevisiae: role of membrane potential and cellular ATP levels

Influx of Ca2+ into cells of Saccharomyces cerevisiae was measured under non-steady-state conditions, which enable measurements of the initial rate of transport across plasma membranes without interference by the vacuolar Ca2+ transport system. Removal of glucose from the incubation medium led to inactivation of Ca2+ influx within 5 min. Readdition of glucose led to a transient increase in the rate of Ca2+ transport, reaching a peak after 3-5 min. A second increase was observed 60-80 min later. To examine whether the first transient activation of Ca2+ influx by glucose was mediated by membrane hyperpolarization, influx of 45Ca2+ was measured in the presence and absence of metabolic substrates (glucose, glycerol, and glucose plus antimycin A) in cells hyperpolarized to different values of membrane potential (delta psi). Logarithms of the rate of Ca2+ influx were plotted against values of delta psi. Two different slopes were obtained, depending upon whether the metabolic substrate was present or absent. Ca2+ influx in the presence of the metabolic substrates was always higher than expected by their effect on delta psi. Glycerol plus antimycin A did not affect Ca2+ influx. It was concluded that metabolized substrates activate Ca2+ influx not only by effects on delta psi but also by additional mechanism(s). Since no simple correlation between Ca2+ influx and intracellular ATP levels was observed, it was concluded that ATP levels do not affect the initial rates of Ca2+ transport across the plasma membrane of S. cerevisiae.     

Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans

The characteristics of the uptake of lipophilic cations tetraphenylphosphonium (TPP+) into Candida albicans have been investigated to establish whether TPP+ can be used as a membrane potential probe for this yeast. A membrane potential (delta psi, negative inside) across the plasma membrane of C. albicans was indicated by the intracellular accumulation of TPP+. The steady-state distribution of TPP+ was reached within 60 min and varied according to the expected changes of delta psi. Agents known to depolarize membrane potential caused a rapid and complete efflux of accumulated TPP+. The initial influx of TPP+ was linear over a wide range of TPP+ concentrations (2.5-600 microM), indicating a non mediated uptake. Thus, TPP+ is a suitable delta psi probe for this yeast.     

Regulation of Ca2+ efflux in rat liver mitochondria. Role of membrane potential

The paper analyzes the relationship between membrane potential (delta psi), steady state pCao (-log [Ca2+] in the outer aqueous phase) and rate of ruthenium-red-induced Ca2+ efflux in liver mitochondria. Energized liver mitochondria maintain a pCao of about 6.0 in the presence of 1.5 mM Mg2+ and 0.5 mM Pi. A slight depression of delta psi results in net Ca2+ uptake leading to an increased steady state pCao. On the other hand, a more marked depression of delta psi results in net Ca2+ efflux, leading to a decreased steady-state pCao. These results reflect a biphasic relationship between delta psi and pCao, in that pCao increases with the increase of delta psi up to a value of about 130 mV, whereas a further increase of delta psi above 130 mV results in a decrease of pCao. The phenomenon of Ca2+ uptake following a depression of delta psi is independent of the tool used to affect delta psi whether by inward K+ current via valinomycin, or by inward H+ current through protonophores or through F1-ATP synthase, or by restriction of e- flow. The pathway for Ca2+ efflux is considerably activated by stretching of the inner membrane in hypotonic media. This activation is accompanied by a decreased pCao at steady state and by an increased rate of ruthenium-red-induced Ca2+ efflux. By restricting the rate of e- flow in hypotonically treated mitochondria, a marked dependence of the rate of ruthenium-red-induced Ca2+ efflux on the value of delta psi is observed, in that the rate of Ca2+ efflux increases with the value of delta psi. The pCao is linearly related to the rate of Ca2+ efflux. Activation of oxidative phosphorylation via addition of hexokinase + glucose to ATP-supplemented mitochondria, is followed by a phase of Ca2+ uptake, which is reversed by atractyloside. These findings support the view that Ca2+ efflux in steady state mitochondria occurs through an independent, delta psi-controlled pathway and that changes of delta psi during oxidative phosphorylation can effectively modulate mitochondrial Ca2+ distribution by inhibiting or activating the delta psi-controlled Ca2+ efflux pathway.     

Ca2+ transport across the platelet plasma membrane. A role for membrane glycoproteins IIB and IIIA

Human platelets maintain a low cytosolic free Ca2+ concentration in part by controlling plasma membrane Ca2+ transport. The present studies examine the role in this process of two well-characterized membrane proteins: glycoproteins IIb and IIIa. These glycoproteins form a Ca2+-dependent complex which serves as both the platelet fibrinogen receptor and the principle site for high affinity Ca2+ binding on the platelet surface. The kinetics of plasma membrane Ca2+ exchange were compared in normal platelets and in thrombasthenic platelets, which lack the IIb X IIIa complex. Under steady-state conditions, the maximum rate of plasma membrane Ca2+ exchange in the thrombasthenic platelets was half the rate observed in normal platelets. The size of the cytosolic exchangeable Ca2+ pool and the cytosolic free Ca2+ concentration, however, were normal. A quantitatively similar decrease in plasma membrane Ca2+ exchange was seen in normal platelets after incubation with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 37 degrees C, conditions that dissociate the IIb X IIIa complex. This decrease in the Ca2+ exchange rate in normal platelets could be prevented by preincubating platelets with a complex-specific anti-IIb X IIIa monoclonal antibody, but not by preincubating platelets with an anti-IIIa monoclonal antibody. In order to determine whether loss of the IIb X IIIa complex primarily affects Ca2+ influx or Ca2+ efflux, both processes were also examined under nonsteady-state conditions. An immediate decrease in the 45Ca2+ influx rate was seen when Ca2+ was added back to platelets preincubated with EGTA at 37 degrees C. The 45Ca2+ efflux rate, on the other hand, was not immediately affected. These data suggest, therefore, that an intact IIb X IIIa complex is necessary for normal Ca2+ homeostasis in platelets.     

Tetraphenylphosphonium ion is a true indicator of negative plasma-membrane potential in the yeast Rhodotorula glutinis. Experiments under osmotic stress and at low external pH values.

In the yeast Rhodotorula glutinis, accumulation of the tetraphenylphosphonium ion (TPP+) was increased under conditions of osmotic stress, indicating a hyperpolarization of the negative membrane potential (delta psi). The following observations were consistent with the occurrence of hyperpolarized delta psi: enhanced accumulation of glucosamine, the uptake of which is also driven by delta psi; increased respiratory rate. The accumulation of TPP+ was gradually decreased by lowering the pH of cell suspensions. At pH values below 4.5, no TPP+ was taken up, but instead thiocyanate (SCN-) was accumulated, indicating a positive delta psi. The pH-dependent influx of glucosamine followed the pattern of TPP+ accumulation both in the wild-type and in the nystatin-resistant mutant, M67, which displayed a negative delta psi down to pH 3. Thus TPP+ accumulation in Rh. glutinis reflected actual electrical potential difference across the plasma membrane.     

Delta9-tetrahydrocannabinol induces apoptosis in human prostate PC-3 cells via a receptor-independent mechanism

During hypoxia of isolated cardiomyocytes, Ca2+ entry into mitochondria may occur via the Na/Ca exchanger, the normal efflux pathway, and not the Ca-uniporter, the normal influx route. If this is the case, then depletion of myocyte Na+ should inhibit Ca2+ uptake, and collapse of the mitochondrial membrane potential (delta psi(m)) would inhibit the uniporter. To test these hypotheses, isolated rat myocytes were exposed to metabolic inhibition, to mimic hypoxia, and [Ca2+]m and [Ca2+]c determined by selective loading of indo-1 into these compartments. Delta psi(m) was determined using rhodamine 123. Following metabolic inhibition, [Ca2+]m was significantly lower in Na-depleted cells than controls (P<0.001), [Ca2+]c was approximately the same in both groups, and mitochondria depolarised completely. Thus Na-depletion inhibited mitochondrial Ca2+ uptake, suggesting that Ca2+ entry occurred via Na/Ca exchange, and the collapse of delta psi(m) during metabolic inhibition is consistent with inactivity of the Ca-uniporter.     

Ammonium Uptake by Rice Roots (III. Electrophysiology)

The transmembrane electrical potential differences ([delta][psi]) were measured in epidermal and cortical cells of intact roots of 3-week-old rice (Oryza sativa L. cv M202) seedlings grown in 2 or 100 [mu]M NH4+ (G2 or G100 plants, respectively). In modified Johnson's nutrient solution containing no nitrogen, [delta][psi] was in the range of -120 to -140 mV. Introducing NH4+ to the bathing medium caused a rapid depolarization. At the steady state, average [delta][psi] of G2 and G100 plants were -116 and -89 mV, respectively. This depolarization exhibited a biphasic response to external NH4+ concentration similar to that reported for 13NH4+ influx isotherms (M.Y. Wang, M.Y. Siddiqi, T.J. Ruth, A.D.M. Glass [1993] Plant Physiol 103: 1259-1267). Plots of membrane depolarization versus 13NH4+ influx were also biphasic, indicating distinct coupling processes for the two transport systems, with a breakpoint between two concentration ranges around 1 mM NH4+. The extent of depolarization was also influenced by nitrogen status, which was larger for G2 plants than for G100 plants. Depolarization of [delta][psi] due to NH4+ uptake was eliminated by a protonophore (carboxylcyanide-m-chlorophenylhydrazone), inhibitors of ATP synthesis (sodium cyanide plus salicylhydroxamic acid), or an ATPase inhibitor (diethylstilbestrol). The results of these observations are discussed in the context of the mechanisms of NH4+ uptake by high- and low-affinity transport systems operating across the plasma membranes of root cells.     

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.     

NMR studies on Na+ transport in Synechococcus PCC 6311.

The freshwater cyanobacterium Synechococcus PCC 6311 is able to adapt to grow after sudden exposure to salt (NaCl) stress. We have investigated the mechanism of Na+ transport in these cells during adaptation to high salinity. Na+ influx under dark aerobic conditions occurred independently of delta pH or delta psi across the cytoplasmic membrane, ATPase activity, and respiratory electron transport. These findings are consistent with the existence of Na+/monovalent anion cotransport or simultaneous Na+/H+ +anion/OH- exchange. Na+ influx was dependent on Cl-, Br-, NO3-, or NO2-. No Na+ uptake occurred after addition of NaI, NaHCO3, or Na2SO4. Na+ extrusion was absolutely dependent on delta pH and on an ATPase activity and/or on respiratory electron transport. This indicates that Na+ extrusion via Na+/H+ exchange is driven by primary H+ pumps in the cytoplasmic membrane. Cells grown for 4 days in 0.5 m NaCl medium, "salt-grown cells," differ from control cells by a lower maximum velocity of Na+ influx and by lower steady-state ratios of [Na+]in/[Na+]out. These results indicate that cells grown in high-salt medium increase their capacity to extrude Na+. During salt adaptation Na+ extrusion driven by respiratory electron transport increased from about 15 to 50%.     

Changes in membrane potential during calcium ion influx and efflux across the mitochondrial membrane.

1. A depolarisation of the membrane of rat liver mitochondria, as measured with the safranine method, is seen during Ca2+ uptake. The depolarisation is followed by a slow repolarisation, the rate of which can be increased by the addition of EGTA or phosphate. 2. Plots relating the initial rate of calcium ion (Ca2+) uptake and the decrease in membrane potential (delta psi) to the Ca2+ concentration show a half-maximal change at less than 10 micron Ca2+ and a saturation above 20 micron Ca2+. 3. Plots relating the initial rate of Ca2+ uptake to delta psi are linear. 4. Addition of Ca2+ chelators, nitriloacetate or EGTA, to deenergized mitochondria equilibrated with Ca2+ causes a polarisation of the mitochondrial membrane due to a diffusion potential created by electrogenic Ca2+ efflux. 5. If the extent of the response induced by different nitriloacetate concentrations is plotted against the expected membrane potential a linear plot is obtained up to 70 mV with a slope corresponding to two-times the extent of the response induced by valinomycin in the presence of different potassium ion gradients. This suggests that the Ca2+ ion is transferred across the membrane with one net positive charge in present conditions.     

Transport of sodium, potassium, and calcium across rabbit polymorphonuclear leukocyte membranes. Effect of chemotactic factor

The transport properties of the rabbit peritoneal polymorphonuclear leukocyte (PMN) plasma membrane to Na+, K+, and Ca2+ have been characterized. The use of a silicone oil centrifugation technique provided a rapid and reliable method for measuring ion fluxes in these cells. Na+ and K+ movements across PMN membranes were found to be rapid. The value for the unifirectional steady-state fluxes (in meq/liter cell X min) were of the order of 3.0 for Na+ and 7.4 for K+. Ouabian inhibited both K+ influx and Na+ efflux, the latter being also dependent on the presence of extracellular potassium. The rate constant (in min-1) for 45Ca influx was found to be .05 and that for 45Ca efflux .04. The synthetic chemotactic factor formyl-methionyl-leucyl-phenylalanine (FMLP) was found to affect the fluxes of Na+, K+, and Ca2+ at concentrations as low as 10(-10)M. FMLP induced a large and rapid increase in the permeability of the PMN plasma membrane to 22Na. Smaller and delayed enhancements of 42K influx and 22Na efflux were also noted. Some evidence that the latter findings are a consequence of the increased 22Na influx is presented. 45Ca influx and efflux were also stimulated by FMLP. In the presence of 0.25 mM extracellular calcium, FMLP induced an increase in the steady-state level of cell-associated 45Ca. In the presence of .01 mM extracellular calcium, however, a transient decrease in the steady-state level of cell-associated 45Ca was induced by FMLP. The curves relating the concentration of FMLP to its effects on cation fluxes are very similar to those found for its enhancement of migration.     

Mechanism of Na+ deprivation-induced catecholamine secretion from freshly isolated bovine adrenal chromaffin cells

We have studied the mechanism of Na+ deprivation-induced catecholamine secretion from freshly isolated bovine adrenal chromaffin cells. Na+ deprivation-induced catecholamine secretion depended on free extracellular Ca2+ concentrations and was almost parallel to 45Ca2+ influx into the cells under various experimental conditions. Furthermore, Na+ deprivation-induced 45Ca2+ influx and catecholamine secretion were actually induced by a relative Na+ concentration gradient across the plasma membrane, but not by simple omission of Na+ from the medium. These results indicate that the deprivation of Na+ from the medium changes the relative Na+ gradient across the plasma membrane and results in Ca2+ influx via a reverse mode of Na(+)-Ca(2+) exchange rather than by inducing Ca2+ entry through Ca2+ channels by eliminating the competition between extracellular Na+ and Ca2+.     

Commitment to differentiation of murine erythroleukemia cells involves a modulated plasma membrane depolarization through Ca2+-activated K+ channels

The role of the plasma membrane potential (delta psi p) in the commitment to differentiation of murine erythroleukemia (MEL) cells has been studied by analyzing the ionic basis and the time course of this potential in the absence or the presence of different types of inducers. delta psi p was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane and displayed a 22-hour depolarization phase (from -28 to +5 mV) triggered by factors contained in foetal calf serum (FCS) and followed by a nearly symmetrical repolarization phase. After measuring the electrochemical equilibrium potential of Na+, K+, and Cl-, the relative contribution of these ions to delta psi p was evaluated by means of ion substitution experiments and by the addition of ion flux inhibitors (tetrodotoxin [TTX], 4-acetoamide-4'-isothiocyanostilbene-2,2'-disulfonate [SITS]) and ionophores (Valinomycin, A23187). The Na+ contribution to delta psi p appeared negligible, the potential being essentially generated by K+ and Cl- fluxes. When evaluated by a new mathematical approach, the effects of Valinomycin and A23187 at different times of incubation provided evidence that both the depolarization and the repolarization phase were due to variations of the K+ permeability across the plasma membrane (PK) mediated by Ca2+-activated K+ channels. All the inducers tested (dimethylsulfoxide [DMSO], hexamethylen-bis-acetamide [HMBA], diazepam), although they did not modify the ionic basis of delta psi p, strongly attenuated the depolarization rate of this potential. This attenuation was not brought about when the inducers were added to noninducible MEL cell clonal sublines. Cell commitment occurred only during the depolarization phase and increased proportionally to the attenuation of this phase up to a threshold beyond which the further increase of the attenuation was associated with the inhibition of commitment. The major role of the inducers apparently consisted of the stabilization of the Ca2+-activated K+ channels, suggesting that a properly modulated delta psi p depolarization through these channels is primarily involved in the signal generation for MEL cell commitment to differentiation.     

Effect of extracellular Ca2+ on plasma membrane Ca2+ inflow and cytoplasmic free Ca2+ in isolated hepatocytes

An initial rapid phase and a subsequent slow phase of 45Ca2+ uptake were observed following the addition of 45Ca2+ to Ca2+-deprived hepatocytes. The magnitude of the rapid phase increased 15-fold over the range 0.1-11 mM extracellular Ca2+ (Ca2+o) and was a linear function of [Ca2+]o. The increases in the rate of 45Ca2+ uptake were accompanied by only small increases in the intracellular free Ca2+ concentration. In cells made permeable to Ca2+ by treatment with saponin, the rate of 45Ca2+ uptake (measured at free Ca2+ concentrations equal to those in the cytoplasm of intact cells) increased as the concentration of saponin increased from 1.4 to 2.5 micrograms per mg wet weight cells. Rates of 45Ca2+ uptake by cells permeabilized with an optimal concentration of saponin were comparable with those of intact cells incubated at physiological [Ca2+o], but were substantially lower than those for intact cells incubated at high [Ca2+o]. It is concluded that Ca2+ which enters the hepatocyte across the plasma membrane is rapidly removed by binding and transport to intracellular sites and by the plasma membrane (Ca2+ + Mg2+)-ATPase and the plasma membrane Ca2+ inflow transporter is not readily saturated with Ca2+o.     

The relationship between extramitochondrial Ca2+ concentration, respiratory rate, and membrane potential in mitochondria from brown adipose tissue of the rat

The ability of isolated mitochondria from rat brown-adipose tissue to regulate extramitochondrial Ca2+ (measured by arsenazo) was studied in relation to their ability to produce heat (measured polarographically). The energetic state of the mitochondria was expressed as a membrane potential, delta psi (estimated with safranine), and was varied semi-physiologically by the use of different GDP concentrations. In these mitochondria GDP binds to the 32-kDa polypeptide, thermogenin, which regulates coupling. Ca2+ uptake (at 5 microM extramitochondrial Ca2+) was maximal at delta psi greater than 150 mV. Basal Ca2+ release increased from 1 to 2 nmol x min-1 x mg-1 below 150 mV. Na+ -stimulated rate of Ca2+ release was stable within the investigated delta psi span (100-160 mV). Initial Ca2+ levels were maintained below 0.2 microM for 100 mV less than delta psi less than 160 mV. Ca2+ levels maintained after Ca2+ challenge (20 nmol Ca2+ x mg-1) were below 0.4 microM for delta psi greater than 135 mM. Respiration was unstimulated for delta psi greater than 150 mV and was maximal at delta psi less than or equal to 135 mV. In the presence of well-oxidised substrates, the respiration at maximally activated thermogenin was markedly below fully uncoupled respiration and was probably limited by thermogenin activity--i.e. by a limited H+ reentry (OH- exit) and therefore by a membrane potential maintained at about 135 mV. It is concluded that at membrane potentials of 135 mV and above the mitochondria exhibit full Ca2+ control and are able to regulate thermogenic output up to maximum without interfering with this Ca2+ control. Membrane potential probably does not decrease below 135 mV in vivo. Therefore, Ca2+ homeostasis and thermogenesis are non-interfering and can be hormonally independently regulated, e.g. by alpha-adrenergic and beta-adrenergic stimuli, respectively.     

Parathyroid hormone secretion in the absence of extracellular free Ca2+ and transmembrane Ca2+ influx

The involvement of extracellular Ca2+ and Ca2+ influx across the plasma membrane in parathyroid hormone (PTH) secretion was investigated in vitro using a new preparation of bovine parathyroid cells. Incubation of these cells in the presence of 25 microM or 2.5 microM free ambient Ca2+ induced a maximal rate of PTH secretion. Low free Ca2+ secretion is not associated with changes in membrane permeability, requires metabolic energy, and is reversible. The Ca2+ channel blocker D600 had no effect on either 45Ca-influx or PTH secretion in these cells. These results, showing that extracellular Ca2+ and Ca2+ influx across the plasma membrane are not required for PTH secretion by parathyroid cells, emphasize the differences in the cellular mechanisms underlying the secretion of PTH vs that of other secretory cells.     

Coupling of Li+ distribution to the plasma membrane potential of rat cortical synaptosomes

The effect of the plasma membrane potential delta psi p on the transport rate and steady state distribution of Li+ was assessed in rat cortical synaptosomes. Up to 15 mM Li+ failed to saturate Li+ influx into polarized synaptosomes in a Na+-based medium with 3 mM external K+. Veratridine increased and tetrodotoxin, ouabain, or high external K+ decreased the rate of Li+ influx. At steady state, Li+ was concentrated about 3-fold in resting synaptosomes at 0.3 to 1 mM Li+ externally. Subsequent depolarization of the plasma membrane by veratridine or high external K+ induced an immediate release of Li+. When graded depolarizations were imposed onto the plasma membrane by varying concentrations of ouabain, veratridine, or external K+, steady state distribution of Li+ was linearly related with K+ distribution or electrochemical activity coefficients. It was concluded that uptake rate and steady state distribution of Li+ depend significantly on delta psi p. However, Li+ gradients were lower than predicted from delta psi p, suggesting that (secondary) active transport systems counteracted passive equilibration by uphill extrusion of Li+. The electrochemical potential difference delta mu Li+ maintained at a delta psi p of -72 mV was calculated to 4.2 kJ/mol of Li+. At physiological external K+, Li+ was not actively transported by the sodium pump. The ouabain sensitivity resulted from the coupling of Li+ uptake to the pump-dependent K+ diffusion potential. In low K+ and K+-free media, however, active transport of Li+ by the sodium pump contributed to total uptake. In the absence of K+, Li+ substituted for K+ in generating a delta psi p of -64 mV maximally, as calculated from TPMP+ distribution at 40 mM external Li+. Since Li+ gradients were far too low to account for a diffusion potential, it was assumed that Li+ gave rise to an electrogenic pump potential.     

Active extrusion of potassium in the yeast Saccharomyces cerevisiae induced by low concentrations of trifluoperazine

Trifluoperazine (TFP), the antipsychotic drug, induces substantial K+ efflux, membrane hyperpolarization and inhibition of H+-ATPase in the yeast Saccharomyces cerevisiae. Investigations on the mechanism of these effects revealed two different processes observed at different incubation conditions. At an acidic pH of 4.5 and an alkaline pH of 7.5, K+ efflux was accompanied by substantial proton influx which led to intracellular acidification and dissipation of delta psi formed by cation efflux. The results indicated nonspecific changes in membrane permeability. Similar results were also observed when cells were incubated at pH 5.5-6.0 with higher concentrations of TFP (above 75 microM). On the other hand, low concentrations of TFP (30-50 microM) at pH 5.5-6.0 caused marked membrane hyperpolarization and K+ efflux unaccompanied by the efflux of other cations and by H+ influx. Our experiments indicate that under these conditions K+ efflux was an active process. (1) K+ efflux proceeded only in the presence of a metabolic substrate and was inhibited by metabolic inhibitors. (2) When 0.3-0.9 mM-KCl was present in the medium at pH 6.0, the concentration of K+ within the cells (measured at the end of the incubation with TFP) was much lower than the theoretical concentration of Kin+ if the distribution of K+ between medium and cell water was at equilibrium (at zero electrochemical gradient). (3) Valinomycin decreased the net K+ efflux and decreased the membrane hyperpolarization induced by TFP, probably by increasing the flux of K+ into the cells along its electrochemical gradient. (4) Conditions which led to active K+ efflux also led to a marked decrease in cellular ATP level. The results indicate that under a specific set of conditions TFP induces translocation of K+ against its electrochemical gradient.     

Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.     

Extracellular Ca²+ alleviates NaCl-induced stomatal opening through a pathway involving H₂O₂-blocked Na+ influx in Vicia guard cells

To gain further insights into the function of extracellular Ca2+ in alleviating salt stress, Vicia faba guard cell protoplasts (GCPs) were patch-clamped in a whole-cell configuration. The results showed that 100 mM NaCl clearly induced Na+ influx across the plasma membrane in GCPs and promoted stomatal opening. Extracellular Ca2+ at 10 mM efficiently blocked Na+ influx and inhibited stomatal opening, which was partially abolished by La3+ (an inhibitor of plasma membrane Ca2+ channel) or catalase (CAT, a H?O? scavenger), respectively. These results suggest that the plasma membrane Ca2+ channels and H?O? possibly mediate extracellular Ca2+-blocked Na+ influx in GCPs. Furthermore, extracellular Ca2+ activated the plasma membrane Ca2+ channels under NaCl stress, which was partially abolished by CAT. These results, taken together, indicate that hydrogen peroxide (H?O?) likely regulates Na+ uptake by activating plasma membrane Ca2+ channels in GCPs. In accordance with this hypothesis, H?O? could mimic extracellular Ca2+ to activate Ca2+ channels and block Na+ influx in guard cells. A single-cell analysis of cytosolic free Ca2+ ([Ca2+](cyt)) using Fluo 3-AM revealed that extracellular Ca2+ induced the accumulation of cytosolic Ca2+ under NaCl stress, but had few effects on the accumulation of cytosolic Ca2+ under non-NaCl conditions. All of these results, together with our previous studies showing that extracellular Ca2+ induced the generation of H?O? in GCPs during NaCl stress, indicate that extracellular Ca2+ alleviates salt stress, likely by activating the H?O?-dependent plasma membrane Ca2+ channels, and the increase in cytosolic Ca2+ appears to block Na+ influx across the plasma membrane in Vicia guard cells, leading to stomatal closure and reduction of water loss.