Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Metabolic Inhibition Strongly Inhibits Na-Dependent Mg Efflux in Rat Ventricular Myocytes

We measured intracellular Mg²⁺ concentration ([Mg²⁺]_i) in rat ventricular myocytes using the fluorescent indicator furaptra (25°C). In normally energized cells loaded with Mg²⁺, the introduction of extracellular Na⁺ induced a rapid decrease in [Mg²⁺]_i: the initial rate of decrease in [Mg²⁺]_i (initial Δ[Mg²⁺]_i/Δt) is thought to represent the rate of Na⁺-dependent Mg²⁺ efflux (putative Na⁺/Mg²⁺ exchange). To determine whether Mg²⁺ efflux depends directly on energy derived from cellular metabolism, in addition to the transmembrane Na⁺ gradient, we estimated the initial Δ[Mg²⁺]_i/Δt after metabolic inhibition. In the absence of extracellular Na⁺ and Ca²⁺, treatment of the cells with 1 μM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, an uncoupler of mitochondria, caused a large increase in [Mg²⁺]_i from ∼0.9 mM to ∼2.5 mM in a period of 5-8 min (probably because of breakdown of MgATP and release of Mg²⁺) and cell shortening to ∼50% of the initial length (probably because of formation of rigor cross-bridges). Similar increases in [Mg²⁺]_i and cell shortening were observed after application of 5 mM potassium cyanide (KCN) (an inhibitor of respiration) for ≥90 min. The initial Δ[Mg²⁺]_i/Δt was diminished, on average, by 90% in carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone-treated cells and 92% in KCN-treated cells. When the cells were treated with 5 mM KCN for shorter times (59-85 min), a significant decrease in the initial Δ[Mg²⁺]_i/Δt (on average by 59%) was observed with only a slight shortening of the cell length. Intracellular Na⁺ concentration ([Na⁺]_i) estimated with a Na⁺ indicator sodium-binding benzofuran isophthalate was, on average, 5.0-10.5 mM during the time required for the initial Δ[Mg²⁺]_i/Δt measurements, which is well below the [Na⁺]_i level for half inhibition of the Mg²⁺ efflux (∼40 mM). Normalization of intracellular pH using 10 μM nigericin, a H⁺ ionophore, did not reverse the inhibition of the Mg²⁺ efflux. From these results, it seems likely that a decrease in ATP below the threshold of rigor cross-bridge formation (∼0.4 mM estimated indirectly in the this study), rather than elevation of [Na⁺]_i or intracellular acidosis, inhibits the Mg²⁺ efflux, suggesting the absolute necessity of ATP for the Na⁺/Mg²⁺ exchange.     

Kinetic characterization of Na+/H+ antiport of Plasmodium falciparum membrane

Intraerythrocytic malaria parasites produce vast amounts of lactic acid through glycolysis. While the egress of lactate is very rapid, the mode of extrusion of H⁺ is not known. The possible involvement of a Na⁺/H⁺ antiport in the extrusion of protons across the plasma membrane of Plasmodium falciparum has been investigated by using the fluorescent pH probe 6-carboxyfluorescein. The resting cytosolic pH was 7.27 ± 0.1 in ring stage parasites and 7.31 ± 0.12 in trophozoites. Spontaneous acidification of parasite cytosol was observed in Na⁺-medium and realkalinization occurred upon addition of Na⁺ to the medium in a concentration-dependent manner, with no apparent saturation. The rate of H⁺-at the ring stage was higher than that at the trophozoite stage due to the larger surface/volume ratio of the young parasite stage. Na⁺-H⁺-was: 1) inhibited by the Na⁺/H⁺ inhibitors amiloride and 5-(N-ethyl-isopropyl) amiloride (EIPA), though at relatively high concentrations; 2) augmented with rising pH₆ (pH_i = 6.2 [Na⁺]_o = 30 mM); and 3) decreased with increasing pH_i (pH_o = 7.4; [Na⁺]_o = 30 mM). The pH_i and the pH_o dependencies of H⁺-were almost identical at all parasite stages. Only at pH_i > 7.6 efflux was totally obliterated. The target of this inhibitory effect is probably other than the antiport. Results indicate that H⁺-is mediated by a Na⁺/H⁺ antiport which is regulated by host and parasite pH and by the host cytosol sodium concentration. The proton transport capacity of the antiport can easily cope with all the protons of lactic acid produced by parasite's glycolysis. © 1993 Wiley-Liss, Inc.     

Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons

Inastrocytes, as [K⁺]_o was increased from 1.2 to 10 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. As [K⁺]_o was increased from 10 to 60 mM, intracellular concentration of these three ions showed no significant change. When [K⁺]_o was increased from 60 to 122 mM, an increase in [K⁺]_i and [Cl^–]_i and a decrease in [Na⁺]_i were observed.Inneurons, as [K⁺]_o was increased from 1.2 to 2.8 mM, [Na⁺]_i and [Cl^–]_i were decreased, whereas [K⁺]_i was increased. As [K⁺]_o was increased from 2.8 to 30 mM, [K⁺]_i, [Na⁺]_i and [Cl^–]_i showed no significant change. When [K⁺]_o was increased from 30 to 122 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. Inastrocytes, pH_i increased when [K⁺]_o was increased. Inneurons, there was a biphasic change in pH_i. In lower [K⁺]_o (1.2–2.8 mM) pH_i decreased as [K⁺]_o increased, whereas in higher [K⁺]_o (2.8–122 mM) pH_i was directly related to [K⁺]_o. In bothastrocytes andneurons, changes in [K⁺]_o did not affect the extracellular water content, whereas the intracellular water content increased as the [K⁺]_o increased. Transmembrane potential (E_m) as measured with Tl-204 was inversely related to [K⁺]_o between 1.2 and 90 mM, a ten-fold increase in [K⁺]_o depolarized the astrocytes by about 56 mV and the neurons about 52 mV. The E_m values measured with Tl-204 were close to the potassium equilibrium potential (E_k) except those in neurons at lower [K⁺]_o. However, they were not equal to the chloride equilibrium potential (E_Cl) at [K⁺]_o lower than 30 mM in both astrocytes and neurons. Results of this study demonstrate that alteration of [K⁺]_o produced different changes in [K⁺]_i, [Na⁺]_i, [Cl^–]_i, and pH_i in astrocytes and neurons. The data show that astrocytes can adapt to alterations in [K⁺]_o, in such a way to maintain a more suitable environment for neurons.     

Intracellular free sodium concentrations in GH4C1 cells

In the present investigation, intracellular sodium ([Na⁺]_i) levels were determined in GH₄C₁ cells using the fluorescent probe SBFI. Fluorescence was determined by excitation at 340 nm and 385 nm, and emission was measured at 500 nm. Intracellular free sodium ([Na⁺]_i) was determined by comparing the ratio 340/385 to a calibration curve. The ratio was linear between 10 and 60 mM Na⁺. Resting [Na⁺]_i in GH₄C₁ cells was 26 ± 6.2 mM (mean ± SD). In cells incubated in Na⁺-buffer [Na⁺]_i decreased to 3 ± 3.6 mM. If Na⁺/K⁺ ATPase was inhibited by incubating the cells with 1 mM ouabain, [Na⁺]_i increased to 47 ± 12.8 mM in 15 min. Stimulating the cells with TRH, phorbol myristyl acetete, or thapsigargin had no effect on [Na⁺]_i. Incubating the cells in Ca²⁺-buffer rapidly increased [Na⁺]_i. The increase was not inhibited by tetrodotoxin. Addition of extracellular Ca²⁺, nimodipine, or Ni²⁺ to these cells immediately decreased [Na⁺]_i, whereas Bay K 8644 enhanced the influx of Na⁺. In cells where [Na⁺]_i was increased the TRH-induced increase in intracellular free calcium ([Ca²⁺]_i) was decreased compared with control cells. Our results suggest that Na⁺ enters the cells via Ca²⁺ channels, and [Na⁺]_i may attenuate TRH-induced changes in [Ca²⁺]_i in GH₄C₁ cells. © 1993 Wiley-Liss, Inc.     

Effects of ouabain on proliferation of human endothelial cells correlate with Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and intracellular ratio of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript>

Side-by-side with inhibition of the Na⁺,K⁺-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na⁺ and K⁺ ratio ([Na⁺]_i/[K⁺]_i). Thus, we compared the doseand time-dependences of the effect of ouabain on intracellular [Na⁺]_i/[K⁺]_i ratio, Na⁺,K⁺-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na⁺]_i/[K⁺]_i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na⁺,K⁺-ATPase activity by 25-30%, as measured by the rate of ⁸⁶Rb⁺ influx. Higher ouabain concentrations inhibited Na⁺,K⁺-ATPase, increased [Na⁺]_i/[K⁺]_i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na⁺]_i/[K⁺]_i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na⁺]_i/[K⁺]_i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na⁺ and K⁺ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na⁺,K⁺-ATPase and decrease in the [Na⁺]_i/[K⁺]_i ratio.     

Stimulation of active potassium transport in LK sheep red cells by blood group-L-antiserum

Summary Anti-L serum prepared by immunization of a high-potassium-type (HK) (blood type MM) sheep with blood from a low-potassium-type (LK) (blood type ML) sheep contained an antibody which stimulated four- to sixfold K⁺-pump influx in LK (LL) sheep red cells. In long-termin vitro incubation experiments, LK sheep red cells sensitized with anti-L showed a net increase in K⁺ after two days of incubation at 37°C, whereas HK-nonimmune (NI)-serum-treated control cells lost K⁺. The antibody could be absorbed by LK (LL) sheep red cells but not by HK sheep red cells. Kinetic experiments showed that the concentration of external K⁺ ([K⁺]₀) required to produce halfmaximum stimulation of the pump ([Na⁺]₀=0, replaced by Mg⁺⁺) was the same (0.25 mM) in L-antiserum-treated or untreated LK cells. LK cells with different [K⁺]_i (Na⁺ replacement) were prepared by the p-chloromercuribenzene sulfonate (PCMBS) method. At [K⁺]₀=5 mM, pump influx decreased as [K⁺]_i increased from 1 to 70 mM in L-antiserum-treated LK cells, whereas LK cells treated with HK-NI-serum ceased to pump at [K⁺]_i=35 mM. Exposure to anti-L serum produced an almost twofold increase in the number of pump sites of LK cells as measured by the binding of tritiated ouabain by LK sheep red cells. These findings indicate that the formation of a complex between the L-antigen and its antibody stimulates active transport in LK sheep red cells both by changing the kinetics of the pump and by increasing the number of pump sites.     

The Death of Ouabain-Treated Renal Epithelial C11-MDCK Cells is Not Mediated by Swelling-Induced Plasma Membrane Rupture

This study examined the role of cell volume modulation in plasma membrane rupture and death documented in ouabain-treated renal epithelial cells. Long-term exposure to ouabain caused massive death of C11-MDCK (Madin-Darby canine kidney) epithelial cells, documented by their detachment, chromatin cleavage and complete loss of lactate dehydrogenase (LDH), but did not affect the survival of vascular smooth muscle cells (VSMCs) from the rat aorta. Unlike the distinct impact on cell survival, 2-h exposure to ouabain led to sharp elevation of the [Na⁺]_i/[K⁺]_i ratio in both cell types. A similar increment of Na_i⁺ content was evoked by sustained inhibition of Na⁺,K⁺-ATPase in K⁺-free medium. However, in contrast to ouabain, C11-MDCK cells survived perfectly during 24-h exposure to K⁺-free medium. At 3 h, the volume of ouabain-treated C11-MDCK cells and VSMCs, measured by the recently developed dual-image surface reconstruction technique, was increased by 16 and 12%, respectively, whereas 5–10 min before the detachment of ouabain-treated C11-MDCK cells, their volume was augmented by ~30–40%. To examine the role of modest swelling in the plasma membrane rupture of ouabain-treated cells, we compared actions of hypotonic medium on volume and LDH release. We observed that LDH release from hyposmotically swollen C11-MDCK cells was triggered when their volume was increased by approximately fivefold. Thus, our results showed that the rupture of plasma membranes in ouabain-treated C11-MDCK cells was not directly caused by cell volume modulation evoked by Na⁺,K⁺-ATPase inhibition and inversion of the [Na⁺]_i/[K⁺]_i ratio.     

Control of aerobic glycolysis in the brain in vitro

Protoveratrine-(5 M) stimulated aerobic glycolysis of incubated rat brain cortex slices that accompanies the enhanced neuronal influx of Na⁺ is blocked by tetrodotoxin (3 M) and the local anesthetics, cocaine (0.1 mM) and lidocaine (0.5 mM). On the other hand, high [K⁺]-stimulated aerobic glycolysis that accompanies the acetylcholine-sensitive enhanced glial uptakes of Na⁺ and water is unaffected by acetylcholine (2 mM). Experiments done under a variety of metabolic conditions show that there exists a better correlation between diminished ATP content of the tissue and enhanced aerobic glycolysis than between tissue ATP and the ATP-dependent synthesis of glutamine. Whereas malonate (2 mM) and amino oxyacetate (5 mM) suppress ATP content and O₂ uptake, stimulate lactate formation, but have little effect on glutamine levels, fluoroacetate (3 mM) suppresses glutamine synthesis in glia, presumably by suppressing the operation of the citric acid cycle, with little effect on ATP content, O₂ uptake, and lactate formation. Exogenous citrate (5 mM), which may be transported and metabolized in glia but not in neurons, inhibits lactate formation by cell free acetone-dried powder extracts of brain cortex but not by brain cortex slices. These results suggest that the neuron is the major site of stimulated aerobic glycolysis in the brain, and that under our experimental conditions glycolysis in glia is under lesser stringent metabolic control than that in the neuron. Stimulation of aerobic glycolysis by protoveratrine occurs due to diminution of the energy charge of the neuron as a result of stimulation of the sodium pump following tetrodotoxin-sensitive influx of Na⁺; stimulation by high [K⁺, NH₄ ⁺, or Ca²⁺ deprivation occurs partly by direct stimulation of key enzymes of glycolysis and partly by a fall in the tissue ATP concentration.     

Relationship between Intracellular Na+ Concentration and Reduced Na+ Affinity in Na+,K+-ATPase Mutants Causing Neurological Disease

The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na⁺,K⁺-ATPase α₂ and α₃ isoforms, expressed in glial and neuronal cells, respectively. Although these disorders are distinct, they overlap in phenotypical presentation. Two Na⁺,K⁺-ATPase mutations, extending the C terminus by either 28 residues (“+28” mutation) or an extra tyrosine (“+Y”), are associated with FHM2 and RDP, respectively. We describe here functional consequences of these and other neurological disease mutations as well as an extension of the C terminus only by a single alanine. The dependence of the mutational effects on the specific α isoform in which the mutation is introduced was furthermore studied. At the cellular level we have characterized the C-terminal extension mutants and other mutants, addressing the question to what extent they cause a change of the intracellular Na⁺ and K⁺ concentrations ([Na⁺]_i and [K⁺]_i) in COS cells. C-terminal extension mutants generally showed dramatically reduced Na⁺ affinity without disturbance of K⁺ binding, as did other RDP mutants. No phosphorylation from ATP was observed for the +28 mutation of α₂ despite a high expression level. A significant rise of [Na⁺]_i and reduction of [K⁺]_i was detected in cells expressing mutants with reduced Na⁺ affinity and did not require a concomitant reduction of the maximal catalytic turnover rate or expression level. Moreover, two mutations that increase Na⁺ affinity were found to reduce [Na⁺]_i. It is concluded that the Na⁺ affinity of the Na⁺,K⁺-ATPase is an important determinant of [Na⁺]_i.     

Effects of cytoskeletal modifications on Ca2+ influx after cerebral ischemia

Summary.  The fungal toxin cytochalasin D as well as endogenous gelsolin depolymerize filamentous actin which may induce dynamic uncoupling of membrane ion channels. In vitro application of cytochalasin D reduced NMDA-induced [³H]noradrenaline release from mouse brain neocortical slices by 38%. In gsn deficient neocortical synaptosomes [Ca²⁺]_i increase in response to K⁺ (30 mM) depolarization was 33% higher than in wild-type. After transient focal cerebral ischemia K⁺-induced [Ca²⁺]_i increase in neocortical synaptosomes was 56% lower than in synaptosomes prepared from the non-ischemic contralateral hemisphere. After in vivo pretreatment with cytochalasin D 10 min before MCA occlusion K⁺-induced [Ca²⁺]_i increase in synaptosomes in vitro prepared 1 h after reperfusion from the ischemic hemisphere was only 25% lower than in contralateral synaptosomes, while cytochalasin D pretreatment in vivo did not reduce K⁺-induced [Ca²⁺]_i increase in vitro. Hence, presynaptic Ca²⁺ influx and subsequently neuronal vulnerability are attenuated by increased and are aggravated by decreased F-actin depolymerization. Received June 29, 2001 Accepted August 6, 2001 Published online August 9, 2002     

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.     

Extracellular ATP stimulates inositol phospholipid turnover and calcium influx in C6 glioma cells

Extracellular ATP caused a dose-dependent accumulation of inositol phosphates and a rise in cytosolic free Ca²⁺ ([Ca²⁺]_i) in C₆ glioma cells with an EC₅₀ of 60±4 and 10±5 M, respectively. The threshold concentration of ATP (3 M) for increasing [Ca²⁺]_i was approximately 10-fold less than that for stimulating phosphoinositide (PI) turnover. The PI response showed a preference for ATP; ADP was about 3-fold less potent than ATP but had a comparable maximal stimulation (11-fold of the control). AMP and adenosine were without effect at concentrations up to 1 mM. ATP-stimulated PI metabolism was found to be partially dependent on extracellular Ca²⁺ and Na⁺ but was resistant to tetrodotoxin, saxitoxin, amiloride, ouabain, and inorganic blockers of Ca²⁺ channels (Co²⁺, Mn²⁺, La³⁺, or Cd²⁺). In Ca²⁺-free medium, ATP caused only a transient increase in [Ca²⁺]_i as opposed to a sustained [Ca²⁺]_i increase in normal medium. The ATP-induced elevation of [Ca²⁺]_i was resistant to Na⁺ depletion and treatment with saxitoxin, verapamil and nisoldipine, but was attentuated by La³⁺. The differences in the characteristics of ATP-caused P₁ hydrolysis and [Ca²⁺]_i rise suggest that ATP receptors are independently coupled to phospholipase C and receptor-gated Ca²⁺ channels. Because of the robust effect of ATP in stimulating PI turnover and the apparent absence of P₁-purinergic receptors, the C₆ glioma cell line provides a useful model for investigating the transmembrane signalling pathway induced by extracellular ATP. The mechanisms underlying the unexpected finding of [Na⁺]_o dependency for ATP-induced PI turnover require further investigation.Abbreviations PI phosphoinositide - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - PDBu phorbol 12, 13-dibutyrate - PSS physiological saline solution - IP inositol phosphates - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - IP₄ inositol (1,3,4,5) tetrakisphosphate - PLC phospholipase C     

Changes in free-calcium levels and pH in synaptosomes during transmitter release

The free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in rat brain synaptosomes estimated by the indicator quin 2 is 104±8 nM (S.D.) in artificial cerebrospinal fluid (1.2 mM Ca²⁺), but decreases at lower Ca²⁺ concentrations in the medium. The presence of quin 2 in the synaptosomes does not affect either the spontaneous release of transmitter (γ-aminobutyric acid) or the release induced by K⁺ depolarisation. In quin 2-loaded synaptosomes, depolarisation by K⁺ causes an abrupt increase in [Ca]_i (less than 2-fold) that is approximately proportional to the extent of depolarisation, whereas depolarisation by veratrine alkaloids produces a slow rise in [Ca]_i. The increase in [Ca]_i produced by K⁺ depolarisation does not occur in the absence of Ca²⁺ in the medium. The data are consistent with a direct correlation between [Ca_i] and transmitter release in functional synaptosomes. The pH in synaptosomes estimated by the indicator quene 1 is 7.04±0.07 and is stable in media containing 5 mM bicarbonate. The pH in synaptosomes was decreased by protoveratrine but not by K⁺ depolarisation.     

Death of ouabain-treated renal epithelial cells: evidence for p38 MAPK-mediated Na i + /K i + -independent signaling

Recent studies demonstrate that cytotoxic actions of ouabain and other cardiotonic steroids (CTS) on renal epithelial cells (REC) are triggered by their interaction with the Na⁺,K⁺-ATPase α-subunit but not the result of inhibition of Na⁺,K⁺-ATPase-mediated ion fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio. This study examined the role of mitogen-activated protein kinases (MAPK) in the death of ouabain-treated REC. Exposure of C7-MDCK cells that resembled principal cells from canine kidney to 3 μM ouabain led to phosphorylation of p38 without significant impact on phosphorylation of ERK and JNK MAPK. Maximal increment of p38 phosphorylation was observed at 4 h followed by cell death at 12 h of ouabain addition. In contrast to ouabain, neither cell death nor p38 MAPK phosphorylation were affected by elevation of the [Na⁺]_i/[K⁺]_i ratio triggered by Na⁺,K⁺-ATPase inhibition in K⁺-free medium. p38 phosphorylation was noted in all other cell types exhibiting death in the presence of ouabain, such as intercalated cells from canine kidney and human colon rectal carcinoma cells. We did not observe any action of ouabain on p38 phosphorylation in ouabain-resistant smooth muscle cells from rat aorta and endothelial cells from human umbilical vein. Both p38 phosphorylation and death of ouabain-treated C7-MDCK cells were suppressed by p38 inhibitor SB 202190 but were resistant to its inactive analogue SB 202474. Our results demonstrate that death of CTS-treated REC is triggered by Na_i⁺,K_i⁺—independent activation of p38 MAPK.     

An Interaction Between ATP and High K+: Mutual Impairment of ATP- and High K+-Evoked [Ca2+]i Increase in NG 108–15 Cells

The interaction between ATP- and high K⁺-evoked increase in intracellular free calcium concentration ([Ca²⁺]_i) was investigated to gain an insight into the mechanism of interaction of ATP with voltage-sensitive calcium channels. [Ca²⁺]_i was measured in the neuronal model, neuroblastoma × glioma hybrid cells (NG 108–15), using the fluorescence indicator fura-2. In the presence of 1.8 mM extracellular Ca²⁺, ATP induced a rapid, concentration-dependent increase in [Ca²⁺]_i. High K⁺ (50 mM) evoked a [Ca²⁺]_i rise from 109 ± 11 nM to 387 ± 81 nM (n = 16). The application of either of these two [Ca²⁺]_i-increase provoking agents in sequence with the other caused impairment of the latter effect. The mutual desensitization of the responses to ATP and high K⁺ strongly suggests that both agents rely at least in part on the same source of Ca²⁺ for elevation of [Ca²⁺]_i in NG 108–15 cells.     

Exchange between inorganic phosphate and adenosine triphosphate in (Na+,K+)-ATPase

(Na⁺,K⁺)-ATPase is able to catalyze a continuous ATP?P_i exchange in the presence of Na⁺ and in the absence of a transmembrane ionic gradient. At pH 7.6 the Na⁺ concentration required for half-maximal activity is 85 mM and at pH 5.1 it is 340 mM. In the presence of optimal Na⁺ concentration, the rate of exchange is maximal at pH 6.0 and varies with ADP and P_i concentration in the assay medium. ATP?P_i exchange is inhibited by K⁺ and by ouabain.     

ATP-Induced Inhibition of Na+, K+, Cl− Cotransport in Madin-Darby Canine Kidney Cells: Lack of Involvement of Known Purinoceptor-Coupled Signaling Pathways

P_2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca²⁺] _i , activation of phospholipases C (PLC) and A₂ (PLA₂), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na⁺,K⁺,Cl⁻ cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier (∼50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca²⁺]_i that was abolished by a chelator of Ca²⁺ _i , BAPTA. However, neither BAPTA nor the Ca²⁺ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca²⁺-pump, thapsigargin, modified ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4,5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na⁺,K⁺,Cl⁻ cotransport activity. Inhibitors of PLA₂ (AACOCF₃), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na⁺ channels (amiloride), Cl⁻ channels (NPPB) and Na⁺/H⁺ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport and suggest the presence of a novel P₂-receptor-coupled signaling mechanism. Received: 29 July 1998/Revised: 19 October     

Acceleration of Sodium-Calcium Exchange Activity during ATP-induced Calcium Release in Transfected Chinese Hamster Ovary Cells

The P_2U purinergic agonist ATP (0.3 mM) elicited an increase in [Ca²⁺]_i due to Ca²⁺ release from intracellular stores in transfected Chinese hamster ovary cells that express the bovine cardiac Na⁺/Ca²⁺ exchanger (CK1.4 cells). The following observations indicate that ATP-evoked Ca²⁺ release was accompanied by a Ca²⁺- dependent regulatory activation of Na⁺/Ca²⁺ exchange activity: Addition of extracellular Ca²⁺ (0.7 mM) 0–1 min after ATP evoked a dramatic rise in [Ca²⁺]_i in Na⁺-free media (Li⁺ substitution) compared to Na⁺-containing media; no differences between Na⁺- and Li⁺-based media were observed with vector-transfected cells. In the presence of physiological concentrations of extracellular Na⁺ and Ca²⁺, the ATP-evoked rise in [Ca²⁺]_i declined more rapidly in CK1.4 cells compared to control cells, but then attained a long-lived plateau of elevated [Ca²⁺]_i which eventually came to exceed the declining [Ca²⁺]_i values in control cells. ATP elicited a transient acceleration of exchange-mediated Ba²⁺ influx, consistent with regulatory activation of the Na⁺/Ca²⁺ exchanger. The acceleration of Ba²⁺ influx was not observed in vector-transfected control cells, or in CK1.4 cells in the absence of intracellular Na⁺ or when the Ca²⁺ content of the intracellular stores had been reduced by prior treatment with ionomycin. The protein kinase C activator phorbol 12-myristate 13-acetate attenuated the exchange-mediated rise in [Ca²⁺]_i under Na⁺-free conditions, but did not inhibit the ATP-evoked stimulation of Ba²⁺ influx. The effects of PMA are therefore not due to inhibition of exchange activity, but probably reflect the influence of protein kinase C on other Ca²⁺ homeostatic mechanisms. We conclude that exchange activity is accelerated during ATP-evoked Ca²⁺ release from intracellular stores through regulatory activation by increased [Ca²⁺]_i. In the absence of extracellular Ca²⁺, the stimulation of exchange activity is short-lived and follows the time course of the [Ca²⁺]_i transient; in the presence of extracellular Ca²⁺, we suggest that the exchanger remains activated for a longer period of time, thereby stabilizing and prolonging the plateau phase of store-dependent Ca²⁺ entry.     

Kinetic parameters and mechanism of active cation transport in HeLa cells as studied by Rb+ influx

On incubation of HeLa cells in chilled isotonic medium, intracellular Na⁺ (Na_c⁺) increased and K⁺ (K_c⁺) decreased with time, reaching steady levels after 3 h. The steady levels varied in parallel with the extracellular cation concentrations ([Na⁺]_e, [K⁺]_e). The cell volumes and the protein and water contents, respectively, of cells kept for 3 h in chilled media of various [Na⁺]_e and [K⁺]_e were not significantly different. Ouabain-sensitive Rb⁺ influx took place at the initial rate for a certain period which depended on [Na⁺]_c at the beginning of the assays. The existence of two external K⁺ loading sites per Na⁺/K⁺-pump was demonstrated. The affinities of the sites for Rb⁺ as a congener of K⁺ were almost the same. Na_e⁺ inhibited ouabain-sensitive Rb⁺ influx competitively, whereas K_c⁺ was not inhibitory. Kinetic parameters were determined: the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for Rb_e⁺ in the absence of Na_e⁺ was 0.16 mM and the K_i for Na_e⁺ was 36.8 mM; the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was 19.5 mM and the K_i for K_c⁺ seemed to be extremely large. The rate equation of the ouabain-sensitive Rb⁺ influx suggests that Na⁺ and K⁺ are exchanged alternately through the pump by a binary mechanism.