Effect of calcium on the energy status of rat brain synaptosomes under acidosis.

Incubation of rat brain synaptosomes at pH 6.0 in Ca2+-containing medium is associated with a decrease in the ATP content and the rate of oxygen consumption. ATP/ADP ratio decreased from 6.6 +/- 0.24 at pH 7.4 to 3.2 +/- 0.17 at pH 6.0. The content of 86Rb+ and [3H]tetraphenylphosphonium measured at pH 7.4 did not change after preincubation at pH 6.0, indicating the absence of lesion of synaptosomal plasma membranes and intrasynaptosomal mitochondria. Incubation with 1 mM EGTA in Ca2+-free medium as well as addition of 1 mM ouabain or 10 microM ruthenium red prevents the effect of acidosis. Similar results were obtained when 5 mM pyruvate was used as a mitochondrial substrate instead of glucose. It is suggested that acidosis-induced decrease in the ATP level is associated with the increase in Ca2+ concentration in the cytoplasm and its transport into mitochondria. Ouabain reverses this process due to activation of Na+/Ca2+ exchange.

[Acidosis inhibits oxidative phosphorylation in intrasynaptosomal mitochondria by releasing calcium from cytoplasmic store]. / Kal'tsii, osvobozhdaemyi iz vnutrikletochnykh depo, ingibiruet okislitel'noe fosforilirovanie mitokhondrii v sinaptosomakh mozga krys pri atsidoze.

The origin of calcium responsible for earlier observed acidosis-induced decrease in ATP content and inhibition of respiration in rat brain synaptosomes was studied. Acidosis (pH 6.0) inhibits both basal and potassium-stimulated 45Ca2+ uptake (60 mM KCl). Calcium channel blockers verapamil (100 microM) and 45Ca2+ (100 microM) have no effect on the level of ATP and respiration rate at pH 6.0. Theophylline (10 mM) releasing calcium from intracellular stores lowered ATP and O2 consumption rate at pH 7.4 but not at pH 6.0 being effective only in calcium-containing medium. Inhibitor of calcium transport in mitochondria ruthenium red (10 microM) prevented acidosis-induced ATP decrease. It is suggested that acidosis inhibits oxidative phosphorylation by releasing calcium from cytoplasmic stores with its subsequent transport into intrasynaptosomal mitochondria.

Na+/H+ exchange in vascular smooth muscle cells is controlled by GTP-binding proteins.

This study examines the involvement of GTP-binding proteins (Gps) in the regulation of Na+/H+ exchange and Ca2+ influx, which are increased in vascular smooth muscle cells from spontaneously hypertensive rats. Gp activity was modulated by fluoride, GTPgammaS, GDPbetaS, and antisense oligodeoxynucleotides complementary to conserved regions of the alpha- and beta-subunits of Gps (alpha-comm and beta-comm, respectively). Beta-adrenergic-induced Gs-mediated cAMP production was used as a positive control to estimate the efficiency of these compounds. Na+/H+ exchange, measured as ethylisopropyl amiloride-sensitive 22Na influx, was activated by 5- to 6-fold by a 30-minute preincubation of cells with 10 mmol/L NaF with a K0.5 for NaF of approximately 13 mmol/L. In contrast, no activation of 45Ca influx was observed under preincubation of vascular smooth muscle cells with NaF in Ca2+-free medium, whereas at [Ca2+]o >0.5 mmol/L, simultaneous addition of 45Ca and 10 mmol/L NaF led to sharply increased isotope uptake. NaF-induced 45Ca influx did not reach saturation up to 3 mmol/L [Ca2+]o and 20 mmol/L NaF and was correlated with the formation of calcium-fluoride complexes measured by light scattering. GTPgammaS increased basal cAMP production and Na+/H+ exchange, whereas GDPbetaS decreased isoproterenol-induced cAMP production and Na+/H+ exchange. Alpha-comm reduced whereas beta-comm augmented isoproterenol-induced cAMP production by 70%. Both oligodeoxynucleotides decreased basal Na+/H+ exchange by 40% to 50%. NaF-induced Na+/H+ exchange was not sensitive to alpha-comm but was inhibited by 60% in beta-comm-loaded cells. Neither basal nor NaF-induced 45Ca uptake was affected by GTPgammaS, GDPbetaS, and the oligodeoxynucleotides. Our results show that 45Ca uptake is activated by NaF in vascular smooth muscle cells by nonspecific accumulation of calcium-fluoride complexes and is not related to modification of Gps. On the contrary, the Na+/H+ exchanger is controlled by Gps, and Gp beta-subunits are involved in [Ca2+]o-independent activation of this carrier by NaF.

Hypoosmotic shock activates Ca2+ channels in isolated nerve terminals.

Influence of hypotonic swelling on Ca2+ (45Ca2+) uptake in rat brain synaptosomes was studied. A decrease in medium osmolality from 310 to 260-180 mOsm led to a progressive stimulation of 45Ca2+ accumulation. The effect was blocked by verapamil (IC50 = 5 microM), CoCl2 (IC50 = 58 microM) and retained at a fixed concentration of external sodium indicating the involvement of Ca2+ channels rather than Na+/Ca2+ exchange in swelling-induced Ca2+ influx. The populations of calcium channels observed in hypoosmotic and depolarizing conditions are different in three aspects: (i) kinetics of 45Ca2+ entry; (ii) insensitivity to dihydropyridines and omega-conotoxin GVIA; (iii) insensitivity to preliminary depolarization by high potassium. The effects of swelling and depolarization on Ca2+ uptake were additive. No change in membrane potential monitored with diS-C3-(5) was recorded during synaptosome hypotonic swelling. The results suggest the existence in synaptosomal plasma membrane of volume-dependent calcium-permeable channels with properties distinct from those of the voltage-dependent calcium channels. Activation of these channels may constitute an early event in volume regulation of nerve terminals in anisoosmotic conditions.

[The regulation of cell volume: the mechanisms of intracellular signalling]. / Reguliatsiia ob''ema kletok: mekhanizmy vnutrikeltochnoi signalizatsii.

The mechanisms of intracellular [correction of intercellular] signalling responsible for cell volume regulation are elucidated in the 2nd part of the review. Data on a nature of the "volume" sensor and signals acting on it, the properties of structures and mechanisms regulating the cell volume, are discussed as well as genetic regulation of the cell volume responses.

Volume-dependent regulation of ion carriers in human and rat erythrocytes: role of cytoskeleton and protein phosphorylation.

This study examines the effect of heat-induced cytoskeleton transitions and phosphoprotein phosphatase inhibitors on the activity of shrinkage-induced Na+, K+, 2Cl- cotransport and Na+/H+ exchange in rat erythrocytes and swelling-induced K+, Cl- cotransport in human and rat blood cells. Preincubation of human and rat erythrocytes at 49 degrees C drastically activated K+, Cl- cotransport and completely (rat) or partly (human) abolished its volume-dependent regulation. The same procedure did not affect basal activity of Na+, K+, 2Cl- cotransport but completely abolished its activation by shrinkage thus suggesting the involvement of a thermosensitive element of cytoskeleton network in the volume-dependent regulation of cotransporters. Both the shrinkage- and electrochemical proton gradient-induced Na+/H+ exchange was inhibited by the heat treatment to the same extent (50-70%), thus indicating the different signaling pathways involved in the activation of Na+, K+, 2Cl- cotransport and Na+/H+ exchange by cell shrinkage. This suggestion is in accordance with data on the different kinetics of volume-dependent activation and inactivation of these carriers as well as on their sensitivity to medium osmolality. Both swelling- and heat-induced increments of K+, Cl- cotransport activity were diminished by inhibitors of phosphoprotein phosphatases (okadaic acid and calyculin). In rat erythrocytes these compounds potentiate shrinkage-induced Na+/H+ exchange. On the contrary, neither basal nor shrinkage-induced Na+, K+, 2Cl- cotransport was affected by these compounds. Our results indicate a key role of cytoskeleton network in volume-dependent activation of K+, Cl- and Na+, K+, 2Cl- cotransport and the involvement of protein phosphorylation-dephosphorylation cycle in regulation of the activity of K+, Cl- cotransport and Na+/H+ exchange.

[Effect of acidosis on membrane potential and calcium transport in rat brain synaptosomes]. / Vliianie atsidoza na membrannyi potentsial i transport kal'tsiia v sinaptosomakh mozga krys.

The influence of acidosis on the transmembrane potential, sodium pump and membranous systems of calcium transport was studied on isolated presynaptic nerve terminals (synaptosomes) from rat brain. It is established that acidic shift causes a decrease of membrane potential, a large inhibition of the sodium pump (by three times at pH 6.0). All the systems controlling both inward- and outward-directed calcium fluxes are partially blocked by low pH. At pH 6.0 the basal influx and calcium pump are reduced two-fold while the voltage-sensitive calcium channels and Na+/Ca2+ exchanger are inhibited by three and four to five times, respectively. We have no found any evidence of acidosis-induced net flux of calcium directed inwards.

Swelling-induced activation of Na+,K+,2Cl- cotransport in C6 glioma cells: kinetic properties and intracellular signalling mechanisms.

Swelling of C6 glioma cells in hypotonic medium (180 mOsm) results in two- to three-fold activation of K+ (86Rb+) influx suppressed by 10 microM bumetanide. Bumetanide-sensitive transport of 86Rb+ is dependent on extracellular K+, Na+ and Cl- both in iso-osmotic conditions and under hypo-osmotic shock, supporting the notion that it is mediated by Na+,K+,2Cl- cotransport. Inhibitors of protein kinase C (10 microM polymyxin B and l microM staurosporine) had no significant effect on basal cotransport but reduced its hypotonic stimulation by 70-80%. Similar results were obtained with calmodulin antagonist R24571 (10 microM), indicating Ca2+/calmodulin-dependence of the process. Influence of polymyxin B and R24571 was not additive. Swelling-activated Na+,K+,2Cl- cotransport was also suppressed by protein kinase C activator PMA (l microM). By contrast, preincubation of cells with inhibitors of protein phosphatases (100 microM vanadate, 5 mM fluoride and 0.5 microM okadaic acid) activated greatly the bumetanide-sensitive 86Rb+ uptake in isotonic conditions, while a subsequent hypotonic swelling led to smaller or no increment. These results indicate the involvement of Ca2+/calmodulin-dependent staurosporine/polymyxin B-sensitive protein kinase other than protein kinase C in swelling-induced activation of Na+,K+,2Cl- cotransport in glial cells.

[Response of the rat erythrocyte glycolytic system to hyperosmotic shrinkage]. / Otvet glikoliticheskoi sistemy éritrotsitov krysy na giperosmoticheskoe szhatie.

Hyperosmotic shrinkage (adding 300 microM sucrose to isotonic medium) stimulates lactate and ATP accumulation in rat but not human erythrocytes in which ATP pool was preliminary depleted. Inhibitors of Na(+)-K(+)-2Cl(-)-cotransport, Na+/H(+)- exchange and Na(+)- pump known to be activated by hypertonic medium had no influence on volume-induced effect. EGTA (1 microM), Ca(2+)-ATPase inhibitor vanadate (200 microM) and antagonist of calmodulin R24571 (10 microM) suppressed the stimulation of glycolysis by 80 and 30%, respectively. Addition of 1 microM Ca2+ and 1 mM Ca(2+)-ionophore A23187 in Ca2+ free medium stimulated glycolysis by 20% at isotonic conditions while additional hyperosmotic shrinkage resulted in a two-fold activation. It is suggested that shrinkage regulates activity of glycolytic enzymes through the mechanism of intracellular signaling involving Ca2+.

Effect of proteolysis on the state of lipid phase in rat brain synaptosomal membranes.

Enzymatic proteolysis of the proteins of synaptic membranes has been found to be accompanied by the promotion of lipid peroxidation probably mediated by the liberation of membrane-bound iron. As fluorescent probes pyrene and diphenylhexatriene show, the microviscosity and micropolarity of membrane lipid phase rise as a result of lipid peroxidation. Different structural changes induced by proteolysis are displayed under inhibition of lipid peroxidation. Thus, the microviscosity of the bulk lipid phase appears to be lowered and the annular lipid microviscosity raised. Another explanation of the fluorescent data for annular lipids is the exclusion of pyrene molecules from this lipid pool, leading to a reduction of the probe local concentration. The changes observed in membrane lipid phase are considered as primary structural effects of proteolysis, not mediated by a phospholipase activation.

Swelling-induced K+ influx in cultured primary astrocytes.

The effect of swelling of cultured primary astrocytes from rat brain in hypotonic medium on K+ influx has been studied. A decrease in osmolality from 310 to 180 mOsm increased the activity of sodium pump (ouabain-inhibited 86Rb+ influx) and Na+,K+,2Cl- cotransport (ouabain-insensitive bumetanide-inhibited 86Rb+ influx) by 70 and 35%, respectively. It is suggested that activation of these transport systems makes it possible to retain a high potassium concentration in the cells under regulatory volume decrease.

Osmotic regulation of sodium pump in rat brain synaptosomes: the role of cytoplasmic sodium.

The effect of hypoosmolality of incubation medium on the rat of ouabain-sensitive 86Rb+ transport in rat brain synaptosomes was studied. A decreased osmolality from 310 to 250 mOsm increased the rate of 86Rb+ uptake from 3.72 to 6.23 nmol/mg of protein min. To evaluate the involvement of cytoplasmic sodium in sodium pump stimulation inhibitors of ion channels and transport pathways able to increase [Na+]in were used. Tetrodotoxin (1 microM), amiloride (0.5 mM) and verapamil (0.1 mM) had no influence on the osmotic response of the sodium pump. The decrease of sodium concentration in incubation medium to 15 mM, leading to a practical loss of its transmembrane gradient, did not abolish stimulation of pump. No increase in 22Na+ influx or intrasynaptosomal sodium content was registered at hypotonic conditions. It is suggested that osmotic regulation of Na+,K(+)-ATPase is not connected with an increase of internal sodium through opening of sodium channels, or with activation of other membrane sodium-transporting systems.

Kinetics and peculiarities of thermal inactivation of volume-induced Na+/H+ exchange, Na+,K+,2Cl- cotransport and K+,Cl- cotransport in rat erythrocytes.

The kinetics of the volume-dependent activation of Na+/H+ exchange, Na+,K+,2Cl(-)-cotransport and K+,Cl(-)-cotransport in rat erythrocytes was studied. The significant increase in the rate of Na+/H+ exchange is observed within 15 min after hypertonic shrinkage while the maximum transport rate is reached by 20 min. A delay of about 5 min was found in activation of Na+,K+,2Cl(-)-cotransport, the maximum transport rate being reached 10 min after shrinkage. Activation of K+,Cl(-)-cotransport by hypotonic swelling was registered within 10 min after cell swelling, with a simultaneous achievement of the constant transport rate. Preincubation of cells at 49 degrees C has no effect on the basal Na+/H+ exchange and Na+,K+,2Cl(-)-cotransport but suppresses the activation of these systems by osmotic shrinkage. On the contrary, the rate of K+,Cl(-)-cotransport in isosmotic medium is raised 10-fold after preincubation at 49 degrees C. The thermal treatment at 49 degrees C blocks the activation of K+,Cl(-)-cotransport by swelling. On the basis of the data on thermal denaturation of spectrin at the same temperature it was suggested that the cytoskeleton of erythrocyte membrane is involved in volume regulation of the ion-transporting systems and that the molecular mechanisms which underlie the activation of Na+/H+ exchange, Na+,K+,2Cl(-)-cotransport and K+,Cl(-)-cotransport are essentially different.

Effect of gamma-irradiated carbohydrates on isolated synaptic membranes.

The effect of gamma-irradiated solutions of carbohydrates, mainly glucose, upon Na+, K(+)-ATPase and lipid peroxidation in rat brain synaptosomal membranes was studied. The membrane damage by irradiated glucose was enhanced in the presence of Fe2+ and was diminished when a free-radical scavenger (BHT) or metal chelators (EDTA, EGTA) were present. It is suggested that a key element in the free-radical membrane damage by irradiated carbohydrates is an Fe(2+)-complex of some species of the radiolysis products. Participation of radiotoxins of carbohydrate origin in radiobiological effects is discussed.

Sensitivity of the brain synaptosomal membrane Mg(2+)-ATPase activity to arachidonic acid is under control of the Na+,K(+)-ATPase state.

Evidence is presented for the sensitivity of the synaptosomal plasma membrane Mg(2+)-ATPase activity to arachidonic acid being dependent on the functional state of Na+,K(+)-ATPase. An "Inversion effect" was observed at arachidonic acid concentrations exceeding 80 mumol/l when the Mg(2+)-ATPase activity (after ouabain addition) is higher than the total ATPase activity (without ouabain). The "Inversion effect" is reduced by cyclooxygenase inhibitor indomethacin or acetylsalicylic acid and restored by prostaglandin PGA2 or PGD2.

[Osmotic regulation of furosemide-sensitive K+ (86Rb+) transport in rat brain synaptosomes]. / Osmoticheskaia reguliatsiia furosemid-chuvstvitel'nogo transporta K+ (86Rb+) v sinaptosomakh mozga krys.

In the rat brain synaptosomes the furosemide-sensitive component of 86Rb+ uptake constituted 30.8% of the total uptake in the medium containing 132 mM Na+. A decrease in the medium tonicity from 310 to 230 mOsm increased the rate of 86Rb+ uptake from 2.38 +/- 0.58 to 7.12 +/- 0.52 nMoles/mg of protein/min.

[Osmotic regulation of the sodium pump in rat brain synaptosomes]. / Osmoticheskaia reguliatsiia natrievogo nasosa v sinaptosomakh golovnogomozga krys.

Effect of medium osmolarity on 3H-ouabain binding and the rate of ouabain-sensitive 86Rb+ transport in the rat brain synaptosomes was studied. A decrease in tonicity to 230 mOsm increases both parameters indicating the activation of the sodium pump upon synaptosome swelling. The effect is retained in the absence of inside-oriented Na+ gradient, i. e. a rise in Na(in) is not responsible for hypoosmotic activation. Colchicine (5mM) decreased and cytochalasin B (40 microM) increased the ouabain binding. In the presence of cytochalasin B the inhibition of binding observed under hypotonic conditions was shifted to higher osmolarity values. It is suggested that volume regulation of the sodium pump is controlled by the cytoskeleton elements.

[Effect of temperature on polarity of an annular and bilayer synaptic membrane lipid]. / Vliianie temperatury na poliarnost' anuliarnogo i bisloinogo lipida sinapticheskikh membran.

Using fluorescent probe pyrene a study was carried out of temperature dependence of the annular and lipid bilayer polarity in synaptic membranes. Polarity of microenvironment was evaluated by a ratio of intensities of vibronic bands in pyrene fluorescence spectra at direct excitation of probe or through energy transfer from protein molecules. At the temperature range 10-50 degrees C the polarity of bilayer was shown to increase while that of annular lipid underwent biphasic changes. Above 20 degrees C polarity of the bilayer was higher than of the annular zone. From experiments on pyrene fluorescence quenching with potassium iodide the conclusion has been made that changes in polarity correlate with pyrene accessibility to water.

On the mechanism of shrinkage-induced potassium influx in rat and human erythrocytes.

The rates of 86Rb influx into human and rat erythrocytes were studied in media of various tonicity. At sucrose concentrations below 0.3 mol/l, the ouabain-insensitive, furosemide-inhibited component of influx increased in rat but not in human erythrocytes; this may be explained by a rise in the rate of Na+, K+, Cl-- and/or K+, Cl-cotransport. An increase in osmolarity resulted in a reduction of this as well as of the ouabain and furosemide-insensitive component in rat erythrocytes. At the same conditions a drastic inhibition of Na+, K(+)-pump occurred both in rat and human erythrocytes. We failed to observe a lag-phase in the activation of the cotransport in rat erythrocytes; i. e. the process of activation parallels the shrinkage of cells. In rat erythrocyte ghosts, the shrinkage-induced stimulation of the cotransport was lost, and the direction of their osmotic reaction (inhibition of transport pathways) was similar to that in human erythrocyte ghosts. It is suggested that the mechanism of volume regulation of ion transport in intact cells involves a step of physical amplification via a change in interactions between the protein carcass and the lipid bilayer.