Voltage Dependence of the Activity of Inositol Trisphosphate Receptors of the Nuclear Envelope of Hippocampal Pyramidal Neurons

In our previous studies, we demonstrated that the inner membrane of the nuclear envelope of pyramidal neurons of the СА1 hippocampal area possesses inositol trisphosphate receptors (ІР₃Rs). In this study, we analyzed in more detail the kinetic properties of the channels of these receptors and examined the dependence of the probability of their open state (Р _о) on the voltage inside the lumen of the nuclear envelope. At positive potentials, the activity of channels of the above type was significantly more intense than at negative potentials. In the case where the potential was positive, the Р _о of channels of ІР3Rs increased at the expense of enhancement of both the frequency of their triggering and the duration of the open state. We believe that the voltage dependence of the activity of ІР₃Rs in the inner nuclear envelope is a significant factor influencing the regulation of calcium signals in the nucleus and exerts a noticeable effect on the functioning of pyramidal hippocampal cells.     

Spontaneously active ion channels of membranes of the nuclear envelope of hippocampal pyramidal neurons

The genetic apparatus of an eukaryotic cell is surrounded by two membranes of the nuclear envelope that forms a half-permeable barrier for the movement of molecules and ions. Using a patch-clamp technique in experiments on isolated nuclei of pyramidal neurons from the hippocampal CA1 area, we describe the biophysical properties of spontaneously active ion channels in the nuclear membranes of these cells. In the external nuclear membrane, we found anion channels with a unitary conductance of 156 pS and with very rapid kinetics of fluctuation, while in the inner membrane we recorded cationic channels with a unitary conductance of 248 pS and very slow kinetics. Channels of both types demonstrated clear voltage dependences. We hypothesize that the physiological importance of these channels is related to the function of the intermembrane space of the nuclear envelope of these cells forming a considerable calcium store. It seems possible that such channels in the nuclear membranes are necessary for the maintenance of the ion balance between the cytoplasm and perinuclear space and between the latter and karyoplasm, and also for neutralization of voltage shifts in the course of Ca²⁺ release. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 3–8, January–February, 2007.     

Importance of Cationic Channels for Functioning of the Nuclear Envelope of Neurons as a Calcium Store

The membrane of the endoplasmic reticulum is, in fact, an extension of the nuclear envelope of eukaryotic cells; both these compartments can fulfill the function of intracellular calcium stores. Using a patch-clamp technique, we studied the biophysical properties of the channels expressed in the inner nuclear membrane of pyramidal neurons of the rat hippocampal CA1 area, in particular of large-conductance cationic channels and calcium channels of inositol trisphosphate receptors (the main channels in membranes of this type). As the results of the measurements showed, the activity of channels of both types demonstrates clearly pronounced voltage dependences. The probability of their open state (P _o) depends on the voltage inside the nuclear envelope lumen. At positive potentials, the activity of these channels is significantly more intense than at negative potentials. Moreover, channels of both types are reversibly blocked at considerable negative potentials. We believe that this property of ion channels in the nuclear envelope is an important factor responsible for the control of calcium signals in the cell nucleus. We propose a hypothesis on the mechanism underlying termination of Ca²⁺ release from such intracellular stores, which is based on the specificity of the voltage dependence of ion channels of the above-mentioned types.     

Impact of Geometrical Characteristics of the Organellar Store and Organelle-Free Cytosol on Intracellular Calcium Dynamics in the Dendrite: a Simulation Study

The dependence of intracellular calcium dynamics on geometrical size relations between calcium-exchanging parts of the intracellular space was studied in mathematical models corresponding to a thin fragment of the Purkinje neuron spiny dendrite. The plasma membrane contained ion channels typical of this cell type, including channels that conduct an excitatory synaptic current, and ion pumps. The model equations took into account calcium exchange between the cytosol, extracellular medium, intracellular store (a cistern of the endoplasmic reticulum, ER), endogenous calcium buffers, and an exogenous buffer (fluorescent dye used in the experiments). The ER membrane contained the calcium pump and channels of calcium-dependent and inositol-3-phosphate-dependent calcium release, as well as leakage channels. With the compartment size fixed, the ER cistern diameter was varied so that the proportion of the organelle in the total volume changed from 1 to 36%. Under these conditions, identical synaptic excitation caused similar electrical reactions (calcium spikes) but different concentration responses. Equal increments in the ER diameter led to unequal, more pronounced at thicker diameters, increments of the peak cytosolic concentrations of Са²⁺ ([Ca²⁺] _i ) and of a Са²⁺-fluorescent dye complex [CaD], as well as those of the Са²⁺ concentration in the dendrite ER (characterized by a shift from the basal level, Δ[Ca²⁺]_ER). The changes in [Ca²⁺] _i and [CaD] followed more adequately those in the volume of the organelle-free cytosol, while Δ[Ca²⁺]_ER changes were more similar to those in the ER membrane area. Therefore, the relative occupancy of the intracellular volume by organellar calcium stores and their sizes in a dendritic compartment are important structural factors that essentially modulate the calcium dynamics, and this structural dependence can be adequately reflected in the experiments using fluorophores. Neirofiziologiya/Neurophysiology, Vol. 41, No. 1, pp. 19–31, January–February, 2009.     

Effects of Chronic Introduction of Antidepressants on NMDA-Induced Damage to Neurons of the Rat Hippocampus and Cerebral Cortex

In in vitro studies on superfused slices obtained from the rat hippocampus and cortex, we found that 50 μM N-methyl-D-aspartate (NMDA) applied to the slices in the presence of 10 μM glycine for 15 min exerts a significant damaging action to neurons of these structures. One hour after termination of the action of NMDA, this was manifested in more than a twofold decrease in the synaptic reactivity of pyramidal neurons of the hippocampal СА1 area and layers II/III of the cerebral cortex. The excitotoxic effect of NMDA was prevented by application of competitive (D-2-amino-5-phosphonovaleric acid, 50 μM) and noncompetitive (ketamine, 100 μM) blockers of NMDA receptors. A blocker of glycine-binding sites of NMDA receptors (compound ТСВ 24.15, 10 μM) weakened NMDA-induced damage to the neurons. A competitive blocker of glutamate АМРА receptors, 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 μM), and a local anesthetic, lidocaine hydrochloride (50 μM), did not modify the excitotoxic effect of NMDA. A blocker of voltagedependent L-type calcium channels, verapamil (20 μM), demonstrated some trend to intensification of NMDA excitotoxic action. An inhibitor of tyrosine-protein phosphatases, sodium vanadate, when i.p. injected into rats in a dose of 15 mg/kg 6 h prior to the electrophysiological experiment, decreased the damaging action of NMDA. Two-hour-long treatment of cerebral slices with 1 μM genistein, an inhibitor of tyrosine kinases, weakened the neuroprotective effect of sodium vanadate. Chronic injections (14 days in daily doses of 20 mg/kg) of antidepressants belonging to different functional classes (imipramine, fluoxetine, and pyrazidol) into rats decreased (similarly to blockers of NMDA receptors) the excitotoxic action of NMDA receptors. Neuroprotective effects of antidepressants were weakened upon the action of genistein. We conclude that the neuroprotective activity of antidepressants under conditions of excitotoxic action of NMDA is mainly determined by an increase in the activity of tyrosine kinases in the cytoplasm and/or neuronal nucleus.     

The Involvement of Calcium Transport Systems of the Plasma Membrane in Calcium Exchange in Neurons of the <Emphasis Type="Italic">Carassius gibelio</Emphasis> Cerebellum

We studied the role of Na⁺/Ca²⁺ exchanger (NCX) and Ca²⁺-ATPase of the plasma membrane (РМСА), known to be the most important intracellular systems controlling calcium exchange in cerebellar neurons of a fish species tolerant to hypoxia, Carassius gibelio. In our experiments, we used the corresponding blockers of these transport systems, ions of lithium and lanthanum. The intracellular Ca2+ concentration ([Ca²⁺] _і ) was measured using a calcium-sensitive dye, Fura-2AM, and a microfluorescence technique. We found that neurons of the Carassius cerebellum possess an effective system of cleaning of the cytoplasm from excessive Ca²⁺, which is provided by both NCX and РМСА functioning in the plasma membrane. Under conditions of the blockade of functioning of РМСА using lanthanum, the basal Ca²⁺ level in the cells increased, on average, by 31.4% with respect to the control, independently of the duration of test depolarizations. After switching off of the NCX functioning by the replacement of sodium ions in the extracellular solution by lithium ions, the Ca²⁺ level in the cell increased by 36.6% with respect to the control (also independently of the duration of depolarization). The obtained data indicate that the functioning of РМСА and NCX in Carassius cerebellar neurons significantly influences the intracellular calcium exchange providing the maintenance of an adequate basal Ca2+ level in these neurons.     

Comparative Model Analysis of Calcium Exchange between the Cytosol and Stores of Mitochondria or Endoplasmic Reticulum

The objects of the study were single-compartment mathematical models corresponding to a fragment of the dendrite of a cerebellar Purkinje neuron containing the mitochondria (model 1) or a cistern of the endoplasmic reticulum, ER, (model 2) as the calcium stores. We investigated the dependence of the intracellular Ca²⁺ dynamics on geometrical sizes of calcium exchanging parts of the intracellular space and the difference between the kinetic characteristics of storing in two types of stores occupying different portions of the compartment volume. The plasma membrane of the compartment bore the ion channels, particularly those conducting excitatory synaptic current, and the calcium pump typical of this neuron type. The model equations took into account Ca²⁺ exchange between the cytosol, extracellular medium, organelle stores, non-organelle endogenous buffers, and an exogenous buffer (fluorescent dye), and also the diffusion of Са²⁺ into adjacent regions of the dendrite. In model 1, the mitochondria exchanged Са²⁺ with the cytosol via the uniporter and sodium/calcium exchanger; mitochondrial processes, such as the tricarboxylic acid cycle and aerobic cellular respiration, were also taken into account. In model 2, the ER membrane contained the calcium pump, channels of passive leak, and channels of calcium-induced and inositol-3-phosphate-dependent release of Са²⁺. Increases in the portion of the stores in the total volume of the compartment from 1 to 36% led to a proportional increase in the peak values of the cytosolic calcium concentration ([Ca²⁺] _i ); the concentration of Са²⁺ in the mitochondria ([Ca²⁺]_mit) or ER ([Ca²⁺]_ER) increased correspondingly. During generation of bell-shaped cytosolic calcium signals of equal intensity and duration, the ER (due to a greater rate of storing, as compared with that in the mitochondria) was able to uptake several times more Са²⁺ (four times at 36% filling of the volume by the organelles). It is suggested that the revealed different kinetic characteristics of Са²⁺ storing by different organelles are determined by the rates of binding to transport molecules present in the store membrane and, therefore, are defined by concentrations (surface densities) of these molecules and their saturation at certain levels of [Ca²⁺]i. It has been shown that the occupancy of the intracellular volume by organelle stores of any type is a structural factor, which is able to essentially modulate the values of Ca²⁺ concentration.     

Effects of mibefradil on synaptic transmission in the hippocampus and on voltage-dependent currents in isolated hippocampal and thalamic neurons of the rat

The effects of a novel anti-hypertensive drug, mibefradil, on voltage-dependent currents in isolated thalamic and hippocampal neurons, as well as on synaptic transmission in the hippocampus have been studied. Mibefradil exerted a potent inhibitory action on low-threshold calcium currents in thalamic neurons (IC₅₀=160 nM). In higher concentrations (1–20 μM), this drug blocked not only low-threshold calcium current but also voltage-dependent sodium and delayed potassium currents in pyramidal hippocampal neurons. The amplitude of population action potentials in hippocampal slices decreased by 55% in the presence of 20μM mibefradil. All of the effects of mibefradil were almost completely reversible. In our experiments, the sensitivity of low-threshold calcium channels in thalamic neurons to mibefradil was higher than that observed on other objects. The ability of mibefradil to block not only calcium currents but also other types of voltage-dependent ion conductances in hippocampal neurons may be considered an essential factor that determines the specificity of the pharmacological profile of this drug.     

Role of Carboxylic Groups in the Control of Nonspecific Permeability of Mitochondrial Membranes

Ionized COOH groups are present in molecular structures involved in the process of formation of mitochondrial permeability transition pores (MPTPs), in particular, in the ADP/ATP antiporter and/or voltage-dependent anion channels. In experiments on preparations of isolated mitochondria obtained from rat hepatocytes, we found that, in the case of induction of nonspecific permeability through mitochondrial membranes under the action of Cа²⁺ in a relatively low concentration (15 μM), modulation of the activity of COOH groups with the use of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1 mM) led to unidirectional effects, namely to acceleration of the processes of formation of MPTPs and transport of incubation solution and Са²⁺ through these megachannels, prolongation of the open state of the latter, as well as to increases in the final volume (swelling) of the mitochondria and to a rise in the amount of Са²⁺ released from these organelles. In contrast, when calcium was used in a high concentration (100 μM), the directions of the above processes were dissimilar. Slowing down of the flow of incubation solution through MPTPs and the process of their formation was observed; at the same time, Са²⁺ release from the mitochondria was accelerated. However, the final volume of the mitochondria and the amount of Са²⁺ released from these cellular structures increased. Differences between the effects of the used modulator of the activity of COOH groups on the nonspecific permeability of the mitochondria induced by calcium applied in low and high concentrations are perhaps determined by the following. The process of swelling of the mitochondria is saturable, while Са²⁺ release from these organelles shows an unlimited pattern. The latter process (Са²⁺ release) probably undergoes calcium-initiated inactivation. The mechanisms of induction of nonspecific permeability of the mitochondrial membranes under the action of low and high calcium concentrations differ from each other. The calcium uniporter in the mitochondria is sensitive to the modulator of the activity of COOH groups. Diffusion of water through the inner mitochondrial membrane and/or other systems provides some contribution to the studied processes; this can lead to changes in the transport of liquids in these organelles.     

The actions of the neonicotinoid imidacloprid on cholinergic neurons of Drosophila melanogaster

The neonicotinoid insecticide imidacloprid is an agonist on insect nicotinic acetylcholine receptors (nAChRs). We utilised fura-2-based calcium imaging to investigate the actions of imidacloprid on cultured GFP-tagged cholinergic neurons from the third instar larvae of the genetic model organism Drosophila melanogaster. We demonstrate dose-dependent increases in intracellular calcium ([Ca²⁺]_i) in cholinergic neurons upon application of imidacloprid (10 nM–100 μM) that are blocked by nAChR antagonists mecamylamine (10 μM) and α-bungarotoxin (α-BTX, 1 μM). When compared to other (untagged) neurons, cholinergic neurons respond to lower concentrations of imidacloprid (10–100 nM) and exhibit larger amplitude responses to higher (1–100 μM) concentrations of imidacloprid. Although imidacloprid acts via nAChRs, increases in [Ca²⁺]_i also involve voltage-gated calcium channels (VGCCs) in both groups of neurons. Thus, we demonstrate that cholinergic neurons express nAChRs that are highly sensitive to imidacloprid, and demonstrate a role for VGCCs in amplifying imidacloprid-induced increases in [Ca²⁺]_i.     

Voltage-dependent calcium channels in cultivated neurons of the rat hippocampus

Calcium currents through the somatic membrane of cultivated (a low-density culture) hippocampal neurons of rats were studied with the use of a patch-clamp technique in the whole-cell configuration. Low- and high-threshold components of calcium currents were found in the somata of all studied cells. Low-threshold currents were activated at a membrane potential of about−75 mV and reached the maximum amplitude at −45±4 mV, while the maximum amplitude of high-threshold currents was observed at 17±6 mV. Low-threshold calcium currents differed from high-threshold current in weak suppression by low Cd²⁺ concentration (10–20 μM), while Ni²⁺ inhibited both types of calcium currents to an equal extent. Experiments with organic channel blockers showed that in most neurons at least four channel types were expressed: these were L, N, P, and channels insensitive to the used blockers (presumably, R-type). A blocker of L-type calcium channels, nifedipine (10 μM), blocked, on the average, 22.7±5.2%; a blocker of N-type channels, ω-CTx-GVIA (1.0 μM), blocked 30.0±5.0% and a blocker of P/Q channels, ω-Aga-IVA (200 nM), blocked 37.2±13.3% of the integral high-threshold current. A resistive component equalled 15.7±5.1% of the latter current. It is concluded that hippocampal neurons cultivated with a low density express a pharmacologically heterogeneous population of calcium channels, and the relative proportions of different type channels are close to the earlier described channel type composition in rat hippocampal slices. Our study shows that the low-density culture can be used as an adequate model for studying calcium channels in the somatic membrane of hippocampal neurons.     

Increased contribution of N-methyl-D-aspartate receptors to synaptic transmission inCA1 area of the rat hippocampus after short-term episodes of hypoxia/aglycemia

Effect of hypoxia/aglycemia episodes on excitatory postsynaptic currents (EPSC) evoked in pyramidal neurons of the rat hippocampalCA1 area by electrical stimulation of Schaffer collaterals was studied using voltage-clamp and intracellular perfusion techniques. By 60–80 min after a 10-min-long hypoxia/aglycemia episode, the EPSC amplitude increased and the EPSC decay was considerably slowed down, if compared with control. In contrast to control conditions, under which EPSC decay kinetics did not depend on the stimulus strength, hypoxia/aglycemia was followed by slowing down of the EPSC decay when stimulus intensity increased. The stimulus-dependent posthypoxic “slow” EPSC component was depressed both by D-(−)-2-amino-5-phosphonovaleric acid, an NMDA receptor blocker, and by 6-cyano-7-nitroquinoline-2,3-dion, a non-NMDA receptor blocker, which suggested possible polysynaptic origin of the above EPSC component. We suggest that short-term hypoxia/aglycemia transforms into an active state the NMDA receptors in the synapses of excitatory reccurrent collaterals of theCA1 hippocampal area, which had not functioned before. An increase in the intracellular calcium concentration from 1.5 to 5.0 mM resulted in the effect similar to that produced by hypoxia/aglycemia, which suggests that calcium channels play an important role in the mechanisms responsible for hypoxia-related activation of “silent” NMDA receptors.     

Involvement of L-type calcium channels and mitochondria in post-tetanic potentiation: Is it a general rule for different types of synapses?

Our experiments and studies of a few other authors demonstrated that L-type calcium channels and mitochondria are involved in the induction of post-tetanic potentiation (PTP) in a number of preparations (Aplysia central nervous system, hippocampal cell cultures, crayfish neuromuscular junctions, etc.). We extend this conclusion on cortical synapses by the demonstration that inhibitors of mitochondrial Ca²⁺ uptake and release suppress PTP in rat neocortical cell cultures. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 403–404, July–October, 2007.     

Changes in the Expression of <Emphasis Type="Italic">с-fos</Emphasis> and NADPH-Diaphorase Activity in Rat Hippocampal Structures Related to Food Deprivation and Realization of Operant Food-Procuring Movements

The expression of early c-fos gene (marker of neuronal activation) and NADPH-diaphorase reactivity (NADPH-dr) was studied in various hypothalamic structures of rats in the norm, in the state of starvation, and after realization of long-lasting (repeated 4 to 12 times per minute for 30 min) motivated stereotyped food-procuring forelimb movements. In rats in the starving state, as compared with the control, the densities (number of units within a 200 × 200 μm² test area of a 40-μm-thick slice) of Fos-immunoreactive (Fos-ir) neurons in the parvicellular part of the paraventricular nucleus (Ра), supraoptic (SO), and medial preoptic (МРО) nuclei, anterior hypothalamic region (АН), and lateral hypothalamic nucleus (LH) were significantly greater (Р < 0.05) than in the control. In the dorsomedial (DMD) and ventromedial (VMHD) hypothalamic nuclei, this index did not differ from control values. After the performance of intense unilateral operant movements, higher densities of labeled neurons (as compared with that in control and starving animals) were observed in the PаAP, SO, МРО, and DMD, while smaller densities were observed in the LH and VMH. NADPH-dr neurons (i.e., NO synthase-containing cells) were observed in many hypothalamic nuclei; the maximum density of such NO-generating neurons was found in the Pa, SO, MPO, and DMD. The overwhelming majority of Fos-ir and NADPH-dr neurons in neurons was observed after realization of stereotyped food-procuring movements in the Ра and SO. This specificity of changes in the number of Fos-irand NADPH-dr neurons in the hypothalamic nuclei reflects, perhaps, the involvement of these structures in the control of autonomic functions in the course of realization of operant reflexes and adaptation of the function of the cardiovascular system to the corresponding intense physical and emotional loading.     

Properties of Large-Conductance Cationic Channels in the Neuronal Nuclear Envelope

The genetic apparatus of the nucleus of eukaryotic cells is surrounded by the nuclear envelope containing ion channels with different biophysical properties. The channels responsible for calcium release from the nuclear envelope into the intranuclear space have attracted special attention. As we found, a great number of large-conductance ion channels selective with respect to monovalent cations and impermeable for bivalent cations are present in all membranes where inositol trisphosphate receptors are expressed. We showed that channels of this type are insensitive to blockers of potassium channels and, according to their properties, cannot be attributed to one well-known type of ion channels or another. In addition, since their blockers remain unknown, these channels at present cannot be isolated and cloned. Channels of this type deserve further investigation; we hypothesize that large-conductance cationic channels can be involved in the control of the duration of Ca²⁺ release from the calcium store.     

Characteristics of store-operated calcium channels activated by depletion of the endoplasmic reticulum ryanodine-sensitive calcium stores in rat dorsal root ganglion neurons

In neurons of the rat dorsal root ganglia (DRG), using a patch-clamp technique in the whole-cell configuration, we studied the characteristics of calcium channels activated by depletion of the ryanodine-sensitive calcium stores of the endoplasmic reticulum. Current-voltage (I-V) relationships of these store-operated calcium channels were obtained by subtraction of the integral I-V characteristics after application of caffeine from the integral I-V characteristics of calcium channels in the control. Currents through store-operated calcium channels could be induced by application of a series of hyperpolarization current pulses to the cell under conditions of replacement of a calcium-free solution containing caffeine by a caffeine-free solution containing 2 mM Ca²⁺. In this case, the following two main conditions were abserved: Voltage-operated calcium channels were inactivated, while a gradient of the electrochemical potential for calcium ions was increased, which made easier passing of these currents through store-operated calcium channels. Therefore, we found that in DRG neurons, despite the presence of great numbers of both voltage-operated and receptor-dependent calcium channels, one more mechanism underlying the entry of calcium through store-operated channels does exist. Neirofiziologiya/Neurophysiology, Vol. 39, No. 3, pp. 195–200, May–June, 2007.     

Early Anoxic Damage to the Hippocampus and Its Modifications Resulting From Chronic Influences of Antidepressants

We examined the actions of 5- or 7.5-min-long episodes of oxygen/glucose deprivation, OGD (temperature, 37°C), on pyramidal neurons of the CA1 hippocampal area and granular neurons of the gyrus dentatus. The respective damage to these neurons was manifested as an irreversible decrease in the amplitude of field EPSPs developing in such neuronal populations. Antagonists of NMDA receptors, D-2-amino-5 phosphonovaleric acid (50 μM), ketamine (50 μM), and compound TSB 24.15 (10 μM), demonstrated neuroprotective activities under these conditions, but only in the case of the 5-min-long exposure to OGD. A blocker of AMPAreceptors, DNQX (10 μM), combined with a local anesthetic, lidocaine hydrochloride (50 μM), induced comparable effects at the 7.5-min-long exposure. A blocker of calcium channels, verapamil (20 μM), exerted no effect on the level of injury of the neurons induced by the OGD influence. An inhibitor of tyrosine phosphoprotein phosphatases, sodium orthovanadate (15 mg/ml), demonstrated protective activities at both exposures, 5 and 7.5 min long. Chronic (during two weeks) preliminary injections of imipramine, fluoxetine, and pyridazol (everyday doses 20 mg/kg) into experimental animals resulted in noticeable weakening of the OGD-induced impairments of hippocapmal slices in the case of both exposures used, 5 and 7.5 min. The neuroprotective effects of chronically introduced antidepressants were augmented under the action of sodium orthovanadate. It is supposed that neuroprotective actions of preliminarily chronically introduced antidepressants with respect to the anoxic damage to hippocampal neurons is determined (at least to a considerable extent) by intensification of expression of neurotrophins. Under the influence of the latter, the functional activity of NMDA receptors decreases, and consequences of OGD-induced increase in the intracellular Ca²⁺ concentration are weakened.     

Kinetic Analysis of the Calcium- and Cadmium-Induced Development of Nonspecific Permeability of the Mitochondrial Inner Membrane

We studied the Са²⁺- and Cd²⁺-induced development of the nonspecific permeability of the mitochondrial inner membrane in preparations obtained from rat liver tissue, which is accompanied by swelling of these organelles and intensification of light dispersion of their suspension. Addition of 5 to 100 μM Са²⁺ or 1 to 50 μM Сd²⁺ to the medium caused swelling of the mitochondria. With increase in concentrations of Са²⁺ and Cd²⁺, the latency of the effect decreased, and the rate of swelling of these organelles increased. Upon isolated action of Са²⁺, the intensity of the process (amplitude of changes) did not depend significantly on the concentration of the above ions, while upon isolated action of Cd²⁺, it was the maximum at the concentration of 1 mM and noticeably decreased with increase in the concentration. The dependence of the rate of Са²⁺- and Cd²⁺-induced swelling of the mitochondria on the concentration of these ions was described by power and sigmoid functions, respectively. The calculated maximum rate and the constant of 50% saturation of these processes were equal to 0.609 and 1.084 extinction units/min⋅mg protein and 19.85 and 7.28 μM for Са²⁺- and Cd²⁺-induced swelling of the mitochondria, respectively. Cyclosporine A (10 μM) suppressed completely the Са²⁺-induced swelling of the mitochondria and decreased only partly the Cd²⁺-induced swelling. Dithiothreitol (1 mM) inhibited completely the latter effect but did not influence significantly the Са²⁺-stimulated process. Therefore, the distinctions between the kinetics of Са²⁺- and Cd²⁺-induced swelling of the mitochondria, as well as the different sensitivity of these processes to cyclosporine A and dithiothreitol, prove that the mechanisms underlying interactions between the cations of the above metals and the inner mitochondrial membrane in the course of the development of nonspecific permeability of these organelles are dissimilar. *Deceased     

A method for detection and characterization of GABA(A) receptor ligands using calcium-sensitive fluorescent probes

A method for detecting and characterizing possible ligands of neuronal GABA(A) receptors has been developed, which is based on measuring the calcium response to GABA by the fluorescence of a two-wavelength Ca-sensitive probe Fura-2. In a young (2–4 days) rat hippocampal cell culture, GABA induced depolarization and a transient increase in Ca²⁺ concentration in the cytosol of neurons due to activation of voltage-dependent calcium channels. A brief application of GABA could attenuate the calcium response to a subsequent addition of depolarizing agents (GABA or KCl). However, at modest amplitudes of calcium response to GABA, the reduction of the subsequent effect of KCl was insignificant, and the amplitudes of responses to KCl and to GABA proved to be linearly correlated, with a slope of ∼3.4. Therefore, the GABA calcium signals could be normalized in order to compare experiments performed on different days and different cultures. With such normalization, we estimated the EC₅₀ for GABA in neurons at ∼2.23 μM and the Hill coefficient at ∼1.9. A blocker of voltage-dependent calcium channels nifedipine suppressed the calcium responses both to KCl and to GABA, so that the linear relationship between their amplitudes was retained. To further validate the method, the IC50 and the type of inhibition were verified for known noncompetitive and competitive antagonists of GABA(A) receptors.