Metabolic differences in hippocampal 'Rett' neurons revealed by ATP imaging.

Understanding metabolic control of neuronal function requires detailed knowledge of ATP handling in living neurons. We imaged ATP in organotypic hippocampal slices using genetically encoded sensor Ateam 1.03 modified to selectively transduce neurons in the tissue. ATP imaging indicated distinct differences in ATP production and consumption in dentate gyrus and cornu ammonis (CA) areas. Removal of extracellular Mg(2+) from the bath evoked epileptiform-like activity that was accompanied by ATP decline from 2-3 to 1-2mM. The slices fully recovered from treatment and showed persistent spontaneous activity. Neuronal discharges were followed by transient ATP changes and periodic activation of ATP-sensitive K(+) (K-ATP) channels. The biggest ATP decreases during epileptiform-like episodes of activity were observed in CA1 and CA3 neurons. Examination of neurons from the Rett model mice MeCP2(-/y) showed that seizure-like activity had earlier onset and subsequent spontaneous activity demonstrated more frequent discharges. Hippocampal MeCP2(-/y) neurons had higher resting ATP levels and showed bigger ATP decreases during epileptiform-like activity. More intense ATP turnover in MeCP2(-/y) neurons may result from necessity to maintain hippocampal function in Rett syndrome. Elevated ATP may make, in turn, Rett hippocampus more prone to epilepsy due to inadequate activity of K-ATP channels.

Single KATP channel opening in response to stimulation of AMPA/kainate receptors is mediated by Na+ accumulation and submembrane ATP and ADP changes.

Excessive stimulation of glutamatergic receptors (GluRs) can overexcite neurons. This can be dampened by KATP channels linking metabolic and neuronal activities, but the cross-talk has not yet been examined on the single channel level. In the brainstem and hippocampal neurons, GluR agonists augmented the open state probability (Popen) of KATP channels with relative efficacy: kainate AMPA > NMDA > t-ACPD. Inhibition of calcium influx and chelation of intracellular calcium did not modify the effects. Kainate did not augment production of reactive oxygen species measured with roGFP1. H2O2 slightly increased Popen, but GluR effects were not modified. GluR actions were abolished in Na(+)-free solutions and after blockade of Na(+)-K(+)-ATPase. KATP channels in open-cell patch-clamp measurements were inhibited by ATP, stimulated by ADP, and kainate was effective only in the presence of ATP. GluR stimulation enhanced ATP consumption that decreased submembrane ATP levels, whereas metabolic poisoning diminished bulk ATP. Modelling showed strong ATP depletion and ADP accumulation near the membrane, and both effects contributed to Popen increases after GluR stimulation. Kainate and hypoxia activated KATP channels in the functional brainstem slices. Inhibition of aerobic ATP production and GluR stimulation were about equally effective in KATP channel opening during hypoxia. Induction of seizure-like activity in hippocampal slices with Mg(2+)-free solutions was accompanied by ATP decrease and KATP channel opening. We propose that KATP channels and GluRs are functionally coupled that can regulate long-lasting changes of neuronal activity in the CNS neurons.

Calmodulin and calmodulin kinase II mediate emergent bursting activity in the brainstem respiratory network (preBötzinger complex).

Emergence of persistent activity in networks can be controlled by intracellular signalling pathways but the mechanisms involved and their role are not yet fully explored. Using calcium imaging and patch-clamp we examined the rhythmic activity in the preBötzinger complex (preBötC) in the lower brainstem that generates the respiratory motor output. In functionally intact acute slices brief hypoxia, electrical stimulation and activation of AMPA receptors transiently depressed bursting activity which then recovered with augmentation. The effects were abrogated after chelation of intracellular calcium, blockade of L-type calcium channels and inhibition of calmodulin (CaM) and CaM kinase (CaMKII). Rhythmic calcium transients and synaptic drive currents in preBötC neurons in the organotypic slices showed similar CaM- and CaMKII-dependent responses. The stimuli increased the amplitude of spontaneous and miniature excitatory synaptic currents indicating postsynaptic changes at glutamatergic synapses. In the acute and organotypic slices, CaM stimulated and ADP inhibited calcium-dependent TRPM4 channels and CaMKII augmented synaptic drive currents. Experimental data and simulations show the role of ADP and CaMKII in the control of bursting activity and its relation to intracellular signalling. I propose that CaMKII-mediated facilitation of glutamatergic transmission strengthens emergent synchronous activity within preBötC that is then maintained by periodic surges of calcium during the bursts. This may find implications in restoration and consolidation of autonomous activity in the respiratory disorders.

Epac-mediated cAMP-signalling in the mouse model of Rett Syndrome.

Rett Syndrome (RTT) is a neurodevelopmental disease thought to be caused by deficits in synaptogenesis and neuronal circuitry. cAMP is one of the key factors for neuronal outgrowth, plasticity and regeneration. We examined its homeostasis in RTT during early postnatal development of the essential part of the respiratory network, pre-Bötzinger complex. Using targeted expression of Epac1-camps sensor in neurons we quantified cAMP levels and their fluctuations in MeCP2-/y mice, an established model of RTT. Resting cAMP levels in the mutant were smaller than in the wild-type. cAMP transients elicited by depolarisation and stimulation of adenylate cyclase had also smaller amplitudes and faster time-courses. The anomalies in MeCP2 -/y mice were removed after inhibition of phosphodiesterase PDE4 with rolipram. Brief cAMP elevations triggered elongation of neuronal processes that was significantly bigger in the wild-type. The effects were observed after inhibition of protein kinase A and mimicked by activation of a guanine nucleotide exchange factor, Epac, with 8-(4-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pCPT). The agonist reinforced bursting in preBötC neurons in the mutant and converted it to the wild-type. All actions of 8-pCPT were not reproduced by its non-active analogue and abolished by Epac signalling inhibitor Brefeldin A. We propose that disturbances in cAMP homeostasis in MeCP2 -/y mice can lead to inadequate Epac signalling. Concomitant defective development of respiratory circuits may be responsible for irregular breathing activity in RTT.

Imaging cytoplasmic cAMP in mouse brainstem neurons.

BACKGROUND: cAMP is an ubiquitous second messenger mediating various neuronal functions, often as a consequence of increased intracellular Ca2+ levels. While imaging of calcium is commonly used in neuroscience applications, probing for cAMP levels has not yet been performed in living vertebrate neuronal tissue before. RESULTS: Using a strictly neuron-restricted promoter we virally transduced neurons in the organotypic brainstem slices which contained pre-Bötzinger complex, constituting the rhythm-generating part of the respiratory network. Fluorescent cAMP sensor Epac1-camps was expressed both in neuronal cell bodies and neurites, allowing us to measure intracellular distribution of cAMP, its absolute levels and time-dependent changes in response to physiological stimuli. We recorded [cAMP]i changes in the micromolar range after modulation of adenylate cyclase, inhibition of phosphodiesterase and activation of G-protein-coupled metabotropic receptors. [cAMP]i levels increased after membrane depolarisation and release of Ca2+ from internal stores. The effects developed slowly and reached their maximum after transient [Ca2+]i elevations subsided. Ca2+-dependent [cAMP]i transients were suppressed after blockade of adenylate cyclase with 0.1 mM adenylate cyclase inhibitor 2'5'-dideoxyadenosine and potentiated after inhibiting phosphodiesterase with isobutylmethylxanthine and rolipram. During paired stimulations, the second depolarisation and Ca2+ release evoked bigger cAMP responses. These effects were abolished after inhibition of protein kinase A with H-89 pointing to the important role of phosphorylation of calcium channels in the potentiation of [cAMP]i transients. CONCLUSION: We constructed and characterized a neuron-specific cAMP probe based on Epac1-camps. Using viral gene transfer we showed its efficient expression in organotypic brainstem preparations. Strong fluorescence, resistance to photobleaching and possibility of direct estimation of [cAMP] levels using dual wavelength measurements make the probe useful in studies of neurons and the mechanisms of their plasticity. Epac1-camps was applied to examine the crosstalk between Ca2+ and cAMP signalling and revealed a synergism of actions of these two second messengers.

Imaging of respiratory network topology in living brainstem slices.

Topology of neuronal networks contributes to their functioning but the structure-function relationships are not yet understood. In order to reveal the spatial organisation of the respiratory network, we expressed enhanced green fluorescent proteins in neurons in brainstem slices containing the respiratory kernel (pre-Bötzinger complex). The expression was neuron specific due to use of adeno-associated viral vector driving transgene expression from synapsin 1 promoter. Both neuronal cell bodies and their dendrites were labelled with high efficacy. This labelling allowed for enhanced spatial resolution as compared to conventional calcium-sensitive dyes. Neurons occupied about 10% of tissue volume and formed an interconnected network. Using custom-developed software, we quantified the network structure that had a modular structure consisting of clusters having transverse (dorso-ventral) orientation. They contained in average seven neurons and connections between the cells in different clusters were less frequent. This novel in situ imaging technique is promising to gain new knowledge about the fine structure and function of neuronal networks in living slice preparations.

Dynamic activation of K(ATP) channels in rhythmically active neurons.

1. The respiratory centre within the brainstem is one of the most active neuronal networks that generates ongoing rhythmic activity. Stabilization of such vital activity requires efficient processes for activity-correlated adjustment of neuronal excitability. Recent investigations have shown that a regulatory factor coupling electrical activity with cell metabolism comprises ATP-dependent K(+) channels (K(ATP) channels), which continuously adjust the excitability of respiratory neurons during normoxia and increasingly during hypoxia. 2. We used the single-cell antisense RNA amplification-polymerase chain reaction (PCR) technique to demonstrate that respiratory neurons co-express the sulphonylurea receptor SUR1 with the Kir6.2 potassium channel protein. 3. Single channel measurements on rhythmically active inspiratory neurons of the brainstem slice preparation of newborn mice revealed that K(ATP) channels are periodically activated in synchrony with each respiratory cycle. 4. The Na(+)-K(+)-ATPase was inhibited with ouabain to demonstrate that oscillations of the channel open probability disappear, although respiratory activity persists for a longer time. Such findings indicate that K(ATP) channel open probability reflects activity-dependent fluctuations in the ATP concentration within submembrane domains. 5. We also examined the effects of extracellular [K(+)] and hypoxia. All changes in the respiratory rhythm (i.e. changes in cycle length and burst durations) affected the periodic fluctuations of K(ATP) channel activity. 6. The data indicate that K(ATP) channels continuously modulate central respiratory neurons and contribute to periodic adjustment of neuronal excitability. Such dynamic adjustment of channel activity operates over a high range of metabolic demands, starting below physiological conditions and extending into pathological situations of energy depletion.

Intrinsic optical signals in respiratory brain stem regions of mice: neurotransmitters, neuromodulators, and metabolic stress.

In the rhythmic brain stem slice preparation, spontaneous respiratory activity is generated endogenously and can be recorded as output activity from hypoglossal XII rootlets. Here we combine these recordings with measurements of the intrinsic optical signal (IOS) of cells in the regions of the periambigual region and nucleus hypoglossus of the rhythmic slice preparation. The IOS, which reflects changes of infrared light transmittance and scattering, has been previously employed as an indirect sensor for activity-related changes in cell metabolism. The IOS is believed to be primarily caused by cell volume changes, but it has also been associated with other morphological changes such as dendritic beading during prolonged neuronal excitation or mitochondrial swelling. An increase of the extracellular K(+) concentration from 3 to 9 mM, as well as superfusion with hypotonic solution induced a marked increase of the IOS, whereas a decrease in extracellular K(+) or superfusion with hypertonic solution had the opposite effect. During tissue anoxia, elicited by superfusion of N(2)-gassed solution, the biphasic response of the respiratory activity was accompanied by a continuous rise in the IOS. On reoxygenation, the IOS returned to control levels. Cells located at the surface of the slice were observed to swell during periods of anoxia. The region of the nucleus hypoglossus exhibited faster and larger IOS changes than the periambigual region, which presumably reflects differences in sensitivities of these neurons to metabolic stress. To analyze the components of the hypoxic IOS response, we investigated the IOS after application of neurotransmitters known to be released in increasing amounts during hypoxia. Indeed, glutamate application induced an IOS increase, whereas adenosine slightly reduced the IOS. The IOS response to hypoxia was diminished after application of glutamate uptake blockers, indicating that glutamate contributes to the hypoxic IOS. Blockade of the Na(+)/K(+)-ATPase by ouabain did not provoke a hypoxia-like IOS change. The influences of K(ATP) channels were analyzed, because they contribute significantly to the modulation of neuronal excitability during hypoxia. IOS responses obtained during manipulation of K(ATP) channel activity could be explained only by implicating mitochondrial volume changes mediated by mitochondrial K(ATP) channels. In conclusion, the hypoxic IOS response can be interpreted as a result of cell and mitochondrial swelling. Cell swelling can be attributed to hypoxic release of neurotransmitters and neuromodulators and to inhibition of Na(+)/K(+)-pump activity.

Cytoskeleton mediates inhibition of the fast Na+ current in respiratory brainstem neurons during hypoxia.

Whole-cell Na+ currents (INa) were recorded in inspiratory neurons in a medullary slice preparation from neonatal mouse that contains the functional respiratory network. Hypoxia and metabolic poisoning with KCN rapidly inhibited INa by reducing the number of Na+ channels available for opening during depolarization. Application of agents specific for G-proteins, protein kinase C and A, intracellular Ca2+ and pH did not prevent the hypoxic inhibition of INa. The effects of hypo-osmolarity and hypoxia were additive, whereas hyperosmolarity partially prevented a subsequent hypoxic inhibition of INa. Cytochalasin B and colchicine decreased, and taxol or phalloidin increased INa and reduced its hypoxic inhibition. We conclude that cytoskeleton rearrangements during hypoxia are responsible for suppression of a fast INa in brainstem respiratory neurons, which could be mediated by the uncoupling of channel inactivation gates from cytoskeletal elements.

A1 adenosine receptors modulate respiratory activity of the neonatal mouse via the cAMP-mediated signaling pathway.

The effects of adenosine and its analogs on the function of the respiratory center were studied in the spontaneously active rhythmic slice of neonatal and juvenile mice (4-14 days old). Whole cell, spontaneous postsynaptic currents (sPSCs) and single channel KATP currents were recorded in inspiratory neurons of the pre-Bötzinger complex. Adenosine (50-600 microM) inhibited the respiratory rhythm. This was accompanied by increase in the activity of KATP channels in cell-attached patches. The A1 adenosine receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA, 0.3-2 microM), inhibited the respiratory rhythm, sPSCs, and enhanced activity of KATP channels. The A1 adenosine receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 1-3 microM), showed opposite effects and occluded the CCPA actions. Agents specific for A2 adenosine receptors (CGS 21860 and NECA, both applied at 1-10 microM) were without effect. Elevation of intracellular cAMP concentration ([cAMP]i) by 8-Br-cAMP (200-500 microM), forskolin (0.5-2 microM), or isobutylmethylxantine (IBMX, 30-90 microM) reinforced the rhythm, whereas NaF (100-800 microM) depressed it. The open probability of single KATP channels in cell-attached patches decreased after application of forskolin and increased in the presence of NaF. [cAMP]i elevation reversed the effects of A1 receptors both on the respiratory rhythm and KATP channels. A1 receptors and [cAMP]i modified the hypoxic respiratory response. In the presence of A1 agonists the duration of hypoxic augmentation shortened, and depression of the respiratory rhythm occurred earlier. Elevation of [cAMP]i prolonged augmentation and delayed the development of the depression. We conclude that A1 adenosine receptors modulate the respiratory rhythm via inhibition of intracellular cAMP production and concomitant activation of KATP channels.

Hypoxia activates ATP-dependent potassium channels in inspiratory neurones of neonatal mice.

1. The respiratory centre of neonatal mice (4 to 12 days old) was isolated in 700 micro(m) thick brainstem slices. Whole-cell K+ currents and single ATP-dependent potassium (KATP) channels were analysed in inspiratory neurones. 2. In cell-attached patches, KATP channels had a conductance of 75 pS and showed inward rectification. Their gating was voltage dependent and channel activity decreased with membrane hyperpolarization. Using Ca2+-containing pipette solutions the measured conductance was lower (50 pS at 1.5 mM Ca2+), indicating tonic inhibition by extracellular Ca2+. 3. KATP channel activity was reversibly potentiated during hypoxia. Maximal effects were attained 3-4 min after oxygen removal from the bath. Hypoxic potentiation of open probability was due to an increase in channel open times and a decrease in channel closed times. 4. In inside-out patches and symmetrical K+ concentrations, channel currents reversed at about 0 mV. Channel activity was blocked by ATP (300-600 microM), glibenclamide (10-70 microM) and tolbutamide (100-300 microM). 5. In the presence of diazoxide (10-60 microM), the activity of KATP channels was increased both in inside-out, outside-out and cell-attached patches. In outside-out patches, that remained within the slice after excision, the activity of KATP channels was enhanced by hypoxia, an effect that could be mediated by a release of endogenous neuromodulators. 6. The whole-cell K+ current (IK) was inactivated at negative membrane potentials, which resembled the voltage dependence of KATP channel gating. After 3-4 min of hypoxia, K+ currents at both hyperpolarizing and depolarizing membrane potentials increased. IK was partially blocked by tolbutamide (100-300 microM) and in its presence, hypoxic potentiation of IK was abolished. 7. We conclude that KATP channels are involved in the hypoxic depression of medullary respiratory activity.

Plasmalemmal and intracellular Ca2+ pumps as main determinants of slow Ca2+ buffering in rat hippocampal neurones.

Using the Ca(2+)-sensitive fluorescent indicator dye fura-2, the mechanisms by which cytoplasmic free Ca2+ concentration, [Ca]i, decays to resting levels were studied in neurones cultured from the rat hippocampus. The time-course of [Ca]i restoration after transient elevations due to CaCl2 injections or brief exposures to 50 mM K Cl were biexponential. Application of specific inhibitors of systems participating in Ca2+ removal from cytoplasm changed both basal [Ca]i and the slow phase of recovery, but the fast phase was unaltered by any treatment. Inhibition of the plasmalemmal Ca2+ pump by external alkalinization or intracellular acidification was reversible, whereas calmodulin inhibitors (calmidazolium and triftazine, W-13) acted irreversibly. The net effects of blockers of the intracellular Ca2+ pump, thapsigargin (Tg) and t-BuHQ, were similar. Suppression of mitochondrial Ca2+ uptake or Ca2+ extrusion due to Na+/Ca2+ exchange, reversibly increased [Ca]i but the time-course of [Ca]i clearance was marginally changed. After glutamate application [Ca]i restoration was prolonged which was mediated by concomitant intracellular acidification causing inhibition of plasmalemmal Ca2+ ATPase. It is concluded that Ca2+ homeostasis in rat hippocampal neurones is mainly determined by Ca2+ pumps in both the surface membrane and internal stores, whereas Na+/Ca2+ exchange and mitochondria play a minor role.

Metabotropic ATP receptor in hippocampal and thalamic neurones: pharmacology and modulation of Ca2+ mobilizing mechanisms.

Changes in cytoplasmic free Ca2+ concentration, [Ca]i, elicited by ATP, were studied in neurones cultured from rat hippocampus and thalamus. ATP evoked [Ca]i increases in about 30% of all cells tested and suppressed [Ca]i transients in responsive cells. The number of responses to ATP markedly increased after pretreatment of cells with inhibitors of protein kinase C, H-7 or staurosporine. The potentiation was blocked by a phorbol ester and by dioleylglycerol. In pretreated cells both once peak [Ca]i and the number of successive trials were augmented by an [ATP] increase. The former effect can be described by the Michaelis-Menten equation whereas the latter one has a steeper, leftward-shifted dependence. Both concentration dependences are explained with a model, describing Ca2+ release as a threshold phenomena. ATP analogues had the rank of potency: ATP approximately ADP >> AMP > alpha, beta-MeATP. A single ATP application depleted internal Ca2+ stores which could be replenished by brief membrane depolarization with high-K+. ATP- and caffeine-induced [Ca]i transients were independent, indicating two non-overlapping Ca2+ storage sites. Only caffeine effects were potentiated at an elevated [Ca]i level, showing a Ca(2+)-induced Ca2+ release. Inhibitors of the Ca2+ pump in internal stores, ryanodine and sulphydryl reagents suppressed the ATP-induced [Ca]i transients, acting via different mechanisms.

Spatial and temporal control of intracellular free Ca2+ in chick sensory neurons.

Digital imaging of fura-2 fluorescence and the voltage-clamp technique were combined to study cytoplasmic free Ca2+ concentration, [Ca]i, in neurons cultured from chick dorsal root ganglia. Depolarizing pulses raised [Ca]i to a new steady-state level which was achieved earlier in neurites than in the soma. The rise in [Ca]i during stimulated bursting or rhythmic activity was also faster in neurites. After stimulation [Ca]i recovered monoexponentially in the soma and biexponentially in neurites. Application of 50 mM KCl produced membrane depolarization and a concomitant increase of [Ca]i. During wash-out [Ca]i often declined to an intermediate steady-state level at which it stayed for several minutes. Thereafter the resting level of [Ca]i was quickly restored. [Ca]i recovery was delayed after treating the cell with 2 microM thapsigargin, an inhibitor of the Ca2+ pump of internal Ca2+ stores. Caffeine (10 mM) transiently increased [Ca]i. A second caffeine application produced smaller [Ca]i changes due to the prior depletion of Ca2+ stores, which could be replenished by brief exposure to KCl. Thapsigargin (2 microM) transiently increased [Ca]i both in the standard and Ca(2+)-free solution. [Ca]i transients due to caffeine and thapsigargin started in the cell interior, in contrast to [Ca]i changes evoked by membrane depolarization, which were noticed first at the cell edge. Caffeine and thapsigargin induced a transient inward current which persisted in the presence of 1 mM La3+ and in Ca(2+)-free solutions, but which was greatly diminished in Na(+)-free solutions. The effects of caffeine and thapsigargin were mutually exclusive both in the generation of [Ca]i transients and in the inward current induction.

[Changes in the intracellular calcium concentration and transmembrane currents, evoked by iontophoretic injections of cyclic AMP, in Helix pomatia neurons]. / Izmeneniia vnutrikletochnoi kontsentratsii kal'tsiia transmembrannykh tokov, vyzvannye ionoforeticheskoi in"ektsiei tsAMF, v neironakh vinogradnoi ulitki.

Transmembrane currents and intracellular concentration of free Ca2+ were measured in voltage-clamped isolated neurons of Helix pomatia following the injection of cAMP. In most neurons in the range of membrane potentials from -40 to -100 mV cAMP injection induced both inward current and a long-lasting increase in [Ca2+]in. In the Ca-free external medium and after addition of EGTA (a Ca chelator) to it, the cAMP-induced inward current and [Ca2+]in increase remained unchanged. In most cases in Na-free external solution the cAMP-induced inward current markedly decreased, whereas [Ca2+]in changes remained as it were. Cd2+ (2 mM) did not affect the cAMP-induced current and [Ca2+]in increase. Both procaine++ and ryanodine (inhibitors of Ca release from intracellular stores) did not change the cAMP-induced effects. La3+ (1 mM) blocked both the inward current and an increase of [Ca2+]in. Obtained data confirm the hypothesis of cAMP-mediated Ca release from the intracellular stores.

[Relation between the surface potential of mouse neuroblastoma clone C1300 cells and the phase of the cell cycle]. / Zavisimost' poverkhnostnogo potentsiala kletok neiroblastomy myshi klona C1300 ot fazy kletochnogo tsikla.

The surface charge of neuroblastoma cells in different phases of the cell cycle was studied by the microelectrophoresis method. The surface charge increased by 50% on the average after addition of colchicine to the culture medium, by 20-30% after addition of dibutyryl-cAMP or removal of the serum from the medium and decreased by 30% after addition of DMSO. These changes correlated well with variations of the protein content per cell.

Surface charge of mammalian neurones as revealed by microelectrophoresis.

The surface charge of isolated rat dorsal root ganglion neurones was studied by microelectrophoresis technique. The increase of Ca concentration caused greater reduction of the electrophoretic mobility compared to that produced by an equivalent amount of divalent organic cations, dimethonium or hexamethonium. No charge reversal for Ca concentrations up to 80 mM was observed. These data fit the suggestion that two anion groups of the outer membrane surface can bind one Ca ion with apparent binding constant of about 50 M-1. In solutions of low pH the electrophoretic mobility of cells decreased corresponding to titration of acidic groups with apparent pK = 4.2. Trypsin treatment in mild conditions markedly reduced the surface charge; however, neuraminidase and hyaluronidase did not change it. N-bromosuccinimide (a specific reagent for carboxylic groups of proteins) decreased the electrophoretic mobility about 60%. However, no increase of the surface charge after the action of specific reagents for amino groups (2,4,6-trinitrobenzene-sulfonic acid and maleic anhydride) was observed. It was shown that the surface charge depends also on the intracellular metabolism. If 1 mM dibutyryl cAMP or theophilline was added to the culture medium (thus, raising the concentration of cAMP inside the cell) the surface charge increased. This effect developed slowly and reached its maximum on the third day of incubation. Treatment of cells by 5 mM tolbutamide (an inhibitor of some protein kinases) did not change cell mobility. Addition of 5 mM N-ethylmaleimide (an inhibitor of adenylate cyclase) to the culture medium produced some decrease of the surface charge.(ABSTRACT TRUNCATED AT 250 WORDS)

[Surface charge of mouse neuroblastoma cells and changes during growth and morphologic differentiation of the cell population]. / Poverkhnostnyi zariad kletok neiroblastomy myshi i ego izmenenie v protsesse rosta i morfologicheskoi differentsirovki kletochnoi populiatsii.

Microelectrophoresis method was used to study the surface charge of murine neuroblastoma cells (clone C1300-N18TG2). It was shown that the surface charge of these cells was determined mainly by anionic groups of the membrane which were distributed with density 0.2 e/nm3 in the layer covering its outer surface. The thickness of this layer was about 10 nm. These groups interacted with Ca ions (binding constant Kca-10-50 l/mol) and were titrated according to pK-3.8. Trypsin, neuraminidase and N-bromosuccinimide (which irreversibly neutralize the carboxylic groups of proteins) decreased the electrophoretic mobility of neuroblastoma cells while tosylchloride (a specific reagent for aminogroups) slightly increased it. The surface charge depended also on the conditions of cultivation of cell population. Morphological cell differentiation induced by removing the serum from the culture medium increased their mobility by about 30%. During the cultivation of cells in a medium with 10 or 50% of serum variations of the their mean electrophoretic mobility value were observed which were opposite-phase to the value of the daily increment of the number of cells. It was assumed that these effects were connected with partial self-synchronization of cell population. It is concluded that the surface charge of the neuroblastoma cells determined by microelectrophoresis method is mainly determined by the carboxylic groups of membrane periphery proteins and gangliosides and the content of these membrane components depends on the stage of cell development.

[2 calcium currents in the somatic membrane of neurons of Helix pomatia]. / Dva kal'tsievykh toka v somaticheskoi membrane neironov vinogradnoi ulitki.

Two different calcium currents were revealed in the somatic membrane of Helix pomatia neurons. In addition to the main current described in literature, depolarizing the membrane from the holding potential level (-120 divided by -100 mV) an additional calcium current was observed. It was activated at depolarizations to -80 divided by -40 mV. Contrary to the main calcium current it did not deteriorate during intracellular perfusion by solutions containing fluoride. Time-dependence of this current could be described in the framework of the Hodgkin-Huxley model with time constants for activation and inactivation equal to tau m = 6-8 ms and tau h = 300-600 ms, respectively. The amplitude of this current increased with increase of extracellular Ca2+ concentration and decreased after addition of Co2+, Ni2+, Cd2+, nifedipine and verapamil. Dissociation constants of these substances with corresponding channels determined for the maximum of current-voltage relationship were 2 (Ca2+), 3 (Co2+), 0.06 (nifedipine) and 0.2 mmol/l (verapamil). Properties of the fluoride-insensitive calcium current and data obtained for other calcium channels are compared. Its possible functional role is also discussed.