Biomedical subjects
Publications and source records attributed to O A Krishtal.
The development of electrophysiology is traced from the early beginnings represented by the work of the Dutch microscopist, Jan Swammerdam, in the 17th century through the first notion of an aqueous transmembrane pore as a substrate of excitability made by Luigi Galvani in late 18th century to the invention late in the 20th century of the patch-clamp technique by Erwin Neher and Bert Sakmann.
Neuroprotective action of MK-801 administrated pre- and postischemically, in vivo or in vitro, respectively, was studied on hippocampal slices using decapitation ischemia model. Recovery of orthodromic population spikes in CA1 region was measured during postischemic incubation of the slices with oxygenated artificial cerebrospinal fluid (ACSF). The ability of postischemically applied MK-801 to restore the electrical activity dramatically depended on the timing of its application during the reoxygenation period. When applied in vitro, together with the start of reoxygenation, MK-801 was as effective as in the case of in vivo administration before the ischemia. The delay in the in vitro administration for only a few minutes led to a dramatic decrease in the drug effectiveness. When applied in 30 min after the start of reoxygenation, MK-801 was totally ineffective. The dose/response relationship between MK-801 concentration and the amplitude of recovered orthodromic population spikes of hippocampal pyramidal neurons is logarithmic. The ED(50) value for the action of "postischemic" MK-801 is ca. 10(-5) M. Preischemic in vivo application of the drug [intraperitoneal (i.p.) injection 15 min prior to decapitation] results in ED(50) ca. 0,2 mg/kg. The slope of both dose/concentration-response curves is similar. The time course of population spike recovery after 90-min ischemia is identical for pre- and postischemic action of MK-801 (estimated for ED(50) in both cases). These data allow to suggest that "preischemic" MK-801 is predominantly active as a neuroprotector only after ischemia, within a short therapeutic window at the start of the reoxygenation period.
The constituents of Ginkgo biloba leaf extract, ginkgolides A, B, C and J are known as effective antagonists of platelet-activating factor (PAF). Here, we will demonstrate that these substances are also effective blockers of glycine-activated chloride channels in the hippocampal neurons of rat. As examined in several other voltage- and ligand-operated channels, this ginkgolide action is selective. The blocking action of all tested ginkgolides is use-dependent--they block open glycine-activated channels. The IC (50) values for saturating blocking action of ginkgolides B and C are 0.273 microM and 0.267 microM, respectively, while ginkgolides A and J are less effective--IC (50) values are 1.97 microM and 2.0 microM. Corresponding dose-response relationships are close to single-site binding isotherms. Another constituent of EGb 761, bilobalide, is a weak inhibitor of NMDA receptor-activated current. Its synthetic analogue, NV-31, demonstrates a weak facilitatory action on Gly-activated conductance. Novel findings have indicated the possibility that the unique modulating activity profiles of the EGb 761 (definition see editorial) constituents examined are due to their effects on the anion homeostasis of central neurons.
We investigated modulation of GABA(A) receptor-mediated whole-cell currents in cerebellar Purkinje neurones by several derivatives of benzophenone. A metabolite of phenazepam, 5-bromo-2'-chloro-2-aminobenzophenone (I), caused dual modification of peak amplitudes of GABA-gated currents that depended upon the concentration of applied GABA and incubation time. Following short 10 s pre-incubations, 1-30 microM I facilitated activation and delayed deactivation of currents evoked by 500 ms pulses of 20 microM GABA. In addition, 10 microM I prominently enhanced desensitisation of currents during applications of 500 microM GABA mainly by decreasing the value of the fast time constant of the desensitisation. Continuous 6 min incubation with 10 microM I during GABA stimulation or its administration between but not during 1 s pulses of 500 microM GABA led to a gradual, partly reversible attenuation of GABA-activated currents. This inhibition was not observed when I was applied only during pulses of GABA, indicating that the blockade was not use-dependent. One of the possible mechanisms of this down-modulation could be an intracellular effect of I, because when applied intracellularly it caused slow inhibition of responses to consecutive GABA pulses. When 3-30 microM I was applied on the background of small 'plateau'-like current 5-7 s after application of 500 microM GABA, it was able to block open channels with on and off rates similar to those observed with 30 microM picrotoxin but much slower than in the case of 500 microM benzylpenicillin. At a concentration of 10 microM, 5-substituted benzophenones, but not 2-aminobenzophenone or benzophenone itself, exhibited modulatory properties similar to I and distinct from those of picrotoxin and benzylpenicillin. Therefore, we conclude that derivatives of benzophenone are a novel class of GABA(A) receptor modulators with a unique pharmacological profile.
We examined effects of omega-conotoxin previously known as a selective blocker of N-type calcium channels, on the adenosine triphosphate (ATP)-induced currents in the rat dorsal root ganglion neurons. These neurons express at least two types of ionotropic purinoreceptors: P2X3 receptors that have very rapid desensitization kinetics and P2X2/X3 heterooligomeric receptor, which exhibits slow desensitization. We have found that omega-conotoxin GVIA potently inhibits the inward currents mediated by both receptor types. This effect was specific for the receptor subtypes: the IC(50) value for responses evoked by 10 microM ATP was 21.2 +/- 1.7 nM for the P2X3 receptor-mediated responses and 3.84 +/- 0.43 microM for slower responses mediated by P2X2/X3 heteropolymers. The efficacy of another type of omega-conotoxin, MVIIC, is much lower: at 10 microM the latter toxin inhibited the rapidly desensitizing response by 65% and the slowly desensitizing response by 18%. The effects of both toxins were reversible and independent on the membrane potential. Omega-Conotoxin GVIA shifted the dose dependence for the agonistic action of ATP on P2X3 receptors to higher concentrations without producing any effect on the kinetics of the response. It is suggested that omega-conotoxin allosterically modulates the receptor properties, rather than competes for the agonist binding site.
Recovery of orthodromic and antidromic population spikes in CA1 hippocampal slices of 30-day-old Wistar rats has been studied in the reperfusion period after prolonged (90 min) decapitation ischemia with and without preceding 15 min long non-voluntary motor activity of intact animals. The preconditioning motor activity significantly enhances the resistance of pyramidal neurons to ischemia at a temperature of 30 degrees C. The period of protection lasts for up to 40 min after the end of motor activity. In case the ischemia was started within 5-10 min after the preconditioning, complete restoration of the field potentials to preischemic control level could be achieved. These data are the first indication of the neuroprotective effect of preconditioning motor activity in CA1 damage after prolonged global ischemia.
The effects of 7-bromo-5-(2-chlorophenyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one (phenazepam, PNZ), a 1,4-benzodiazepine derivative, and its physiological metabolites on GABA-activated whole-cell currents were studied in enzymatically isolated rat Purkinje neurones. PNZ, its hydroxylated metabolite (HPNZ) and a reference benzodiazepine, diazepam, potently enhanced (up to 200% of control) peak amplitude of currents activated by 10 microM GABA with EC50s of 6.1 +/- 0.8, 10.3 +/- 1.4 and 13.5 +/- 1.9 nM respectively. Both PNZ and HPNZ caused a parallel leftwards shift of the concentration/effect relationship for GABA. Another metabolite, 6-bromo-(2-chlorophenyl) quinazoline-2-one (QNZ), augmented responses to 10 microM GABA with a maximal efficacy similar to that of the 1,4benzodiazepines tested, although its EC50 was 2.4 +/- 0.2 microM. A further metabolite, 5-bromo-(2-chlorophenyl)-2-aminobenzophenone (ABPH), had only minimal effects on the responses elicited by 10 microM GABA. Incubation with QNZ and ABPH had biphasic effects on the concentration/effect relationship for GABA. These compounds enhanced peak amplitudes of currents activated by low concentrations of GABA, but inhibited responses to saturating concentrations of the agonist. This effect could, in part, be explained by the acceleration of the desensitisation process by those substances. It is concluded that both PNZ and HPNZ can be referred to as full positive modulators of GABAA receptors and that they are primarily responsible for GABAergic effects of therapeutic doses of PNZ.
A simple and reproducible animal model of global ischemia, induced by decapitation in 30-day-old Wistar rats, has been developed. It allows to perform electrophysiological analysis of the postischemic reperfusion period in the brain slices. Periods of ischemia up to 40 min increase population spikes measured in the CA1 area of the hippocampus during 2-5 h of reperfusion. Thus after 30-min decapitation-induced ischemia (at t(ischem)=25 degrees C), the mean amplitude of the recorded maximum orthodromic population spikes was 159% of the control obtained in the non-ischemic animals. Longer ischemic episodes result in the depression of the population spikes. After 2 h of ischemia, the amplitude of population spikes was about 89% of control. After 3 h of decapitation ischemia, the neurons could not be reactivated. The duration of ischemic episode needed for the irreversible depression of the electrical activity of the brain neurons drastically depends on the temperature at which the ischemic brain is maintained. Thus, only 2 h were needed at 30 degrees C as compared to nearly 3 h at 25 degrees C. We have found that intraperitoneal injection of neuroprotectors which precedes decapitation enables reactivation of the post-ischemic neurons even after very long periods of global ischemia. Thus, MK-801, a non-competitive NMDA receptors antagonist, or NBQX, a blocker of AMPA receptors, administrated 15 min before the long-term (90 min) decapitation ischemia (30 degrees C), induced dose-dependent recovery of population spike with ED(50) values 0.2 mg/kg and 3 mg/kg respectively. Our results demonstrate that, in spite of the high vulnerability of hippocampal neurons to hypoxia and ischemia, their electrical activity can be restored after prolonged (more then 1 h) decapitation ischemia. Administration of NMDA or AMPA antagonists enhances recovery.
Properties of excitatory postsynaptic currents during increased glutamate release were investigated by means of a whole-cell voltage-clamp in CA1 pyramidal neurons of rat hippocampal slices. Enhancement of transmitter release by 50 microM 4-aminopyridine or by elevated extracellular Ca2+ (up to 5 mM) resulted in a substantial increase in the peak excitatory postsynaptic current amplitude and in the significant stimulus-dependent prolongation of the excitatory postsynaptic current decay. The stronger the stimulus, the slower the excitatory postsynaptic current decay became. The pharmacologically isolated N-methyl-D-aspartate, but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid component of the excitatory postsynaptic current exhibited this phenomenon. The possible connection of such behaviour of the N-methyl-D-aspartate component to the loss of voltage control was tested in the following way: the peak of the N-methyl-D-aspartate component was enhanced under 50 microM 4-aminopyridine and then returned back to the control level by a low dose of D-2-amino-5-phosphonopentanoic acid. However, the decay of the decreased N-methyl-D-aspartate component remained slow suggesting another origin of the stimulus-dependent kinetics. Dihydrokainate, a non-competitive inhibitor of glutamate uptake, did not influence the kinetics of the N-methyl-D-aspartate component in control but induced its dramatic stimulus-dependent prolongation when applied on the background of a low dose of 4-aminopyridine (10 microM) which did not affect the decay by itself. We propose that the delayed stimulus-dependent kinetics of the N-methyl-D-aspartate component is due to the saturation of uptake mechanisms and subsequent activation of extrasynaptic N-methyl-D-aspartate receptors. Our present observations therefore support the hypothesis that N-methyl-D-aspartate receptors may play a role in the cross-talk between synapses by means of the transmitter spillover.
KA-672.HCl (KA-672) is a new substance demonstrating anti-dementia properties. It shows modulatory effects on several neurotransmitter systems known to be affected in patients with Alzheimer's disease. In this study the action of KA-672 on the NMDA receptors was examined by applying patch clamp techniques to acutely isolated hippocampal neurons. KA-672 antagonizes NMDA responses in a voltage-dependent manner. At a holding potential of -90 mV the IC50 value for the blocking action of KA-672 was 20+/-7 microM. This action of KA-672 is independent on the concentration either of agonist or coagonist of NMDA receptor. Ketamine, which interacts with the PCP center, does not occlude the action of KA-672. Evidently, KA-672.HCl is a weak NMDA receptor-operated channel blocker. This property may account for its pharmacological profile.
Somato-dendritic subthreshold transient potassium current [I(SA)] was measured in acutely isolated rat hippocampal CA1 pyramidal neurons. The inactivation of this current was insensitive to externally applied H2O2 (20 mM) which causes cysteine oxidation. This result suggests that Shal-channels not Shaker Kv1.4 channels underlie the somato-dendritic I(SA) in rat CA1 pyramidal neurons. The kinetics of the I(SA) inactivation was measured at various [K+]out. Increase in [K+]out leads to acceleration of Shal-channel inactivation. Thus, the shift in [K+]out from 1 to 50 mM results in decreased inactivation time constant from 37 to 19 ms. This effect of [K+]out on the I(SA) is opposite to the previously described action of [K+]out on the inactivation of Shaker K+ channels.
The action of synthetic kava pyrones, (+)-methysticin and (+/-)-kavain, on voltage-operated Na(+)-channels was studied in whole-cell patch-clamped CA1 hippocampal neurons. In doses of 1-400 microM, both compounds exerted a rapid and reversible inhibition of the peak amplitude of Na(+)-currents. Shifting holding membrane potential (Vhold) to more positive values enhanced their blocking effect. The drugs studied did not demonstrate use-dependent properties at 10 Hz stimulation but shifted H infinity curve toward more negative potentials, accelerated time-course of inactivation and slowed down the recovery from inactivation. Voltage-dependence of Na(+)-channel inhibition can be explained by interaction of (+)-methysticin and (+/-)-kavain with resting closed and inactivated states of Na(+)-channel.
The action of diadenosine polyphosphates on Ca2+ channels was studied in two preparations: isolated hippocampal neurons and synaptosomes, both from the rat brain. High-voltage-activated Ca2+ channels were recorded in freshly isolated CA3 neurons using a whole-cell patch-clamp technique. Current-voltage relationships were measured in the control and after incubation in 5 microM diadenosine pentaphosphate. In the majority of tested pyramidal neurons, the latter procedure led to a reversible increase in the high-voltage-activated current through Ca2+ channels when measured at the holding potential of -100 mV but not at -40 mV. In experiments on synaptosomes from the whole brain, diadenosine pentaphosphate taken at a concentration of 100 microM increased the intrasynaptosomal calcium level measured by means of spectrofluorimetry for 26 +/- 1.8 nM (by 24 +/- 2%). Nifedipine failed to block this effect both in synaptosomes and hippocampal neurons. Potentiation of the current through Ca2+ channels in hippocampal neurons as well as the increase in intrasynaptosomal Ca2+ were irreversibly blocked by 5 microM omega-conotoxin, but not by 200 nM omega-Agatoxin-IVA. These data indicate that diadenosine polyphosphates enhance the activity of N-type Ca2+ channels in many central neurons of the rat brain.
Patch- and concentration-clamp techniques were used to compare the effects of the uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists (+)-MK-801 (dizocilpine, (+)-5-methyl-10, 11-dihydro-5H-dibenzocyclohepten-5, 10-imine maleate), ketamine, memantine (1-amino-3,5-dimethyladamantane) and amantadine (1-amino-adamantane) on agonist-induced inward currents in freshly dissociated rat hippocampal and striatal neurons. In hippocampal neurons, ketamine (5 microM), menantine (10 microM) and amantadine (100 microM) selectively antagonized inward current responses to NMDA (500 microM plus glycine 5 microM) in a voltage-dependent manner without affecting responses to (s)-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (100 microM) or gamma-aminobutyric acid (10 microM). The NMDA receptor antagonistic effect of all four agents was typical of open channel blockade. The kinetics of blockade/unblockade was inversely related to antagonist affinity. In hippocampal neurons amantadine was the least potent NMDA receptor antagonist (IC50 18.6 +/- 0.9 microM) and showed the fastest blocking kinetics, whereas (+)-MK-801 was the most potent (IC50 0.12 +/- 0.01 microM) and showed the slowest blocking kinetics. Memantine (IC50 1.04 +/- 0.26 microM) and ketamine (IC50 0.43 +/- 0.10 microM) were almost equipotent and had similar, intermediate blocking kinetics. In striatal neurons recorded under identical conditions (+)-MK-801, ketamine and memantine were 3- to 4-fold less potent whereas amantadine was somewhat more potent than on hippocampal neurons. This could offer an explanation for the better clinical profile of amantadine in Parkinson's disease, as therapeutically relevant concentrations of amantadine are likely to be more active in the striatum whereas memantine is likely to be more active in other structures.
Effects of capsaicin, an essential ingredient of hot peppers, on high voltage-activated (HVA) calcium channels were investigated in voltage-clamped and internally perfused acutely dissociated rat trigeminal and hippocampal neurones. In micromolar concentrations capsaicin inhibited the whole HVA Ca2+ current without affecting resting membrane conductance in both types of neurones. IC50 values of 14.5 microM and 21.2 microM (n = 5) were obtained in sensory and hippocampal neurones, respectively. Capsaicin-induced inhibition became irreversible after prolonged incubation (30 s) with the drug. It is concluded that capsaicin is a nonspecific blocker of HVA Ca2+ channels in different types of nerve cells.
Long-term potentiation is currently a leading candidate for a physiological memory mechanism in CNS. The interpretation of this phenomenon is contradictory in many respects. However, there is clear evidence that long-term potentiation is critically dependent on activation of N-methyl-D-aspartate receptors. Recently it has been shown that extracellularly applied glutamate also induces long-lasting changes in the properties of synaptic transmission in the hippocampus that can be attributed to long-term potentiation. The involvement of presynaptic mechanisms has been reported. Here we demonstrate a definite increase in both the open time and open-state probability of N-methyl-D-aspartate-operated channels, induced by prolonged application of glutamate to hippocampal slices.
Dose-related blocking effects of R56865, flunarizine and nimodipine on voltage-activated Na+ currents recorded in the whole-cell voltage clamp mode were studied in acutely isolated Purkinje neurons of rat cerebellum. The dose-dependences of blocking action were obtained for all drugs at a holding potential of -110 mV and rare stimulation. At stimulation frequencies 5 and 15 Hz the block produced by R56865 was increased showing a shift of dose-dependence to lower concentrations of antagonist. This shift was less pronounced for flunarizine, practically absent for nimodipine, and increased for all drugs with an increase in the amplitude of stimulating voltage pulse. With the change in holding potential to -80 mV the block produced by R56865 and flunarizine increased showing a dose-dependence shift to lower concentrations of antagonists. All the drugs tested induced parallel shifts of the steady-state voltage-dependence of inactivation of Na+ channels to more negative membrane potentials. R56865, and to a lesser extent flunarizine, slowed down the recovery of Na+ channels from steady-state inactivation increasing the relative number of channels which showed slow recovery. In the absence of Na+ current inactivation (treatment by intracellular pronase) R56865 at a concentration of 1 microM blocked modified channels preferentially in the open state, while the block produced by flunarizine showed no dependence on voltage pulse protocol. R56865 was shown to decrease the cell leakage while other drugs produced little or no effect. It is concluded that R56865 and flunarizine block Na+ currents predominantly by interacting with inactivated Na+ channels. The higher ability of R56865 to block open channels and to increase slow inactivation underlies its higher frequency-dependence. These characteristics suggest the use of R56865 and flunarizine in the treatment of cerebral ischemia.
The blocking action of recently synthesized benzothiazolamine derivative R56865 was compared with that of dihydropyridine (nimodipine) and diphenylalkylamine (flunarizine) on low-voltage-activated and non-inactivating high-voltage-activated Ca2+ currents. The experiments were carried out on freshly isolated Purkinje neurons of rat cerebellum using patch-clamp technique in the whole-cell configuration. Among the substances tested R56865 was found to be the most effective blocker of the Ca2+ current. In the sequence R56865, flunarizine and nimodipine, apparent Kd values for low-voltage-activated current are 0.1, 0.9 and 3.5 microM, and for high-voltage-activated current 3.1, 9.5 and 38 microM, respectively. The current-voltage relationships for both types of currents displayed little or no shift under either flunarizine or R56865 but showed a 10-mV shift in the positive direction under the action of nimodipine. The steady-state inactivation curves for low-voltage-activated calcium currents were shifted under the action of R56865, flunarizine and nimodipine (in concentrations which blocked 50-60% of the current) to more negative membrane potentials for 20, 10 and 6 mV, respectively. In contrast to R56865, flunarizine blocked both types of Ca2+ channel in a use-dependent manner. It is concluded that the order of potency of Ca2+ antagonist for both types of channels studied is R56865 > flunarizine > nimodipine. Strong shift of steady-state inactivation relationship by R56865 can further facilitate its blocking action in in vivo conditions.