Novel mechanism for temperature-independent transitions in flexible molecules: role of thermodynamic fluctuations.

A novel physical mechanism is proposed to explain the temperature-independent transition reactions in molecular systems. The mechanism becomes effective in the case of conformation transitions between quasi-isoenergetic molecular states. It is shown that at room temperatures, stochastic broadening of molecular energy levels predominates the energy of low-frequency vibrations accompanying the transition. This leads to a cancellation of temperature dependence in the stochastically averaged rate constants. As an example, a physical interpretation of temperature-independent onset of P2X{3} receptor desensitization in neuronal membranes is provided.

P2X receptors and synaptic plasticity.

Adenosine triphosphate (ATP) is released in many synapses in the CNS either together with other neurotransmitters, such as glutamate and GABA, or on its own. Postsynaptic action of ATP is mediated through metabotropic P2Y and ionotropic P2X receptors abundantly expressed in neural cells. Activation of P2X receptors induces fast excitatory postsynaptic currents in synapses located in various brain regions, including medial habenula, hippocampus and cortex. P2X receptors display relatively high Ca2+ permeability and can mediate substantial Ca2+ influx at resting membrane potential. P2X receptors can dynamically interact with other neurotransmitter receptors, including N-methyl-D-aspartate (NMDA) receptors, GABA(A) receptors and nicotinic acetylcholine (ACh) receptors. Activation of P2X receptors has multiple modulatory effects on synaptic plasticity, either inhibiting or facilitating the long-term changes of synaptic strength depending on physiological context. At the same time precise mechanisms of P2X-dependent regulation of synaptic plasticity remain elusive. Further understanding of the role of P2X receptors in regulation of synaptic transmission in the CNS requires dissection of P2X-mediated effects on pre-synaptic terminals, postsynaptic membrane and glial cells.

[ROLE PHOSPHOINOSITID SIGNALING PATHWAY IN OPIOIDS CONTROL OF P2X3 RECEPTORS IN THE PRIMARY SENSORY NEURONS].

Homomeric P2X3 receptors expressed in primary nociceptive neurons are crucial elements in the pain signal generation. In turn, opioid system regulates the intensity of this signal in both CNS and PNS. Here we describe the effects of opioids on P2X3 receptors in DRG neurons studied by using patch clamp technique. Activation of G-protein coupled opioid receptors by endogenous opioid Leu-enkephalin (Leu), resulted in the two opposite effects on P2X3 receptor-mediated currents (P2X3 currents). In particular, application of 1 µM Leu lead to the complete inhibition of P2X3 currents. However, after pretreatment of the neurons with a Gi/o-protein inhibitor pertussis toxin (PT), the same concentration of Leu caused facilitation of P2X3 currents. PLC inhibitor U-73122 at concentration of 1 µM completely eliminated both facilitating and inhibitory effects of Leu on P2X3 currents. Thus, opioid receptor agonists cause two oppositely directed effects on P2X3 receptors in DRG neurons of rats and both of them are mediated through PLC signaling pathway. Our results point to a possible molecular basis of the mechanism for the well-known transition inhibitory action of opioids (analgesia) to facilitating (hyperalgesia).

Cross-desensitization Reveals Pharmacological Specificity of Excitatory Amino Acid Receptors in Isolated Hippocampal Neurons.

Ionic currents elicited by excitatory amino acids were studied, using the concentration clamp method, in enzymatically isolated rat hippocampal neurons. Cross-desensitization between the responses to various agonists was applied to separate the activity of two types of receptors, N-methyl-d-aspartate (NMDA) and non-NMDA. NMDA receptors were selectively activated by NMDA, l- and d-aspartate, d-glutamate and quinolinate. Kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate appeared to be selective, and quisqualate relatively less selective non-NMDA agonists, acting on the same receptor type. l-Glutamate, l- and d-homocysteate activated both receptor types. It is supposed that two receptor sites, activation site and desensitization site, control the action of agonists at the non-NMDA receptor. When examined in the cross-desensitization experiments, NMDA and non-NMDA receptors appear to be represented by the two homogeneous and independent receptor populations operating different ionic channels.

Modulation of GABA(A) receptor-mediated currents by benzophenone derivatives in isolated rat Purkinje neurones.

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.

Glutamate induces long-term increase in the frequency of single N-methyl-D-aspartate channel openings in hippocampal CA1 neurons examined in situ.

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.

R56865 as Ca(2+)-channel blocker in Purkinje neurons of rat: comparison with flunarizine and nimodipine.

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.

Cationic channels activated by extracellular ATP in rat sensory neurons.

Single channels activated by externally applied ATP were investigated in cultured sensory neurons from nodosal and spinal ganglia of rat using patch clamp and concentration clamp methods. Mean conductance of single ATP-activated channels was 17 pS when measured at a holding potential of -75 mV in saline containing 3 mM Ca2+ and 1 mM Mg2+. Sublevels of conductance were detected in some cases. The current-voltage relationship for a single channel is highly non-linear and demonstrates inwardly directed rectification. The I-V curve obtained for single channels was identical to that for macroscopic current. ATP activated the channels in the absence of divalent cations (in ethylenediaminetetra-acetate-containing medium) as well as in their presence. This indicates that ATP as a free anion can activate the receptor. Ca2+ ions decreased both macro- and microscopic ATP-activated currents. The concentration dependence of this Ca2+ effect does not fit a single site binding isotherm. The single channel current demonstrated prominent fluctuations. When measured in the 0-4 kHz frequency band the amplitude of fluctuations evaluated as a double r.m.s. was about 30% of the mean amplitude of current. The autocorrelation function for the current fluctuations in an open channel could be approximated by a single exponential with the time constant of 0.4 ms. These fluctuations did not depend on the presence of divalent cations in the external medium. The open time distribution for the investigated channels could be described by a sum of two exponentials. Presumably this reflects the existence of two subtypes of ATP-activated channels.

R56865 and flunarizine as Na(+)-channel blockers in isolated Purkinje neurons of rat cerebellum.

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.

A highly potent and selective N-methyl-D-aspartate receptor antagonist from the venom of the Agelenopsis aperta spider.

Agatoxin-489, extracted from the venom of the Agelenopsis aperta spider, was studied on acutely isolated perfused hippocampal neurons of rat using the concentration clamp technique. Agatoxin-489 proved to be a selective N-methyl-D-aspartate antagonist; responses to applications of N-methyl-D-aspartate or L-aspartate were blocked by concentrations of agatoxin-489 ranging between 0.1 nM and 1 microM, while responses to kainate were not affected by agatoxin-489 at concentrations up to 10 microM. The actions of agatoxin-489 against responses to N-methyl-D-aspartate or L-aspartate were use- and voltage-dependent, being less pronounced with an increase in the holding potential from -100 to -30 mV. The action of agatoxin-489 could be completely or partially reversed only after washout in the presence of an N-methyl-D-aspartate agonist. The washout was more effective at positive membrane potentials ranging from 0 to +20 mV. These results imply that the spider toxin agatoxin-489, like dizocilpine, is a potent and selective N-methyl-D-aspartate antagonist which preferentially interacts with activated N-methyl-D-aspartate receptors and/or open N-methyl-D-aspartate-activated ionic channels.

Enhancement of glutamate release uncovers spillover-mediated transmission by N-methyl-D-aspartate receptors in the rat hippocampus.

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.

Kava extract ingredients, (+)-methysticin and (+/-)-kavain inhibit voltage-operated Na(+)-channels in rat CA1 hippocampal neurons.

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.

Diadenosine polyphosphates selectively potentiate N-type Ca2+ channels in rat central neurons.

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.

Receptors for ATP in rat sensory neurones: the structure-function relationship for ligands.

1. The pharmacological properties of the ATP-activated conductance in isolated sensory neurones of the rat were investigated by use of voltage clamp and concentration clamp techniques. 2. Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), cytidine 5'-triphosphate (CTP), cytidine 5'-diphosphate (CDP) and some derivatives activate these receptors, whereas adenosine 5'-monophosphate (AMP), cytidine 5'-monophosphate (CMP) and other naturally-occurring nucleotides are competitive blockers. 3. In the sequence of substances, adenosine 5'-(beta,gamma-methylene)-triphosphonate (APPCP), adenosine 5'-(beta,gamma-difluoromethylene)- triphosphonate (APPCF2P), adenosine 5'-(beta,gamma-dichloromethylene)-triphosphonate (APPCC12P) and adenosine 5'-(beta,gamma-dibromomethylene)triphosphonate (APPCBr2P), the properties of ligands depend on the radius of the atom linked to the carbon of the diphosphonate group. Thus, APPCP is an agonist, APPCF2P is a partial agonist, while dichloromethylene and dibromomethylene analogues of adenosine 5'-(beta,gamma-methylene)triphosphonate demonstrate features of competitive blockers. APPCC12P is the most effective blocker of ATP-receptors (inhibition constant Ki = 21 +/- 4 microM). An adenosyl or adenylyl radical, when connected to the terminal phosphate of ATP, converts the agonist into a partial agonist. 4. Two especially important parts of the ATP molecule are crucial for the interactions with receptors. They are: (1) the vicinity of C6 of the purine ring and (2) the polyphosphate chain. Some modifications in these regions of the molecule result in the transformation of an agonist into an antagonist.

[K+]out accelerates inactivation of Shal-channels responsible for A-current in rat CA1 neurons.

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.

A novel selective NMDA agonist, N-phthalamoyl-L-glutamic acid (PhGA).

Ionic currents elicited by N-phthalamoyl-L-glutamic acid (PhGA) were investigated on freshly isolated hippocampal neurons with the whole-cell voltage clamp and concentration clamp techniques. PhGA elicited desensitizing inward currents in Mg(2+)-free salines only in the presence of glycine. The dose-response relationship for PhGA was close to a Langmuir isotherm with Kd = 3.7 mM and saturating level 0.75 of that for L-aspartate (L-Asp). PhGA-activated currents were blocked by Mg2+, D-2-amino-5-phosphonovalerate and kynurenate, and had the same reversal potential as L-Asp-activated currents. Complete cross-desensitization was obtained between the responses to PhGA and L-Asp. We conclude that PhGA is a new selective 'superacidic' agonist of the N-methyl-D-aspartate type of glutamate receptor.

Capsaicin blocks Ca2+ channels in isolated rat trigeminal and hippocampal neurones.

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.

The putative cognitive enhancer KA-672.HCl is an uncompetitive voltage-dependent NMDA receptor antagonist.

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.

Receptor for protons in the membrane of sensory neurons.

The neurons isolated from spinal ganglia and from the ganglion of trigeminal nerve of a rat were investigated in the conditions of intracellular perfusion and voltage clamp. Many cells responded to rapid changes of the external pH from 7.4 to l.9 and lower values by pH-dependent desensitizing inward current. This current was saturated at pH 5.4 (pKa 6.2) and carried by Na+ and K+ ions )PK:PNa approximately equal to 0.1). The discovered mechanism is capable of producing depolarization of the neuronal membrane in response to a weak acidification of the external medium.

Therapeutic time window for the neuroprotective action of MK-801 after decapitation ischemia: hippocampal slice data.

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.