Acid-sensing ion channels regulate spontaneous inhibitory activity in the hippocampus: possible implications for epilepsy.

Acid-sensing ion channels (ASICs) play an important role in numerous functions in the central and peripheral nervous systems ranging from memory and emotions to pain. The data correspond to a recent notion that each neuron and many glial cells of the mammalian brain express at least one member of the ASIC family. However, the mechanisms underlying the involvement of ASICs in neuronal activity are poorly understood. However, there are two exceptions, namely, the straightforward role of ASICs in proton-based synaptic transmission in certain brain areas and the role of the Ca(2+)-permeable ASIC1a subtype in ischaemic cell death. Using a novel orthosteric ASIC antagonist, we have found that ASICs specifically control the frequency of spontaneous inhibitory synaptic activity in the hippocampus. Inhibition of ASICs leads to a strong increase in the frequency of spontaneous inhibitory postsynaptic currents. This effect is presynaptic because it is fully reproducible in single synaptic boutons attached to isolated hippocampal neurons. In concert with this observation, inhibition of the ASIC current diminishes epileptic discharges in a low Mg(2+) model of epilepsy in hippocampal slices and significantly reduces kainate-induced discharges in the hippocampus in vivo Our results reveal a significant novel role for ASICs.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'.

[VOLTAGE-GATED CALCIUM CHANNELS: CLASSIFICATION AND PHARMACOLOGICAL PROPERTIES (PART I).]

Calcium influx though voltage-gated calcium channels mediate a huge amount of physiological events and cellular responses. Numerous scientific reports indicate that calcium channels are involved in synaptic transmission, neurotransmitter release, regulation of gene expression, cellular membrane voltage oscillations, pacemaker activity, secretion of specific substances from nerve and secretory cells, morphological differentiation, activation of calcium-dependent enzymes, etc. This review represents the modern classification, molecular structure, physiological and pharmacological properties of voltage-gated calcium channels expressed in mammalian cells.

Thrombin modulates persistent sodium current in CA1 pyramidal neurons of young and adult rat hippocampus.

Serine protease thrombin, a key factor of blood coagulation, participates in many neuronal processes important for normal brain functioning and during pathological conditions involving abnormal neuronal synchronization, neurodegeneration and inflammation. Our previous study on CA3 pyramidal neurons showed that application ofthrombin through the activation of specific protease-activated receptor 1 (PAR1) produces a significant hyperpolarizing shift of the activation of the TTX-sensitive persistent voltage-gated Na+ current (I(Nap)) thereby affecting membrane potential and seizure threshold at the network level. It was shown that PAR1 is also expressed in CA1 area of hippocampus and can be implicated in neuronal damage in this area after status epilepticus. The aim of the present study was to evaluate the effect of thrombin on I(NaP) in CA1 pyramidal neurons from adult and young rats. Using whole cell patch-clamp technique we demonstrate that thrombin application results in the hyperpolarization shift of I(NaP) activation as well as increase in the I(NaP) amplitude in both age groups. We have found that I(NaP) in pyramidal neurons of hippocampal CA 1 region is more vulnerable to the thrombin action than I(NaP) in pyramidal neurons of hippocampal CA3 region. We have also found that the immature hippocampus is more sensitive to thrombin action which emphasizes the contribution of thrombin-dependent pathway to the regulation of neuronal activity in immature brain.

[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).

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction.

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.

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.

Extracellular cAMP inhibits P2X receptors in rat sensory neurones through G protein-mediated mechanism.

AIM: To identify the mechanisms of P2X(3) receptor inhibition by extracellular cyclic adenosine monophosphate (cAMP) in rat dorsal root ganglion (DRG) neurones. METHODS: Whole-cell currents were measured in cultured DRG neurones using the combination of voltage and concentration clamp. RESULTS: We have found that extracellular cAMP inhibits P2X(3)-mediated currents in a concentration- and use-dependent manner. The P2X(3) currents, activated by ATP applied every 4 min, were inhibited by 55% in the presence of 10 microm cAMP and by 81% in the presence of 30 microm cAMP. At 8 min interval between ATP applications the same concentration of cAMP did not alter the currents. Addition of 0.5 mm of guanosine 5'-O-(2-thiodiphosphate) to intracellular solution blocked the inhibitory action of cAMP. The inhibitory effects of cAMP were not mimicked by extracellular application of 30 mum adenosine. CONCLUSIONS: In this paper, we demonstrate, for the first time, that extracellular application of cAMP to rat sensory neurones inhibits P2X(3) receptors via a G protein-coupled mechanism in a use-dependent manner, thus indicating the neuronal expression of specific plasmalemmal cAMP receptor.

P2X receptors in sensory neurons co-cultured with cancer cells exhibit a decrease in opioid sensitivity.

Opioids are known to control the activity of P2X receptors in the sensory neurons of rats. These receptors are important in persistent pain signaling. However, there are extremely severe pain states, such as those associated with metastatic diseases, that are refractory to opioid treatment. We have tested the possibility that cancer cells affect the sensitivity of P2X(2/3) and P2X(2) receptors to opiates. The sensitivity of ATP-activated currents to the selective mu-opioid receptor agonist endomorphin-1 was evaluated in rat nodose neurons co-cultured (on separate coverslips) with fibrosarcoma cells (NCTC 2472) using whole-cell patch-clamp recordings. Both in control and in co-cultured neurons, P2X-mediated responses exhibited highly variable biphasic desensitization kinetics with fast and slow components. However, ATP-activated currents in co-cultured neurons acquired a new feature: the degree of their inhibition by endomorphin-1 demonstrated strong dependence on their desensitization kinetics. The neurons with 'slower' responses were subject to a smaller inhibitory effect of the opioid. The 'ultra-slow' responses completely lost their sensitivity to the opioid. The occurrence of such responses, rarely observed in the control neurons, was considerably increased with the duration of co-culturing. Application of endomorphin-1 to nodose neurons, co-cultured with rapidly proliferating but non-malignant cells (fibroblasts), resulted in data similar to those for the control. In summary, fibrosarcoma cells release diffusible factors altering the properties of desensitization kinetics of P2X receptors and, in particular, decrease their sensitivity to opioid inhibitory control. These phenomena may increase neuronal excitability initiated by peripheral ATP release and thereby contribute to the decreased sensitivity of cancer pain to opioids.

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.

P2X3 receptor gating near normal body temperature.

P2X3 purinoreceptors expressed in mammalian sensory neurons are involved in nociception, mechanosensory transduction, and temperature sensation. Homomeric P2X3 receptors desensitize rapidly (<500 ms after activation by an agonist) and recover from desensitization very slowly (20-25 min at room temperature). They are susceptible to use-dependent inhibition by low nanomolar concentrations of ATP through developing the "high-affinity binding site" (HABS), which traps ATP molecules, thus keeping receptors in a desensitized state (Pratt et al., J Neurosci 25:7359-7365, 2005). Indeed, here we demonstrated directly that the desensitization of the receptor, after being activated by ATP, proceeds independently of the presence of agonist. We found that the temperature sensitivity of P2X3 receptors is abnormal: development of desensitization does not depend on temperature within the range between 25 and 40 degrees C, whereas the recovery from desensitization is greatly \accelerated with temperature increase (Q10 approximately 10). The sensitivity of HABS to low nanomolar ATP near normal body temperature (35 degrees C) is substantially lower than at 25 degrees C (IC50 is 3.2+/-0.3 nM at 35 degrees C and 0.79+/-0.09 nM at 25 degrees C). HABS itself is subjected to slow desensitization partially loosing its sensitivity to ATP: at 35 degrees C the response completely recovers in 10 min in the presence of 3 nM ATP, making the receptor operational in the presence of up to 30 nM ATP. Unusual combination of temperature sensitivity/insensitivity of P2X3 receptors may be related to their pivotal role in the processing of thermal sensitivity as revealed by recent knockout experiments.

From Galvani to patch clamp: the development of electrophysiology.

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.

The agonists for nociceptors are ubiquitous, but the modulators are specific: P2X receptors in the sensory neurons are modulated by cannabinoids.

P2X2 and P2X3 receptors expressed in mammalian sensory neurons participate in nociception. Cannabinoid receptors modulate nociceptive processing in various models of pain. They are also expressed in nociceptive sensory neurons. We have examined the effect of cannabinoids on the slow P2X2 and P2X2/3 receptors in the cells isolated from nodosal and dorsal root ganglia of rat. The study was carried out by means of the whole-cell patch clamp and rapid superfusion methods. We have found that both endogenous and synthetic cannabinoids (anandamide, WIN55,212-2, and (R)-(+)-methanandamide) inhibit the slow response to ATP mediated by P2X2 and P2X2/3 receptors in a majority of tested neurons. This inhibition was significant but only partial: anandamide (0.5-1 microM) inhibited the response to 51+/-21% of control. In the remaining minority of tested neurons, the response was transiently facilitated. The effect of cannabinoids appears to be mediated via cannabinoid CB(1) receptors: it was reversibly inhibited by selective CB(1) antagonist, SR141716A (10 microM). Introduction of cyclic AMP (0.5 mM) into the cell potently facilitated the inhibitory effect of cannabinoids: the ATP-activated current was inhibited to 13+/-10% of control. These data indicate that cannabinoids may inhibit nociceptive responses produced by P2X receptors.

Opioids inhibit purinergic nociceptors in the sensory neurons and fibres of rat via a G protein-dependent mechanism.

We have found that opioid and P2X receptors are functionally coupled in the sensory nerve fibres and neurons of rat. When examined in the skin-nerve preparation, the ATP-evoked discharges of nerve fibres belonging to n. saphenous were inhibited by various opiates in a naloxone-dependent manner. The functional coupling between opioid and purinergic receptors was studied in the neuronal cell bodies isolated from dorsal root and nodose ganglia. Both fast (mediated by P2X(3) receptors) and slow (P2X(2/3) heteromeric receptors) responses of sensory neurons to ATP were inhibited by opioids. The inhibition of slow responses developed in a characteristic biphasic manner: an initial short phase of potentiation (lasting for 300-400 s) was followed by long-lasting inhibition of the response (for about 50% when saturated). Both phases of the response were initiated by the application of the highly selective ligand for mu-receptors, endomorphin 1 (30 nM). Intracellular GTPgammaS caused a partial inhibition of the ATP responses and opioids were not effective against the residual response. Intracellular GDP eliminated the effects of opioids, while pertussis toxin (PTX) abolished only the inhibition phase. Thus, P2X receptors in the sensory neurons are affected by opioids via multiple G protein-dependent pathways.

Modulatory action of RFamide-related peptides on acid-sensing ionic channels is pH dependent: the role of arginine.

Acid-sensing ionic channels (ASICs) are involved in such functions of the sensory nervous system as mechanoreception, nociception and perception of acid taste. Phe-Met-Arg-Phe amide-related (FMRFa-related) peptides in micro m concentrations slow down the rate of ASICs desensitization. Here we report that this effect is strongly pH dependent: the lower the pH used to activate ASICs, the larger is the modulatory effect of Arg-Phe amide-related (RFa-related) peptides. Pre-application of the peptides results in a change to the desensitization kinetics of the ASICs-operated current from monoexponential to biexponential: the fast component retains the control kinetics, whereas the slow one is induced by the peptide. The lower the pH, the larger is the slow component, whereas there is practically no modulation at pH 6.6. Phe-Met-Val-Phe amide (FMVFa), which has neutral valine instead of arginine, similarly modulates the kinetics of ASICs, but does not reveal pH dependence of this action. Thus, positively charged arginine regulates the access of the RFa-related peptides to the modulatory site. We suggest that the pH dependence of the modulatory action of RFa-related peptides can be associated with the interaction of a positively charged arginine with histidine residues in the molecule of ASIC.

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.

P2X receptor-mediated excitatory synaptic currents in somatosensory cortex.

Fast P2X receptor-mediated excitatory postsynaptic current (EPSC) was found in pyramidal neurones of layer V of somatosensory cortex in slices acutely isolated from the brain of 17- to 22-day-old rats. The EPSCs were elicited by field electrical stimulation in the layer VI at 0.1 Hz in the presence of picrotoxin. When the glutamatergic EPSC was blocked by glutamate receptors inhibitors NBQX and D-AP5, a residual EPSC (rEPSC) was recorded from 85% of neurones tested. This rEPSC was not affected by blockers of nicotinic (hexamethonium) and serotonin (Y25130) receptors; however, it was reversibly inhibited by P2X receptors antagonists (NF023, NF279, and PPADS). An application of ATP (20 microM), beta,gamma-methylene ATP (25 microM), and alpha,beta-methylene ATP (20 microM) to acutely isolated pyramidal neurones of layer V evoked inward currents (30 to 400 pA) in 75% of cells tested. We concluded that several subtypes of P2X purinoreceptors participate in synaptic transmission in neocortex.

Structure-activity studies with Ginkgo biloba extract constituents as receptor-gated chloride channel blockers and modulators.

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.

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.

Ionotropic P2X purinoreceptors mediate synaptic transmission in rat pyramidal neurones of layer II/III of somato-sensory cortex.

Fast P2X receptor-mediated excitatory postsynaptic current (EPSC) was identified in pyramidal neurones of layer II/III of somato-sensory cortex in acutely isolated slices obtained from the brain of 17- to 22-day-old rats. The EPSCs were elicited by electrical stimulation of vertical axons originating from layer IV-VI neurones at 0.1 Hz in the presence of bicuculline. When the glutamatergic EPSC was blocked by saturating concentrations of glutamate receptor inhibitors 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo-[f]-quinoxaline-7-sulphonamide (NBQX) and D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5), a small EPSC component was recorded from 90 % of neurones tested. This residual EPSC was not affected by selective blockers of nicotinic (hexamethonium) or serotonin (N-(1-azabicyclo-[2.2.2]oct-3-yl)-6-chloro-4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride, Y-25130) receptors, but it was reversibly inhibited by the antagonists of P2X receptors NF023 (8,8'-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis-1,3,5-naphthalene-trisulphonic acid), NF279 (8,8'-[carbonylbis (imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalene-trisulphonic acid) and PPADS (pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid). Application of ATP (10 microM) or alpha,beta-methylene ATP (10 microM) to pyramidal neurones, acutely isolated from cortical slices, evoked inward currents (30 to 200 pA) in 65 % of cells tested. The relative calcium/caesium permeability (P(Ca)/P(Cs)) of P2X receptors was 12.3 as estimated from the reversal potential of ATP-induced current measured at different extracellular calcium concentrations. We concluded that P2X purinoreceptors are activated during synaptic transmission in neocortex.

Modulation of ion channels in rat neurons by the constituents of Hypericum perforatum.

Despite almost forty years of widespread use of antidepressant drugs, their mode of action is still unknown. Hyperforin, a phloroglucinol derivative, is a major pharmacologically and therapeutically active constituent of Hypericum perforatum extract that is widely used as an herbal antidepressant drug. However, the mechanism or mechanisms of action of these naturally abundant, non-toxic extracts remain unclear. Enzymatically isolated patch-clamped rat central and peripheral neurons exposed to rapid changes in the composition of external medium (concentration clamp) were used in our experiments to investigate the modulation of the various voltage- and ligand-gated channels by hyperforin, as well as by other constituents of Hypericum perforatum. At nanomolar concentrations, hyperforin induced significant inhibition of various ion channels. In the case of P-type Ca2+ channels, we established that hyperforin acts via interaction with calmodulin or through calmodulin-activated pathways involving at least one second messenger. The results presented here indicate that multiple mechanisms and extract constituents may be involved in the antidepressant action of Hypericum extracts, and that they could also possess neuroprotective and analgesic effects.