Site of action of lubeluzole on voltage-sensitive Ca(2+) channels in isolated dorsal root ganglion cells of the rat: influence of pH.

Besides other pharmacological effects, the neuroprotective compound lubeluzole blocks low-voltage-activated (iLVA) and high-voltage-activated (iHVA) calcium channel currents. We investigated the site of action of lubeluzole on Ca(2+) channels in isolated dorsal root ganglion cells of the rat, using whole-cell voltage clamp. Experiments with extracellular application of 3 microM lubeluzole (pK(a) = 7.6) at different values of extracellular pH suggest that the protonated form of lubeluzole contributes to the block of iLVA and iHVA from the extracellular side. The partial block of iLVA and iHVA by 3 microM lubeluzole at extracellular pH 9 and intracellular pH (pH(i)) 9 indicates that the uncharged form of lubeluzole (L) may contribute to the block as well. The voltage-dependent acceleration of the apparent inactivation of iHVA by lubeluzole was much more pronounced at lower pH(i), which is consistent with membrane penetration of L and an open channel block of iHVA by the prononated form of lubeluzole acting from the intracellular side. Decreasing pH(i) induced a negative shift of the half-inactivation potential of iLVA and increased the lubeluzole-induced block of iLVA. Experiments with extracellular or intracellular application of a quaternary ammonium derivative of lubeluzole (R133121), which was less potent than lubeluzole, support the above conclusions on the side of action of lubeluzole. Application of lubeluzole via the patch pipette affected iLVA and iHVA only minimally compared with extracellular application, probably partly due to efflux of L through the cell membrane. These experiments suggest that lubeluzole blocks Ca(2+) channels from both the extracellular and the intracellular side.

Analysis of the human KCNH2(HERG) gene: identification and characterization of a novel mutation Y667X associated with long QT syndrome and a non-pathological 9 bp insertion.

Long QT (LQT) syndrome is a potentially life-threatening disorder, characterized by a distinct cardiac arrhythmia known as torsades de pointes. Mutations within a number of genes linked to the familial form, including that coding for a cardiac potassium channel called KCNH2 (HERG), have been described based on the characterized genomic organization. A standardized method was developed to screen the entire gene for gene variants. We report a single base pair substitution, introducing a premature STOP codon at codon 667 of the gene in a healthy individual with an extended QTc interval (460 msec). In vitro expression of the codon Y667X variant in Xenopus oocyte suggests that the autosomal dominant variant does not function in a dominant/negative manner and cannot co-assemble to form a channel, resulting in a reduction of the KCNH2 current, and an extension of the QT interval. This indicates that pathogenic LQT gene variants exist in the apparently normal population, the prognosis and clinical consequences of which remain to be determined. The assays described should facilitate future studies into this area.

Influence of lubeluzole on voltage-sensitive Ca++ channels in isolated rat neurons.

Lubeluzole is neuroprotective in a photochemical stroke model, whereas the (R)-enantiomer of the same molecule is not [De Ryck M, Keersmaekers R, Duytschaever H, Claes C, Clincke G, Janssen M and Van Reet G (1996) J Pharmacol Exp Ther 279:748-758]. We investigated the effects of lubeluzole and the (R)-enantiomer on voltage-sensitive Ca++ channels of isolated rat dorsal root ganglion cells, using whole-cell voltage-clamp, with Ba++ as the charge carrier. Both compounds blocked the low-voltage-activated Ba++ current (iLVA or T current) with an IC50 value of 1.2 microM. Lubeluzole and the (R)-enantiomer also blocked the high-voltage-activated calcium channel current (iHVA), with IC50 values of 2.6 and 3.5 microM, respectively, and accelerated the apparent inactivation of iHVA. This acceleration was more pronounced with lubeluzole than with the (R)-enantiomer at 3 and 10 microM. Both compounds produced a clear tonic block of iLVA and iHVA, even in the absence of previous stimulation. Lubeluzole and the (R)-enantiomer induced a negative shift of the inactivation curve of iLVA and showed down the recovery from inactivation. This resulted in a stronger inhibition of iLVA at more depolarized conditioning potentials and higher stimulation frequencies. The block of iHVA was voltage and frequency dependent. Lubeluzole and the (R)-enantiomer also blocked iHVA in isolated rat superior cervical ganglion cells and cerebellar Purkinje cells. The Ca++ channel-blocking properties of lubeluzole may contribute to its neuroprotective effect. However, the small difference between the two enantiomers in inhibition of Ca++ channel currents does not explain the stereospecificity of the neuroprotective properties of lubeluzole in vitro and in vivo.

Altered Na(+)-channel function as an in vitro model of the ischemic penumbra: action of lubeluzole and other neuroprotective drugs.

Veratridine blocks Na(+)-channel inactivation and causes a persistant Na(+)-influx. Exposure of hippocampal slices to 10 microM veratridine led to a failure of synaptic transmission, repetitive spreading depression (SD)-like depolarizations of increasing duration, loss of Ca(+)-homeostasis, a large reduction of membrane potential, spongious edema and metabolic failure. Normalization of the amplitude of the negative DC shift evoked by high K+ ACSF 80 min after veratridine exposure was taken as the primary endpoint for neuroprotection. Compounds whose mechanisms of action includes Na(+)-channel modulation were neuroprotective (IC50-values in microM): tetrodotoxin 0.017, verapamil 1.18, riluzole 1.95, lamotrigine > or = 10, and diphenylhydantoin 16.1. Both NMDA (MK-801 and PH) and non-NMDA (NBQX) excitatory amino acid antagonists were inactive, as were NOS-synthesis inhibitor (nitro-L-arginine and L-NAME) Ca(2+)-channel blockers (cadmium, nimodipine) and a K(+)-channel blocker (TEA). Lubeluzole significantly delayed in time before the slices became epileptic, postponed the first SD-like depolarization, allowed the slices to better recover their membrane potential after a larger number of SD-like DC depolarizations, preserved Ca2+ and energy homeostasis, and prevented the neurotoxic effects of veratridine (IC50-value 0.54 microM). A concentration of lubeluzole, which was 40 x higher than its IC50-value for neuroprotection against veratridine, had no effect on repetitive Na(+)-dependent action potentials induced by depolarizing current in normal ACSF. The ability of lubeluzole to prevent the pathological consequences of excessive Na(+)-influx, without altering normal Na(+)- channel function may be of benefit in stroke.

Extracellular ions during veratridine-induced neurotoxicity in hippocampal slices: neuroprotective effects of flunarizine and tetrodotoxin.

Veratridine, by blocking Na+ channel inactivation and shifting activation to more negative membrane potentials, causes Na(+)-influx and a persistent tendency for depolarization. Veratridine is neurotoxic to cultured neurones, and this neurotoxicity can be blocked by the class IV calcium antagonist, flunarizine. We were interested to know whether similar effects could be found in a functional differentiated tissue containing adult neurones and glial cells. We examined this in hippocampal slices using extracellular potential recordings and ion-selective microelectrodes sensitive to [Na+]o, [Ca2+]o and [K+]o. Veratridine blocked synaptic transmission in CA1, and induced several episodes of spreading depression (SD). This was followed by a long-lasting increase in [K+]o and a continuous decrease in [Ca+]o. Following veratridine exposure to hypoxia only revealed a small negative DC shift and small shifts in extracellular ions; indicating that the cells had lost the ability to maintain ion homeostasis before the hypoxia, and that veratridine had been neurotoxic. In hippocampal slices obtained from guinea pigs which had been pretreated with 40 mg/kg x 2 flunarizine orally the time before the first SD induced by veratridine was doubled. Although the ion shifts during the first SD were similar to controls, flunarizine reduced the time of recovery of [Ca2+]o, [K+]o and DC potential. The increase in [K+]o baseline and the massive decrease in [Ca2+]o baseline seen following the SDs in the solvent group were smaller in the flunarizine-treated slices. During the subsequent hypoxic period the negative DC shift was 8x larger in the flunarizine group, and the shifts in [K+]o, [Na+]o and [Ca2+]o were bigger. Tetrodotoxin also delayed the first SD during veratridine and increased the size of the DC shift during the subsequent hypoxic period. Both flunarizine and tetrodotoxin therefore protected adult brain tissue containing glia from the neurotoxicity of veratridine. These findings suggest that persistent Na(+)-influx and the consequent Ca2(+)-influx produce neurotoxicity, and that the ability to attenuate this neurotoxicity may be important in the mechanism of action of cerebroprotective drugs from different pharmacological classes.

Functional role of 5-HT2 receptors in the regulation of sleep and wakefulness in the rat.

Recently developed agents specifically acting on different 5-hydroxytryptamine (5-HT) receptor populations were used to analyze the functional role of 5-HT2 receptor subtypes in the sleep-wakefulness cycle of the rat. The 5-HT2 receptor antagonist ritanserin injected intraperitoneally (IP) (0.04-2.5 mg/kg) induced an increase in deep slow wave sleep (SWS2) duration at the expense of wakefulness (W), light slow wave sleep (SWS1) and paradoxical sleep (PS). The stimulation of 5-HT2 receptors by 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) produced a dose-related increase in W and a dose-dependent decrease in both SWS2 and PS. Pretreatment with ritanserin (0.16-2.5 mg/kg) or with cinanserin (2.5-5 mg/kg), another 5-HT2 receptor antagonist, dose-dependently reversed the W enhancement and the SWS2 deficit produced by DOM, but not the PS deficit. Sleep-wakefulness alterations (increase in W and SWS1 combined with a suppression of SWS2 and PS) observed after IP injection of two putative 5-HT1 receptor agonists, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) (2.5 mg/kg) and 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) (0.63 mg/kg), were not modified by ritanserin pretreatment (0.16-2.5 mg/kg). These results further support the hypothesis that the serotonergic system plays an active role in the regulation of the sleep-wakefulness cycle in the rat and that 5-HT2 receptors are involved in this action. In addition, it is suggested that 5-HT1 receptor subtypes are unlikely to interact with 5-HT2 receptors in the sleep-wakefulness modulation mediated through 5-HT2 receptors.

Episodic barrel rotations induced by intrastriatal injection of quinolinic acid in rats. Inhibition by anticonvulsants.

Unilateral intrastriatal injection of quinolinic acid (2,3 pyridine dicarboxylate; QUIN) in the rat produces episodic barrel rotations and tonic-clonic forepaw movements, lasting for several hours. We investigated whether intraperitoneal posttreatment with anticonvulsants could abolish this phenomenon when it is already fully developed, and whether their potency ratio was similar in models of epilepsy. All 8 tested antiepileptics, namely carbamazepine, clonazepam, diazepam, diphenylhydantoin, ethosuximide, flunarizine, phenobarbital and sodium valproate decreased this behaviour in a dose-dependent way. Six other drugs with anticonvulsant properties were also effective: DL-2-amino-7-phosphonoheptanoic acid, desipramine, etomidate, ketamine, meprobamate and sabeluzole. The ED50-values for halving the frequency of the episodes of barrel rotation correlated well with published ED50-values for inhibition of tonic hindpaw extension in the maximal metrazol seizure test (rs = .95, p less than 0.001) and with the ED50-values for halving the duration of the forepaw clonus in the rat-kindling model (rs = .93, p less than 0.001). This quinolinic acid test allows visualization of the onset of action of anticonvulsants, with each animal as its own control. In order to assess whether this test is also sensitive to drugs influencing the symptoms of Huntington's disease, the effect of the dopamine antagonists haloperidol and pimozide, the acetylcholinesterase inhibitor physostigmine and the anticholinergics atropine and dexetimide were investigated as well. The experiments suggested that the barrel rotations and clonic forepaw movements, only 3-6 hours after intrastriatal injection of QUIN respond to anticonvulsants, but are not specifically sensitive to drugs used in the symptomatic treatment of Huntington's disease.

Effects of temperature and temperature gradients on ion-sensitive microelectrodes.

Ion-sensitive microelectrodes are widely used in studies of mammalian tissues. Often the tissue is maintained at 37 degrees C, some 10-15 degrees C above room temperature. The temperature difference between the room and the preparation was found to be capable of altering the measured ion potential by as much as 10 mV. The change depended on 3 factors: the temperature dependence of the Nernst slope, the temperature dependence of the interference factor, and the thermoelectric potential induced by the temperature difference between the two ends of the ion-exchanger column. Certain combinations of these changes can cancel each other, resulting in spurious but apparently temperature-insensitive readings. The first two factors can produce errors when the temperature of calibration differs from the temperature of the tissue being measured. Serious errors in measurements of ion concentration can also occur, due to all 3 factors, if a temperature gradient exists across the ion exchanger column; this situation can easily occur when recording from exposed mammalian tissues. The use of a short ion-exchanger column will reduce but not eliminate effects due to a temperature gradient.

Evidence for a role of the N-methyl-D-aspartate (NMDA) receptor in cortical spreading depression in the rat.

The neurotransmitter glutamate activates the N-methyl-D-aspartate (NMDA), quisqualate and kainate receptors. It has been proposed, but also disputed, that local release of glutamate would play a pivotal role in cortical spreading depression (SD). We tested this hypothesis by investigating the influence of NMDA antagonists on SD, using the non-competitive NMDA antagonists ketamine, phencyclidine (PCP) and MK-801 and the competitive NMDA antagonist DL-2-amino-7-phosphonoheptanoate (2-APH), injected intraperitoneally in rats anesthetized with alfentanil. SD was elicited by cathodal DC-stimulation of the frontal cortex. SD propagation was followed using two ion-sensitive microelectrodes placed in the parietal and occipital cortex. The NMDA antagonists increased SD threshold, decreased the propagation velocity and decreased the duration of the accompanying extracellular DC, K+ and Ca2+ changes at the following doses: 40 mg/kg ketamine, 10 mg/kg PCP, 0.63 mg/kg MK-801, 10 and 40 mg/kg 2-APH. With each NMDA antagonist failure of SD propagation between both microelectrodes could be observed. SD elicitation (or propagation) was inhibited completely with 80 mg/kg ketamine, 3.1 mg/kg MK-801 and 160 mg/kg 2-APH. These NMDA antagonists have also anticonvulsant properties. None of these effects on SD were observed with high doses of other anticonvulsants such as 80 mg/kg phenytoin or 40 mg/kg diazepam. These experiments indicate that endogenous release of excitatory amino acids and their action on the NMDA receptor play an important role in the initiation, propagation and duration of SD.

Spreading depression and central nervous system pharmacology.

Spreading depression is a reversible response of brain tissue to a local insult. It has been postulated to be the physiological substrate for the aura phase of classic migraine. The properties and mechanisms of spreading depression were studied in the parietal neocortex of the alfentanil-anesthesized rat, by using a cup electrode that provided close control of the electrical stimulus while allowing specific ion (K+) and potential recordings to be made directly beneath the cathode, the region of origin of the stimulus-induced spreading depression. Cathodal stimulation caused the extracellular K+ concentration to rise, and spreading depressions were observed when this concentration exceeded 8-12 mM in the upper 100-200 microns of cortex. In some experiments extracellular [K+] continued to increase for 5-10 sec after termination of the stimulus, without detectable after-discharge in the potential record, before subsiding. While spreading depression could easily be induced by pressure on the cortex, local damage incidental to opening the dura rarely induced spreading depression. This suggests that a local (1-mm2) neurovascular injury is not likely to induce spreading depression--at least in normal cortex--and so is probably not the source of the spreading depression postulated to generate the aura of classic migraine. Mechanisms of spreading depression, and drugs that influence spreading depression, are reviewed, and possible uses of spreading depression in the pharmacology of the central nervous system are considered.

Effects of phenytoin and flunarizine on the rise in extracellular potassium induced by repetitive stimulation of rat cerebral cortex.

At a critical intensity of repetitive stimulation, [K+]0 in cerebral cortex increases rapidly to a "ceiling" value of 10-12 mM. Phenomena related to this rapid regenerative increase in [K+]0 were investigated using K+-sensitive microelectrodes. The critical intensity required to induce this rise was found to be increased by phenytoin (90 mg/kg preload + 30 mg/kg presurgery) but not by flunarizine (30 mg/kg preload + 10 mg/kg presurgery), when the stimulus (20 Hz, 0.4 ms pulse duration, 5 or 40 s train duration) was applied to a 1 mm2 area of parietal neocortex in alfentanil-anesthesized rats. Flunarizine and phenytoin appear to differ in their mechanisms of anti-epileptic action.

Potassium translocation and spreading depression induced by electrical stimulation of the brain.

Cathodal current pulses with durations from 20 ms to 80 s were applied to the surface of rat parietal neocortex, and the strength-duration properties of the threshold stimulus for initiation of spreading depression was determined. In one series, extradural stimulation was used. In a second series, a liquid electrode was used, the dura was opened, and K+-sensitive microelectrodes were used to determine the time course of extracellular K+ concentration during and after each stimulus pulse. With the dura open, the strength-duration curve was of the form It0.55 = constant. With extradural stimulation, the slope of the log-log plot relating current intensity to pulse duration changed gradually as the pulse duration increased, averaging 0.63. Theoretical analysis suggests that diffusion of K+ away from a zone of current-induced accumulation can account for these slope data. Applicability of this mechanism of K+ accumulation to observed changes in sensitivity of neurons to repeated stimulation at subcortical sites is considered.

Surface pH and the control of intracellular pH in cardiac and skeletal muscle.

Both surface pH (pHs) and intracellular pH (pHi) were measured using single- and double-barreled pH-sensitive microelectrodes in isolated sheep cardiac Purkinje strands, rabbit and cat papillary muscle, and mouse and rat soleus muscle. Superfusion of the preparations with a relatively low buffered solution (containing 5 mM HEPES buffered to control pH) causes surface acidosis that correlates with efflux of metabolically produced acids in the unstirred layer of fluid surrounding the tissue. Acidification of the surface layer induces a slower acid change of pHi and depresses the rate of proton extrusion following an imposed intracellular acid load. In cardiac preparations, the lowering of pHi correlates with depression of twitch tension. Transient changes of pHs and pHi are seen when a weak acid or base is suddenly added to, or removed from the superfusion solution. Indirect evidence of the presence of carbonic anhydrase in the extracellular surface layer is obtained from analysis of transient pHs changes in presence and absence of acetazolamide.

Strength-duration properties of cathodal pulses eliciting spreading depression in rat cerebral cortex.

Strength-duration curves for the threshold stimulus for initiation of spreading depression were determined for durations of 2 ms to 120 s, using cathodal surface stimulation through a well-defined area. With the dura open, the strength-duration curve was of the form I square root t = constant. With extradural stimulation, the slope of the log-log plot relating current intensity to pulse duration increased gradually as the pulse duration increased.

Measurement of ischemic changes in cerebral blood flow by the hydrogen clearance technique and brain cortical temperature. Influence of flunarizine.

In dogs global cerebral ischemia was produced by clamping reversibly the left subclavian and brachiocephalic arteries, supplying the head. The intercostal arteries were ligated permanently. Cerebral blood flow (CBF) was measured discontinuously using a hydrogen saturation-desaturation technique. Clamping of the former two vessels caused an increase in systemic blood pressure. When this increase was not blunted by previous splenectomy and blood withdrawal a still important CBF remained during the clamp. However, if this rise in blood pressure was impaired, CBF decreased to 9 +/- 8% (mean +/- S.D., n = 14) of the pre-ischemic value. Flunarizine is known to have anti-hypoxic/ischemic properties. The influence of this drug (0.1 mg/kg i.v.), injected 10 min after the beginning of a 30-min ischemia period, on the post-ischemic CBF was investigated. Two-three hour after ischemia CBF was significantly lower in the solvent-treated animals than in the flunarizine-treated group, in which CBF approached the preischemic values. Changes in CBF were also followed continuously by measurement of the variations of brain versus aortic temperature. It was analyzed what information this can provide on CBF.

The effects of flunarizine in experimental models related to the pathogenesis of migraine.

Two new hypotheses suggest that the key pathology in migraine has a neuronal origin. A pivotal role is assigned to brain hypoxia (1) and spreading depression (SD) (neuronal depolarization spreading gradually over the cortex) (2). Flunarizine has been tested both against brain hypoxia and SD. Its potent antihypoxic properties in animal models led to its use as a prophylactic drug in migraine therapy. Earlier experiments suggested that flunarizine shortened recovery after neuronal depolarization. Recent experiments suggest that flunarizine may enhance the threshold for the elicitation of SD. Finally, it is often unclear whether the effects observed with flunarizine are due to a vascular or a direct neuronal effect. Therefore, a study was made to show whether flunarizine affected hypoxia-induced alterations in synaptic function in slices of hippocampus held in vitro. At physiological drug concentrations in brain, flunarizine improved post-hypoxic recovery of synaptic function. A direct neuronal protective effect was thus demonstrated.

Influence of organic acids on intracellular pH.

By use of double-barreled pH-sensitive microelectrodes, intracellular pH was measured in isolated sheep cardiac Purkinje strands. After equimolar substitution of 20 mmol/l Cl- by several organic anions at constant extracellular pH 6.8, the rate of induced intracellular acidification was measured. For many organic acids tested, a relation was found between the rate of intracellular acidification and the product of their dissociation constant (pK'a) and diisopropylether-to-water partition ratio (p'). L-Lactate and pyruvate, and also cyanoacetate and alpha-ketobutyrate, caused faster acidifications than anticipated from their pK'a and p'. The rate of intracellular acidification, induced by L-lactate and pyruvate, was markedly depressed in the presence of 4 mmol/l alpha-cyano-4-hydroxycinnamate, a known inhibitor of the carrier-mediated pyruvate transport. The drug also had an effect on the acidification produced by cyanoacetate, alpha-ketobutyrate, glycolate, alpha-hydroxybutyrate, and alpha-chloropropionate, but not on that produced by propionate and acetate. L-Lactate caused a faster acidification than D-lactate. Our results suggest the existence of a facilitated diffusion for L-lactate, pyruvate, and some other organic acids in sheep Purkinje cells.

pH aspects of transient changes in conduction velocity in isolated heart fibers after partial replacement of chloride with organic anions.

Conduction velocity in isolated rabbit atrial fibers was continuously measured in solutions having a different anionic composition. When 20mmol/l of chloride was replaced by 20 mmol/l lactate or other anions of weak organic acids at constant pH 6.8, biphasic initial transient changes in conduction velocity were observed. The produced transient changes had a greater amplitude with organic acids which have a greater pK and lipid/water partition ratio. The magnitude of the transients was also greater at pH 6.8 than at pH 7.5, and also when the buffering capacity of the superfusion solution was smaller. Measurements of intracellular pH (pHi) in sheep Purkinje fibers and of pH at the surface (pHs) of sheep Purkinje and rabbit atrial fibers with pH sensitive microelectrodes, showed a transient increase of pH and a sustained decrease of pHi on replacement of 20mmol/l chloride by organic anions of weak acids (at constant pH of the superfusion solution). A combined influence of the transient pHs change and the sustained pHi modification seems to be important in the explanation of the biphasic changes in conduction velocity.