Short- and long-term differential effects of neuroprotective drug NS-7 on voltage-dependent sodium channels in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited veratridine-induced (22)Na(+) influx via voltage-dependent Na(+) channels (IC(50)=11.4 microM). The inhibition by NS-7 occurred in the presence of ouabain, an inhibitor of Na(+),K(+) ATPase, but disappeared at higher concentration of veratridine, and upon the washout of NS-7. NS-7 attenuated veratridine-induced (45)Ca(2+) influx via voltage-dependent Ca(2+) channels (IC(50)=20.0 microM) and catecholamine secretion (IC(50)=25.8 microM). Chronic (>/=12 h) treatment of cells with NS-7 increased cell surface [(3)H]-STX binding by 86% (EC(50)=10.5 microM; t(1/2)=27 h), but did not alter the K(D) value; it was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, but was not associated with increased levels of Na(+) channel alpha- and beta(1)-subunit mRNAs. In cells subjected to chronic NS-7 treatment, (22)Na(+) influx caused by veratridine (site 2 toxin), alpha-scorpion venom (site 3 toxin) or beta-scorpion venom (site 4 toxin) was suppressed even after the extensive washout of NS-7, and veratridine-induced (22)Na(+) influx remained depressed even at higher concentration of veratridine; however, either alpha- or beta-scorpion venom, or Ptychodiscus brevis toxin-3 (site 5 toxin) enhanced veratridine-induced (22)Na(+) influx as in nontreated cells. These results suggest that in the acute treatment, NS-7 binds to the site 2 and reversibly inhibits Na(+) channels, thereby reducing Ca(2+) channel gating and catecholamine secretion. Chronic treatment with NS-7 up-regulates cell surface Na(+) channels via translational and externalization events, but persistently inhibits Na(+) channel gating without impairing the cooperative interaction between the functional domains of Na(+) channels.     

Donepezil attenuates excitotoxic damage induced by membrane depolarization of cortical neurons exposed to veratridine

Long-lasting membrane depolarization in cerebral ischemia causes neurotoxicity via increases of intracellular sodium concentration ([Na+]i) and calcium concentration ([Ca2+]i). Donepezil has been shown to exert neuroprotective effects in an oxygen-glucose deprivation model. In the present study, we examined the effect of donepezil on depolarization-induced neuronal cell injury resulting from prolonged opening of Na+ channels with veratridine in rat primary-cultured cortical neurons. Veratridine (10 microM)-induced neuronal cell damage was completely prevented by 0.1 microM tetrodotoxin. Pretreatment with donepezil (0.1-10 microM) for 1 day significantly decreased cell death in a concentration-dependent manner, and a potent NMDA receptor antagonist, dizocilpine (MK801), showed a neuroprotective effect at the concentration of 10 microM. The neuroprotective effect of donepezil was not affected by nicotinic or muscarinic acetylcholine receptor antagonists. We further characterized the neuroprotective properties of donepezil by measuring the effect on [Na+]i and [Ca2+]i in cells stimulated with veratridine. At 0.1-10 microM, donepezil significantly and concentration-dependently reduced the veratridine-induced increase of [Ca2+]i, whereas MK801 had no effect. At 10 microM, donepezil significantly decreased the veratridine-induced increase of [Na+]i. We also measured the effect on veratridine-induced release of the excitatory amino acids, glutamate and glycine. While donepezil decreased the release of glutamate and glycine, MK801 did not. In conclusion, our results indicate that donepezil has neuroprotective activity against depolarization-induced toxicity in rat cortical neurons via inhibition of the rapid influx of sodium and calcium ions, and via decrease of glutamate and glycine release, and also that this depolarization-induced toxicity is mediated by glutamate receptor activation.     

Lithium inhibits function of voltage-dependent sodium channels and catecholamine secretion independent of glycogen synthase kinase-3 in adrenal chromaffin cells

Lithium has been proven to be effective in the therapy of bipolar disorder, but its mechanism of pharmacological action is not clearly defined. We examined the effects of lithium on voltage-dependent Na(+) channels, nicotinic acetylcholine receptors, and voltage-dependent Ca(2+) channels, as well as catecholamine secretion in cultured bovine adrenal chromaffin cells. Lithium chloride (LiCl) reduced veratridine-induced (22)Na(+) influx in a concentration-dependent manner, even in the presence of ouabain, an inhibitor of Na(+), K(+)-ATPase. Glycogen synthase kinase-3 (GSK-3) inhibitors (SB216763, SB415286 or the GSK-3 inhibitor IX) did not affect veratridine-induced (22)Na(+) influx, as well as inhibitory effect of LiCl on veratridine-induced (22)Na(+) influx. Enhancement of veratridine (site 2 toxin)-induced (22)Na(+) influx caused by alpha-scorpion venom (site 3 toxin), beta-scorpion venom (site 4 toxin), or Ptychodiscus brevis toxin-3 (site 5 toxin), still occurred in the presence of LiCl in the same manner as in the control cells. LiCl also reduced veratridine-induced (45)Ca(2+) influx and catecholamine secretion. In contrast, LiCl (< or = 30 mM) had no effect on nicotine-induced (22)Na(+) influx, (45)Ca(2+) influx and catecholamine secretion, as well as on high K(+)-induced (45)Ca(2+) influx and catecholamine secretion. Chronic treatment with LiCl at 100mM (but not at < or = 30 mM) significantly reduced cell viability in a time-dependent manner. These results suggest that lithium selectively inhibits Na(+) influx thorough Na(+) channels and subsequent Ca(2+) influx and catecholamine secretion, independent of GSK-3 inhibition.     

Astroglial cell death induced by excessive influx of sodium ions

Na(+) influx has been implicated to play an important role in the mechanisms of neuronal cell damage under ischemia as well as in neurodegenerative disorders. Thus far, however, the effects of Na(+) influx on astrocytic damage have not been studied extensively. In the present study, we have examined the effects of Na(+) influx induced by veratridine (Na(+) channel opener), monensin (Na(+) ionophore), and glutamate (co-transportation with Na(+)) on rat cultured astroglial damage. Cells were incubated with bicarbonate buffer with 25 mM glucose containing either 100 microM veratridine, 10 microM monensin, or 1 mM glutamate with or without 1 mM ouabain for 20 h. Cellular damage was evaluated quantitatively by lactate dehydrogenase (LDH) release or 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction. Veratridine, monensin, or glutamate alone did not induce significant astroglial damage. Veratridine and monensin co-incubated with ouabain, which inhibits active extrusion of Na(+) by Na(+),K(+)-ATPase, thereby enhances intracellular Na(+) accumulation, caused significant cell death (P<0. 001, approximately 50% cell damage), whereas glutamate did not. Na(+)-free solution substituted by choline (impermeable cation) attenuated cell damage induced by veratridine and monensin markedly, while Li(+) substitution (permeable cation) rather exacerbated. Nifedipine (100 microM), a blocker of L-type Ca(2+) channel, reduced veratridine-induced glial damage by 50%. Neither bepridil nor benzamil, a blocker of Na(+)-Ca(2+) exchanger, had any protection. Cyclosporin A (1 or 10 microM), an inhibitor of mitochondrial permeability transition or 10 microM N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethyl ketone (zVAD-fmk), which inhibits a broad range of caspases, did not show protective effects.     

Veratridine induces apoptotic death in bovine chromaffin cells through superoxide production

The molecular mechanisms involved in veratridine-induced chromaffin cell death have been explored. We have found that exposure to veratridine (30 microM, 1 h) produces a delayed cellular death that reaches 55% of the cells 24 h after veratridine exposure. This death has the features of apoptosis as DNA fragmentation can be observed. Calcium ions play an important role in veratridine-induced chromaffin cell death because the cell permeant Ca(2+) chelator BAPTA-AM and extracellular Ca(2+) removal completely prevented veratridine-induced toxicity. Following veratridine treatment, there is a decrease in mitochondrial function and an increase in superoxide anion production. Veratridine-induced increase in superoxide production was blocked by tetrodotoxin (TTX; 10 microM), extracellular Ca(2+) removal and the mitochondrial permeability transition pore blocker cyclosporine A (10 microM). Veratridine-induced death was prevented by different antioxidant treatments including catalase (100 IU ml(-1)), N-acetyl cysteine (100 microM), allopurinol (100 microM) or vitamin E (50 microM). Veratridine-induced DNA fragmentation was prevented by TTX (10 microM). Veratridine produced a time-dependent increase in caspase activity that was prevented by Ca(2+) removal and TTX (10 microM). In addition, calpain and caspases inhibitors partially prevented veratridine-induced death. These results indicate that chromaffin cells share with neurons the molecular machinery involved in apoptotic death and might be considered a good model to study neuronal death during neurodegeneration.     

Protective effects of a selective L-type voltage-sensitive calcium channel blocker, S-312-d, on neuronal cell death

Amyloid beta protein (Abeta)- and human group IIA secretory phospholipase A(2) (sPLA(2)-IIA)-induced neuronal cell death have been established as in vitro models for Alzheimer's disease (AD) and stroke. Both sPLA(2)-IIA and Abeta causes neuronal apoptosis by increasing the influx of Ca(2+) through L-type voltage-sensitive Ca(2+) channel (L-VSCC). In the present study, we evaluated effects of a selective L-VSCC blocker, S-(+)-methyl 4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitro-phenyl)thieno[2,3-b]pyridine-5-carboxylate (S-312-d), on Abeta- and sPLA(2)-IIA-induced neuronal apoptosis in primary cultures of rat cortical neurons. S-312-d significantly rescued cortical neurons from Abeta- and sPLA(2)-IIA-induced cell death. Both cell death stimuli caused the appearance of apoptotic features such as plasma membrane blebs, chromatin condensation, and DNA fragmentation. S-312-d completely suppressed these apoptotic features. Before apoptosis, the two death ligands markedly enhanced an influx of Ca(2+) into neurons. S-312-d significantly prevented neurons from sPLA(2)-IIA- and Abeta-induced Ca(2+) influx. Furthermore, the neuroprotective effect of S-312-d was more potent than that of another L-VSCC blocker, nimodipine. On the other hand, blockers of other VSCCs such as the N-type and P/Q-type calcium channels had no effect on the neuronal cell death, apoptotic features and Ca(2+) influx. In conclusion, we demonstrated that S-312-d rescues cortical neurons from Abeta- and sPLA(2)-IIA-induced apoptosis.     

Effects of resveratrol, a grape polyphenol, on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells

We report the effects of resveratrol, a polyphenol found in the skins of red grapes, on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. Resveratrol suppressed catecholamine secretion and (22)Na(+) and (45)Ca(2+) influx induced by acetylcholine, an agonist of nicotinic acetylcholine receptors, in a concentration-dependent manner (IC(50)=20.4, 11.0, and 62.8 microM, respectively). Resveratrol also inhibited catecholamine secretion induced by veratridine, an activator of voltage-dependent Na(+) channels, and 56 mM K(+), an activator of voltage-dependent Ca(2+) channels, at concentrations similar to those for (45)Ca(2+) influx. Resveratrol directly inhibited the current evoked by acetylcholine in Xenopus oocytes expressing alpha3beta4 neuronal nicotinic acetylcholine receptors (IC(50)=25.9 microM). Furthermore, resveratrol (IC(50)=5.32 microM) attenuated (14)C-catecholamine synthesis induced by acetylcholine. The present findings suggest that resveratrol inhibits acetylcholine-induced catecholamine secretion and synthesis through suppressing ion influx in cultured bovine adrenal medullary cells.     

Incorporation of sodium channel blocking and free radical scavenging activities into a single drug, AM-36, results in profound inhibition of neuronal apoptosis

AM-36 is a novel neuroprotective agent incorporating both antioxidant and Na(+) channel blocking actions. In cerebral ischaemia, loss of cellular ion homeostasis due to Na(+) channel activation, together with increased reactive oxygen species (ROS) production, are thought to contribute to neuronal death. Since neuronal death in the penumbra of the ischaemic lesion is suggested to occur by apoptosis, we investigated the ability of AM-36, antioxidants and Na(+) channel antagonists to inhibit toxicity induced by the neurotoxin, veratridine in cultured cerebellar granule cells (CGC's). Veratridine (10 - 300 microM) concentration-dependently reduced cell viability of cultured CGC's. Under the experimental conditions employed, cell death induced by veratridine (100 microM) possessed the characteristics of apoptosis as assessed by morphology, TUNEL staining and DNA laddering on agarose gels. Neurotoxicity and apoptosis induced by veratridine (100 microM) were inhibited to a maximum of 50% by the antioxidants, U74500A (0.1 - 10 microM) and U83836E (0.03 - 10 microM), and to a maximum of 30% by the Na(+) channel blocker, dibucaine (0.1 - 100 microM). In contrast, AM-36 (0.01 - 10 microM) completely inhibited veratridine-induced toxicity ( IC(50) 1.7 (1.5 - 1.9) microM, 95% confidence intervals (CI) in parentheses) and concentration-dependently inhibited apoptosis. These findings suggest veratridine-induced toxicity and apoptosis are partially mediated by generation of ROS. AM-36, which combines both Na(+) channel blocking and antioxidant activity, provided superior neuroprotection compared with agents possessing only one of these actions. This bifunctional profile of activity may underlie the potent neuroprotective effects of AM-36 recently found in a stroke model in conscious rats.     

Protective effect of DY-9760e, a calmodulin antagonist, against neuronal cell death

An excessive elevation of intracellular Ca(2+) levels is known to play a key role in the pathological events following cerebral ischemia. DY-9760e, 3-[2-[4-(3-chloro-2-methylphenylmethyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, is a potent calmodulin antagonist that attenuates brain damage in focal ischemia models. In the present study, we investigated the effect of DY-9760e on neuronal cell death induced by a variety of cell-toxic stimuli that increase intracellular Ca(2+). Cell death was induced by the exposure of primary cultured neurons to excitotoxic agents such as glutamate and N-methyl-D-aspartate, membrane-depolarizing agents such as veratridine and high KCl, or thapsigargin an endoplasmic reticulum Ca(2+)-ATPase inhibitor. Treatment with DY-9760e resulted in a dose-dependent prevention of neuronal cell death elicited by excitotoxicity, voltage-gated channel opening, and inhibition of endoplasmic reticulum Ca(2+)-ATPase. These results indicate that DY-9760e can rescue neurons from various types of cell-toxic stimuli, which may contribute to attenuation of brain injury after cerebral ischemia.     

Depolarization preconditioning produces cytoprotection against veratridine-induced chromaffin cell death

The hypothesis that K(+) channels and cell depolarization are involved in neuronal death and neuroprotection was tested in bovine chromaffin cells subjected to two treatment periods: the first period (preconditioning period) lasted 6 to 48 h and consisted of treatment with high K(+) solutions or with tetraethylammonium (TEA), a K(+) channel blocker; the second period consisted of incubation with veratridine for 24 h, to cause cell damage. Preconditioning with high K(+) (20-80 mM) or TEA (10-30 mM) for 24 h caused 20-60% cytoprotection against veratridine-induced cell death in bovine chromaffin cells. The absence of Ca(2+) ions during the first 9 h of an 18-h preconditioning period abolished the cytoprotection. Preconditioning with K(+) or TEA increased by 2.5-fold the expression of brain-derived neurotrophic factor and by nearly 2-fold the expression of the antiapoptotic protein Bcl-2. However, preconditioning did not modify the veratridine-evoked Ca(2+) signal. High K(+) shifted the Em by about 10 mV and TEA evoked a transient burst of action potentials superimposed on a sustained depolarization. We conclude that preconditioning may protect chromaffin cells from death by blocking K(+) channels that depolarize the cell and cause a cytosolic Ca(2+) signal, leading to enhanced expression of BDNF and Bcl-2.     

Selective inhibition by riluzole of voltage-dependent sodium channels and catecholamine secretion in adrenal chromaffin cells

We examined the effects of riluzole, a neuroprotective drug, on voltage–dependent Na channels, nicotinic receptors, and voltage-dependent Ca channels, as well as catecholamine secretion, in comparison with those of verapamil and nicardipine, in primary cultures of bovine adrenal chromaffin cells. Riluzole inhibited veratridine-induced ²²Na influx via voltage-dependent Na channels even in the presence of ouabain, an inhibitor of Na,K-ATPase. Blockade of Na channels by riluzole was concentration-dependent with an IC₅₀ of 5.3 μM. It was associated with a similar concentration-related reduction of veratridine-induced ⁴⁵Ca influx via voltage-dependent Ca channels, and of catecholamine secretion. Riluzole had no effect on ⁴⁵Ca influx caused by high K, which directly activates voltage-dependent Ca channels, and on nicotine-induced ²²Na influx, which passes through the nicotinic receptors. Verapamil and nicardipine attenuated ²²Na influx caused by veratridine or nicotine at the same concentrations as they suppressed high K-induced ⁴⁵Ca influx. The inhibitory effect of riluzole on veratridine-induced ²²Na influx disappeared at high concentrations of veratridine. A potentiation of veratridine (site 2 toxin)-induced ²²Na influx caused by α-scorpion venom (site 3 toxin), β-scorpion venom (site 4 toxin), or brevetoxin PbTx-3 (site 5 toxin), occurred in the presence of riluzole in the same manner as in control cells. These results suggest that riluzole binds to the veratridine site in voltage–dependent Na channels. It does not impair the cooperative interaction between the functional peptide segments of Na channels, but selectively inhibits gating of Na channels, thereby reducing Ca influx via Ca channels and catecholamine secretion. In contrast, verapamil and nicardipine suppress Na influx both Na channels and nicotinic receptors. Received: 4 November 1997 / Accepted: 11 February 1998     

Effects of carbamazepine, phenytoin, lamotrigine, oxcarbazepine, topiramate and vinpocetine on Na+ channel-mediated release of [3H]glutamate in hippocampal nerve endings

Several of the most effective antiepileptic drugs are believed to stop the paroxysmal neuronal activity acting as Na(+) channel blockers. However, no single study comparing in parallel the potency and efficacy of the most commonly used antiepileptic drugs on brain Na(+) channel-mediated responses is available. In the present study the effects of increasing concentrations of carbamazepine, phenytoin, lamotrigine, oxcarbazepine and topiramate, which are among the most frequently used antiepileptic drugs, and of the new putative antiepileptic drug, vinpocetine, on the release of glutamate (Glu) elicited by the Na(+) channel opener, veratridine were investigated in hippocampal isolated nerve endings preloaded with the labeled excitatory amino acid neurotransmitter. The present results show that carbamazepine, phenytoin, lamotrigine and oxcarbazepine, in the range from 150 to 1500 microM, progressively inhibit [(3)H]Glu release induced by veratridine. Also vinpocetine progressively inhibits the veratridine-induced response, but in a much lower range of concentrations (from 1.5 to 15 microM), whereas topiramate only exerts a modest inhibition (20%) of Glu release to veratridine at the highest dose tested (1500 microM). These results indicate that the mechanism of action of several of the most widely used antiepileptic drugs involves reduction in cerebral presynaptic voltage sensitive Na(+) channels permeability. Considering that the high doses of antiepileptic drugs required to control seizures are frequently accompanied by adverse secondary effects, the higher potency of vinpocetine to reduce Na(+) channels permeability might be advantageous.     

Sodium- and calcium-dependent steps in the mechanism of neonatal rat cardiac myocyte killing by ionophores. II. The calcium-carrying ionophore, A23187.

Several lines of evidence indicate a role for elevated intracellular Ca2+ in mechanisms of cell killing induced by a wide variety of agents. Cardiac myocytes are susceptible to killing under various conditions, including ischemia and exposure to monensin. In order to delineate the Ca(2+)-dependent cell killing mechanism(s) to which cardiac myocytes are susceptible, we have investigated the mechanism by which they are killed by Ca2+ plus the divalent cation ionophore A23187. Evidence has been obtained for two Ca(2+)-mediated injury steps followed by a Na(+)-mediated step leading to cell death detected as membrane permeabilization to trypan blue dye. The first Ca(2+)-mediated step requires the presence of A23187 and low extracellular Ca2+ concentrations (1-100 microM) and is inhibited by Mn2+ and Ni2+ ions. The second Ca(2+)-dependent step requires extracellular Ca2+ concentrations in approximately the physiological range (greater than 1 mM), is not dependent on ionophore, and is not inhibited by Mn2+. Arachidonic acid release occurs during both Ca(2+)-mediated steps, but mostly during the second step. The second injury step is characterized by visible cell swelling and release of lactate dehydrogenase enzyme activity. The Na(+)-dependent step requires extracellular Na+ equal to or greater than half the physiological concentration (i.e., greater than or equal to 75 mM). Li+, which has a smaller ionic radius than Na+, can partially substitute for its in the Na(+)-dependent step, whereas K+, Cs+, Rb+, and NH4+ (which have larger ionic radii) cannot.     

Inhibition of different pathways influencing Na(+) homeostasis protects organotypic hippocampal slice cultures from hypoxic/hypoglycemic injury

A prominent feature of cerebral ischemia is the excessive intracellular accumulation of both Na(+) and Ca(2+), which results in subsequent cell death. A large number of studies have focused on pathways involved in the increase of the intracellular Ca(2+) concentration [Ca(2+)](i), whereas the elevation of intracellular Na(+) has received less attention. In the present study we investigated the effects of inhibitors of different Na(+) channels and of the Na(+)/Ca(2+) exchanger, which couples the Na(+) to the Ca(2+) gradient, on ischemic damage in organotypic hippocampal slice cultures. The synaptically evoked population spike in the CA1 region was taken as a functional measure of neuronal integrity. Neuronal cell death was assessed by propidium iodide staining. The Na(+) channel blocker tetrodotoxin, and the NMDA receptor blocker MK 801, but not the AMPA/kainate receptor blocker NBQX prevented ischemic cell death. The novel Na(+)/Ca(2+) exchange inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), which preferentially acts on the reverse mode of the exchanger, leading to Ca(2+) accumulation, also reduced neuronal damage. At higher concentrations, KB-R7943 also inhibits Ca(2+) extrusion by the forward mode of the exchanger and exaggerates neuronal cell death. Neuroprotection by KB-R7943 may be due to reducing the [Ca(2+)](i) increase caused by the exchanger.     

Modulation by ouabain and diphenylhydantoin of veratridine-induced 22Na influx and its relation to 45Ca influx and the secretion of catecholamines in cultured bovine adrenal medullary cells

Summary The effects of ouabain and diphenylhydantoin on the secretion of catecholamines induced by veratridine were investigated in cultured bovine adrenal medullary cells with special reference to ion fluxes. Veratridine itself induced an influx of ²²Na and ⁴⁵Ca as well as secretion of catecholamines, which were antagonized by tetrodotoxin, a selective inhibitor of voltage dependent Na channels. The secretion of catecholamines caused by veratridine was not observed either in Na free or Ca free medium. Veratridineinduced influx of ⁴⁵Ca did not occur in Na free medium, while veratridine-induced influx of ²²Na occurred even in Ca free medium. Veratridine-induced influx of ²²Na, ⁴⁵Ca and secretion of catecholamines were all potentiated by ouabain, a potent inhibitor of Na, K-ATPase. Omission of K from the medium, a condition which suppresses the Na, K-ATPase activity, also augmented these cell responses caused by veratridine. On the contrary, diphenylhydantoin, which is known to decrease the intracellular concentration of Na, reduced the veratridine-induced influx of ²²Na, ⁴⁵Ca and secretion of catecholamines. The potentiating effects of ouabain on the veratridine-induced cell responses were all abolished by diphenylhydantoin. These findings imply that veratridine, ouabain and K removal as well as diphenylhydantoin modulate the intracellular accumulation of ²²Na which is involved in the influx of ⁴⁵Ca and the secretion of catecholamines.     

Investigation Of AM-36: A Novel Neuroprotective Agent

1. The neurochemical sequelae following cerebral ischaemia are complex, involving excess release of excitatory amino acids, particularly glutamate, disruption of ionic homeostasis due to Na+ and Ca2+ influx and generation of toxic free radicals, ultimately leading to cell death by both necrosis and apoptosis. 2. Drugs that block components of this biochemical cascade, such as glutamate receptor antagonists, sodium channel blockers and free radical scavengers, have been investigated as putative neuroprotective agents. The knowledge that multiple mechanisms contribute to neuronal injury in ischaemia have led to the general recognition that a single drug treatment is unlikely to be beneficial in the treatment of cerebral ischaemia. 3. AM-36 [1-(2-(4-chlorophenyl)-2-hydroxy)ethyl-4-(3,5-bis(1,1-dimethyl)-4-hydroxyphenyl)methylpiperazine] is one of a series of hybrid molecules designed to incorporate multiple neuroprotective mechanisms within the one structure. Primary screening tests demonstrated that AM-36 inhibited binding to the polyamine site of glutamate receptors, blocked neuronal sodium channels and had potent anti-oxidant activity. In neuronal cell cultures, AM-36 inhibited toxicity induced by N-methyl-D-aspartate (NMDA) and the sodium channel opener veratridine and, in addition, inhibited veratridine-induced apoptosis. 4. In a middle cerebral artery occlusion model of stroke in conscious rats, systemic administration of AM-36 markedly reduced both cortical and striatal infarct volume and significantly improved functional outcome in motor performance, neurological deficit and sensorimotor neglect tests. AM-36 was neuroprotective even when administration was delayed until 3 h systemically, or 5 h intravenously, after induction of stroke. 5. These studies indicate that AM-36 is a unique neuroprotective agent with multiple modes of action, making it an attractive candidate for the treatment of acute stroke in humans.     

A novel Na+ and Ca2+ channel blocker, T-477, prevents brain edema following microsphere-induced permanent occlusion of cerebral arterioles in rats

One of the most common acute complications of stroke is brain edema. Treatment of edema is recommended when the condition of the patients is deteriorating. The present study was undertaken to evaluate the effect of T-477 [(R)-(+)-2-(4-chlorophenyl)-2,3-dihydro-4-diethyl aminoacetyl-4H-1,4-benzorthiazine hydrochloride], a novel neuronal Na+ and Ca2+ channel blocker, on brain edema in rats. Cerebral ischemia was induced by intra-arterial infusion of 1000 microspheres into the forebrain of freely moving rats, resulting in brain edema. T-477 was intravenously infused continuously for 24 h or twice for 3 h with a 3-h interval between infusions immediately after microsphere injection. T-477 dose-dependently inhibited the increase in brain water content by both infusion procedures; the inhibition was statistically significant at doses of 25 mg/kg per 24 h and 14 mg/kg per 9 h. Additionally, infusion of T-477 at a dose of 14 mg/kg per 9 h significantly inhibited the decrease in K content and the increase in Ca content of the forebrain. In conclusion, T-477 prevents brain edema following microsphere-induced cerebral embolism in rats.     

Elevated extracellular K(+) concentrations inhibit N-methyl-D-aspartate-induced Ca(2+) influx and excitotoxicity.

Although extracellular [K(+)] ([K(+)](E)) is highly elevated during brain ischemia, in vitro studies aimed at explaining the mechanisms of excitotoxicity have been conducted at low [K(+)](E). Whether high [K(+)](E) affects excitotoxicity has not been formally addressed. Therefore this study, using digital fluorescence microscopy, tested how the elevation of [K(+)](E) from 5.6 to 60 mM affects N-methyl-D-aspartate (NMDA)-induced Ca(2+) and Na(+) influx, plasma membrane (PM) potential, mitochondrial Ca(2+) load, and viability of primary cultures of rat cerebellar granule cells. High [K(+)](E) curtailed the NMDA-induced Ca(2+) and Na(+) influx and mitochondrial Ca(2+) overload, and prevented neuronal death. Surprisingly, the inhibitory effect of high [K(+)](E) on the NMDA-induced Ca(2+) influx could not be linked to depolarization of the PM. Apparently, the PM of cerebellar granule cells exposed to NMDA was more depolarized at low than at high [K(+)](E), probably because the NMDA-induced Na(+) influx was greatly enhanced when the extracellular [Na(+)]/[K(+)] ratio was increased. When this ratio was small, i.e., at high [K(+)](E), the NMDA-induced increase in cytoplasmic [Na(+)] was suppressed, preventing Ca(2+) influx via the reverse operation of the Na(+)/Ca(2+) exchanger, which may explain the inhibitory effect of high [K(+)](E) on NMDA-induced Ca(2+) influx and excitotoxicity.     

Electrophysiological effect of l-cis-diltiazem, the stereoisomer of d-cis-diltiazem, on isolated guinea-pig left ventricular myocytes

l-cis-Diltiazem, the stereoisomer of the L-type Ca(2+) channel blocker d-cis-diltiazem, protects cardiac myocytes from ischemia and reperfusion injury in the perfused heart and from veratridine-induced Ca(2+) overload. We determined the effect of l-cis-diltiazem on the voltage-dependent Na(+) current (I(Na)) and lysophosphatidylcholine-induced currents in isolated guinea-pig left ventricular myocytes by a whole-cell patch-clamp technique. l-cis-Diltiazem inhibited I(Na) in a dose-dependent manner without altering the current-voltage relationship for I(Na) (K(d) values : 729 and 9 microM at holding potentials of -140 and -80 mV, respectively). A use-dependent block of I(Na), the leftward shift of the steady-state inactivation curve and the delay of recovery from inactivation suggest that l-cis-diltiazem has a higher affinity for the inactivated state of Na(+) channels. In addition to I(Na), the lysophosphatidylcholine-induced currents were inhibited by l-cis-diltiazem in a similar concentration range. It is suggested that inhibition of both Na(+) channels and lysophosphatidylcholine-activated non-selective cation channels contributes to the cardioprotective effect of l-cis-diltiazem.     

Heterogeneous increases of cytoplasmic calcium: distinct effects on down-regulation of cell surface sodium channels and sodium channel subunit mRNA levels

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by G?6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.