A novel voltage-sensitive Na and Ca channel blocker, NS-7, prevents suppression of cyclic AMP-dependent protein kinase and reduces infarct area in the acute phase of cerebral ischemia in rat

Binding of cyclic AMP to the regulatory subunit of cyclic AMP-dependent protein kinase (PKA) is an essential step in cyclic AMP-mediated intracellular signal transduction. This binding is, however, rapidly inhibited in the acute phase of cerebral ischemia, indicating that the signal transduction via PKA is very vulnerable to ischemia, although this signal pathway is very important for neuronal survival in the brain. Several lines of evidence suggest that the activation of voltage-sensitive Na+ and Ca(2+) channels is an important mediator of acute ischemic brain damage. In the present study, therefore, we examined the effect of a novel Na+ and Ca(2+) channel blocker, NS-7 (4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride), on changes in the binding activity of PKA to cyclic AMP in permanent focal cerebral ischemia, which was induced by occlusion of the middle cerebral artery by the intraluminal suture method for 5 h in the rat. NS-7 (1 mg/kg) or saline was intravenously infused 5 min after occlusion. The binding activity of PKA to cyclic AMP and local cerebral blood flow were assessed by the in vitro [(3)H]cyclic AMP binding and the [(14)C]iodoantipyrine methods, respectively. NS-7 significantly suppressed inhibition of the binding activity of PKA to cyclic AMP in the ischemic regions such as the frontal and parietal cortices and the medial region of the caudate-putamen without affecting cerebral blood flow or arterial blood pressure. Infarct area measured in the brain slices stained with cresyl violet was significantly smaller in animals treated with NS-7 than in those treated with saline. Blockade of voltage-sensitive Na+ and Ca(2+) channels by NS-7 was expected to reduce ischemia-induced depolarization and thus prevent a massive formation of free radicals, which is known to inhibit the binding activity of PKA to cyclic AMP. These data clearly indicate that NS-7 provides very efficient neuroprotection in the acute phase of cerebral ischemia, and sustains the normal function of PKA.     

Cerebroprotective action of a Na/Ca channel blocker NS-7: II. Effect on the cerebral infarction, behavioral and cognitive impairments at the chronic stage of permanent middle cerebral artery occlusion in rats

We have previously shown that NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride] reduces the size of cerebral infarction measured by 2,3,5-triphenyltetrazolium chloride staining at 48 h after permanent middle cerebral artery occlusion (MCAO) in rats. To determine whether NS-7 improves the pathological and behavioral changes at the chronic stage of MCAO, the effect of this compound on the cerebral infarction as well as the neurological and cognitive impairments was investigated 7 days after MCAO. Single or five daily injections of NS-7 (0.125–0.5 mg/kg, i.v.) significantly reduced the infarct volume and improved the neuronal dysfunction including the hind leg paralysis, walking disability and motor incoordination, and the deficit of passive avoidance task, although the neuroprotective efficacy was not different among these dosing regimens. On the other hand, the effects of single versus repeated injections of NS-7 at 0.1 or 0.2 mg/kg on the neurological symptoms were compared at 4 weeks after MCAO. At a lower dose, repeated but not single injection of NS-7 significantly improved the neurological symptoms, although the single injection was effective at a higher dose. From these findings, it is suggested that NS-7 reverses the behavioral and cognitive dysfunction observed at the chronic stage of cerebral ischemia by suppressing the cerebral infarction.     

Effects of blockade of voltage-sensitive Ca/Na channels by a novel phenylpyrimidine derivative, NS-7, on CREB phosphorylation in focal cerebral ischemia in the rat

NS-7 is a novel blocker of voltage-sensitive Ca²⁺ and Na⁺ channels, and it significantly reduces infarct size after occlusion of the middle cerebral artery. Persistent activation of cyclic AMP response element binding protein (CREB), which can be induced by increase in intracellular Ca²⁺ concentrations or other second messengers, has recently been found to be closely associated with neuronal survival in cerebral ischemia. The present study was therefore undertaken to evaluate the neuroprotective effects of NS-7 by analyzing changes in CREB phosphorylation in a focal cerebral ischemia model. CREB phosphorylation in the brain of rats was investigated immunohistochemically at 3.5–48-h recirculation after 1.5-h occlusion of the middle cerebral artery. NS-7 (1 mg/kg; NS-7 group) or saline (saline group) was intravenously injected 5 min after the start of recirculation. The NS-7 group showed significantly milder activation of CREB phosphorylation in various cortical regions after 3.5 h of recirculation than the saline group. The inner border zone of ischemia in the NS-7 group subsequently exhibited a moderate, but persistent, increase in number of phosphorylated CREB-positive neurons with no apparent histological damage. By contrast, the saline group displayed a marked, but only transient, increase in number of immunopositive neurons in this border zone after 3.5 h of recirculation, and this was followed by clear suppression of CREB phosphorylation and subsequent loss of normal neurons. These findings suggest that: (1) the marked enhancement of CREB phosphorylation in the acute post-ischemic phase may be triggered largely by an influx of calcium ions as a result of activation of the voltage-sensitive Ca²⁺ and Na⁺ channels; and that (2) the neuroprotective effects of NS-7 may be accompanied by persistent activation of CREB phosphorylation in the inner border zone of ischemia.     

Cerebroprotective action of a Na/Ca channel blocker NS-7: I. Effect on the cerebral infarction and edema at the acute stage of permanent middle cerebral artery occlusion in rats

The effect of a novel Na⁺/Ca²⁺ channel blocker NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride] on the cerebral infarction, edema and brain energy metabolism was investigated in rats after permanent middle cerebral artery occlusion (MCAO). The infarction and brain water content were evaluated at 48 h and 24 h after MCAO, respectively. A single bolus injection of NS-7 (0.03125–0.25 mg/kg) immediately after MCAO produced a dose-dependent reduction in the infarct volume as well as edema both in the cerebral cortex and striatum. Glycerol (4 g/kg) also decreased water content both in the occluded and non-occluded brain, but it did not reduce the size of cerebral infarction. Unlike glycerol, NS-7 did not change the water content in non-occluded brain. Moreover, a significant protective action was still observed even when NS-7 was injected once at 12 h after occlusion. In addition, NS-7 significantly reversed the decrease in tissue ATP content observed at 3 h but not at 0.5 h after MCAO. These findings suggest that a Na⁺/Ca²⁺ channel blocker NS-7 protects cerebral tissues against ischemic insults by improving the disturbance of cerebral energy metabolism and suppressing the cerebral edema.     

Coupling of AMPA receptors with the Na/Ca exchanger in cultured rat astrocytes

Astrocytes exhibit three transmembrane Ca²⁺ influx pathways: voltage-gated Ca²⁺ channels (VGCCs), the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na⁺/Ca²⁺ exchangers. Each of these pathways is thought to be capable of mediating a significant increase in Ca²⁺ concentration ([Ca²⁺]_i); however, the relative importance of each and their interdependence in the regulation astrocyte [Ca²⁺]_i is not known. We demonstrate here that 100 μM AMPA in the presence of 100 μM cyclothiazide (CTZ) causes an increase in [Ca²⁺]_i in cultured cerebral astrocytes that requires transmembrane Ca²⁺ influx. This increase of [Ca²⁺]_i is blocked by 100 μM benzamil or 0.5 μM U-73122, which inhibit reverse-mode operation of the Na⁺/Ca²⁺ exchanger by independent mechanisms. This response does not require Ca²⁺ influx through VGCCs, nor does it depend upon a significant Ca²⁺ influx through AMPA receptors (AMPARs). Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive intracellular Ca²⁺ stores, although depletion of these Ca²⁺ stores does not decrease the peak [Ca²⁺]_i response to AMPA. We propose that activation of AMPARs in astrocytes can cause [Ca²⁺]_i to increase through the reverse mode operation of the Na⁺/Ca²⁺ exchanger with an associated release of Ca²⁺ from intracellular stores. This proposed mechanism requires neither Ca²⁺-permeant AMPARs nor the activation of VGCCs to be effective.     

Effect of nicardipine, a Ca channel blocker, on pyruvate dehydrogenase activity and energy metabolites during cerebral ischemia and reperfusion in gerbil brain

The purpose of this study was to determine if nicardipine, a calcium ion channel blocker, affects pyruvate dehydrogenase (PDH) activity and improves energy metabolism during cerebral ischemia and reperfusion. Cerebral ischemia was induced, using the bilateral carotid artery occlusion method, for 60 min followed by reperfusion up to 120 min in gerbils. Nicardipine (1 mg/kg) or saline (vehicle-treated) was given to gerbils 30 min prior to the occlusion of the common carotid arteries. PDH activity and metabolites (ATP, PCr, and lactate) were measured in cortex prior to ischemia, immediately following ischemia, and after each reperfusion period. After 60 min ischemia, PDH activity increased in both groups, and was significantly higher in the nicardipine-treated group. After 20 min reperfusion, PDH activity in the nicardipine-treated group recovered to control levels, whereas, the PDH activity in the vehicle-treated group remained elevated, and was higher than the nicardipine-treated animals. At 60 and 120 min reperfusion, the activities in the vehicle-treated group were significantly below control levels, there were no differences, however, between the two groups. ATP and PCr concentrations were markedly depleted immediately after ischemia in both groups. ATP levels at 20 min reperfusion and PCr levels at 60 min reperfusion were significantly higher in the nicardipine-treated group. Lactate concentrations in both groups increased 7–8 fold, similarly, immediately after ischemia. During reperfusion, the lactate remained elevated in both groups, though the levels in the nicardipine-treated group were lower than those in the vehicle-treated group, but not significantly. Nicardipine treatment normalized PDH activity quickly and improved energy metabolism after reperfusion.     

Neuroprotective effects of NS-7, voltage-gated Na+/Ca2+ channel blocker in a rodent model of transient focal ischaemia

The aim of the present study was to characterize neuroprotective activity of NS- 7, a mixed voltage-gated sodium and calcium channel blocker in a model of transient focal ischaemia in rats. Ischaemia was induced by a 75 min reversible occlusion of middle cerebral artery (MCAo) using a nylon filament. NS-7 (0.5 mg/kg i.v.) or 0.9% NaCl (1 ml/kg i.v.) were infused over 3 min. starting 30 min after the MCAo. Infarct analysis was performed 72 h after ischaemia. Application of NS- 7 produced significant protection seen in neurological tests and diminished brain damage by 37% in total infarct (17.7+/- 3.0% vs. 27.9 +/- 3.2% control; [p < 0.01]; t-test), 47.8% in cortical infarct size by (8.5 +/- 2.4% vs. 16.2 +/- 2.4% control; [p < 0.01]), and by 21.5% in striatal infarction (9.2 +/- 0.8% vs. 11.7 +/- 0.9% control; [p < 0.05]). The results indicate that NS- 7 has potential for neuroprotection against transient ischaemic insult.     

Immunologic localization and kinetic characterization of a Na/Ca exchanger in neuronal and non-neuronal cells

The plasma membrane Na⁺/Ca²⁺ exchanger is believed to play a role in the regulation of Ca²⁺ fluxes in neurons, though the lack of specific inhibitors has limited the delineation of its precise contribution. We recently reported the development of antibodies against a 36-kDa brain synaptic membrane protein which immunoprecipitated exchanger activity from solubilized membranes. In the present study we examined the kinetics of the Na⁺/Ca²⁺ exchanger in primary neurons in culture, in a neuronal hybrid cell line (NCB-20), and in a fibroblast-like cell line (CV-1) to see whether the level of exchanger activity correlated with the degree of immunostaining produced by our antibodies. The V_max was determined for each cell type and found to be highest in primary neurons. Exchanger activity increased in primary neurons between days 1 and 6 in culture, but no such time-dependent change occurred in either of the cell lines. Immunoblot analysis of the three cell types probed with the anti-36-kDa protein antibodies revealed significantly greater immunostaining in the primary neurons compared with the other two cell types. Intensity of staining of neurons also increased significantly between days 1 and 6 in culture. Immunocytochemistry showed significant labelling of the primary neurons on the neuritic processes and points of contact between cells. The NCB-20 and CV-1 cells showed considerably lower levels of immunoreactivity. The antibodies immunoextracted 90% of the exchanger activity in the primary neurons and 70 and 50% of the activity in NCB-20 and CV-1 cells respectively. Thus the expression of the 36-kDa protein appears to be closely associated with the Na⁺/Ca²⁺ exchanger in neuronal cells and, possibly to a lesser extent, in non-neuronal cells.     

Inhibition by neuroprotective drug NS-7 of nicotine-induced Na influx, Ca influx and catecholamine secretion 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 nicotine-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀=15.5 μM); the suppression by NS-7 was observed in the presence of ouabain, an inhibitor of Na⁺,K⁺-ATPase, and was not attenuated upon the washout of NS-7. NS-7 decreased nicotine-induced maximum influx of ²²Na⁺ without altering the EC₅₀ value of nicotine. Also, NS-7 diminished nicotine-induced ⁴⁵Ca²⁺ influx via nicotinic receptors and voltage-dependent Ca²⁺ channels (IC₅₀=14.1 μM) and catecholamine secretion (IC₅₀=19.5 μM). These results suggest that NS-7 produces noncompetitive and long-lasting inhibitory effects on neuronal nicotinic receptors in adrenal chromaffin cells, and interferes with the stimulus-secretion coupling.     

A new type of Ca channel blocker, NC-1100, inhibits the low- and high-threshold Ca currents in the rat CNS neurons

The effect of a new type of organic Ca²⁺ channel blocker, NC-1100 [(±)-1-(3,4-dimethoxyphenyl)-2-(4-diphenylmethylpiperazinyl)ethanol dihydrochloride], on both low- and high-threshold Ca²⁺ currents was studied in the whole-cell mode of the pyramidal neurons freshly dissociated from rat hippocampal CA1 region under voltage-clamp condition. The NC-1100 reversibly reduced the high-threshold Ca²⁺ current (HVAI_Ca) in a concentration-dependent manner without affecting the current-voltage relationship. The values of half-inhibition (IC₅₀) were 1.3 × 10⁻⁵ and 9.1 × 10⁻⁶M in external solution containing 10 and 2.5 mM Ca²⁺, respectively. The NC-1100 also decreased the low-threshold Ca²⁺ current (LVAI_Ca) in a concentration-dependent manner. The inhibitory potency was augmented by increasing the stimulation frequency and / or decreasing the extracellular Ca²⁺ concentration to a physiological range (2.5 mM). The IC₅₀ value decreased to 7.7 × 10⁻⁷M in external solution containing 2.5 mM Ca²⁺ at a stimulation frequency of 1 Hz. The NC-1100 delayed the reactivation of LVA Ca²⁺ channel and enhanced voltage-dependently the steady-state inactivation, suggesting that this drug bound not only the resting LVA Ca²⁺ channel but also the inactivated one.     

Influence of lactate accumulation on Na,K-ATPase activity of ischemic and post-ischemic brain

In this study the cerebral Na+, K+-ATPase activity as well as selected parameters of oxidative metabolism and electrophysiological function were assessed in normoglycemic and hyperglycemic rats which were exposed to ischemia produced by electrocautery of the vertebral arteries and reversible occlusion of the carotid arteries. In hyperglycemic animals 0.5 h of ischemia was associated with massive accumulation of lactate (34 mumol X g-1) and enhanced Na+, K+-ATPase activity (116% control), whereas normoglycemic animals showed more moderate lactate accumulation (17 mumol X g-1) and normal Na+, K+-ATPase activity (102% control). In normoglycemic animals release of the carotid clamps and recirculation for 0.5-1.5 h was associated with a normalization of the lactate levels and a decrease in Na+, K+-ATPase activity (68-72% control). Restituted hyperglycemic animals showed metabolic changes which seemed related to the blood pressure, with hypotensive hyperglycemic animals showing continuing massive lactacidosis (30-35 mumol X g-1) and enhanced Na+, K+-ATPase activity (108-110% control), whereas normotensive hyperglycemic animals showed progressive decreases in lactate level (14-20 mumol X g-1) and normal or mildly suppressed Na+, K+-ATPase activity (88-97% control). These patterns of change suggest that the reperfusion of the post-ischemic hyperglycemic-hyperlactacidotic brain was inadequate or non-homogeneous.     

Effect of dantrolene on KCl- or NMDA-induced intracellular Ca2+ changes and spontaneous Ca2+ oscillation in cultured rat frontal cortical neurons

Summary Dantrolene has been known to affect intracellular Ca²⁺ concentration ([Ca²⁺]_i) by inhibiting Ca²⁺ release from intracellular stores in cultured neurons. We were interested in examining this property of dantrolene in influencing the [Ca²⁺]_i affected by the NMDA receptor ligands, KCl, L-type Ca²⁺ channel blocker nifedipine, and two other intracellular Ca²⁺-mobilizing agents caffeine and bradykinin. Effect of dantrolene on the spontaneous oscillation of [Ca²⁺]_i was also examined. Dantrolene in M concentrations dose-dependently inhibited the increase in [Ca²⁺]_i elicited by NMDA and KCl. AP-5, MK-801 (NMDA antagonists), and nifedipine respectively reduced the NMDA and KCl-induced increase in [Ca²⁺]_i. Dantrolene, added to the buffer solution together with the antagonists or nifedipine, caused a further reduction in [Ca²⁺]_i to a degree similar to that seen with dantrolene alone inhibiting the increase in [Ca₂₊]_i caused by NMDA or KCl. At 30 M, dantrolene partially inhibited caffeine-induced increase in [Ca²⁺]_i whereas it has no effect on the bradykinin-induced change in [Ca²⁺]_i. The spontaneous oscillation of [Ca²⁺]_i in frontal cortical neurons was reduced both in amplitude and in base line concentration in the presence of 10 M dantrolene. Our results indicate that dantrolene's mobilizing effects on intracellular Ca²⁺ stores operate independently from the influxed Ca²⁺ and that a component of the apparent increase in [Ca²⁺]_i elicited by NMDA or KCl represents a dantrolene-sensitive Ca₂₊ release from intracellular stores. Results also suggest that dantrolene does not affect the IP₃-gated release of intracellular Ca²⁺ and that the spontaneous Ca²⁺ oscillation is, at least partially, under the control of Ca²⁺ mobilization from internal stores.Abbreviations AP-5 (±)-2-amino-5-phosphonopentanoic acid - AMPA amino-3-hydroxy-5-methyl-isoxazole-4-propionate - BSS balanced salt solution - CNS central nervous system - CICR Ca²⁺-induced Ca²⁺ release - DCKA 5,7-dichlorokynurenate - DNasel deoxyribonuclease I - DMEM Dulbecco's Modified Eagle's Medium - EGTA ethylene glycol-bis(-aminoethyl ether)N,N,N,N,-tetraacetic acid - FCS fetal calf serum - fura-2-AM 1-(2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy-2-ethane-N,N,N,N-te-traacetic acid, pentaacetoxymethyl ester - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - [Ca ²⁺] _i intracellular free Ca²⁺ concentration - LTP long-term potantiation - MK-801 (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]-cyclohepten-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate     

Brevetoxin derivatives act as partial agonists at neurotoxin site 5 on the voltage-gated Na+ channel

Brevetoxins (PbTx-1 to PbTx-10) are potent lipid-soluble polyether neurotoxins produced by the marine dinoflagellate Karina brevis, an organism associated with 'red tide' blooms in the Gulf of Mexico. Ingestion of shellfish contaminated with K. brevis produces neurotoxic shellfish poisoning (NSP) in humans. NSP symptoms emanate from brevetoxin activation of neurotoxin site 5 on voltage-gated sodium channels (VGSC) [Toxicon 20 (1982) 457]. In primary cultures of rat cerebellar granule neurons (CGN), brevetoxins produce acute neuronal injury and death. The ability of a series of naturally occurring and synthetic brevetoxins to trigger Ca(2+) influx in CGN was explored in the present study. Intracellular Ca(2+) concentration was monitored in fluo-3-loaded CGN using a fluorescent laser imaging plate reader. The naturally occurring derivatives PbTx-1, PbTx-2 and PbTx-3 all produced a rapid and concentration-dependent increase in cytosolic [Ca(2+)]. The maximum response to PbTx-1 was approximately two-fold greater than that of either PbTx-2 or PbTx-3. Two synthetic derivatives of PbTx-3, alpha-naphthoyl-PbTx-3 and beta-naphthoyl-PbTx-3, were also tested. Both alpha- and beta-naphthoyl-PbTx-3 stimulated a rapid and concentration-dependent Ca(2+) influx that was, however, less efficacious than that of PbTx-3. These data indicate that, analogous to neurotoxin site 2 ligands, activators of neurotoxin site 5 display a range of efficacies, with PbTx-1 being a full agonist and other derivatives acting as partial agonists.     

SM-20220, a Na(+)/H(+) exchanger inhibitor: effects on ischemic brain damage through edema and neutrophil accumulation in a rat middle cerebral artery occlusion model

The Na(+)/H(+) exchanger (NHE) is activated during ischemia-reperfusion in an effort to restore intracellular pH to normal levels. The NHE is recognized to exist as a distinct protein in the plasma membranes of a variety of cells. We investigated the pharmacological effects of a Na(+)/H(+) exchanger inhibitor, SM-20220 (N-(aminoiminomethyl)-1-methyl-1-H-indole-2-carboxamide methanesulfonate), on ischemic brain damage, edema and neutrophil accumulation at 72 h after middle cerebral artery (MCA) occlusion in a rat MCA occlusion model. SM-20220 was intravenously administered as a bolus injection immediately after occlusion, followed by a continuous infusion over 2.5 h. At 72 h after occlusion, the infract area was measured using hematoxylin-eosin staining and, using the same slices, neutrophils in the brain were immuno-stained with anti-myeloperoxidase (n=11). In a separate study, rat behavior was scored and scaled, and brains removed for the determination of water content by the dry-weight method. SM-20220 significantly (P<0.05) attenuated cerebral infarct volume, water content, and the neutrophil accumulation at 72 h after the MCA occlusion, and ameliorated neurological deficits. SM-20220, an NHE inhibitor prevented the progress of cerebral ischemic damage and edema following MCA occlusion in rats though a possible mechanism that may be due to the inhibition of neutrophil accumulation. The NHE in neutrophils may enhance the progress of cerebral damage following cerebral ischemia-reperfusion.     

Apamin, a blocker of the calcium-activated potassium channel, induces neurodegeneration of Purkinje cells exclusively

Following acute intracerebroventricular injections of 1 ng of apamin and chronic apamin infusion (0.4 ng/μl, 0.5 μl/h, 14 days), the rat brains exhibited bilateral damage only in the cerebellum. The argyrophilic cells were Purkinje cells in copula pyramis, flocculus, paraflocculus, and paramedian lobules. These data demonstrate that the inactivation of small conductance Ca²⁺-activated K⁺ channels by apamin induces a non-limbic neurodegeneration.     

A pharmacological model for studying the role of Na gradients in the modulation of synaptosomal free [Ca]i levels and energy metabolism

Lactate production (J_lac), oxygen consumption rate (Q_O2), plasma membrane potentials (E_m) and cytosolic free calcium levels [Ca²⁺]_i were studied on symaptosomes isolated from rat brains, incubated in presence of high doses of nicardipine (90 μM), diltiazem (0.5 mM) and verapamil (0.25 mM), and submitted to depolarizing stimulation or inhibition of mitochondrial respiration. Nicardipine was able to completely prevent the veratridine-induced stimulation ofJ_lac, Q_O2andE_m depolarization, whereas diltiazem and verapamil were less effective, although the concentrations used were 5 and 3 times higher, respectively, than nicardipine. Diltiazem, verapamil and nicardipine (9 μM) also prevented the veratridine-induced increase in [Ca²⁺]_i, this effect being much less pronounced if the drugs were added after veratridine. Monensin (20 μM) was also able to increase [Ca²⁺]_i but this effect was not affected by verapamil. Synaptosomes were also submitted to an inhibition of respiration of intrasynaptic mitochondria by incubation with rotenone (5 μM); in this condition of mimicked hypoxiaE_m was more positive of about 11 mV; none of the drugs utilized modified this situation. The rotenone-induced 3-fold increase inJ_lac was barely modified by diltiazem and verapamil but it was completely abolished by nicardipine. The possible mechanism of the counteracting action of the drugs towards veratridine stimulation and rotenone inhibition and the involvement of Na⁺/Ca²⁺ exchanger in affecting [Ca²⁺]_i are discussed.     

Effect of the removal of extracellular Ca on the response of cytosolic concentrations of Ca to ouabain in carotid body glomus cells of adult rabbits

Effect of the removal of extracellular Ca²⁺ on the response of cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) to ouabain, an Na⁺/K⁺ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca²⁺]_i (mean±S.E.M.; 38±5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca²⁺]_i increase was abolished by the removal of extracellular Na⁺. D600 (50 μM), an L-type voltage-gated Ca²⁺ channel antagonist, inhibited the [Ca²⁺]_i increase by 57±7% (n=4). Removal of extracellular Ca²⁺ eliminated the [Ca²⁺]_i increase, but subsequent washing out of ouabain in Ca²⁺-free solution produced a rise in [Ca²⁺]_i (62±8% increase, n=6, P<0.05), referred to as a [Ca²⁺]_i rise after Ca²⁺-free/ouabain. The magnitude of the [Ca²⁺]_i rise was larger than that of ouabain-induced [Ca²⁺]_i increase. D600 (5 μM) inhibited the [Ca²⁺]_i rise after Ca²⁺-free/ouabain by 83±10% (n=4). These results suggest that ouabain-induced [Ca²⁺]_i increase was due to Ca²⁺ entry involving L-type Ca²⁺ channels which could be activated by cytosolic Na⁺ accumulation. Ca²⁺ removal might modify the [Ca²⁺]_i response, resulting in the occurrence of a rise in [Ca²⁺]_i after Ca²⁺-free/ouabain which mostly involved L-type Ca²⁺ channels.     

Temporal alterations in voltage gated Ca2+ channel immunoreactivities in the gerbil hippocampus following ischemic insults

In the present study, temporal changes of voltage-gated Ca²⁺ channel (VGCC) immunoreactivities were evaluated in the gerbil hippocampus following ischemia. P/Q-type VGCC immunoreactivity was elevated in the hippocampus in the 3 h post-ischemic group. In the 30 min post-ischemic group, N-type VGCC immunoreactivity began to increase only in the CA1 region. L-type (1C) VGCC immunoreactivity was significantly increased in the 12 h post-ischemic group. L-type (1D) VGCC immunoreactivity began to increase in the CA1 region in the 30 min post-ischemic group and peaked in the 12 h post-ischemic group. These findings suggest that the altered VGCC immunoreactivities following ischemia may play an important role in the ischemic neuronal injury.     

Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+-dependent mechanism in cultured rat sympathetic neurons

Superior-cervical ganglion (SCG) cells dissociated from newborn rats depend on nerve growth factor (NGF) for survival. Membrane depolarization with elevated K+ is known to prevent neuronal death following NGF deprivation and/or to promote survival via a Ca2+-dependent mechanism. Here we have exploited the possibility of whether or not a Na+-dependent pathway for neuronal survival is present in these cells. Veratridine (ec50=40 nM), a voltage-dependent Na+ channel activator, significantly delayed the onset of apoptotic cell death in NGF-deprived SCG neurons that had been cultured for 7 days in the presence of NGF. This effect was blocked completely by Na+ channel blockers including tetrodotoxin (TTX, 1 μM), benzamil (25 μM) and flunarizine (1 μM), but was not attenuated by nimodipine (1 μM), an L-type Ca²⁺ channel blocker. The saving effect of veratridine on cultured neurons was observed even in low Ca²⁺ media (0–1.0 mM), but was completely abolished in a low Na⁺ medium (38 mM). Sodium-binding benzofuran isophthalate was employed as a fluorescent probe for monitoring the level of cytoplasmic free Na⁺, which revealed a sustained increase in its level (12.9 mM, 307% of that of control) in response to veratridine (0.75 μM). The TTX or flunarizine completely blocked veratridine-induced Na⁺ influx in these cultured neurons. Moreover, no appreciable increase in intracellular Ca²⁺ was detected under these conditions. Though Na⁺ channels were effectual in SCG neurons which were freshly isolated from newborn rats, the Na⁺-dependent saving effect of veratridine was not observed in these young neurons. These lines of evidence suggest that the death-suppressing effect of veratridine on cultured SCG neurons depends on the Na⁺ influx via voltage-dependent Na⁺ channels, and suggests the presence of Na⁺-dependent regulatory mechanism(s) in neuronal survival.     

NCX3 knockout mice exhibit increased hippocampal CA1 and CA2 neuronal damage compared to wild-type mice following global cerebral ischemia

There is uncertainty as to whether the plasma membrane Na(+)/Ca(2+)exchanger (NCX) has a neuroprotective or neurodamaging role following cerebral ischemia. To address this issue we compared hippocampal neuronal injury in NCX3 knockout mice (Ncx3(-/-)) and wild-type mice (Ncx3(+/+)) following global cerebral ischemia. Using a bilateral common carotid artery occlusion (BCCAO) model of global ischemia we subjected NCX3 knockout and wild-type mice to 17 and 15 minutes of ischemia. Following the 17 minute period of ischemia, wild-type mice exhibited approximately 80% CA1 neuronal loss and approximately 40% CA2 neuronal loss. In contrast, NCX3 knockout mice displayed >95% CA1 neuronal loss and approximately 95% CA2 neuronal loss. Following the 15 minute period of ischemia, wild-type mice did not exhibit any significant hippocampal neuronal loss. In contrast, NCX3 knockout mice displayed approximately 45% CA1 neuronal loss and approximately 25% CA2 neuronal loss. The results clearly demonstrate that mice deficient in the NCX3 protein are more susceptible to global cerebral ischemia than wild-type mice. Our findings suggest NCX3 has a positive role in maintaining neuronal intracellular calcium homeostasis following ischemia, and that when exchanger function is compromised neurons are more susceptible to calcium deregulation and cell death.