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.     

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.     

Late expression of Na+/H+ exchanger 1 (NHE1) and neuroprotective effects of NHE inhibitor in the gerbil hippocampal CA1 region induced by transient ischemia

Although acidosis may be involved in neuronal death, the participation of Na⁺/H⁺ exchanger (NHE) in delayed neuronal death in the hippocampal CA1 region induced by transient forebrain ischemia has not been well established. In the present study, we investigated the chronological alterations of NHE1 in the hippocampal CA1 region using a gerbil model after ischemia/reperfusion. In the sham-operated group, NHE1 immunoreactivity was weakly detected in the CA1 region. Two and 3 days after ischemia/reperfusion, NHE1 immunoreactivity was observed in glial components, not in neurons, in the CA1 region. Four days after ischemia/reperfusion, NHE1 immunoreactivity was markedly increased in CA1 pyramidal neurons as well as glial cells. These glial cells were identified as astrocytes based on double immunofluorescence staining. Western blot analysis also showed that NHE protein level in the CA1 region began to increase 2 days after ischemia/reperfusion. The treatment of 10 mg/kg 5-(N-ethyl-N-isopropyl) amiloride, a NHE inhibitor, significantly reduced the ischemia-induced hyperactivity 1day after ischemia/reperfusion. In addition, NHE inhibitor potently protected CA1 pyramidal neurons from ischemic damage, and NHE inhibitor attenuated the activation of astrocytes and microglia in the ischemic CA1 region. In addition, NHE inhibitor treatment blocked Na⁺/Ca²⁺ exchanger 1 immunoreactivity in the CA1 region after transient forebrain ischemia. These results suggest that NHE1 may play a role in the delayed death, and the treatment with NHE inhibitor protects neurons from ischemic damage.     

Involvement of calmodulin in neuronal cell death

A large body of evidence indicates that disturbances of Ca(2+) homeostasis may be a causative factor in the neurotoxicity following cerebral ischemia. However, the mechanisms by which Ca(2+) overload leads to neuronal cell death have not been fully elucidated. Calmodulin, a major intracellular Ca(2+)-binding protein found mainly in the central nervous system, mediates many physiological functions in response to changes in the intracellular Ca(2+) concentration, whereas Ca(2+) overload in neurons after excitotoxic insult may induce excessive activation of calmodulin signaling pathways, leading to neuronal cell death. To determine the role of calmodulin in the induction of neuronal cell death, we generated primary rat cortical neurons that express a mutant calmodulin with a defect in Ca(2+)-binding affinity. Neurons expressing the mutant had low responses of calmodulin-dependent signaling to membrane depolarization by high KCl and became resistant to glutamate-triggered excitotoxic neuronal cell death compared with the vector or wild-type calmodulin-transfected cells, indicating that blocking calmodulin function is protective against excitotoxic insult. These results suggest that calmodulin plays a crucial role in the processes of Ca(2+)-induced neuronal cell death and the possibility that the blockage of calmodulin attenuates brain injury after cerebral ischemia.     

Neuroprotective effect of NS-7, a novel Na+ and Ca2+ channel blocker,in a focal ischemic model in the rat

NS-7 is a novel, voltage-dependent Na(+) and Ca(2+) channel blocker. This study evaluated the in vivo neuroprotective effect of NS-7 in a rat transient focal ischemic model when administered during occlusion. Left middle cerebral artery occlusion was induced in adult male Sprague-Dawley rats for 120 min using an intraluminal thread method. The rats received a single intravenous injection of NS-7 or saline (control group) just after the onset of ischemia, and at 30, 60 and 120 min after ischemia. Their brains were removed after 48 h reperfusion, sectioned, and stained with hematoxylin and eosin. Animals were evaluated by neurological examination at 120 min ischemia and 48 h reperfusion. Infarcted cortex and striatum were measured quantitatively and infarction volumes were calculated. Cortical infarction volumes were 128+/-74 (NS-7) and 214+/-64 mm(3) (control) immediately after the ischemia group, 155+/-48 (NS-7) and 225+/-12 mm(3) (control) after the 30 min group, 160+/-54 (NS-7) and 225+/-48 mm(3) (control) after the 60 min group, and 176+/-43 (NS-7) and 223+/-38 mm(3) (control) after the 120 min group. Cortices in NS-7-treated groups were significantly less infarcted than in control groups at all treatment times. There was no significant difference in the striatal infarction volume between the treatment and control groups. Neurological examination showed that hemiparesis and abnormal posture of the NS-7 groups were significantly more improved at 48 h reperfusion than those of the control groups without posture examination in the 120 min group. These observations suggest that NS-7 may be a new potential therapeutic agent for the acute phase of cerebral infarction.     

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     

Taurine-induced modulation of voltage-sensitive Na+ channels in rat dorsal root ganglion neurons

The physiological role of taurine, an abundant free amino acid in the neural system, is still poorly understood. The aim of this study was to investigate its effect on TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na+ currents in enzymatically dissociated neurons from rat dorsal root ganglion (DRG) with conventional whole-cell recording manner under voltage-clamp conditions. A TTX-S Na+ current was recorded preferentially from large DRG neurons and a TTX-R Na+ current preferentially from small ones. For TTX-S Na+ channel, taurine of the concentration > or = 10 mM shifted the activation curve in the depolarizing direction and the inactivation curve in the hyperpolarizing direction. There was no change in the activation curve for TTX-R Na+ channel and the inactivation curve was shifted in the hyperpolarizing direction slightly in the presence of taurine > or = 20 mM. When the recovery kinetics was examined, the presence of taurine resulted in a slower recovery from inactivation of TTX-S currents and no change of TTX-R ones. All the effects of taurine were weakly concentration-dependent and partly recovered quite slowly after washout. Our data indicate that taurine alters the properties of Na+ currents in intact DRG neurons. These may contribute to the understanding of taurine as a natural neuroprotectant and the potential of taurine as a useful medicine for the treatment of sensory neuropathies.     

Cell death in weaver mouse cerebellum

Mice with the weaver mutation exhibit an uneven weave to their gait, ataxia, mild locomotor hyperactivity and, occasionally, tonic-clonic seizures. A single amino acid mutation in a G-protein coupled, inwardly rectifying K+ channel, GIRK2, gives rise to the symptoms seen in the weaver mice. Two areas of the brain are primarily affected. Cerebellar granule cell neurons die soon after birth and dopaminergic neurons are severely depleted in the substantia nigra. In this article we review recent studies of wild-type and mutant GIRK channels found in native cells or introduced into expression systems. We also review two models that explain some of the details leading to the neuronal cell death observed in weaver mice.     

Repetitive application of caffeine sensitizes caffeine-induced Ca release in bullfrog sympathetic ganglion neurons

Cytosolic free calcium concentration ([Ca(2+)](i)) was recorded from cultured bullfrog sympathetic ganglion cells loaded with the Ca(2+)-indicator Fura-2 or Fura-6F. Repetitive application of caffeine at a low concentration, which either failed to produce any [Ca(2+)](i) elevation or induced a small gradual increase in [Ca(2+)](i) at first challenge, produced a drastic increase in the amplitude of Ca(2+) release (caffeine response). The caffeine response eventually reached peak amplitude and then remained constant even if caffeine application were continued. This augmentation was maintained for up to 2 h, and was achieved not only by repetitive application but also by a long exposure of caffeine. However, this augmentation was neither achieved by repetitive administration of high K(+)-solution, nor caused by inhibition of phosphodiesterase by caffeine. The repetitive or sustained application of caffeine is suggested to increase the caffeine sensitivity of the calcium release channel to calcium, thus causing the potentiation of the caffeine response.     

Expression and kinetic properties of Na(+) currents in rat cardiac dorsal root ganglion neurons

The expression and properties of voltage-gated Na(+) currents in cardiac dorsal root ganglion (DRG) neurons were assessed in this study. Cardiac DRG neurons were labelled by injecting the Fast Blue fluorescent tracer into the pericardium. Recordings were performed from 138 cells. Voltage-dependent Na(+) currents were found in 115 neurons. There were 109 neurons in which both tetrodotoxin-sensitive (TTX-S, blocked by 1 microM of TTX) and tetrodotoxin-resistant (TTX-R, insensitive to 1 microM of TTX) Na(+) currents were present. Five cells expressed TTX-R current only and one cell only the TTX-S current. The kinetic properties of Na(+) currents and action potential waveform parameters were measured in neurons with cell membrane capacitance ranging from 15 to 75 pF. The densities of TTX-R (110.0 pA/pF) and TTX-S (126.1 pA/pF) currents were not significantly different. Current threshold was significantly higher for TTX-R (-34 mV) than for TTX-S (-40.4 mV) currents. V(1/2) of activation for TTX-S current (-19.6 mV) was significantly more negative than for TTX-R current (-9.2 mV), but k factors did not differ significantly. V(1/2) and the k constant for inactivation for TTX-S currents were -35.6 and -5.7 mV, respectively. These values were significantly lower than those recorded for TTX-R current for which V(1/2) and k were -62.3 and -7.7 mV, respectively. The action potential threshold was lower, the 10-90% rise time and potential width were shorter before than after the application of TTX. Based on this we drew the conclusion that action potential recorded before adding tetrodotoxin was mainly TTX-S current dependent, while the action potential recorded after the application of toxin was TTX-R current dependent. We also found 23 cells with mean membrane capacitance ranging from 12 to 35 pF (the smallest labelled DRG cells found in this study) that did not express the Na(+) current. The function of these cells is unclear. We conclude that the overwhelming majority of cardiac dorsal root ganglion neurons in which voltage-dependent Na(+) currents were present, exhibited both TTX-S and TTX-R Na(+) currents with remarkably similar expression and kinetic properties.     

Domoic acid neurotoxicity in cultured cerebellar granule neurons is controlled preferentially by the NMDA receptor Ca influx pathway

We have monitored real-time alterations in [Ca(2+)](i) in fluo-3-loaded cerebellar granule neurons exposed to domoate, and ascertained the influence of pharmacological blockers of various Ca(2+) entry pathways on intracellular Ca(2+) accumulation, excitatory amino acid (EAA) release and neuronal death. Domoate produced a rapid and concentration-dependent increase in [Ca(2+)](i), the magnitude of which correlated closely with the severity of neuron loss. The increase in [Ca(2+)](i) was derived from activation of NMDA receptors, L-type voltage-sensitive calcium channels (VSCC) and the reversed mode of operation of the Na(+)/Ca(2+) exchanger. When the level of neuroprotection conferred by pharmacological manipulation of these calcium entry pathways was regressed with the corresponding reductions in [Ca(2+)](i) load, it was observed that neuronal vulnerability is controlled preferentially by NMDA receptors. This observation is consistent with our previous study of brevetoxin-induced autocrine excitotoxicity and with the source specificity hypothesis of others [J. Neurochem. 71 (1998) 2349], which suggests that elevation of [Ca(2+)](i) in the vicinity of the NMDA receptor ion channel activates processes leading to neuronal death.     

Heat shock protects cultured rat astrocytes in a model of reperfusion injury

We have previously found that incubation of cultured rat astrocytes in Ca²⁺-containing medium after exposure to Ca²⁺-free medium caused an increase in intracellular Ca²⁺ ([Ca²⁺_i) followed by delayed cell death. Here, we examined whether thermal stress protects astrocytes from cell death in this model system of reperfusion injury. Cultured astrocytes were preincubated at 40–44°C for 10–20 min in fetal calf serum-free medium, incubated at 37°C for 24 h in serum-containing medium, and subjected to the in vitro reperfusion experiment. Thermal stress attenuated reperfusion-induced cell toxicity. Furthermore, the stress increased cell viability after incubation with serum-free medium containing Ca²⁺. These effects of heat shock required incubation in serum-containing medium for at least 12 h after heat shock, and it was blocked by the protein synthesis inhibitor cycloheximide. Thermal stress increased synthesis of several proteins, and one of the inducible proteins was identified as the 72-kDa heat shock protein by an immunoblot analysis. Neither the increase in [Ca²⁺]_i nor the Na⁺-Ca²⁺ exchange activity in astrocytes induced in this model were affected by thermal stress. These findings suggest that heat shock proteins protect astrocytes from cell death in a model of reperfusion injury and they may affect processes down stream of the increase in [Ca²⁺]_i.     

Overexpression of Bcl-2 attenuates MPP, but not 6-ODHA, induced cell death in a dopaminergic neuronal cell line

In order to investigate the role of Bcl-2 in dopaminergic cells, we established a dopaminergic neuronal cell line (MN9D) stably expressing human Bcl-2 (MN9D/Bcl-2) or neomycin (MN9D/Neo). Overexpression of Bcl-2 in MN9D cells attenuated cell death due to treatment of mitochondrial electron transport inhibitors includingN-methyl-4-phenylpyridinium, whereas it did not prevent cell death induced by reagents generating reactive oxygen species including 6-hydroxy-dopamine. Moreover, the rate of glucose uptake in MN9D/Bcl-2 was significantly lower than that in MN9D/Neo after MPP⁺treatment. Thus, Bcl-2 may counter aberrations in mitochondrial electron transfer processes by altering energy metabolism within the MN9D cells.     

Control of the Na, K -pump by nerve growth factor is essential to neuronal survival

Recently we have shown that nerve growth factor (NGF) controls the performance of the Na⁺, K⁺ -pump in its target ganglionic neurons in suspension cultures. In the present study, enriched neuronal preparations of embryonic day 8 (E8) chick dorsal root ganglia (DRG) were obtained by means of a differential attachment procedure using tissue culture plastic dishes. Neurons were routinely seeded into polyornithine-coated 16 mm culture wells in the presence of NGF. After 18 h, cultures were switched to media with or without NGF, and containing either⁸⁶Rb⁺ (as a tracer for K⁺) or²²Na⁺ (as a tracer for Na ions). Over the next 12–15 h the cultures were assessed for numbers of surviving neurons and accumulated radioactivity. Cultured E8 chick DRG neurons fail to maintain their intracellular K⁺ concentration when deprived of NGF over 4–6 h. The NGF-deprived and K⁺- depleted neurons reaccumulate K⁺ within minutes of delayed NGF administration. The occurrence of this K⁺ response in culture to added NGF parallels the response occurring in E8 neuronal suspensions, including the time of onset of irreversibility. Similar experiments performed with²²Na⁺ indicate corresponding ionic behaviors for cultured E8 DRG neurons. These NGF-controlled ionic responses in monolayer cultures occur for E7 and E10 neurons, but not E14 neurons and parallel the survival response to NGF of the same neurons. Blocking the pump performance by NGF deprivation leads to neuronal death. Identical results are obtained by addition of oubain or omission of external K⁺ in the presence of NGF. Partial reduction of pump performance by any one of these treatments leads to partial survival of the neuronal population in a precisely predictable manner. Therefore, control of the pump by NGF is an essential component of the NGF action on neuronal survival.     

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca and glycolysis

To elucidate the mechanism of pH_i changes induced by membrane depolarization, the variations in pH_i and [Ca²⁺]_i induced by a number of depolarizing agents, including high K⁺, veratridine, N-methyl-

Full-size image

-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pH_i and an elevation of intracellular calcium ([Ca²⁺]_i) in the CA1 pyramidal cell layer. The increases in [Ca²⁺]_i caused by the depolarizing agents almost completely disappeared in the absence of Ca²⁺ (0 mM Ca²⁺ with 1 mM EGTA). In Ca²⁺ free media, pH_i acid shifts produced by high K⁺, veratridine or NMDA were attenuated by 10–25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pH_i acid shifts induced by both high K⁺ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K⁺, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na⁺–K⁺ ATPase activity. A Ca²⁺-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H⁺ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.     

The mechanism of the actions of oxaliplatin on ion currents and action potentials in differentiated NG108-15 neuronal cells

Oxaliplatin (OXAL) is a platinum-based chemotherapeutic agent which is effective against advanced or metastatic gastrointestinal cancer. However, the mechanisms responsible for the development of the neuropathy induced by this agent remain unclear. In this study, we attempted to evaluate the possible effects of OXAL on ion currents and action potentials (APs) in NG108-15 cells differentiated with dibutyryl cyclic-AMP. Application of OXAL decreased the peak amplitude of voltage-gated Na⁺ current (I_Na) with no change in the overall current–voltage relations of the currents. This agent also produced a concentration-dependent slowing of I_Na inactivation. A further application of ranolazine reversed OXAL-induced slowing of I_Na inactivation. Unlike ranolazine or riluzole, OXAL had no effect on persistent I_Na elicited by long ramp pulses. OXAL (100 μM) also had little or no effect on the peak amplitude of L-type Ca²⁺ currents in NG108-15 cells, while it suppressed delayed-rectifier K⁺ current. In current-clamp recordings, OXAL alone reduced the amplitude of APs; however, it did not alter the duration of APs. However, after application of tefluthrin, OXAL did increase the duration of APs. Moreover, OXAL decreased the peak amplitude of I_Na with a concomitant reduction of current inactivation in HEK293T cells expressing SCN5A. The effects of OXAL on ion currents presented here may contribute to its neurotoxic actions in vivo.     

Slight impairment of Na+,K+-ATPase synergistically aggravates ceramide- and beta-amyloid-induced apoptosis in cortical neurons

Dysfunction of the Na(+),K(+)-ATPase (Na(+),K(+)-pump), due to reduced energy supply or increased endogenous ouabain-like inhibitors, likely occurs under pathological conditions in the central nervous system. In cultured mouse cortical neurons, we examined the hypothesis that a mild non-toxic inhibition of the Na(+),K(+)-ATPase could synergistically sensitize the vulnerability of neurons to normally non-lethal apoptotic signals. Ouabain at a low concentration of 0.1 microM slightly lessened the Na(+),K(+)-pump activity measured as an ouabain-sensitive current, yet did not affect K(+) homeostasis and viability of cortical neurons. Co-exposure to 0.1 microM ouabain plus non-lethal C(2)-ceramide (5 microM) or beta-amyloid 1-42 (5 microM), however, induced marked intracellular K(+) loss, caspase-3 cleavage, DNA laddering, and synergistically triggered neuronal death. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) predominantly blocked the caspase activation and neuronal death. These results suggest that slight impairment of Na(+),K(+)-pump activity may amplify the disruption of K(+) homeostasis in the presence of a non-lethal apoptotic insult, leading to activation of apoptotic cascade and substantial neuronal injury.     

Effect of systemic zinc administration on delayed neuronal death in the gerbil hippocampus

The divalent cation zinc has been reported to possess several physiological properties such as blocking apoptotic cell death through an inhibitory effect on Ca²⁺-Mg²⁺ endonuclease activity, or modulating the neurotoxicity via glutamate receptor subtypes. In the present study, we investigated the effect of peripherally injected zinc on delayed neuronal death seen in the hippocampus after transient global ischemia, in order to elucidate a possible beneficial role on zinc in ischemic neuronal cell death. Forty-five adult Mongolian gerbils of both sexes underwent transient bilateral clipping of the common carotid arteries for 3 min. In the pretreated animals, ZnCl₂ (20 mg/kg) was injected subcutaneously once, 1 h before ischemia (superacute group; n=6) or twice at 24 and 48 h before ischemia (subacute group; n=14). Histological survey was carried out 3 days later by in situ DNA fragmentation method and 4 days later by hematoxylin-eosin staining by semiquantatively counting dead neurons in the CA1 sector. Subacute zinc pre-administration significantly reduced the nuclear damage and subsequent neuronal death; however, superacutely pre-administered zinc did not protect hippocampal neurons against ischemia but it did not aggravate the effect of ischemia, either. The present study suggested that transfer of exogenous zinc into the intracellular space is required for neuroprotection, presumably via the anti-endonuclease activity.     

Does intracellular release of Ca participate in the afterhyperpolarization of a sympathetic neurone?

The afterhyperpolarization (AHP) of an action potential in the bullfrog sympathetic ganglion cell was highly sensitive to anions (a factor affecting Ca²⁺ release¹⁶) filled in a recording electrode; it was slower for citrate ion than for Cl⁻. The AHP recorded with a ‘KCl-electrode’ was suppressed drastically by D-600 (Ca²⁺-antagonist⁶) and prolonged significantly by caffeine (promoting Ca²⁺ release^4,9), while the AHP recorded with a ‘K₃-citrate-electrode’ was affected only slightly by these agents. Thus, these results suggest that Ca²⁺ entry during an action potential is the main origin of Ca²⁺ for the AHP recorded with a ‘KCl-electrode’, and favour the idea that the intracellular release of Ca²⁺ by an action potential as well as the Ca²⁺ influx participates in the mechanism of the AHP recorded with a ‘K₃-citrate-electrode’.