Increase of intracellular Ca2+ by P2Y but not P2X receptors in cultured cortical multipolar neurons of the rat

The expression and functionality of P2X/P2Y receptor subtypes in multipolar nonpyramidal neurons of mixed cortical cell cultures were investigated by means of immunocytochemistry and fura‐2 microfluorimetry. The morphological studies revealed that most of the neurons are immunoreactive for GABA and express a range of P2X/P2Y receptors, predominantly of the P2X_2,4,6 and P2Y_1,2 subtypes. P2X₁ and P2X₇ receptor immunoreactivity (IR) was found on thin axon‐like processes and presynaptic structures, respectively. Application of ATP caused a small concentration‐dependent increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in most investigated neurons, whereas only about the half of these cells responded to 2′,3′‐O‐(benzoyl‐4‐benzoyl)‐ATP (BzATP), ADPβS, 2MeSADP, or 2MeSATP and even fewer cells to UTP. In contrast, α,β‐meATP, UDP, and UDP‐glucose failed to produce any [Ca²⁺]_i signaling. The response to ATP itself was inhibited by pyridoxal‐5′‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), Reactive Blue 2, 2′‐deoxy‐N⁶‐methyl adenosine 3′,5′‐diphosphate (MRS2179), and suramin (300 μM) as well as by a cyclopiazonic acid‐induced depletion of intracellular Ca²⁺ stores. A Ca²⁺‐free external medium tended to decrease the ATP‐induced [Ca²⁺]_i transients, although this action did not reach statistical significance. Various blockers of voltage‐sensitive Ca²⁺ channels and the gap junction inhibitor carbenoxolone did not interfere with the effect of ATP, whereas a combination of the ionotropic glutamate receptor antagonists D(–)‐2‐amino‐5‐phosphonopentanoic acid (AP5) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) decreased it. Cross‐desensitization experiments between ADPβS or UTP and ATP suggested that ATP acts on the one hand via P2Y_1,2 receptors and on the other hand by additional signaling mechanisms. These mechanisms may involve the release of glutamate (which in consequence activates ionotropic glutamate receptors) and the entry of Ca²⁺ via store‐operated Ca²⁺ channels. Evidence for the presence of functional P2X receptors, in particular P2X₇, remains elusive. J. Comp. Neurol. 516:343–359, 2009. © 2009 Wiley‐Liss, Inc.     

BDNF potentiates spontaneous Ca oscillations in cultured hippocampal neurons

Brain-derived neurotrophic factor (BDNF) is thought to regulate neuronal plasticity in developing and matured neurons, although the molecular mechanisms are less well characterized. We monitored changes in the intracellular calcium (Ca²⁺) levels induced by BDNF using a fluorescence Ca²⁺ indicator (Fluo-3) by means of confocal laser microscopy in rat cultured hippocampal neurons. BDNF acutely potentiated spontaneous Ca²⁺ oscillations in dendrites and also in the soma of several neurons, although it increased intracellular Ca²⁺ in only selective proportion of resting neurons without Ca²⁺ oscillations. The potentiation was observed both in the frequency and the amplitude of Ca²⁺ oscillations, completely blocked by K-252a, and significantly reduced by 2-aminophosphonovaleric acid. These findings suggest that BDNF increases glutamate release and N-methyl-

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-aspartate (NMDA) channel-gated Ca²⁺ influx via TrkB and regulates the frequency and the amplitude of Ca²⁺ oscillations. BDNF may have the potential to modulate spontaneous Ca²⁺ oscillations to regulate neuronal plasticity in developing hippocampal neurons.     

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.     

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’.     

Effects of Hg and CH3Hg on Ca fluxes in rat brain microsomes

A permanent increase in cytosolic Ca²⁺ levels seems to be associated with various pathological situations which may result in cell death. Hg²⁺ and CH₃Hg⁺ are potent neurotoxic agents, but the precise molecular mechanism(s) underlying their effects are not sufficiently understood. In the present study we investigated the potential role of Ca²⁺-ATPase located in the endoplasmic reticulum as a molecular target for mercury. Hg²⁺ and CH₃Hg⁺ inhibited Ca²⁺-ATPase and Ca²⁺ uptake by brain microsomes with similar potencies. However, the inhibitory potency of Hg²⁺ was higher than that of CH3Hg+, probably reflecting differences in the affinity for the sulfhydryl groups of these compounds. Passive or unidirectional Ca²⁺ efflux (measured in the absences of Ca²⁺-ATPase ligands) was increased significantly by CH₃Hg⁺ and Hg²⁺. Again, the potency of Hg²⁺ was higher than that of CH₃Hg⁺. Blockers of Ca²⁺ channels (ruthenium red, procaine, heparin) did not affect the increase in passive Ca t+ efflux induced by mercury compounds, possibly indicating that Ca²⁺ release occurs through Ca²⁺-ATPase. Addition of physiological concentrations of glutathione (GSH) simultaneously with mercury abolished the inhibitory effects of both forms of Hg on Ca Z+-transport. However, if the enzyme was first inhibited with Hg²⁺ or CH₃Hg⁺ and subsequently treated with GSH, the reversal of inhibition was about 50%, suggesting that part of the cysteinyl residues involved in the inhibitory actions of mercury in Ca t+-transport bind to mercury with an extremely high affinity.     

Cytotoxic actions and effects on intracellular Ca2+ and cGMP concentrations of sulphur-containing excitatory amino acids in cultured cerebral cortical neurons

Effects of the sulphur-containing acidic amino acids (SAAs) cysteic acid (CA), homocysteic acid (HCA), cysteine sulphinic acid (CSA), homocysteine sulphinic acid (HCSA), and S-sulphocysteine (SC) on intracellular concentrations of Ca²⁺ ([Ca²⁺]_i) and cGMP ([cGMP]_i) as well as their cytotoxic actions were investigated in cultured cerebral cortical neurons. The glutamate receptor subtype selective antagonists APV (D-(?)-2-amino-5-phosphonopentanoate) acting on N-methyl-D-aspartate (NMDA) receptors and DNQX (6,7-dinitroquinoxaline-2,3-dione) acting on non-NMDA receptors were employed to obtain information about the involvement of glutamate receptor subtypes in these actions of the SAAs. It was found that all SAAs exerted a cytotoxic action on the neurons. The ED₅₀ values for CSA, CA, HCSA, and HCA were around 30 to 50 μM and that for SC was about 150 μM. The glutamate transport blocker L-aspartate-β-hydroxamate increased the efficacy of CSA and CA but had no effect on the cytotoxic actions of the remaining SAAs. In case of CA, HCA, and SC the cytotoxicity could be prevented by APV alone and for HCSA, DNQX could block the toxic action. DNQX reduced the toxicity of HCA somewhat but the presence of APV was required for complete protection. CSA toxicity could only be blocked by the combination of APV and DNQX. All SAAs induced an increase in [cGMP]_i and [Ca²⁺]_i and with regard to [Ca²⁺]_i SC was the most potent and CA the least potent SAA. The effect of all SAAs on [cGMP]_i could be blocked by APV alone whereas DNQX had no effect except in the case of HCSA where the response was blocked completely and HCA where the response was inhibited by 75%. The SAA-induced increase in [Ca²⁺]_i could in all cases be significantly reduced by 0.6 mM Mg²⁺ and in the presence of Mg²⁺, APV dose dependently blocked the remaining SAA induced increase in [Ca²⁺]_i completely. Under these conditions DNQX was also found to block the SAA-induced increase in [Ca²⁺]_i dose dependently. In the absence of Mg²⁺, DNQX (25 μM) inhibited the response of the SAAs only by 65–75%. Under these conditions all SAA responses except that to SC could be fully antagonized by 300 μM APV. The SC-induced increase in [Ca²⁺]_i was inhibited by 60% by APV. The results show that no simple correlation exists between SAA-induced cytotoxicity and their ability to increase intracellular levels of Ca²⁺ and cGMP. However, when both NMDA and non-NMDA receptors were antagonized no toxicity or changes in calcium or cGMP were observed. © 1993 Wiley-Liss, Inc.     

In vivo effects of the Ca2+-antagonist nimodipine on dopamine metabolism in mouse brain

Summary The effects of the Ca²⁺-antagonist nimodipine on central dopamine (DA) neurons in mice were investigatedin vivo. Nimodipine caused a dose-dependent decrease in the DA metabolite 3-methoxytyramine (3-MT) in striatum and the limbic region. If the brains were microwave radiated immediately after decapitation in order to minimize post-mortal accumulation of 3-MT, the effect of nimodipine was less pronounced and statistically not significant.Nimodipine markedly decreased the accumulation of 3-MT induced by pargyline, an inhibitor of monoamine oxidase, a phenomenon that was not attenuated by microwave radiation. Furthermore, whereas nimodipine had no effect on mouse motor activity when given alone it readily blocked the pargyline-induced increase in activity.The concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) in striatum and the limbic region were also reduced by nimodipine as was the accumulation of 3,4-dihydroxyphenylalanine (DOPA) measured after inhibition of the aromatic amino acid decarboxylase by 3-hydroxybenzylhydrazine (NSD 1015). In addition, nimodipine caused decreased concentrations of DA and homovanillic acid (HVA) in the limbic region but not in striatum.Nimodipine caused an increase in the striatal concentrations of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA); these changes were not seen in the limbic region.In conclusion, nimodipine appears to reduce the release as well as the synthesis of DA in mouse brain. These effects are believed to be related to the Ca²⁺-antagonism of nimodipine.     

Selective enhancement by basic fibroblast growth factor of NMDA receptor-mediated increase of intracellular Ca concentration in hippocampal neurons

The short-term effect of bFGF on intracellular Ca²⁺ concentration ([Ca²⁺]_i) of hippocampal neurons was investigated using dissociated cell cultures. Changes in [Ca²⁺]_i were measured by microfluorometrically monitoring the fluorescence intesities from indivudual neurons loaded with fura-2. Perfusion of bFGF (20 ng/ml) alone did not affect the basal level of [Ca²⁺]_i in hippocampal neurons, but clearly enhanced the [Ca²⁺]_i increase induced by NMDA. Quisqualate or KCl-induced [Ca²⁺]_i increase was not influenced by bFGF. These results suggest that bFGF selectively enhances the NMDA receptor-mediated response in hippocampal neurons.     

Ca2+ depletion-induced disconnection of tight junctions in isolated rat brain microvessels

Summary Cerebral microvessels were isolated from rat brains. One part of the microvessel pellets was incubated for 25 or 90 min in Krebs-Henseleit bicarbonate buffer (KHB) at pH 7.5 (control group). The other part of the pellets was treated for the same periods of time with Ca²⁺-free KHB, containing 2.2 mM EGTA and 2 mM glucose (experimental group). Morphological changes of endothelial tight junctions were evaluated in 100 randomly selected interendothelial clefts from isolated cerebral microvessels of each groups by electron microscopy. Following 25 min of incubation time, either with Ca²⁺-containing or with Ca²⁺-free KHB, no significant changes of tight junctions were observed. After 90 min of incubation in Ca²⁺-free medium, 58% of tight junctions were altered (in 42% partial, and in 16% complete disconnection of tight junctions were found). This contrasted the control group, where only 14% of tight junctions were disconnected (12% partially and 2% completely). Our results are consistent with a role for intercellular Ca²⁺ in maintaining structural integrity of cerebral tight junctions.Supported by the Medical Research Council of Canada, grant MT-5958 and the Hungarian Ministry of Health, grant 06/1-44/313     

Mechanism of synchronized Ca oscillations in cortical neurons

Dissociated rat cortical neurons reassociate in vitro to form synaptically connected networks. Removal of Mg²⁺ from the extracellular medium then induces neurons in the network to undergo synchronized oscillations of cytoplasmic calcium. Previous studies have shown that these calcium oscillations involve the activation of NMDA receptors and that the rising phase of each calcium spike is coincident with a brief burst of action potentials (Robinson et al., Jpn. J. Physiol. 43 (Suppl. 1) (1993) S125–130; Robinson et al., J. Neurophysiol. 70 (1993) 1606–1616; Murphy et al., J. Neurosci. 12 (1992) 4834–4845). We have found that these calcium oscillations are dependent on an influx of extracellular calcium but are independent of mobilization of calcium from intracellular stores. The influx of extracellular Ca²⁺ occurs primarily through L-type voltage-gated calcium channels (VGCCs), since diltiazem inhibits calcium oscillations under all conditions. On the other hand, N-, P/Q-, and T-type VGCCs are not required for calcium oscillations, although inhibitors of these channels may act as partial antagonists. In addition to removal of Mg²⁺, oscillations can also be induced by the inhibition of voltage-gated K⁺ channels with 4-aminopyridine (4-AP), a treatment known to increase neurotransmitter release. In the presence of 4-AP, synchronized calcium oscillations become independent of NMDA receptor activation, although they continue to require activation of AMPA/KA receptors. A model for the mechanism of neuronal calcium oscillations and the reason for their synchrony is presented.     

NMDA-evoked consumption and recovery of mitochondrially targeted aequorin suggests increased Ca2+ uptake by a subset of mitochondria in hippocampal neurons

Activation of NMDA receptors produces large increases in cytosolic Ca(2+) that are taken up into mitochondria. We used recombinant aequorin targeted to mitochondria to report changes in matrix Ca(2+) in rat hippocampal neurons in culture. Upon binding Ca(2+), aequorin emits a photon in a one-shot reaction that consumes the indicator. Here we show that stimulation with NMDA produced a mitochondrial Ca(2+) response that rapidly inactivated. However, following a 30-min recovery period the response was restored, suggesting the presence of a pool of indicator that was not exposed to high Ca(2+) during the initial stimulus. We speculate that aequorin distant from the Ca(2+) source was protected from microdomains of high Ca(2+) near the plasmalemma and that this aequorin moved, either by movement of individual mitochondria or via the mitochondrial tubular network, to replenish consumed indicator during the recovery time. A large Ca(2+) increase in a subset of mitochondria could produce local changes in energy metabolism, regional Ca(2+) buffering, and foci that initiate neurotoxic processes.     

Angiotensin II increases intracellular Ca concentration in folliculo-stellate cells of the rat anterior pituitary in primary culture

It has been reported that pituitary gonadotrophs and lactotrophs contain angiotensin II (Ang II) and suggested that Ang II modulates hormone secretion from endocrine cells of the anterior pituitary through paracrine mechanism among the endocrine cells. However, there has been no report on the effect of Ang II on the folliculo-stellate cells (FSC) which are thought to play a regulatory role in the release of hormones from pituitary endocrine cells. We, therefore, examined the effect of Ang II on FCS in primary culture by Ca²⁺ imaging technique. Certain proportion (42%) of FSC responded to 100 nM Ang II by increasing [Ca²⁺]_i. In addition, Ang II elicited the Ca²⁺ response in about 50% of the pituitary endocrine cells. The results indicate that Ang II functions as a paracrine factor among pituitary cells including FSC.     

Signaling pathways involved in Ca2+- and Pb2+-induced vesicular catecholamine release from rat PC12 cells

Since Pb(2+) substitutes for Ca(2+) in essential steps leading to exocytosis, we have investigated whether Ca(2+) and Pb(2+) induce exocytosis through similar pathways. Vesicular catecholamine release was measured from dexamethasone-differentiated PC12 cells using carbon fiber microelectrode amperometry. Effects of drugs known to modulate PKC (PMA, staurosporine), calcineurin (cyclosporin A), calmodulin (W7), and CaM kinase II (KN-62) activity were investigated in intact and in ionomycin-permeabilized PC12 cells. Activation of PKC and inhibition of calmodulin decrease the frequency of exocytotic events evoked by high K(+) stimulation in intact cells. In addition, inhibition of calmodulin enhances the frequency of basal exocytosis from intact cells. Activation of PKC and inhibition of calcineurin enhance the frequency of basal exocytosis in intact as well as in ionomycin-permeabilized cells. Inhibition of PKC and of CaM kinase II cause no significant effects. None of the treatments has a significant effect on vesicle contents. The combined results indicate that PKC and calcineurin enhance and inhibit exocytosis through direct effects on the exocytotic machinery, whereas calmodulin and CaM kinase II exert indirect effects only. Conversely, Pb(2+)-evoked exocytosis in permeabilized cells is strongly reduced by inhibition of CaM kinase II, but is not sensitive to modulation of PKC and calcineurin activity. Inhibition of calmodulin only reduces the delay to onset of Pb(2+)-evoked exocytosis. Synaptotagmin I- and II-deficient PC12-F7 cells exhibit vesicular catecholamine release following depolarization or superfusion with Pb(2+). However, the frequency of exocytosis and the contents of vesicles released are strongly reduced as compared to PC12 cells. It is concluded that Ca(2+)-evoked exocytosis is modulated mainly by PKC and calcineurin, whereas Pb(2+)-evoked exocytosis is mainly modulated by CaM kinase II.     

Flow-cytometric estimation on glutamate- and kainate-induced increases in intracellular Ca of brain neurons: a technical aspect

Effects of glutamate and kainate on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a large population (several thousand) of dissociated cerebellar granule cell neurons were evaluated using a flow-cytometer and a combination of two fluorescent dyes, fluo-3-AM for estimating [Ca²⁺]_i and ethidium bromide for removing neurons that had compromised membranes from the cell population examined. The number of neurons responding to glutamate or kainate in augmenting the fluo-3 fluorescence increased in a dose-dependent manner. The number of neurons responding to kainate was much greater than that to glutamate. CNQX, a blocker of non-NMDA receptors, completely blocked the response elicited by kainate while the complete blockade of this glutamate-induced response was made by a combination of MK-801, a NMDA receptor blocker, and CNQX. Nicardipine, a calcium antagonist, decreased the number of neurons responding to glutamate and kainate, suggesting involvement of voltage-dependent calcium channels. These results indicate that the flow-cytometric measurement of glutamate and kainate responses has the potential to provide answers to such questions as what percentage of the population of neurons respond to these amino acids and what is the resulting distribution of [Ca²⁺]_i.     

Different stimulatory opioid effects on intracellular Ca in SH-SY5Y cells

Present study revealed the stimulatory effects of δ opioid receptor on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in SH-SY5Y cells. Fura-2 based single cell fluorescence ratio (F345/F380) was used to monitor the fluctuation of [Ca²⁺]_i. Application of the selective delta-opioid receptor agonist alone, [D-Pen^2,5]-enkephalin (DPDPE), hardly had any effects on cells cultivated for 3–10 days. However, after the cells had been pre-stimulated with cholinoceptor agonist, carbachol, variable calcium elevation was found in 59% of the cultures. The response was naltridole-reversible and dose-dependent, and was abolished completely by thapsigargin (TG) treatment but not by administration of CdCl₂ or 0-Ca²⁺ bath solutions. DPDPE-mediated [Ca²⁺]_i elevation was abolished by pertussis toxin (PTX) pretreatment but not cholera toxin (CTX), indicating coupling via G proteins of G_i/G_o subfamily. In 17.5% of the responding cells, biphase response was found which may be due to both the stimulatory and the inhibitory effects of opioid. On the other hand, in acutely dissociated cells, DPPDE alone induced [Ca²⁺]_i increase in 50% of the cultures. The probability and the amplitude of the elevation were decreased considerably by application of nifedipine or 0-Ca²⁺ bath solution and was little affected by application of TG. DPDPE activated [Ca²⁺]_i increase via a PTX-insensitive and CTX-sensitive pathway suggesting coupling through G_s subunit. All these indicated the opioid modulated the intracellular Ca²⁺ regulation system through different pathways. SH-SY5Y cell line might be a suitable model for the investigation of the complex mechanism which underlies opioid function.     

Effect of nilvadipine on the voltage-dependent Ca channels in rat hippocampal CA1 pyramidal neurons

Effects of nilvadipine on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) were compared with other organic Ca²⁺ antagonists in acutely dissociated rat hippocampal CA1 pyramidal neurons. The inhibitory effects of nilvadipine, amlodipine and flunarizine on LVA I_Ca were concentration- and use-dependent. The apparent half-maximum inhibitory concentrations (IC₅₀s) at every 1- and 30-s stimulation were 6.3×10⁻⁷ M and 1.8×10⁻⁶ M for flunarizine, 1.9×10⁻⁶ M and 7.6×10⁻⁶ M for nilvadipine, and 4.0×10⁻⁶ M and 8.0×10⁻⁶ M for amlodipine, respectively. Thus, the strength of the use-dependence was in the sequence of nilvadipine>flunarizine>amlodipine. Nilvadipine also inhibited the HVA I_Ca in a concentration-dependent manner with an IC₅₀ of 1.5×10⁻⁷ M. The hippocampal CA1 neurons were observed to have five pharmacologically distinct HVA Ca²⁺ channel subtypes consisting of L-, N-, P-, Q- and R-types. Nilvadipine selectively inhibited the L-type Ca²⁺ channel current which comprised 34% of the total HVA I_Ca. On the other hand, amlodipine non-selectively inhibited the HVA Ca²⁺ channel subtypes. These results suggest that the inhibitory effect of nilvadipine on the neuronal Ca²⁺ influx through both LVA and HVA L-type Ca²⁺ channels, in combination with the cerebral vasodilatory action, may prevent neuronal damage during ischemia.     

Effect of KB-2796, a new diphenylpiperazine Ca antagonist, on voltage-dependent Ca currents and oxidative metabolism in dissociated mammalian CNS neurons

The effects of KB-2796, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4-trimethoxybenzyl)piperazine-2HCl, on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) and on oxidative metabolism were studied in neurons freshly dissociated from rat brain. KB-2796 reduced the peak amplitude of LVA I_Ca in a concentration-dependent manner with a threshold concentration of 10⁻⁷ M when the LVA I_Ca was elicited every 30 s in the external solution with 10 mM Ca²⁺. The concentration for half-maximum inhibition (IC₅₀) was 1.9 × 10⁻⁶M. At 10⁻⁵ M or more of KB-2796, a complete suppression of the LVA I_Ca was observed in the majority of neurons tested. There was no apparent effect on the current-voltage (I-V) relationship and the current kinetics. KB-2796 delayed the reactivation and enhanced the inactivation of the Ca²⁺ channel for LVA I_Ca voltage- and time-dependently, suggesting that KB-2796 preferentially binds to the inactivated Ca²⁺ channel. KB-2796 at a concentration of3.0 × 10⁻⁶M also decreased the peak amplitude of the HVA I_Ca without shifting the I-V relationship. In addition, KB-2796 reduced the oxidative metabolism (the formation of reactive oxygen species) of the neuron in a concentration-dependent manner with a threshold concentration of3 × 10⁻⁶M. It is suggested that the inhibitory action of KB-2796 on the neuronal Ca²⁺ influx and the oxidative metabolism, in combination with a cerebral vasodilatory action, may reduce ischemic brain damage.     

Inhibitory effect of Cd2+ on glycine-induced chloride current in rat hippocampal neurons

The effects of cadmium (Cd(2+)) on glycine-induced Cl(-) current (I(Gly)) were investigated in acutely dissociated rat hippocampal CA1 neurons using the conventional whole-cell patch-clamp technique in this study. We found that Cd(2+) reversibly and concentration-dependently, reduced the amplitudes of I(Gly), with an IC(50) of 1.27 mM and Hill coefficient of 0.45. The depression of I(Gly) by Cd(2+) was independent of membrane voltage between -60 and +40 mV and did not involve a shift in the reversal potential of the current. A non-competitive inhibition was suggested by a double reciprocal plot of the effects of Cd(2+) on the concentration-response curve of the I(Gly). Since intracellular dialysis with 3mM Cd(2+) failed to modify I(Gly), it was suggested that the site of action of Cd(2+) is extracellular. The suppression of I(Gly) by Zn(2+) was unaffected by 3mM Cd(2+), which indicated that Zn(2+) and Cd(2+) bind to independent sites on glycine receptor. The results show that Cd(2+) decreases I(Gly) in acutely dissociated rat hippocampal neurons and the depression of I(Gly) by Cd(2+) may contribute to worsen the neurotoxicological impairment.     

Histochemical study of Ca2+-ATPase activity in ischemic CA1 pyramidal neurons in the gerbil hippocampus

Although cytosolic Ca²⁺ accumulation plays a pivotal role in delayed neuronal death, there have been no investigations on the role of the cellular Ca²⁺ export system in this novel phenomenon. To clarify the function of the Ca²⁺-pump in delayed neuronal death, the plasma membrane Ca²⁺-ATPase activity of CA1 pyramidal neurons was investigated ultracytochemically in normal and ischemic gerbil hippocampus. To correlate enzyme activity with delayed neuronal death, histochemical detection was performed at various recirculation times after 5 min of ischemia produced by occlusion of the bilateral carotid arteries. At 10 min after ischemia, CA1 pyramidal neurons showed weak Ca²⁺-ATPase activity. Although enzyme activity had almost fully recovered 2 h after ischemia, it was reduced again 6 h after ischemia. Thereafter, Ca²⁺-ATPase activity on the plasma membrance of CA1 pyramidal neurons decreased progressively, losing its localization on day 3. On day 4 following ischemia, reaction products were diffusely scattered throughout the whole cell body. Our results indicate that, after once having recovered from ischemic damage, severe disturbance of the membrane Ca²⁺ export system proceeds from the early stage of delayed neuronal death and disturbs the re-export of accumulated cytosolic Ca²⁺, which might contribute to delayed neuronal death. Occult disruption of Ca²⁺ homeostasis seems to occur from an extremely early stage of delayed neuronal death in CA1 pyramidal cells.