Preferential inhibition of L- and N-type calcium channels in the rat hippocampal neurons by cilnidipine

The effect of a dihydropyridine Ca²⁺ antagonist, cilnidipine, on voltage-dependent Ca²⁺ channels was studied in acutely dissociated rat CA1 pyramidal neurons using the nystatin-perforated patch recording configuration under voltage-clamp conditions. Cilnidipine had no effect on low-voltage-activated (LVA) Ca²⁺ channels at the low concentrations under 10⁻⁶ M. On the other hand, cilnidipine inhibited the high-voltage-activated (HVA) Ca²⁺ current (I_Ca) in a concentration-dependent manner and the inhibition curve showed a step-wise pattern; cilnidipine selectively reduced only L-type HVA I_Ca at the low concentrations under 10⁻⁷ and 10⁻⁶ M cilnidipine blocked not only L- but also N-type HVA I_Ca. At the high concentration over 10⁻⁶ M cilnidipine non-selectively blocked the T-type LVA and P/Q- and R-type HVA Ca²⁺ channels. This is the first report that cilnidipine at lower concentration of 10⁻⁶ M blocks both L- and N-type HVA I_Ca in the hippocampal neurons.     

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.     

Possible modulatory role of voltage-activated Ca currents determining the membrane properties of isolated pyramidal neurones of the rat dorsal cochlear nucleus

Voltage-activated Ca²⁺ currents have been studied in pyramidal cells isolated enzymatically from the dorsal cochlear nuclei of 6–11-day-old Wistar rats, using whole-cell voltage-clamp. From hyperpolarized membrane potentials, the neurones exhibited a T-type Ca²⁺ current on depolarizations positive to −90 mV (the maximum occurred at about −40 mV). The magnitude of the T-current varied considerably from cell to cell (−56 to −852 pA) while its steady-state inactivation was consistent (E₅₀=−88.2±1.7 mV, s=−6.0±0.4 mV). The maximum of high-voltage activated (HVA) Ca²⁺ currents was observed at about −15 mV. At a membrane potential of −10 mV the L-type Ca²⁺ channel blocker nifedipine (10 μM) inhibited approximately 60% of the HVA current, the N-type channel inhibitor ω-Conotoxin GVIA (2 μM) reduced the current by 25% while the P/Q-type channel blocker ω-Agatoxin IVA (200 nM) blocked a further 10%. The presence of the N- and P/Q-type Ca²⁺ channels was confirmed by immunochemical methods. The metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (200 μM) depressed the HVA current in every cell studied (a block of approximately 7% on an average). The GABA_B receptor agonist baclofen (100 μM) reversibly inhibited 25% of the HVA current. Simultaneous application of ω-Conotoxin GVIA and baclofen suggested that this inhibition could be attributed to the nearly complete blockade of the N-type channels. Possible physiological functions of the voltage-activated Ca²⁺ currents reported in this work are discussed.     

The cannabinoid agonist Win55,212-2 inhibits calcium channels by receptor-mediated and direct pathways in cultured rat hippocampal neurons

The effects of the cannabinoid receptor agonist Win55,212 on Ca²⁺ channels were studied in rat hippocampal neurons grown in primary culture. Win55,212-2 inhibited whole-cell Ba²⁺ currents through Ca²⁺ channels by both CB1 receptor-mediated and direct mechanisms. The concentration dependent inhibition of the current showed two clear phases, a high-affinity receptor-mediated phase (IC₅₀=14±2 nM) that was stereoselective and sensitive to a CB1 receptor antagonist, 300 nM SR141716, and a non-saturating phase that was neither stereoselective nor inhibited by SR141716. These concentration-dependent effects were paralleled by Win55212-induced inhibition of glutamatergic synaptic transmission. Win55,212-2 (100 nM) inhibited both ω-agatoxin IVA- and ω-conotoxin GVIA-sensitive currents. Thus, activation of cannabinoid receptors inhibits N- and P/Q-type Ca²⁺ channels. Activation of cannabinoid receptors inhibited only a fraction of the whole-cell Ca²⁺ channel current (17±2%) even though more than half of the whole-cell Ba²⁺ current was carried by N- and P/Q-type Ca²⁺ channels. Concentrations of agonist greater than 1 μM inhibited Ca²⁺ channels directly.     

Effects of hypertonia on voltage-gated ion currents in freshly isolated hippocampal neurons, and on synaptic currents in neurons in hippocampal slices

We studied the effects of hypertonia on voltage-gated currents of freshly isolated hippocampal CA1 neurons, using open pipette whole-cell as well as gramicidin-perforated patch-clamp recording. Extracellular osmolarity (π_o) was raised by adding mannitol (50 or 100 mmol/l) to the bathing solution. Hypertonia depressed voltage-gated sodium, potassium and calcium currents in all trials. The threshold activation voltage of the currents did not change during hypertonic depression, but maximal activation of Ca²⁺ current shifted to a more negative potential, suggesting stronger depression of high- compared to low-voltage activated currents. During 30 min high π_o treatment (recorded with open pipette), the depression reached maximum in 10–15 min of exposure. The depression of the computed transient component of the K⁺ current recorded by open pipette was statistically not significant. Following hypertonic treatment recovery of the I_Na, the sustained I_K and sustained I_Ca were incomplete compared to control cells maintained in normal solution for an equal length of time. In hippocampal tissue slices hypertonia (+25, +50 and +100 mmol/l fructose) reversibly depressed excitatory postsynaptic currents (EPSCs). We conclude that the shutdown of membrane ion currents by elevated π_o is not selective, but the degree of the suppression varies among current types. Raising π_o in human patients, possibly combined with mild artificial acidosis, may be useful in the prevention and treatment of acute crises associated with excessive excitation or depolarization of neurons.     

Separation of calcium currents in retinal ganglion cells from postnatal rat

A culture system of the postnatal rat retina was established to investigate Ca²⁺ currents and synaptic transmission in identified neurons. Methods are described that allowed us to select retinal ganglion neurons (RGNs) in short term cultures (up to 48 h in vitro) and in long-term cultures (3 to 21 days in vitro). The specific aim of the present study was to identify channel specific components in whole-cell Ca²⁺ currents of RGNs and to clarify the potential use of the lanthanide Gd³⁺ as a selective Ca²⁺ channel blocker. About one third of freshly dissociated RGNs generated both low voltage activated Ca²⁺ currents (I_Ca(LVA)) and high voltage activated Ca²⁺ currents (I_Ca(HVA)). The remaining 2/3 of RGNs in short term culture and most RGNs in long-term culture displayed only I_Ca(HVA). The latter comprised at least three different components that were functionally rather similar, but could be separated pharmacologically. A significant portion (about 40%) of I_Ca(HVA) was irreversible blocked by the N channel antagonist ω-CgTx (5 μM). The L channel antagonist nifedipine (10 μM) eliminated about 25% of I_Ca(HVA). Thus, about 1/3 of the HVA Ca²⁺ or Ba²⁺ current remained unaffected by either ω-CgTx or nifedipine. ω-AgaTx (200 nM) completely failed to block HVA Ca²⁺ or Ba²⁺ currents in RGNs. Gd³⁺ exerted contrasting actions on LVA and HVA Ca²⁺ currents. While I_Ca(LVA) consistently increased in the presence of Gd³⁺ (0.32–3.2 μM), I_Ca(HVA) always decreased, especially when using higher concentrations of Gd³⁺ (10–32 μM). The blocking action of Gd³⁺ was not restricted to the ω-CgTx-sensitive HVA current component, but also concerned ω-CgTx- and nifedipine-resistant components. The decay of Ca²⁺ currents was accelerated in the presence of Gd³⁺. Even in RGNs lacking I_Ca(LVA), application of 3.2 μM Gd³⁺ significantly reduced the time constant of decay from an average of 64 ms to 36 ms (voltage steps from −90 to 0 mV; 10 mM [Ca²⁺]₀; 26°C). This is in contrast to what had to be expected if an N-type HVA current component was selectively suppressed by Gd³⁺. Gd³⁺ diminished glutamatergic spontaneous synaptic activity in retinal cultures tested during the 3rd week in vitro. Both frequency and amplitude were reduced. Occasionally, the application was followed by a rebound increase of EPSC frequency. A stimulatory effect during application of Gd³⁺ has never been observed. These experiments indicate that RGNs express at least 4 different types of Ca²⁺ currents, that resemble in some aspects T, N and L channel currents. A significant component of the HVA Ca²⁺ current was resistant to the available HVA channel blockers suggesting the presence of a pharmacologically distinct type of HVA Ca²⁺ channel type in RGNs. Our experiments also show that Gd³⁺ is not suitable for isolation of HVA subcomponents in RGNs, but it can be used to distinguish between LVA and HVA Ca²⁺ currents, as these currents reacted to Gd³⁺ in an opposite way. The purely depressive effect of this lanthanide on spontaneous synaptic activity is consistent with the assumption that in retinal neurons LVA Ca²⁺ channels are not involved in the regulation of glutamate release.     

Regulation of Ca-activated nonselective cationic currents in rat pituitary GH3 cells: involvement in L-type Ca current

Ionic currents were investigated by a patch clamp technique in a clonal strain of pituitary (GH₃) cells, using the whole cell configuration with Cs⁺ internal solution. Depolarizing pulses positive to 0 mV from a holding potential of −50 mV activated the voltage-dependent L-type Ca²⁺ current (I_Ca,L) and late outward current. Upon repolarization to the holding potential, a slowly decaying inward tail current was also observed. This inward tail current upon repolarization following a depolarizing pulse was found to be enhanced by Bay K 8644, but blocked by nifedipine or tetrandrine. This current was eliminated by Ba²⁺ replacement of external Ca²⁺ as the charge carrier through Ca²⁺ channels, removal of Ca²⁺ from the bath solution, or buffering intracellular Ca²⁺ with EGTA (10 mM). The reversal potential of inward tail current was approximately −25 mV. When intracellular Cl⁻ was changed, the reversal potential of the Ca²⁺-activated currents was not shifted. Thus, this current is elicited by depolarizing pulses that activate I_Ca,L and allow Ca²⁺ influx, and is referred to as Ca²⁺-activated nonselective cationic current (I_CAN). Without including EGTA in the patch pipette, the slowly decaying inward current underlying the long-lasting depolarizing potential after Ca²⁺ spike was also observed with a hybrid current–voltage protocol. Thus, the present studies clearly indicate that Ca²⁺-activated nonselective cationic channels are expressed in GH₃ cells, and can be elicited by the depolarizing stimuli that lead to the activation of I_Ca,L.     

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 charybdotoxin and leiurotoxon I on potassium currents in bullfrog sympathetic ganglion and hippocampal neurons

The effects of charybdotoxin and leiurotoxin I were examined on several classes of K⁺ currents in bullfrog sympathetic ganglion and hippocampal CA1 pyramidal neurons. Highly purified preparations of charybdotoxin selectively blocked a large voltage- and Ca²⁺-dependent K⁺ current (I_c) responsible for action potential repolarization (IC₅₀ = 6 nM) while leiurotoxin I selectively blocked a small Ca²⁺-dependent K⁺ conductance (I_AHP) responsible for the slow afterhyperpolarization following an action potential (IC₅₀ = 7.5 nM) in bullfrog sympathetic ganglion neurons. Neither of the toxins had a significant effects on other K⁺ currents (M-current [I_M], A-current [I_A] and the delayed rectifier [I_KD] present in these cells. Leiurotoxin I at a concentration of 20 nM had no detectable effect on currents in hippocampal CA1 pyramidal neurons. This lack of effect on I_AHP in central neurons suggests that the channels underlying slow AHPs in those neurons are pharmacologically distinct from analogous channels in peripheral neurons.     

Ca-sensitive inhibition by Pb of α7-containing nicotinic acetylcholine receptors in hippocampal neurons

In the present study the patch-clamp technique was applied to cultured hippocampal neurons to determine the kinetics as well as the agonist concentration- and Ca²⁺-dependence of Pb²⁺-induced inhibition of α7 nicotinic receptors (nAChRs). Evidence is provided that more than two-thirds of the inhibition by Pb²⁺ (3–30 μM) of α7 nAChR-mediated whole-cell currents (referred to as type IA currents) develops rapidly and is fully reversible upon washing. The estimated values for τ_onset and τ_recovery were 165 and 240 ms, respectively. The magnitude of the effect of Pb²⁺ was the same regardless of whether acetylcholine or choline was the agonist. Pre-exposure of the neurons for 800 ms to Pb²⁺ (30 μM) decreased the amplitude and accelerated the decay phase of currents evoked by moderate to high agonist concentrations. In contrast, only the amplitude of currents evoked by low agonist concentrations was reduced when the neurons were exposed simultaneously to Pb²⁺ and the agonists. Taken together with the findings that Pb²⁺ reduces the frequency of opening and the mean open time of α7 nAChR channels, these data suggest that Pb²⁺ accelerates the rate of receptor desensitization. An additional reduction of type IA current amplitudes occurred after 2-min exposure of the neurons to Pb²⁺. This effect was not reversible upon washing of the neurons and was most likely due to an intracellular action of Pb²⁺. Pb²⁺-induced inhibition of α7 nAChRs, which was hindered by the enhancement of extracellular Ca²⁺ concentrations, may contribute to the neurotoxicity of the heavy metal.     

Synthetic ω-conotoxin: a potent calcium channel blocking neurotoxin

The Ca²⁺ channel blocking action of synthetic ω-conotoxin (ωCTX) was studied on isolated frog dorsal root ganglion neurons using a ‘concentration clamp’ technique which enabled internal perfusion and rapid external solution change. At 100 nM, ωCTX showed a time-dependent depression of Ca²⁺ current (I_Ca). At higher concentrations, ωCTX exhibited a dose-dependent depression of I_Ca amplitude without changing the current-voltage relationship. Increases in external Ca²⁺ concentration partly overcame the inhibitory action of ωCTX on the I_Ca amplitude. At 10 μM ωCTX totally blocked I_Ca without effect on the Na⁺ current. It was likely that ωCTX had high selectivity for the Ca²⁺ channel.     

Caffeine rapidly decreases potassium conductance of dissociated outer hair cells of guinea-pig cochlea

The effects of caffeine on the outer hair cells (OHCs) freshly dissociated from guinea-pig cochlea were investigated with the whole-cell patch-clamp technique, in both the conventional and the nystatin perforated patch-clamp configurations under voltage-clamp condition. Application of caffeine (> 1 mM for 10–30 s) induced an inward current (I_caffeine) with decrease of conductance in a dose-dependent manner at a holding potential (V_H) of −60 mV. The reversal potential ofI_caffeine (E_caffeine) was close to the K⁺ equilibrium potential. TheI_caffeine was not affected by Ca²⁺-free external solution. The internal perfusion of the Ca²⁺ chelator BAPTA had no effect onI_caffeine. TheI_caffeine was not modulated by the external application of H-8 or staurosporine and by the internal perfusion of GDP-βS. The amplitude ofI_caffeine was the largest at the basal region of OHCs when caffeine was locally applied by the ‘puffer’ method. These results suggest that caffeine induces a decrease in membrane potassium conductance of the OHCs mainly at the basal region without mediating the intracellular signaling pathway.     

Ionic currents in carotid body type I cells isolated from normoxic and chronically hypoxic adult rats

Whole-cell recordings were used to investigate the effects of a 3-week period of hypoxia (10% O₂) on the properties of K⁺ and Ca²⁺ currents in type I cells isolated from adult rat carotid bodies. Chronic hypoxia significantly increased whole-cell membrane capacitance. K⁺ current amplitudes were not affected by this period of hypoxia, but K⁺ current density was significantly reduced in cells from chronically hypoxic rats as compared with normoxically maintained, age-matched controls. K⁺ current density was separated into Ca²⁺-dependent and Ca²⁺-independent components by bath application of 200 μM Cd²⁺, which blocked Ca²⁺ currents and therefore, indirectly, Ca²⁺-dependent K⁺ currents. Ca²⁺-dependent K⁺ current density was not significantly different in control and chronically hypoxic type I cells. Cd²⁺-resistant (Ca²⁺-insensitive) K⁺ current densities were significantly reduced in type I cells from chronically hypoxic rats. Acute hypoxia (Po₂ 15–22 mmHg) caused reversible, selective inhibition of Ca²⁺-dependent K⁺ currents in both groups of cells and Ca²⁺-insensitive K⁺ currents were unaffected by acute hypoxia. Ca²⁺ channel current density was not significantly affected by chronic hypoxia, nor was the degree of Ca²⁺ channel current inhibition caused by nifedipine (5 μM). Acute hypoxia did not affect Ca²⁺ channel currents in either group. Our results indicate that adult rat type I cells undergo a selective suppression of Ca²⁺-insensitive, voltage-gated K⁺ currents in response to chronic hypoxia in vivo. These findings are discussed in relation to the known adaptations of the intact carotid body to chronic hypoxia.     

Ca-Antagonistic action of bevantolol on hypothalamic neurons in vitro: its comparison with those of other β-adrenoceptor antagonists, a local anesthetic and a Ca-antagonist

The Ca²⁺-antagonistic action of bevantolol, aβ₁-adrenoceptor antagonist, on high- and low-voltage activated Ca²⁺ currents (HVA- and LVA-I_Ca) was examined on neurons dissociated from rat brain. Bevantolol (10⁻⁶ to 10⁻⁴ M) inhibited concentration-dependently bothI_Ca. The IC₅₀ value of bevantolol for LVA-I_Ca was 4 × 10⁻⁵ M, while bevantolol at 10⁻⁴ M inhibited HVA-I_Ca by 28.5 ± 7.7%. The potency of bevantolol in inhibiting bothI_Ca was greater than those of propranolol, labetalol and lidocaine, while the inhibitory action of bevantolol on voltage-activated Na⁺ current was weakest among them. Bevantolol may possess Ca²⁺-antagonistic action that is independent from local anesthetic action.     

Effects of ginsenosides on Ca channels and membrane capacitance in rat adrenal chromaffin cells

We investigated the effects of ginseng total saponins (GTS) and five ginsenosides on voltage-dependent Ca²⁺ channels and membrane capacitance using rat adrenal chromaffin cells. In this study, cells were voltage-clamped in a whole-cell recording mode and a perforated patch-clamp technique was used. The inward Ca²⁺ currents (I_Ca) was elicited by depolarization and the change in cell membrane capacitance (ΔC_m) was monitored. The application of GTS (100 μg/ml) induced rapid and reversible inhibition of the Ca²⁺ current by 38.8 ± 3.6% (n = 16). To identify the particular single component that seems to be responsible for Ca²⁺ current inhibition, the effects of five ginsenosides (ginsenoside Rb₁, Rc, Re, Rf, and Rg₁) on the Ca²⁺ current were examined. The inhibitions to the Ca²⁺ current by Rb₁, Rc, Re, Rf, and Rg₁ were 15.3 ± 2.2% (n = 5); 36.9 ± 2.4% (n = 7); 28.1 ± 1.9% (n = 12); 19.0 ± 2.5% (n = 10); and 16.3 ± 1.6% (n = 15), respectively. The order of inhibitory potency (100 μM) was Rc > Re > Rf > Rg₁ > Rb₁. A software based phase detector technique was used to monitor membrane capacitance change (ΔC_m). The application of GTS (100 μg/ml) induced inhibitory effects on ΔC_m by 60.8 ± 9.7% (n = 10). The inhibitions of membrane capacitance by Rb₁, Rc, Re, Rf, and Rg₁ were 35.3 ± 5.5% (n = 7); 41.8 ± 7.0% (n = 8); 40.5 ± 5.9% (n = 9); 51.2 ± 7.6% (n = 9); and 35.9 ± 5.1% (n = 10), respectively. The inhibitory potencies of the ginsenosides on ΔC_m were Rf > Rc > Re > Rg₁ > Rb₁. Therefore, we found that GTS and ginsenosides exerted inhibitory effects on both Ca²⁺ currents and ΔC_m in rat adrenal chromaffin cells. These results suggest that ginseng saponins regulate catecholamine secretion from adrenal chromaffin cells and this regulation could be the cellular basis of antistress effects induced by ginseng.     

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.     

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.     

Spinal cord neurons are vulnerable to rapidly triggered kainate neurotoxicity in vitro

Initial studies found glutamate injury to murine spinal cultures (14–17 days in vitro) to reflect contributions of both NMDA and AMPA/kainate receptors. Subsequent experiments found the spinal cultures to be more sensitive than cortical cultures to injury from prolonged low level kainate exposures, and, unlike cortical cultures, to be significantly damaged by relatively brief (30–60 min) kainate exposures. This rapidly triggered kainate damage to spinal neurons is Ca²⁺-dependent. Also, more than 40% of spinal neurons (in comparison to about 15% of cortical neurons) are subject to kainate-activated Co²⁺ uptake (CoPsu2+ (+) neurons), a histochemical technique that labels neurons with Ca²⁺-permeable AMPA/kainate channels. These spinal Co²⁺ (+) neurons are very sensitive to Ca²⁺-dependent kainate injury, and show greater kainate-induced elevations in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) than other spinal neurons during low level kainate exposures. Thus, the heightened vulnerability of spinal neurons to kainate toxicity may at least in part reflect the large proportion that possess Ca²⁺ permeable AMPA/kainate channels, permitting receptor activation to trigger rapid Ca²⁺ influx and overwhelm the cells Ca²⁺ homeostatic capabilities.     

Evidence That Actin Depolymerization Protects Hippocampal Neurons against Excitotoxicity by Stabilizing [Ca]i

Calcium influx through glutamate receptors and voltage-dependent channels mediates an array of functional and structural responses in neurons. However, unrestrained Ca²⁺ influx can injure and kill neurons; a mechanism implicated in both acute and chronic neurodegenerative disorders. Data reported here indicate that depolymerization of actin filaments can stabilize intracellular free calcium levels ([Ca²⁺]_i) and protect hippocampal neurons against excitotoxic injury. Studies with fluorescein-labeled phalloidin showed that cytochalasin D and glutamate each induced actin filament depolymerization. The microfilament-disrupting agent cytochalasin D protected cultured rat hippocampal neurons against glutamate toxicity, whereas the actin filament-stabilizing agent jasplakinolide potentiated glutamate toxicity. The microtubule-disrupting agent colchicine was ineffective in protecting neurons against glutamate toxicity. Cytochalasin D did not protect neurons against calcium ionophore toxicity or iron toxicity, indicating that its actions were not due to nonspecific effects on Ca²⁺ or free radical metabolism. Cytochalasin D markedly attenuated kainate-induced damage to hippocampus of adult rats, suggesting an excitoprotective role for actin depolymerization in vivo. Elevations of [Ca²⁺]_i induced by glutamate were attenuated in cultured hippocampal neurons pretreated with cytochalasin D and potentiated in neurons pretreated with jasplakinolide. The [Ca²⁺]_i response to a Ca²⁺ ionophore was unaffected by cytochalasin D, suggesting that actin depolymerization reduced Ca²⁺ influx through membrane channels. Taken together with previous patch clamp data, our findings suggest that depolymerization of actin in response to Ca²⁺ influx may serve as a feedback mechanism to attenuate potentially toxic levels of Ca²⁺ influx.     

Modulation of calcium current, intracellular calcium levels and cell survival by glucose deprivation and growth factors in hippocampal neurons

Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) can protect CNS neurons against ischemic/excitotoxic insults, but the mechanism of action is unknown. Imaging of the calcium indicator dye fura-3 and whole-cell patch clamp recordings of calcium currents were used to examine the mechanisms whereby hypoglycemia damages and growth factors protect cultured rat hippocampal neurons. When cultures were deprived of glucose, massive neuronal death occured 16–24 h following the onset of hypoglycemia. Early hypoglycemia-induced changes included calcium current inhibition and a reduction in intracellular free calcium levels ([Ca²⁺]_i) without morphological signs of neuronal damage. Later changes included a large elevation of [Ca²⁺]_i which was causally involved in neuronal damage. NGF and bFGF prevented or reduced both early and later responses to hypoglycemia. The growth factors increased calcium (barium) current and [Ca²⁺]_i to normal limits during the early stages of hypoglycemia and prevented the later elevation in [Ca²⁺]_i and neuronal damage. Nifedipine, but not omega-conotoxin, blocked calcium currents. The increased calcium current caused by the growth factors was apparently not sufficient to protect neurons against hypoglycemic damage since K⁺ depolarization during the early stages of hypoglycemia did not prevent and, in fact exacerbated, the subsequent neuronal damage. In addition, exposure of neurons to K⁺, NGF or bFGF only during the first 1 h of hypoglycemia did not protect against hypoglycemic damage. Taken together, the data suggest that neurons initially respond to hypoglycemia with a reduction in calcium currents which may provide a means to maintain [Ca²⁺]_i within a concentration range conducive to cell survival. Prolonged energy deprivation eventually results in a failure of calcium extrusion systems, glutamate receptor activation and a loss of neuronal calcium homeostasis. Taken together, the data indicate that the mechanism of growth factor protection against energydeprivation involves of the late prevention rise in [Ca²⁺]_i.