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.     

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.     

Erratum to: Differential alterations in the distribution of voltage-gated calcium channels in aged rat cerebellum: [Brain Research 903 (2001) 247–252]

In the present study, we have investigated the spatial and temporal distribution of voltage-gated calcium channels in the gerbil model of global cerebral ischemia using immunohistochemistry. Distinct localizations of P-type (α_1A), N-type (α_1B), and L-type (α_1C and α_1D) Ca²⁺ channels were observed in the hippocampus at days 1–5 after ischemic injury. However, increased expression of N-type Ca²⁺ channels was detectable in brain regions vulnerable to ischemia only at days 2 and 3 after ischemic injury. The pyramidal cell bodies of CA1-3 areas and the granule cell bodies of the dentate gyrus were intensely stained at days 2 and 3 following ischemic injury. Transient changes in N-type Ca²⁺ channel expression were also observed in the affected cerebral cortex and striatum at days 2 and 3 after ischemic injury. Although the present study has not addressed the multiple mechanisms contributing to the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase in the ischemic brain, the first demonstration of the transient increase in N-type Ca²⁺ channels may prove useful for future investigations.     

Kinetic and pharmacological properties of Ca currents in postganglionic sympathetic neurones projecting to muscular and cutaneous effectors

Voltage-gated Ca²⁺ channels are expressed in neurones and greatly influence neuronal activity by activating Ca²⁺-dependent K⁺ channels. The whole cell patch-clamp technique was used to compare the kinetic and pharmacological properties of voltage-dependent Ca²⁺ currents in two groups of sympathetic neurones identified by the fluorescent tracer Fast Blue: putative muscular sympathetic neurones (MSN) and putative cutaneous sympathetic neurones (CSN). The tracer was injected into the muscular part of the diaphragm (to mark MSN) and into the skin of the ear (to mark CSN). The capacitance of MSN (23.0 pF) was larger than the capacitance of CSN (12.6 pF). The maximum current in MSN (1.3 nA) was also larger than in CSN (0.93 nA). However, the current density was larger in CSN (77.3 pA/pF) than in MSN (57.7 pA/pF) and the current activation rate was faster in CSN (0.27 nA/ms) than in MSN (0.19 nA/ms). V_1/2 and slope factors of activation and inactivation were not significantly different for MSN and CSN. The majority of Ca²⁺ current was available for activation in both categories of neurones at resting membrane potential. Ca²⁺ currents in MSN and CSN were blocked by nifedipine (7.0 and 3.6%, respectively), ω-Agatoxin-IVA (23.0 and 25.6%, respectively) and ω-conotoxin-GVIA (67.0 and 65.1%, respectively). We found that CSN are twice as small, have higher Ca²⁺ current density and their Ca²⁺ activation rate is faster in comparison to MSN. Such properties may lead to faster rise of Ca²⁺ concentration in the cytoplasm of the CSN comparing to MSN and more effectively dampen their activity due to more effective activation of Ca²⁺-dependent K⁺ current. Both kinds of neurones express high proportion of N and P/Q Ca²⁺ current.     

Antagonists‐resistant calcium currents in rat embryo motoneurons

Ca²⁺ channels diversity of cultured rat embryo motoneurons was investigated with whole-cell current recordings. In 5–20 mm Ba²⁺, the whole-cell currents were separated in low- (LVA) and high-voltage-activated (HVA) current. The LVA current was evident since the first day in culture, while the HVA component was small and increased with time. Recordings after 4 days revealed ≈ 20% L-, ≈ 45% N- and ≈ 35% P- and R-type currents. P-type currents were revealed only in 40% of motoneurons, in which 20–200 nm ω-Aga-IVA caused 20% irreversible block of total current. The remaining 60% of cells were insensitive even to higher doses of the toxin (500 nm in 5 mm Ba²⁺), suggesting weak expression and heterogeneous distribution of P-type channels compensated by high densities of HVA Ca²⁺ channels resistant to all the antagonists (R-type). A significant residual current could also be resolved after prolonged applications of 5 μm ω-CTx-MVIIC, which allowed separation of N- and P-type currents by the distinct onset of toxin block. The antagonists-resistant current reveals biophysical characteristics typical of HVA channels, but distinct from the α_1E channel. The current activates around ?20 mV in 20 mm Ba²⁺; inactivates slowly and independently of Ca²⁺; is blocked by low [Cd²⁺] and high [Ni²⁺]; and is larger with Ba²⁺ than Ca²⁺. The uncovered R-type calcium current can account for part of the presynaptic Ca²⁺ current controlling neurotransmitter release at the mammalian neuromuscular junction whose activity is resistant to DHP- and ω-CTx-GVIA, and displays anomalous sensitivity to ω-Aga-IVA and ω-CTx-MVIIC ( 1 ) J. Physiol. (Lond.), 482, 283–290; 2 ) Eur. J. Neurosci., 9, 817–823].     

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.     

Glutamate selectively increases the high-threshold Ca channel current in sensory and hippocampal neurons

Previous studies resulted in conflicting conclusions that glutamate application either decreases or increases the activity of Ca²⁺ channels in hippocampal neurons. We studied whole-cell Ca²⁺ currents (I_Ca) in chick dorsal root ganglion neurons and rat hippocampal cells. For both cell types glutamate (1–30 μM) increased high-threshold Ca²⁺ current. It was independent of the charge carriers, Ca²⁺ or Ba²⁺. Low-threshold Ca²⁺ channel current and the fast sodium current were not changed with glutamate application. The effect developed within 1–2 min and then further facilitated after washout of the agonist. A second application of glutamate produced no additional increase in _ICa. No changes in the time-course of whole-cell currents were observed, suggesting that glutamate recruits ‘sleepy’ Ca²⁺ channels. Whatever its mechanism, overlasting increase of I_Ca by glutamate may be important in neuronal plasticity.     

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K⁺ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about −30 mV by clamping cells from −50 mV to different test pulses (−80 to 50 mV). Negative to −30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs⁺ (5 mM) and partially blocked by TEA (5 mM) and Co²⁺ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca²⁺ inward component partially superimposed to a Ca²⁺-dependent outward current. Inward currents were further characterized by replacing K⁺ with Cs⁺ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca²⁺ or 10.8 mM Ba²⁺ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from −50 to 40 mV (holding potential −80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I–V curve, amounted to 247 ± 103pA(n = 3). This inward current was insensitive to 3 μM TTX, but blocked by 1 mM Co²⁺ and partially reduced by 10 μM D600 and 3 μM PN 200-110. In contrast to outward currents, the inward currents exhibited a ‘run-down’ within about 10 min. Lowering the pO₂ from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K⁺ and Ca²⁺ channels which might be essential for chemoreception in the carotid body.     

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 tacrine on intracellular calcium in cholinergic SN56 neuronal cells

We have found earlier that the depolarization-induced release of acetylcholine from the brain could be inhibited by tacrine (tetrahydroaminoacridine) but the mechanism of this action of tacrine was not clarified (S. Tu?ek, V. Dole?al, J. Neurochem. 56 (1991) 1216). We have now investigated whether tacrine has an effect on the changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) induced by depolarization. Experiments were performed on the cholinergic SN56 neuronal cell line with Fura-2 fluorescence technique of calcium imaging. The depolarization by 71 mmol/l K⁺ evoked minimum increases of [Ca²⁺]_i up to day 5 in culture. Then the response gradually increased and reached a plateau after 7 days in culture. A similar time course was observed for acetylcholinesterase activity. The effect of K⁺ ions was concentration-dependent and the concentration of 71 mmol/l K⁺ evoked maximum [Ca²⁺]_i responses. The increases of [Ca²⁺]_i did not occur in the absence of extracellular calcium. They were mediated by high voltage-activated calcium channels of the L-type and the N-type. Nifedipine (2 μmol/l; L-type calcium channel blocker) and ω-conotoxin GVIA (100 nmol/l; N-type calcium channel blocker) diminished the response to 71 mmol/l K⁺ by 53% and 39%, respectively, and their effects were additive (decrease to 8% of controls). Non-selective inorganic blocker of voltage-activated calcium channels LaCl₃ (0.1 mmol/l) decreased the response by 83%. Tacrine attenuated the [Ca²⁺]_i response in a concentration-dependent manner. At a concentration of 10 μmol/l it inhibited the [Ca²⁺]_i response by 55% and its inhibitory effect was additive with that of ω-conotoxin GVIA but not with that of nifedipine. An equimolar concentration of paraoxon, an irreversible inhibitor of cholinesterases, had no influence on [Ca²⁺]_i response. Tacrine exhibited the same inhibitory effect when paraoxon was present. In conclusion, our data indicate that high-voltage-activated calcium channels of the L-type and the N-type are both present in the SN56 cells but that they are fully expressed only after 6–7 days in culture. Tacrine attenuates the influx of calcium by inhibiting the L-type calcium channels. This inhibitory effect is not a consequence of the anticholinesterase activity of tacrine. The finding that low micromolar concentrations of tacrine may interfere with calcium-dependent events is likely to be of importance for the evaluation of the therapeutic potential of the drug.     

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.     

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.     

Inhibition of N-type Calcium Channels: The Only Mechanism by which Presynaptic α2-Autoreceptors Control Sympathetic Transmitter Release

α₂-Adrenoceptors are known to inhibit voltage-dependent Ca²⁺ channels located at neuronal cell bodies; the present study investigated whether this or alternative mechanisms, possibly downstream of Ca²⁺ entry, underlie the presynaptic α₂-adrenergic modulation of transmitter release from chick sympathetic neurons. Using chick sympathetic neurons, overflow of previously incorporated [³H]noradrenaline was elicited in the presence of extracellular Ca²⁺ by electrical pulses, 25 mM K⁺ or 10μM nicotine, or by adding Ca²⁺ to otherwise Ca²⁺-free medium when cells had been made permeable by the calcium ionophore A23187 or by α-latrotoxin. Pretreatment of neurons with the N-type Ca²⁺ channel blocker ω-conotoxin GVIA and application of the α₂-adrenergic agonist UK 14304 reduced the overflow elicited by electrical pulses, K⁺ or nicotine, but not the overflow caused by Ca²⁺ after permeabilization with α-latrotoxin or A23187. In contrast, the L-type Ca²⁺ channel blocker nitrendipine reduced the overflow due to K⁺ and nicotine, but not the overflow following electrical stimulation or α-latrotoxin- and A23187-permeabilization. The inhibition of electrically evoked overflow by UK 14304 persisted in the presence of nitrendipine and the L-type Ca²⁺ channel agonist BayK 8644, which per se enhanced overflow. In ω-conotoxin GVIA-treated cultures, electrically evoked overflow was also enhanced by BayK 8644 and almost reached the value obtained in untreated neurons. However, UK 14304 lost its effect under these conditions. Whole-cell recordings of voltage-activated Ca²⁺ currents corroborated these results: UK 14304 inhibited Ca²⁺ currents by 33%, nitrendipine caused a 7% reduction, and BayK 8644 increased the currents by 30%. Moreover, the dihydropyridines failed to abolish the inhibition by UK 14304, but pretreatment with ω-conotoxin GVIA, which reduced mean amplitude from 0.95 to 0.23 nA, entirely prevented α₂-adrenergic effects. Our results indicate that the α₂-autoreceptor-mediated modulation of noradrenaline release from chick sympathetic neurons relies exclusively on the inhibition of ω-conotoxin GVIA-sensitive N-type Ca²⁺ channels. Mechanisms downstream of these channels and voltage-sensitive Ca²⁺ channels other than N-type appear not to be important.     

Postnatal development of GABAB receptor-mediated modulation of voltage-activated Ca2+ currents in mouse brain-stem neurons.

GABA_B receptors modulate respiratory rhythm generation in adult mammals. However, little is currently known of their functional significance during postnatal development. In the present investigation, the effects of GABA_B receptor activation on voltage-activated Ca²⁺ currents were examined in rhythmically active neurons of the pre-Bötzinger complex (PBC). Both low- (LVA) and high-voltage-activated (HVA) Ca²⁺ currents were present from the first postnatal day (P1). The density of LVA Ca²⁺ currents increased during the first week, whilst the density of HVA Ca²⁺ currents increased after the first week. In the second postnatal week, the HVA Ca²⁺ currents were composed of L- (47 ± 10%) and N-type (21 ± 8%) currents plus a ‘residual’ current, whilst there were no N-type currents detectable in the first few days. The GABA_B receptor agonist baclofen (30 μm ) increased LVA Ca²⁺ currents (30 ± 11%) at P1–P3, but it decreased the currents (35 ± 11%) at P7–P15 without changing its time course. At all ages, baclofen (30 μm ) decreased the HVA Ca²⁺ currents by ≈ 54%. Threshold of baclofen effects on both LVA and HVA Ca²⁺ currents was 5 μm at P1–P3 and lower than 1 μm at P7–P15. The effect of baclofen was abolished in the presence of the GABA_B receptor antagonist CGP 55845A (50 n m ). We conclude that both LVA and HVA Ca²⁺ currents increased postnatally. The GABA_B receptor-mediated modulation of these currents undergo marked developmental changes during the first two postnatal weeks, which may contribute essentially to modulation of respiratory rhythm generation.     

Regionally specific expression of high-voltage-activated calcium channels in thalamic nuclei of epileptic and non-epileptic rats

The polygenic origin of generalized absence epilepsy results in dysfunction of ion channels that allows the switch from physiological asynchronous to pathophysiological highly synchronous network activity. Evidence from rat and mouse models of absence epilepsy indicates that altered Ca^2 + channel activity contributes to cellular and network alterations that lead to seizure activity. Under physiological circumstances, high voltage-activated (HVA) Ca^2 + channels are important in determining the thalamic firing profile. Here, we investigated a possible contribution of HVA channels to the epileptic phenotype using a rodent genetic model of absence epilepsy. In this study, HVA Ca²⁺ currents were recorded from neurons of three different thalamic nuclei that are involved in both sensory signal transmission and rhythmic-synchronized activity during epileptic spike-and-wave discharges (SWD), namely the dorsal part of the lateral geniculate nucleus (dLGN), the ventrobasal thalamic complex (VB) and the reticular thalamic nucleus (NRT) of epileptic Wistar Albino Glaxo rats from Rijswijk (WAG/Rij) and non-epileptic August Copenhagen Irish (ACI) rats. HVA Ca^2 + current densities in dLGN neurons were significantly increased in epileptic rats compared with non-epileptic controls while other thalamic regions revealed no differences between the strains. Application of specific channel blockers revealed that the increased current was carried by L-type Ca²⁺ channels. Electrophysiological evidence of increased L-type current correlated with up-regulated mRNA and protein expression of a particular L-type channel, namely Ca_v1.3, in dLGN of epileptic rats. No significant changes were found for other HVA Ca²⁺ channels. Moreover, pharmacological inactivation of L-type Ca^2 + channels results in altered firing profiles of thalamocortical relay (TC) neurons from non-epileptic rather than from epileptic rats. While HVA Ca^2 + channels influence tonic and burst firing in ACI and WAG/Rij differently, it is discussed that increased Ca_v1.3 expression may indirectly contribute to increased robustness of burst firing and thereby the epileptic phenotype of absence epilepsy.     

Block of P-type Ca channels in freshly dissociated rat cerebellar Purkinje neurons by diltiazem and verapamil

We investigated the effects of organic Ca²⁺ channel blockers, diltiazem and verapamil, on the high voltage-activated P-type Ca²⁺ channels in freshly isolated rat Purkinje neurons. Both diltiazem and verapamil blocked P-type Ca²⁺ channel current without any change in the current-voltage relation. The block was concentration-dependent. In the presence of these agents, the inactivation curve was shifted to hyperpolarizing potentials. The characteristics of block of P-type Ca²⁺ channels by diltiazem and verapamil are similar to that of L-type Ca²⁺ channels. These results indicate that both benzothiazepine and phenylalkylamine react with P-type Ca²⁺ channels and suggest that some structural features common to which operate in both L-type and P-type Ca²⁺ channels may be involved in drug binding to these channels.     

Enhancement in activities of large conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia

It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca²⁺-activated potassium (BK_Ca) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK_Ca channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K⁺ in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (P_o) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P_o was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca²⁺]_i required to half activate BK_Ca channels was only 1 μM in post-ischemic whereas 2 μM in control neurons, indicating an increase in [Ca²⁺]_i sensitivity after ischemia; and (4) BK_Ca channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK_Ca channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.     

Chronic lead exposure alters presynaptic calcium regulation and synaptic facilitation in Drosophila larvae

Prolonged exposure to inorganic lead (Pb²⁺) during development has been shown to influence activity-dependent synaptic plasticity in the mammalian brain, possibly by altering the regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). To explore this possibility, we studied the effect of Pb²⁺ exposure on [Ca²⁺]_i regulation and synaptic facilitation at the neuromuscular junction of larval Drosophila. Wild-type Drosophila (CS) were raised from egg stages through the third larval instar in media containing either 0 μM, 100 μM or 250 μM Pb²⁺ and identified motor terminals were examined in late third-instar larvae. To compare resting [Ca²⁺]_i and the changes in [Ca²⁺]_i produced by impulse activity, the motor terminals were loaded with a Ca²⁺ indicator, either Oregon Green 488 BAPTA-1 (OGB-1) or fura-2 conjugated to a dextran. We found that rearing in Pb²⁺ did not significantly change the resting [Ca²⁺]_i nor the Ca²⁺ transient produced in synaptic boutons by single action potentials (APs); however, the Ca²⁺ transients produced by 10 Hz and 20 Hz AP trains were larger in Pb²⁺-exposed boutons and decayed more slowly. For larvae raised in 250 μM Pb²⁺, the increase in [Ca²⁺]_i during an AP train (20 Hz) was 29% greater than in control larvae and the [Ca²⁺]_i decay τ was 69% greater. These differences appear to result from reduced activity of the plasma membrane Ca²⁺ ATPase (PMCA), which extrudes Ca²⁺ from these synaptic terminals. These findings are consistent with studies in mammals showing a Pb²⁺-dependent reduction in PMCA activity. We also observed a Pb²⁺-dependent enhancement of synaptic facilitation at these larval neuromuscular synapses. Facilitation of EPSP amplitude during AP trains (20 Hz) was 55% greater in Pb²⁺-reared larvae than in controls. These results showed that Pb²⁺ exposure produced changes in the regulation of [Ca²⁺]_i during impulse activity, which could affect various aspects of nervous system development. At the mature synapse, this altered [Ca²⁺]_i regulation produced changes in synaptic facilitation that are likely to influence the function of neural networks.     

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.     

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.