Critical illness myopathy serum fractions affect membrane excitability and intracellular calcium release in mammalian skeletal muscle

Abstract. The pathogenesis of myopathies occurring in critically ill patients (critical illness myopathy, CIM) is poorly understood. Both local and systemic responses to sepsis and other severe insults to the body are presumed to be involved but the precise mechanisms by which muscle function is impaired are far from clear. To elucidate such mechanisms we investigated the effects of blood serum fractions (5 kDa to 100 kDa molecular weight cut-off, MWCO) from patients with CIM and from control persons on membrane and contractile functions in intact mammalian single skeletal muscle fibres and chemically skinned fibre bundles. In intact fibres, resting membrane potentials were less negative when exposed to CIM serum fractions compared with control serum fractions. Half-width and maximum rise time of action potentials (AP) were smaller in CIM serum low MWCO fractions vs. control serum. Peak amplitudes of fast inward sodium currents (I_Na) were increased by low MWCO-CIM fractions compared with control sera fractions. Additionally, voltage dependent inactivation of I_Na was shifted towards more positive potentials by high MWCO fractions of CIM sera. In skinned fibres, pCa-force relations were similar in CIM and control serum fractions but peak force of Ca²⁺ induced force transients was decreased by low MWCO-CIM vs. control serum fractions. Our results (i) provide the first evidence that serum from CIM patients affects membrane excitability and the excitation-contraction coupling process at the level of the sarcoplasmic reticulum Ca²⁺ release of mammalian muscle fibres and (ii) also show that even control serum fractions per se alter the response to important physiological membrane and contractility parameters compared with physiological saline.     

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.     

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.     

Two types of calcium currents of the mouse bipolar cells recorded in the retinal slice preparation

In the vertebrate retina, the bipolar cell makes reciprocal synapses with amacrine cells at the axon terminal. It has been postulated that amacrine cells may control the transmitter release from bipolar cells by modulating their calcium currents (I_Ca). To clarify this possibility calcium currents were studied in bipolar cells of the mouse retina using a slice preparation. I_Ca was identified by voltage clamp protocols, ionic substitution and pharmacological tools. Depolarization to –30 mV from a holding voltage of –80 mV induced an inward current consisting of an initial transient and a long-lasting sustained component. The transient component was inactivated by holding the membrane at more positive voltages. Addition of 100 μm nifedipine suppressed the sustained component, leaving the transient component almost intact. The sustained component was enhanced when external solution contained 0.1 μm Bay K 8644 or when the external Ca²⁺ was substituted by equimolar Ba²⁺. Omega-conotoxin (10 μm ω-ctxn GVIA) did not alter either component. We concluded that the transient component is a low-voltage activated T-type I_Ca, while the sustained component is a high-voltage activated L-type I_Ca. T-type I_Ca was recorded in all cells tested, while L-type I_Ca was found only in cells that retained axon terminals ramifying in the inner plexiform layer. Thus, it is highly likely that L-type I_Ca is generated at the axon terminal and contributes to the transmitter release from the bipolar cell. The present results confirm that in addition to the T-type I_Ca that had been previously described, bipolar cells of the mammalian retina also contain L-type I_Ca similar to the one that has been reported in bipolar cells of the goldfish. The use of retinal slice preparation allowed us to record this current that was not seen previously in the dissociated mouse bipolar cells.     

Slow calcium current is not reduced in malignant hyperthermic porcine myotubes

Malignant hyperthermia-susceptible (MHS) pigs express a sarcoplasmic reticulum (SR) Ca²⁺-release channel mutation that results in lower than normal contractile thresholds in skeletal muscles. In adult MHS pig muscles the L-type calcium current (I_s) is also reduced. We tested the hypothesis that there is a causal relationship between I_s and the lower contractile threshold by recording I_s from MHs and normal porcine myotubes using the whole cell patch-clamp technique. Current voltage relationships for both MHS and normal myotubes were similar, with peak I_s between +20 and +30 mV. Maximum I_s amplitudes were not different (normal: 4976 ± 566 pA; MHS: 6516 ± 1088 pA) nor was I_s specific density (normal: 9.0 ± 0.8; MHS: 8.8 ± 1.1 pA/pF). In both MHS and normal myotubes, both the dihydropyridine antagonist PN200 –110 (200 nmol/L) and holding the membrane potential at −10 mV for 5 min decreased I_s significantly (by more than 50%). There was no apparent direct relationship between the mutation in the SR Ca²⁺-release channel and lowered I_s. We concluded that reduced I_s in adult MHS pig muscles may be a secondary effect of the SR Ca²⁺-release channel mutation on muscle development. © 1996 John Wiley & Sons, Inc.     

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.     

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.     

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.     

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.     

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.     

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.     

Relation of exocytosis and Ca-activated K current during Ca release from intracellular stores in individual rat chromaffin cells

Measurement of the change in cell membrane capacitance (C_m) along with the change in I_K(Ca) was used to investigate the effects of bradykinin and caffeine on the secretory process in rat adrenal chromaffin cells. In a Ca²⁺-free external solution, bradykinin (100 nM) caused a transient increase in C_m with a concurrent change in I_K(Ca). Extracellular application of neomycin as an inhibitor of phospholipase C activity reversibly inhibited the bradykinin-activated event, implying an IP₃-mediated increase of submembrane-free Ca²⁺. The increases in C_m and I_K(Ca) caused by bradykinin were transient even with the sustained application of bradykinin. Caffeine also caused exocytosis in the Ca²⁺-free solution, and this was irreversibly blocked by ryanodine (1 μM) in a use-dependent manner. Caffeine-sensitive intracellular Ca²⁺ stores were also depleted in several seconds and recovered by an influx of external Ca²⁺. The sequential application of bradykinin and caffeine showed that these are likely to activate Ca²⁺ release from the same or distinct but rapidly equilibrating intracellular Ca²⁺ stores. The single cell assay of exocytosis and the increase in I_K(Ca) revealed cell-to-cell variability in bradykinin- and caffeine-induced exocytotic response. Our results suggest that Ca²⁺ release from intracellular stores potentially increases submembrane Ca²⁺ concentration and modulates simultaneously two submembrane Ca²⁺-dependent processes, exocytosis and I_K(Ca), in rat adrenal chromaffin cells.     

The voltage‐dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na⁺), calcium (Ca²⁺), and potassium (K⁺) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of γ-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca²⁺-binding protein calretinin in a subset of cells, indicated the presence of Cajal–Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na⁺ current activated at about −45 mV, was half-maximal steady-state inactivated at −66.6 mV, and recovery from inactivation followed a two-exponential process (τ₁ = 8.4 ms and τ₂ = 858.8 ms). Na⁺ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (I_fast) activated ≈–40 mV, declined rapidly with a voltage-insensitive time constant (τ≈ 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mm ). I_fast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 μm ) and a second by high concentrations (6 mm ). After elimination of I_fast by a conditioning prepulse (50 ms to −50 mV), a slow K⁺ current (I_KV) could be studied in isolation. I_KV was only moderately affected by 4-AP (6 mm ), while TEA (20 mm ) blocked most (> 80%) of the current. I_KV activated at about −40 mV, declined monoexponentially in a voltage-dependent manner (τ≈ 850 ms at −30 mV), and revealed an incomplete steady-state inactivation. In addition to I_fast and I_KV, indications of a Ca²⁺-dependent outward current component were found. When Na⁺ currents, I_fast, and I_KV were blocked by tetrodotoxin (TTX, 1 μm ), 4-AP (6 mm ) and TEA (20 mm ) an inward current carried by Ca²⁺ was found. Ca²⁺ currents activated at depolarized potentials at about −30 mV, were completely blocked by 50 μm cadmium (Cd²⁺), were sensitive to verapamil (≈ 40% block by 10 μm ), and were not affected by nickel (50 μm ). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (≈ 2 ms, measured at half maximal amplitude) of relative small amplitude (≈ 83 mV, measured from the action potential threshold).     

Gonadotropin-Releasing Hormone Induced Ca Influx in Nonsecreting Pituitary Adenoma Cells: Role of Voltage-Dependent Ca Channels and Protein Kinase C

The action mechanism of gonadotropin-releasing hormone (GnRH) on the cytosolic free calcium concentration ([Ca²⁺]_i) and high-threshold voltage-dependent Ca²⁺ channel activity was studied in human nonsecreting (NS) pituitary adenoma cells. [Ca²⁺]_i was monitored in individual cells by dual emission microspectrofluorimetry using indo1 as intracellular fluorescent Ca²⁺ probe. The whole-cell recording patch-clamp technique was used to study Ca²⁺ channels. A short application of GnRH (1 to 100 nM) induced an increase in [Ca²⁺]_i due to Ca²⁺ entry through plasma membrane voltage-sensitive L-type Ca²⁺ channels. Protein kinase C (PKC) depletion induced by a pretreatment with 1 μM PMA for 24 h abolished spontaneous Ca²⁺ transients and the action of GnRH on [Ca²⁺]_i and Ca²⁺ channels. Phloretin (250 μM and staurosporine (20 nM), two protein kinase C inhibitors, inhibited Ca²⁺ channel activity, thereby suppressing the effect of GnRH. On the other hand, activation of PKC by a short application of phorbol myristate acetate (10 nM) stimulated Ca²⁺ influx through Ca²⁺ channels. These findings demonstrate that, in human NS adenoma cells, GnRH (1 to 100 nM) induces an increase in [Ca²⁺]_i, principally due to Ca²⁺ entry through L-type voltage-activated Ca²⁺ channels. PKC regulates this mechanism as well as basal ion channel activity, thus exerting both positive and negative control of [Ca²⁺]_i in stimulated and unstimulated NS adenoma cells.     

Sodium and calcium mechanisms of rhythmic bursting in excitatory neural networks of the pre‐Bötzinger complex: a computational modelling study

The neural mechanisms generating rhythmic bursting activity in the mammalian brainstem, particularly in the pre‐Bötzinger complex (pre‐BötC), which is involved in respiratory rhythm generation, and in the spinal cord (e.g. locomotor rhythmic activity) that persist after blockade of synaptic inhibition remain poorly understood. Experimental studies in rodent medullary slices containing the pre‐BötC identified two mechanisms that could potentially contribute to the generation of rhythmic bursting: one based on the persistent Na⁺ current (I_NaP), and the other involving the voltage‐gated Ca²⁺ current (I_Ca) and the Ca²⁺‐activated nonspecific cation current (I_CAN), activated by intracellular Ca²⁺ accumulated from extracellular and intracellular sources. However, the involvement and relative roles of these mechanisms in rhythmic bursting are still under debate. In this theoretical/modelling study, we investigated Na⁺‐dependent and Ca²⁺‐dependent bursting generated in single cells and heterogeneous populations of synaptically interconnected excitatory neurons with I_NaP and I_Ca randomly distributed within populations. We analysed the possible roles of network connections, ionotropic and metabotropic synaptic mechanisms, intracellular Ca²⁺ release, and the Na⁺/K⁺ pump in rhythmic bursting generated under different conditions. We show that a heterogeneous population of excitatory neurons can operate in different oscillatory regimes with bursting dependent on I_NaP and/or I_CAN, or independent of both. We demonstrate that the operating bursting mechanism may depend on neuronal excitation, synaptic interactions within the network, and the relative expression of particular ionic currents. The existence of multiple oscillatory regimes and their state dependence demonstrated in our models may explain different rhythmic activities observed in the pre‐BötC and other brainstem/spinal cord circuits under different experimental conditions.     

Modulation of presynaptic Ca2+ currents in frog motor nerve terminals by high pressure

Presynaptic Ca²⁺‐dependent mechanisms have already been implicated in depression of evoked synaptic transmission by high pressure (HP). Therefore, pressure effects on terminal Ca²⁺ currents were studied in Rana pipiens peripheral motor nerves. The terminal currents, evoked by nerve or direct stimulation, were recorded under the nerve perineurial sheath with a loose macropatch clamp technique. The combined use of Na⁺ and K⁺ channel blockers, [Ca²⁺]_o changes, voltage‐dependent Ca²⁺ channel (VDCC) blocker treatments and HP perturbations revealed two components of presynaptic Ca²⁺ currents: an early fast Ca²⁺ current (I_CaF), possibly carried by N‐type (Ca_V2.2) Ca²⁺ channels, and a late slow Ca²⁺ current (I_CaS), possibly mediated by L‐type (Ca_V1) Ca²⁺ channels. HP reduced the amplitude and decreased the maximum (saturation level) of the Ca²⁺ currents, I_CaF being more sensitive to pressure, and may have slightly shifted the voltage dependence. HP also moderately diminished the Na⁺ action current, which contributed to the depression of VDCC currents. Computer‐based modeling was used to verify the interpretation of the currents and investigate the influence of HP on the presynaptic currents. The direct HP reduction of the VDCC currents and the indirect effect of the action potential decrease are probably the major cause of pressure depression of synaptic release.     

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.     

Effects of myasthenia gravis patients' sera with different autoantibodies on slow K+ current at mouse motor nerve terminals

Abstract

The antibodies against pre-synaptic membrane receptor (PsmR) and acetylcholine receptor (AChR) in serum samples of myasthenia gravis (MG) patients and healthy donors were tested by enzyme-linked immunosorbent assays (ELISA). The serum samples of eight MG patients with different autoantibodies and those of six healthy donors without these two kinds of autoantibodies were collected to investigate their effects on the peri-neurially recorded membrane currents at mouse motor nerve terminals. After inhibition of both fast and Ca²⁺-dependent K⁺ currents by tetra-ethylammonium (TEA), a positive wave was revealed, which was a balance of the slow K⁺(I_K,s) and Ca²⁺ currents (I_Ca). Application of anti-PsmR antibody negative MG sera and healthy donor sera, whether anti-AChR antibody positive or negative, did not affect the positive wave. However, the positive wave shifted to prolonged Ca²⁺-plateau when adding two of four anti-PsmR antibody positive serum samples from MG patients, indicating an inhibition of I_K,s by anti-PsmR antibody positive sera. Meanwhile, all serum samples derived from either patients or healthy donors did not affect I_Na.     

Trophic effect of cholera toxin B subunit in cultured cerebellar granule neurons: Modulation of intracellular calcium by GM1 ganglioside

Survival of cerebellar granule cells (CGC) in culture was significantly improved in the presence of cholera toxin B subunit (Ctx B), a ligand which binds to GM1 with specificity and high affinity. This trophic effect was linked to elevation of intracellular calcium ([Ca²⁺]_i), and was additive to that of high K⁺. Survival was optimized when Ctx B was present for several days during the early culture period. ⁴⁵Ca²⁺ and cell survival studies indicated the mechanism to involve enhanced influx of Ca²⁺ through L-type voltage-sensitive channels, since the trophic effect was blocked by antagonists specific for that channel type. Inhibitors of N-methyl-D-aspartate receptor/channels were without effect. During the early stage of culture Ctx B, together with 25 mM K⁺, caused [Ca²⁺]_i to rise to 0.2–0.7 μM in a higher proportion of cells than 25 mM K⁺ alone. A significant change in the nature of GM1 modulation of Ca²⁺ flux occurred after 7 days in culture, at which time Ctx B ceased to elevate and instead reduced [Ca²⁺]_i below the level attained with 25 mM K⁺. GM1 thus appears to serve as intrinsic inhibitor of one or more L-type Ca²⁺ channels during the first 7 days in vitro, and then as intrinsic activator of (possibly other) L-type channels after that period. This is the first demonstration of a modulatory role for GM1 ganglioside affecting Ca²⁺ homeostasis in cultured neurons of the CNS. © 1996 Wiley-Liss, Inc.     

Characterization of a Calcium-dependent Current Generating a Slow Afterdepolarization of CA3 Pyramidal Cells in Rat Hippocampal Slice Cultures

A depolarization-induced, slowly decaying inward current was examined in slice-cultured CA3 pyramidal cells by voltage-clamp techniques and microfluorometric measurements of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). Action potentials elicited by intracellular injection of short-lasting (50 – 100 ms) depolarizing current pulses were followed by a slowly decaying afterhyperpolarization (AHP). After switching to voltage-clamp mode, short-lasting (50 – 100 ms) depolarizing voltage jumps from –60 mV to between –30 and 0 mV induced a slowly decaying outward aftercurrent (I_AHP) which was depressed by bath application of muscarine (0.5 μM). In the presence of muscarine, the same depolarizations induced a slowly decaying afterdepolarization (ADP) or inward aftercurrent (I_ADP)in voltage-clamp mode. This current was also induced in the presence of trans(±)-1-aminc-1,3-cyclopenta-nedicarboxylic acid (t-ACPD, 5 μM), an agonist of metabotropic glutamate receptors, but not in the presence of noradrenalin (5 μM), while both of these agonists depressed I_AHP. I_ADP was depressed by reducing the external Ca²⁺ concentration from 3.8 to 0.5 mM, by external Co²⁺ (1 mM) and by external Cd²⁺ (10 – 100 μM). Combined voltage-clamp recordings and microfluorometric measurements of [Ca²⁺]_i using the Ca²⁺ indicator fura-2 revealed that the amplitude of I_ADP was correlated with the amplitude of depolarization-induced Ca²⁺ influx, I_ADP was absent at membrane potentials < –90 mV, and reached maximal amplitudes at ～–55 mV. Raising the extracellular K⁺ concentration from 2.7 to 13.5 mM increased the amplitude of I_ADP and resulted in a positively directed shift of the apparent reversal potential of I_ADP. When the external Na⁺ concentration was reduced from 157 to 33 or 18 mM the current reversed at more negative potentials and was reduced to 40 and 21%, respectively, of control amplitude. Lowering the external Cl^- concentration from 159 to 20 mM did not affect I_ADP. We conclude that I_ADP most likely represents a Ca²⁺-activated cation current, rather than a Ca²⁺ tail current, or an electrogenic Ca²⁺ extrusion current.