Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+-dependent mechanism in cultured rat sympathetic neurons

Superior-cervical ganglion (SCG) cells dissociated from newborn rats depend on nerve growth factor (NGF) for survival. Membrane depolarization with elevated K+ is known to prevent neuronal death following NGF deprivation and/or to promote survival via a Ca2+-dependent mechanism. Here we have exploited the possibility of whether or not a Na+-dependent pathway for neuronal survival is present in these cells. Veratridine (ec50=40 nM), a voltage-dependent Na+ channel activator, significantly delayed the onset of apoptotic cell death in NGF-deprived SCG neurons that had been cultured for 7 days in the presence of NGF. This effect was blocked completely by Na+ channel blockers including tetrodotoxin (TTX, 1 μM), benzamil (25 μM) and flunarizine (1 μM), but was not attenuated by nimodipine (1 μM), an L-type Ca²⁺ channel blocker. The saving effect of veratridine on cultured neurons was observed even in low Ca²⁺ media (0–1.0 mM), but was completely abolished in a low Na⁺ medium (38 mM). Sodium-binding benzofuran isophthalate was employed as a fluorescent probe for monitoring the level of cytoplasmic free Na⁺, which revealed a sustained increase in its level (12.9 mM, 307% of that of control) in response to veratridine (0.75 μM). The TTX or flunarizine completely blocked veratridine-induced Na⁺ influx in these cultured neurons. Moreover, no appreciable increase in intracellular Ca²⁺ was detected under these conditions. Though Na⁺ channels were effectual in SCG neurons which were freshly isolated from newborn rats, the Na⁺-dependent saving effect of veratridine was not observed in these young neurons. These lines of evidence suggest that the death-suppressing effect of veratridine on cultured SCG neurons depends on the Na⁺ influx via voltage-dependent Na⁺ channels, and suggests the presence of Na⁺-dependent regulatory mechanism(s) in neuronal survival.     

Anoxia induces an increase in intracellular sodium in rat central neurons in vitro

Following our previous observations that anoxia induces a drop in extracellular Na⁺ in the brain slice and that removal of extracellular Na⁺ prevents the anoxia-induced morphological changes in dissociated hippocampal neurons, we hypothesized that intracellular Na⁺ increases during anoxia in isolated neurons. Using the fluorophore Sodium Green in freshly dissociated rat CA1 neurons, and SBFI in cultured cortical neurons, we found that 10 min of anoxia caused an increase in Na_i⁺ in both types of cells, with a latency of about 2 min. In CA1 neurons, fluorescence increased by an average of 20.34% (n = 8). The mean baseline Na_i⁺ level (determined using SBFI) was 25 ± 2.5 mM, whichincreased to about an average of 52 ± 3 mM after 3–4 min. These and our previous results strongly suggest that Na⁺-mediated events are involved in anoxia-induced nerve injury.     

Inhibition of major cationic inward currents prevents spreading depression-like hypoxic depolarization in rat hippocampal tissue slices

Hypoxia-induced spreading depression-like depolarization (hypoxic SD, or anoxic depolarization) is accompanied by the near-loss of membrane potential, severe reduction of membrane resistance, and influx of Na⁺, Ca²⁺, Cl⁻ and water into neurons. The biophysical nature of these membrane changes is incompletely understood. In the present study we applied a pharmacological mixture (10 μM DNQX, 10 μM CPP, 1 μM TTX, and 2 mM Ni²⁺) to rat hippocampal tissue slices to inhibit major Na⁺ and Ca²⁺ inward currents. This inhibitory cocktail slightly depolarized CA1 pyramidal neurons and completely blocked all evoked potentials. In its presence severe hypoxia of up to 20 min duration failed to induce hypoxic SD and the accompanying intrinsic optical signal. Instead, only moderate, very slow negative shifts of the extracellular DC potential were observed. Following 10 min hypoxia and 1 hour wash-out of the inhibitors antidromic and orthodromic responses were still blocked but hypoxic SD with markedly delayed onset could be induced in most slices. In current-clamped CA1 pyramidal cells hypoxia induced a rapid, near-complete depolarization and decreased the input resistance by 89%. In the presence of the cocktail, however, hypoxia caused a gradual, partial depolarization, to about −40 mV; the membrane resistance decreased by only 37%. We conclude that simultaneous blockade of the known major Na⁺ and Ca²⁺ channels consistently prevents hypoxic SD. The hypothesis that SD initiation is the consequence of general loss of selective permeability or general membrane breakdown becomes unlikely. Instead, influx of Na⁺ and Ca²⁺ might play a crucial role in the generation of the rapid SD-like depolarization.     

Anoxia-Induced Neuronal Injury: Role of NaEntry and Na-Dependent Transport

An important cause of anoxia-induced nerve injury involves the disruption of the ionic balance that exists across the neuronal membrane. This loss of ionic homeostasis results in an increase in intracellular calcium, sodium, and hydrogen and is correlated with cell injury and death. Using time-lapse confocal microscopy, we have previously reported that nerve cell injury is mediated largely by sodium and that removing extracellular sodium prevents the anoxia-induced morphological changes. In this study, we hypothesized that sodium enters neurons via specific mechanisms and that the pharmacologic blockade of sodium entry would prevent nerve damage. In cultured neocortical neurons we demonstrate that replacing extracellular sodium with NMDG⁺prevents anoxia-induced morphological changes. With sodium in the extracellular fluid, various routes of sodium entry were examined, including voltage-sensitive sodium channels, glutamate receptor channels, and sodium-dependent chloride–bicarbonate exchange. Blockade of these routes had no effect. Amiloride, however, prevented the morphological changes induced by anoxia lasting 10, 15, or 20 min. At doses of 10 μM–1 mM, amiloride protected neurons in a dose-dependent fashion. We argue that amiloride acts on a Na⁺-dependent exchanger (e.g., Na⁺–Ca²⁺) and present a model to explain these findings in the context of the neuronal response to anoxia.     

Drosophila neurons respond differently to hypoxia and cyanide than rat neurons

Compared with mammalian species, Drosophila melanogaster exhibits marked tolerance to hypoxia or anoxia. However, the underlying cellular mechanisms of tolerance are still largely unknown. In order to assess the electrophysiologic response to O₂ lack in Drosophila neurons and compare them to those in mammals, we used neurons from embryonic cultures of both Drosophila and rat. We studied the effects of hypoxia on membrane potential V_m, input resistance R_m, rheobase, and action potential characteristics before, during and after 3 to 5 min of hypoxia (measured PO₂<20 Torr). In Drosophila neurons, on the one hand, V_m reversibly hyperpolarized with hypoxia by an average of about 20 mV and input resistance decreased by 71% from control. In most cells studied, action potential (AP) amplitude decreased, its duration increased, and its threshold shifted in a hyperpolarized direction before AP generation was attenuated. On the other hand, V_m in rat cortical neurons reversibly depolarized by an average of 10 mV with hypoxia. Input resistance was reversibly reduced by 58% and, in most cells studied, AP amplitude also decreased and its duration increased. In contrast to the effects of hypoxia on V_m, CN caused a depolarization by 22 mV and a slight increase in R_m in Drosophila. In the rat, CN was similar to hypoxia in its effect on R_m. We conclude that (1) rat and Drosophila neurons decrease their excitability in hypoxia by activating different mechanisms; (2) the most likely explanation for the hyperpolarization and the decrease in R_m in Drosophila neurons is the activation of a K⁺ conductance; this activation, by itself, cannot explain the results in rat neurons and (3) hypoxia and cyanide have similar effects in rat neurons but are divergent in their effects in Drosophila neurons.     

Involvement of the glutamate transporter and the sodium–calcium exchanger in the hypoxia-induced increase in intracellular Ca in rat hippocampal slices

Hippocampal slices prepared from adult rats were loaded with fura-2 and the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the CA1 pyramidal cell layer was measured. Hypoxia (oxygen–glucose deprivation) elicited a gradual increase in [Ca²⁺]_i in normal Krebs solution. At high extracellular sodium concentrations ([Na⁺]_o), the hypoxia-induced response was attenuated. In contrast, hypoxia in low [Na⁺]_o elicited a significantly enhanced response. This exaggerated response to hypoxia at a low [Na⁺]_o was reversed by pre-incubation of the slice at a low [Na⁺]_o prior to the hypoxic insult. The attenuation of the response to hypoxia by high [Na⁺]_o was no longer observed in the presence of antagonist to glutamate transporter. However, antagonist to Na⁺–Ca²⁺ exchanger only slightly influenced the effects of high [Na⁺]_o. These observations suggest that disturbance of the transmembrane gradient of Na⁺ concentrations is an important factor in hypoxia-induced neuronal damage and corroborates the participation of the glutamate transporter in hypoxia-induced neuronal injury. In addition, the excess release of glutamate during hypoxia is due to a reversal of Na⁺-dependent glutamate transporter rather than an exocytotic process.     

Effects of the sodium channel blocker tetrodotoxin (TTX) on cellular ion homeostasis in rat brain subjected to complete ischemia

Anoxic depolarization (AD) and failure of the cellular ion homeostasis are suggested to play a key role in ischemia-induced neuronal death. Recent studies show that the blockade of Na⁺ influx significantly improved the neuronal outcome. In the present study, we investigated the effects of 10 μM tetrodotpxin (TTX) on ischemia-induced disturbances of ion homeostasis in the isolated perfused rat brain. TTX inhibited the spontaneous EEG activity, delayed the ischemia-induced tissue acidification, and significantly postponed the occurrece of AD by 65%. The [Ca²⁺]_e elevation prior to AD was attenuated from 17.8% to 6% while the increase of the [Na⁺]_e in this period was enhanced (from 2.9% to 7.3%). These findings implied that the ischemia-induced early cellular sodium load and the corresponding shrinkage of the extracellular space was counteracted by TTX. Our results suggest that the Na⁺ influx via voltage-dependent channels preceding complete breakdown of ion homeostasis is one major factor leading to cell depolarization. The massive Na⁺ influx coinciding with AD, however, may be mainly via non-selective cation channels or/and receptor-operated channels. Persistent Na⁺ influx deteriorates neuronal tissue integrity by favouring Ca²⁺ influx and edema formation. Blockade of ischemia-induced excessive Na⁺ influx is, therefore, a promising pharmacological approach for stroke treatment.     

Removal of extracellular sodium prevents anoxia-induced injury in freshly dissociated rat CA1 hippocampal neurons

Anoxia is believed to cause nerve injury and death in part, by inducing sustained, elevated levels of intracellular Ca²⁺. The increased concentration of intracellular Ca²⁺ is capable, by itself, of inducing nerve injury and death, even without the added stress of anoxia. However, we have recently shown that an increased level of intracellular Ca²⁺ is not necessary for anoxia-induce CA1 nerve injury. Since we have observed that extracellular Na⁺ decreases during anoxia, we studied the role of extracellular Na⁺ in anoxia-induced nerve injury. Removal of extracellular Na⁺ and its replacement with the impermeant cation N-methyl-d-glucamine (NMDG⁺) completely protected freshly dissociated CA1 neurons during and after severe anoxia, for up to 90 min. Intracellular Ca²⁺ decreased during anoxia, recovering during reoxygenation. Propidium iodide was excluded from the neurons for as long as Na⁺ was absent. Addition of Na⁺ (by replacing NMDG⁺) following anoxia resulted in rapid bleb formation, swelling and intracellular Ca²⁺ rise. Removal of Na⁺ before the rupture of blebs caused either shrinkage or pinching off of blebs so that the neuron apparently returned to its previous undisturbed state. We conclude that: (1) replacement of Na_o⁺ with NMDG⁺ totally prevents anoxia-induced nerve injury as manifested by morphological changes, e.g., swelling, bleb formation and membrane injury; (2) upon re-exposure to Na_o⁺ following anoxia in the absence of Na_o⁺, neurons swell and bleb; (3) the appearance of blebs following anoxic exposure in the absence of Na_o⁺ is reversible and Na_o⁺ dependent. (4) Bleb formation itself is not necessarily immediately lethal to the neuron.     

Hypotonic exposure enhances synaptic transmission and triggers spreading depression in rat hippocampal tissue slices

Low extracellular osmotic pressure (π_o) is known to enhance CNS resposiveneess and the chance of seizures, but the mechanism of the hyperexcitability is not clear. We recorded evoked potentials in st. radiatum and st. pyramidale of CA1. Tissue electrical resistance (R_o) was determined from the voltage drop (V_Ro) evoked by constant current pulses. Lowering of π_o by reducing [NaCl] caused a concentration-dependent increase of amplitude and duration of extracellular excitatory postsynaptic potentials (fEPSPs). fEPSPs increased much more than didV_Ro, but antiddromic population spikes increased in proportion toV_Ro. fEPSP increased also in isosmotic low NaCl (fructose or mannitol substituted) olutions, but not as much as in low π_o. In moderately hypotonic solutions orthodromic population spikes increased as expected from the augmented fEPSP, but in strong hypotonia input-output curves shifted to the left and single stimuli evoked multiple population spikes, indicating lowering of threshold of postsynaptic neurons. Blocking N-methyl-d-aspartate (NMDA) receptors did not diminish the enhancement of fEPSP amplitude. Spreading depression (SD) erupted in most slices in very low π_o, but not in isosmotic low [NaCl] solutions. We conclude that the hypotonic enhancement of EPSPs depends, in part, on the lowering of [Na⁺]_o and/or of [Cl⁻]_o, and it may be augmented by dendritic swelling favoring electrotonic spread of EPSPs from dendrites to somata, and buildup of transmitter concentration due to swelling of perisynaptic glia. SD can be initiated by cell swelling, but the depolarization associated with SD is probably not caused by the opening of stretch-gated ion channels.     

In vitro assessment of the effect of methylene blue on voltage-gated sodium channels and action potentials in rat hippocampal CA1 pyramidal neurons

Methylene blue (MB) is a vital dye to allow better visualization and marker of parathyroid glands. The compound causes a toxic encephalopathy in clinical observations and some neuronal adverse effects in experimental studies. Of neurotoxic effects, reduced field excitatory postsynaptic potentials (fEPSPs) in hippocampal slice cultures and apoptosis induced in neurons by MB, suggest that MB may affect electrophysiological properties in neurons. Consequently, studies were undertaken to characterize the effects of MB on voltage-gated sodium currents (I_Na) in hippocampal CA1 neurons. MB was tested at a clinically-relevant concentration (10 μM), of which as a surgical marker of the parathyroid glands, and other concentrations (0.25 μM, 1 μM, and 100 μM). The results showed that MB reduced the amplitude of I_Na at the concentrations of 10 μM and 100 μM. No significant changes were found with the other two concentrations of MB. 10 μM of MB did not produce a shift in the activation–voltage curve of I_Na but produced a hyperpolarizing shift in the inactivation–voltage curve of I_Na and delayed the recovery of I_Na from inactivation. Action potential (AP) properties and the pattern of repetitive firing were examined using whole-cell current-clamp recordings. Peak amplitude, overshoot and maximum velocity of depolarization (V_max) of the evoked single AP decreased in the presence of the 10 μM MB solution. The rate of repetitive firing also decreased. The results suggest MB as a surgical marker of the parathyroid glands, may cause sodium channel inhibition on neurons in the nervous system.     

Glutamine transport in cerebellar granule cells in culture

In the present study, uptake of glutamine by rat cerebellar granule cells, a predominantly glutamatergic nerve cell population, has been investigated. Glutamine is taken up by granule cells via at least three transport systems, A, ASC and L. The L-type low affinity system (K_m=2.6 mM) is the major transport system in the absence of Na⁺. The systems A and ASC represent the Na⁺-dependent transport routes, both with almost identical high affinity for glutamine (K_m=0.26 mM). Similar transport systems for glutamine are also found in cerebral cortical neurons, a predominantly GABAergic nerve cell population, and cerebral cortical astrocytes. The glutamine transport properties in granule cells, however, show a series of differences from that of cortical neurons and astrocytes: (1) uptake of glutamine by granule cells is primarily mediated by system A (54%), while contributions by system A in cortical neurons and astrocytes are less than 30%; (2) granule cells exhibit strikingly higher transport efficiency for glutamine (V_max/K_m=20 min⁻¹ for system A as compared to the V_max/K_m ratio of 5 min⁻¹ in cortical neurons and astrocytes), and (3) the initial uptake rates and the steady-state accumulation levels of glutamine are two- to threefold higher in granule cells than that of cortical neurons and astrocytes. These results taken together suggest that in accordance with the important need to replenish the neurotransmitter pool of glutamate, glutamatergic neurons exhibit highly efficient transport systems to accumulate glutamine, one of the major precursors of glutamate.     

Modulation of junctional conductance between rat carotid body glomus cells by hypoxia, cAMP and acidity

Short-term cultures of glomus cells (up to seven days), were employed to study intercellular electrical communications. Bidirectional electric coupling was established under current clamping after impaling two adjacent glomus cells with microelectrodes, and alternate stimulation and recording. Their resting potential (V_m) and input resistance (R_o) were thus measured. Both coupled cells were then voltage clamped at a level between their V_ms. Current pulses applied to either cell elicited a transjunctional voltage (V_j) and current (I_j), used to calculate the junctional conductance (G_j). G_j was 1.52±0.29 nS (mean±S.E.; n=147). V_j linearly influenced G_j, suggesting ohmic junctions. G_j was not affected by V_m in 50% of the cases. However, there was V_m-dependence in the others, but voltage changes had to be large (>±40 mV from the V_m). Therefore, physiologically or pharmacologically induced glomus cell depolarization or hyperpolarization may not significantly affect intercellular coupling unless there are large variations in V_m. Hypoxia (induced by Na₂S₂O₄ 1 mM or 100% N₂) decreased G_j in 60–80% of the pairs while producing tighter coupling in the rest. Similar effects were obtained when the medium was acidified with lactic acid 1–10 mM. Cobalt chloride (3 mM) prevented, diminished or reversed the changes in G_j observed during low PO₂, suggesting that [Ca²⁺]_i changes are important in hypoxic uncoupling. However, non-specific cationic effects of Co²⁺ have not been ruled out. Applications of the membrane-permeant dB-cAMP 1 mM tightened coupling in almost all cell pairs. This is important because endogenous cAMP increases during hypoxia. Our results suggest that multiple factors modulate junctional conductance between glomus cells. Changes in G_j by ‘natural' stimuli and/or cAMP may play an important role in chemoreception, especially in titrating the release of transmitters toward the carotid nerve terminals.     

Ionic contributions to the oscillatory firing activity of rat Purkinje cells in vitro

Oscillatory firing activity in cerebellar Purkinje cells (PCs) can be maintained by intrinsic ionic conductances in the apparent absence of excitatory and inhibitory synaptic input as demonstrated by application of TTX or antagonists of amino acid-mediated transmission or both. Bursting activity in these cells is associated with a region of ZSR (zero slope resistance, the beginning part of a negative slope resistance region) of the whole cell quasi-steady-state I–V relationship. Blockade of Na⁺ current by TTX unmasked the ZSR region in all PCs tested. Based on current and voltage clamp experiments, hyperpolarization-activated cation current (I_h) participates in the rhythmic firing activity by influencing the amplitude and duration of the interburst interval and the resultant pattern of the burst generation. Blockade of I_h with cesium (Cs⁺) retards the membrane rebound from the after-hyperpolarization and results in longer and more negative hyperpolarization between bursts. However, Cs⁺ did not affect the presence and characteristic of the ZSR region of the whole cell quasi-steady-state I–V curve.     

Correlation of anoxic neuronal responses and calbindin-D28k localization in stratum pyramidale of rat hippocampus

Immunohistochemical staining for the calcium-binding protein calbindin-D_28k (CaBP) was combined with Lucifer Yellow (LY) identification and intracellular recording of changes in membrane parameters of pyramidal neurons in CA2, CA1, and the sebiculum of rat hippocampal slices during brief exposure (4.0 ± 0.19 min) to N₂. Anoxia evoked either a depolarization or hyperpolarization of membrane potential (V_M) (+21.5 ± 2.79 mV above V_M = ?70.5 ± 1.50 mV, n = 30 and ?7.2 ± 0.72 mV below V_M = ?68.2 ± 1.34 mV, n = 24, respectively) and a fall in membrane resistance of =20%. Differences in the response could be correlated with the presence or absence of CaBP and the localization of neurons in different layers of stratum pyramidale and sectors of the hippocampus. For neurons immunopositive for calbindin (CaBP(⁺)), depolarization was observed more frequently (83%) than hyperpolarization (17%); in contrast, 44% of responses of calbindin-negative (CaBP(^?)) neurons were depolarizing and 56% were hyperpolarizing. Depolarizations of CaBP(⁺) neurons were more gradual in slope, and more rapidly reached a plateau in comparison with those recorded in CaBP(^?) neurons. Responses of neurons in the superficial layer of stratum pyramidale (in which 79% of CaBP(⁺) pyramidal neurons were situated) were mainly depolarizing (91%), while for those in the deep layer (which contained 89% of the CaBP(^?) cells) such responses were observed less often (45%). Depolarization was also more common than hyperpolarization for cells located in CA2/CA1c/CA1b (63%) than in the CA1a/subicular region (37%). The depolarizing response of the majority of pyramidal neurons which are CaBP(⁺), superficial, and closer to CA3 may reflect an efficient buffering of intracellular Ca²⁺, which maintains a low [Ca²⁺]_i, steep gradient for Ca²⁺ influx and may facilitate the movement of Ca²⁺ away from points of entry. The neurons which are CaBP(^?), deep, and closer to subiculum and in which N₂ evokes hyperpolarization, on the other hand, may have a sustained elevation/accumulation of cytosolic Ca²⁺ which could activate K⁺ conductance, inhibit Ca²⁺ influx, and stabilize the membrane potential. These experiments provide a functional correlate for CaBP and suggest that it may have a significant role in Ca²⁺ homeostasis and the determination of selective neuronal vulnerability. © 1995 Wiley-Liss, Inc.     

Action potential-like responses in B 104 cells with low Na channel densities

Action potential generation and Na⁺ currents were studied in B104 neuroblastoma cells in vitro using the whole-cell patch-clamp method in voltage-clamp and current-clamp mode. Action potential-like responses were elicited in 38 of 42 cells, with a threshold close to −55 mV for depolarizing stimuli, and −56 mV for anode-break stimuli. Response amplitudes were larger when cells were held at more negative prepulse potentials, and were well fit by a Boltzmann distribution with a midpoint of approx. −75 mV, close to theV_1/2 for Na⁺ current steady-state inactivation in these cells. Cells displaying action potential-like responses exhibited a peak Na⁺ current density of 133 ± 0.14 pA/pF (range, 10.2–296.2 pA/pF) and a lowg_K: g_Na ratio (0.0067 ± 0.0023). Exposure to 0.1 mM Cd²⁺ did not block the generation of action potential-like responses in B104 cells, while 1 μM TTX abolished the responses. We conclude that low densities of Na⁺ channels ( < 3/μm² and < 1/μm² in some cells) can support the generation of action potential-like responses in B104 cells if they are held at hyperpolarized levels to remove inactivation. The low leak and K⁺ conductance of these cells may contribute to their ability to generate action potential-like responses under these circumstances.     

α-Melanocyte-stimulating hormone (α-MSH) release from perifused rat hypothalamic slices

A perifusion system was developed to investigate the control of α-melanocyte-stimulating hormone (α-MSH) release from rat brain. Hypothalamic slices were perifused with Krebs-Ringer bicarbonate (KRB) medium supplemented with glucose, bacitracin and bovine serum albumine. Fractions were set apart every 3 min and α-MSH levels were measured by means of a specific and sensitive radioimmunoassay method. Hypothalamic tissue in normal KRB medium released α-MSH at a constant rate corresponding to 0.1% of the total hypothalamic content per 3 min. The basal release was not altered by Ca²⁺ omission in the medium or addition of the sodium channel blocker tetrodotoxine (TTX). Depolarizing agents such as potassium (50 mM) and veratridine (50 μM), which is known to increase Na⁺ conductance, significantly stimulated α-MSH release in a Ca²⁺-dependent manner. When Na⁺-channels were blocked by TTX (0.5 μM) the stimulatory effect of veratridine was completely abolished whereas the K⁺-evoked release was unaffected. These findings suggest that: (1) voltage-dependent sodium channels are present on α-MSH hypothalamic neurons; (2) depolarization by K⁺ induces a marked stimulation of α-MSH release; (3) K⁺- and veratridine-evoked releases are calcium-dependent. Altogether, these data provide evidence for a neurotransmitter or neuromodulator role for α-MSH in rat hypothalamus.     

Choroid plexus histidine transport

System-N transport plays an important role in l-glutamine uptake into isolated rat choroid plexus but its role in the transport of another System-N substrate, l-histidine, has yet to be determined. Similarly, the possible effects on System-N mediated l-histidine transport of changes in pH and extracellular l-glutamine, such as occur in cerebral ischemia and hepatic encephalopathy, have yet to be examined. In the absence of competing amino acids, l-[³H]histidine uptake in isolated rat choroid plexus was mediated by both Na⁺-independent and Na⁺-dependent transport. The former was inhibited by 2-amino-2-norbornane carboxlic acid, indicating System-L transport, while the latter appears System-N mediated as it was inhibited by three System-N substrates but not substrates for System-A and -ASC. The Na⁺-dependent uptake had a K_m of 0.2 mM and a V_max of 1.4 nmol/mg/min. It accounted for 30% of l-histidine uptake in the presence of physiological concentrations of amino acids. Reductions in pH markedly inhibited Na⁺-dependent but not Na⁺-independent transport indicating that, as in liver but not neurons, System-N mediated transport at the choroid plexus is pH sensitive. Increases in l-glutamine concentration in the pathophysiological range reduced l-histidine uptake via both System-L and -N.     

Blockade of bepridil on IA and IK in acutely isolated hippocampal CA1 neurons

The effects of bepridil, an antianginal agent with antiarrhythmic action, on voltage-dependent K⁺ currents in the CA1 pyramidal neurons acutely isolated from rat hippocampus were studied by means of whole-cell patch clamp techniques. Current recordings were made in the presence of TTX to block Na⁺ current. Depolarizing test pulses activated two components of outward K⁺ currents: a rapidly activating and inactivating component, I_A; and a delayed component, I_K. Results showed that bepridil reduced the amplitude of I_A and I_K, and exerted its inhibitory action in time- and dose-dependent manner. Half-blocking concentrations (IC₅₀) of bepridil on I_A and I_K were 17.8 μM and 1.7 μM, respectively. 10 μM bepridil suppressed I_A and I_K by 46.7% and 77.1% at +30 mV of depolarization, respectively. When I_K was activated nearly uncontaminated with I_A by holding at −50 mV, 10 μM bepridil inhibited I_K by 71.6% at +30 mV of depolarization; 10 μM bepridil positively shifted the voltage-dependent of activation curves of I_A and I_K 12.1 mV and 28.7 mV, respectively. These results suggested that blockade on K⁺ currents by bepridil is preferential for I_K, and contributes to the protection brain against ischemic damage.     

Cationic modulation of 5-HT2 and 5-HT3 receptors in rat sensory neurons: the role of K, Ca and Mg

The effects of changes in external K⁺, Ca²⁺, and Mg²⁺ concentrations on 5-HT₂- and 5-HT₃ receptor-mediated depolarizations of the resting membrane potential in rat dorsal root ganglion (DRG) cells was studied. In cells exhibiting a 5-HT₂-mediated response, 5-HT and α-methyl 5-HT depolarized the resting membrane potential (RMP) and increased the slope of the current–voltage (I/V) relationship. The equilibrium potential (E_r) for the depolarization was linearly related to the logarithm of the [K⁺]_o, indicating the depolarization resulted from a decrease in resting K⁺ conductance. In a subpopulation of large-diameter acutely dissociated DRG neurons recorded from using the whole-cell patch-clamp configuration, 5-HT produced an inward shift in the current required to hold cells at −60 mV. This inward shift in holding current was associated with a reduction in membrane conductance and reversed near E_k. This data suggests that the 5-HT₂ receptor-mediated depolarization and increase in R_in seen in intact DRG preparation is produced by blockade of an outward K⁺ leak current. Increases in [K⁺]_o reduced the increase in R_in and depolarization induced by 5-HT with 50% inhibition of the depolarization occurring at 8.3 mM of [K⁺]_o. Half-normal Ca²⁺ (1.2 mM) produced a downward shift of the 5-HT concentration–response curve, reducing the maximal response by 40%, with minimal effect on the half-maximal response. Mg²⁺ ions did not affect this 5-HT response. In cells exhibiting a 5-HT₃ receptor response, 5-HT and 2-methyl-5-HT produced depolarization with decreased R_in. The E_r for this depolarizing response (−30.2±1.8 mV) became less negative (−11.5 mV) in 10 mM [K⁺]_o with minimal effect on the amplitude of the depolarization. In Na⁺-free superfusate, the 5-HT-induced depolarization was converted to hyperpolarization. This indicated the 5-HT₃ response increased a mixed Na⁺/K⁺ conductance. Elevated Ca²⁺ or Mg²⁺ markedly reduced the 5-HT₃ response. Incubation with 3.5 mM Ca²⁺ shifted the 5-HT concentration–response curve downward and to the right, decreasing the maximal response by 49% and increasing the EC₅₀ by 10-fold. Elevated Mg²⁺ produced similar effects. In cells where both 5-HT₂- and 5-HT₃-mediated responses could be demonstrated, the elevation of K⁺ or the reduction of Ca²⁺ converted a 5-HT₂ response to a 5-HT₃ response. The above data suggest that elevation of [K⁺]_o or reduction of [Ca²⁺]_o produced by rapid firing rates of sensory neurons will favor the expression of 5-HT₃ responses over 5-HT₂ responses.