Stimulus-coupled taurine efflux from cerebellar neuronal cultures: on the roles of Ca++ and Na+

Primary cultures of cerebellar neurons obtained from 7-9-day-old rats and grown 7-9 days in vitro (DIV) were used to study the effects of Na+ and Ca++ on K+-evoked taurine release. These cultures, made up largely of granule neurons (90%) and inhibitory interneurons (5-7%), produced a dose-dependent, depolarization-evoked taurine release that was Ca++-dependent at 40 mM K+, and Ca++-independent at K+ concentrations above 40 mM. The dihydropyridine Ca++ channel agonist BAY K 8644 (1 microM) augmented 30 mM K+-evoked release, while the antagonist nifedipine (5 microM) abolished both the BAY K 8644- and K+-enhanced release. Depolarization with the Na+ channel agonist veratridine (50 microM) stimulated taurine efflux, which was completely blocked by pretreatment with tetrodotoxin (2 microM). However, 50 mM K+-evoked taurine release was not affected by tetrodotoxin pretreatment. Substitution of choline Cl for NaCl partially antagonized 50 mM K+-evoked release, and by itself, the Na+ ionophore monensin (50 microM) stimulated release. These results suggest that both K+-evoked and basal taurine release from primary cerebellar neuronal cultures are sensitive to the levels of both intracellular and extracellular Na+ and Ca++. In contrast to previous findings using cerebellar astrocytes, neuronal L-type Ca++ channels, but not voltage-dependent Na+ channels, also appear to be necessary. The implications of these results on taurine's status as a putative neurotransmitter are discussed.     

Omega-conotoxin- and nifedipine-insensitive voltage-operated calcium channels mediate K(+)-induced release of pro-thyrotropin-releasing hormone-connecting peptides Ps4 and Ps5 from perifused rat hypothalamic slices.

The rat thyrotropin-releasing hormone (TRH) precursor (prepro-TRH) contains five copies of the TRH progenitor sequence linked together by intervening sequences. Recently, we have shown that the connecting peptides prepro-TRH-(160-169) (Ps4) and prepro-TRH-(178-199) (Ps5) are released from rat hypothalamic neurones in response to elevated potassium concentrations, in a calcium-dependent manner. In the present study, the role of voltage-operated calcium channels in potassium-induced release of Ps4 and Ps5 was investigated, using a perifusion system for rat hypothalamic slices. The release of Ps4 and Ps5 stimulated by potassium (70 mM) was blocked by the inorganic ions Co2+ (2.6 mM) and Ni2+ (5 mM). In contrast, the stimulatory effect of KCl was insensitive to Cd2+ (100 microM). The dihydropyridine antagonist nifedipine (10 microM) had no effect on K(+)-evoked release of Ps4 and Ps5. Furthermore, the response to KCl was not affected by nifedipine (10 microM) in combination with diltiazem (1 microM), a benzothiazepine which increases the affinity of dihydropyridine antagonists for their receptor. The dihydropyridine agonist BAY K 8644, at concentrations as high as 1 mM, did not stimulate the basal secretion of Ps4 and Ps5. In addition, BAY K 8644 had no potentiating effect on K(+)-induced release of Ps4 and Ps5. The marine cone snail toxin omega-conotoxin, a blocker of both L- and N-type calcium channels had no effect on the release of Ps4 and Ps5 stimulated by potassium. Similarly, the omega-conopeptide SNX-111, a selective blocker of N-type calcium channels, did not inhibit the stimulatory effect of potassium. The release of Ps4 and Ps5 evoked by high K+ was insensitive to the non-selective calcium channel blocker verapamil (20 microM). Amiloride (1 microM), a putative blocker of T-type calcium channels, did not affect KCl-induced secretion of the two connecting peptides. Taken together, these results indicate that two connecting peptides derived from the pro-TRH, Ps4 and Ps5, are released by K(+)-induced depolarization through activation of voltage-sensitive calcium channels. The calcium channels appear to have a pharmacological profile different from that of L- and N-type channels. Although, their insensitivity to low Cd2+ concentrations and sensitivity to Ni2+ ions would support the involvement of T-type calcium channels, the lack of effect of amiloride suggests that they belong to a yet undefined class of calcium channels.     

Two distinct mechanisms, differentially affected by excitatory amino acids, trigger GABA release from fetal mouse striatal neurons in primary culture

The mechanisms leading to Ca2+-dependent and Ca2+-independent GABA release were studied on highly purified striatal neurons developed in primary culture. Ca2+-dependent GABA release, which represents about 75% of the 56 mM K+ effect was totally inhibited when striatal neurons were first exposed to tetanus toxin (TnTx) (10 micrograms/ml) for 24 hr. The K+ effect was potentiated when 1 mM nipecotic acid (an inhibitor of the GABA uptake system) was added during the stimulation period or when Na+ was replaced by Li+. However, no difference in the GABA release measured under high-K+ conditions was observed after a 22 min preincubation of the neurons in a medium containing nipecotic acid or Li+. Replacement of Cl- ions by SO4(2-) did not modify K+-evoked GABA release. Ca2+-independent GABA release was stimulated by veratridine (20 microM), ouabain (3 mM), and monensin (20 microM), as well as the excitatory amino acids glutamate (100 microM), N-methyl-D-aspartate (100 microM), quisqualate (10 microM), and kainate (1 mM), drugs known to increase intracellular Na+ concentration. The veratridine- or glutamate-evoked GABA release was neither inhibited when intracellular Ca2+ content was reduced by more than 90% nor by treatment of the neurons to TnTx. However, the Ca2+-independent GABA release elicited by veratridine was inhibited by preincubation of the neurons in a medium containing 1 mM nipectotic acid and in a medium containing Li+ instead of Na+ or SO4(2-) instead of Cl-. These results strongly suggest that 2 different GABA release mechanisms exist in striatal neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of voltage-operated calcium channels in alpha-melanocyte-stimulating hormone (alpha-MSH) release from perifused rat hypothalamic slices

The contribution of voltage-operated calcium (VOC) channels in the mechanism of release of alpha-melanocyte-stimulating hormone (alpha-MSH) from hypothalamic neurons was investigated using perifused rat hypothalamic slices. The stimulatory effect of potassium (50 mM) on alpha-MSH release was completely blocked by cadmium (1 mM) a calcium competitor which indifferently blocks T-, L-and N-type VOC channels. To determine the nature of calcium conductances involved in K+-evoked alpha-MSH release, we have investigated the effect of a VOC channel agonist and 3 antagonists on the secretion of the neuropeptide. Administration of synthetic omega-conotoxin fraction GVIA (1 microM), a peptide toxin which blocks both N- and L-type VOC channels, reduced by 33% K+-induced alpha-MSH release. In contrast, the 1,4-dihydropyridine (DHP) antagonist nifedipine, at concentrations up to 100 microM, did not affect the response of hypothalamic alpha-MSH neurons to depolarizing concentrations of KCl. In addition, the secretion of alpha-MSH induced by high K+ concentrations was not reduced by nifedipine (10 microM) in the presence of diltiazem (1 microM), a benzothiazepine derivative which increases the affinity of the DHP antagonist for L-type VOC channels. The DHP agonist BAY K 8644 (0.1-10 microM) did not modify the early phase of the response of alpha-MSH neurons to K+-induced depolarization. In contrast BAY K 8644 (1 or 10 microM) significantly prolonged the duration of K+-induced alpha-MSH release. This sustained release of alpha-MSH induced by BAY K 8644 (10 microM) was totally suppressed by nifedipine (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of prodynorphin peptides release from the rat spinal cord in vitro

The release of immunoreactive (ir-) dynorphin (DYN) and alpha-neoendorphin (alpha-NEO) from spinal cord slices was investigated in rats. A stable, spontaneous, in vitro release of these peptides (6.7 +/- 0.3 of ir-DYN and 15.5 +/- 0.3 fmol/min/g wet tissue of ir-alpha-NEO) was measured in superfusates using highly sensitive radioimmunoassays. The exposure of the slices to the superfusion medium containing 57 mM K+ or 50 microM veratridine increased circa three times the basal release of the peptides. The K(+)-evoked release of ir-alpha-NEO was Ca2(+)-dependent, and the veratridine stimulation was abolished by 1 microM tetrodotoxin. Modulation of the alpha-neoendorphin release from the lumbar enlargement of the rat spinal cord by various neuroactive compounds was studied in vitro. Noradrenaline (1 microM) slightly enhanced the K(+)-induced release of ir-alpha-NEO, but was without effect on the basal release. On the other hand, GABA (10 microM) and muscimol (1 microM) inhibited the K(+)-stimulated release of the peptide. The effect of muscimol was attenuated by bicuculline (10 microM). Other compounds, such as serotonin (1 microM), naloxone (1 microM), U-50, 488H and bicuculline, altered neither the basal nor the K(+)-induced release. These data indicate that both ir-DYN and ir-alpha-NEO are stored in a releasable pool in the spinal cord, which supports the concept that prodynorphin peptides can serve as neurotransmitters in this structure. Furthermore, this study suggests that the spinal cord prodynorphin system may be under an inhibitory gabaergic and an excitatory catecholaminergic control.     

In vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices: involvement of voltage-dependent Ca2+ channels

It was investigated whether Ca2+ is involved in the regulation of basal depolarization-induced dopamine biosynthesis in tuberoinfundibular dopaminergic neurons. The rate of dopamine biosynthesis was estimated by in vitro dihydroxyphenylalanine (DOPA) synthesis in the median eminence following incubation of rat hypothalamic slices with a DOPA decarboxylase inhibitor. Depolarizing agents such as K+ and veratridine increased the synthesis rate of DOPA in the median eminence in a dose-dependent manner with a maximal synthesis rate obtained at concentrations of 50 mM and 50 microM, respectively. Removal of Ca2+ and addition of EGTA (1 mM) into the medium did not influence basal DOPA synthesis in the median eminence but blocked the K+- and veratridine-induced DOPA synthesis. The Ca2+ channel blockers verapamil (100 microM) and Co2+ (4 mM) were effective in reducing the depolarization-induced DOPA synthesis. A23187 (10 microM), a Ca2+ ionophore, stimulated basal DOPA synthesis in the median eminence. On the other hand, tetrodotoxin (2 microM), a Na+ channel blocker, did not change the basal and K+-induced DOPA synthesis in the median eminence whereas it completely inhibited the veratridine-induced DOPA synthesis. These results suggest that depolarization-induced synthesis of dopamine in tuberoinfundibular neurons requires Ca2+ influx through voltage-dependent Ca2+ channels.     

Differential effects of Na-K-ATPase pump inhibition, chemical anoxia, and glycolytic blockade on membrane potential of rat optic nerve

Na(+)-K(+)-ATPase pump failure during either anoxia or ouabain perfusion induces rapid axonal depolarization by dissipating ionic gradients. In this study, we examined the interplay between cation and anion transporting pathways mediating axonal depolarization during anoxia or selective Na(+)-K(+)-ATPase inhibition. Compound resting membrane (V(m)) potential of rat optic nerve was measured in a grease gap at 37 degrees C. Chemical anoxia (2 mM NaCN or NaN(3)) or ouabain (1 mM) caused a loss of resting potential to 42 +/- 11% and 47 +/- 2% of control after 30 min, respectively. Voltage-gated Na(+)-channel blockade was partially effective in abolishing this depolarization. TTX (1 microM) reduced depolarization to 73 +/- 10% (chemical anoxia) and 68 +/- 4% (ouabain) of control. Quaternary amine Na(+) channel blockers QX-314 (1 mM) or prajmaline (100 microM) produced similar results. Residual ionic rundown largely representing co-efflux of K(+) and Cl(-) during chemical anoxia in the presence of Na(+)-channel blockade was further spared with DIDS (500 microM), a broad-spectrum anion transport inhibitor (95 +/- 8% of control after 30 min in anoxia + TTX vs. 73 +/- 10% in TTX alone). Addition of DIDS was slightly more effective than TTX alone in ouabain (74 +/- 5% DIDS + TTX vs. 68 +/- 4% in TTX alone, P < 0.05). Additional Na(+)-entry pathways such as the Na-K-Cl cotransporter were examined using bumetanide, which produced a modest albeit significant sparing of V(m) during ouabain-induced depolarization. Although cation-transporting pathways play the more important role in mediating pathological depolarization of central axons, anion-coupled transporters also contribute to a significant, albeit more minor, degree.     

Presynaptic effect of 7-OH-DPAT on evoked [3H]-acetylcholine release in rat striatal synaptosomes

The objective of the present experiments was to study the presynaptic effect of 7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT, a D(2)-like dopamine receptor agonist) on [3H]-acetylcholine ([3H]-ACh) release induced by potassium (15 mM, 25 mM and 60 mM), potassium channel-blockers (4-aminopyridine, 4-AP; tetraethylammonium, TEA and quinine) and veratridine to gain insight into the mechanisms involved in the activation of the D(2) dopamine-receptor subtype located at striatal cholinergic nerve terminals. 7-OH-DPAT (1 microM) inhibited the evoked [3H]-ACh release induced by K(+) 15 mM in a similar percentage than that obtained during basal conditions (30% and 27%, respectively). Nevertheless, in the presence of 25 mM and 60 mM of K(+) the inhibitory effect of 7-OH-DPAT was completely abolished. 4-AP (1-100 microM) and TEA (1 and 5 mM) significantly enhanced [3H]-ACh release, showing 69.32%+/-7.60% (P<0.001) and 52.27%+/-5.64% (P<0.001), respectively, at the highest concentrations tested. In these conditions, 7-OH-DPAT (1 microM) inhibited the release induced by potassium channel-blockers approximately 25-27%. Quinine (0.1-1 microM) did not alter [3H]-ACh release either in the presence or absence of 7-OH-DPAT. Veratridine 10 microM evoked [3H]-ACh release in the presence of a low-calcium medium, but in such conditions 7-OH-DPAT (1 microM) did not modify the neurotransmitter release in the absence or presence of veratridine. Present data indicate that activation of the presynaptic D(2) dopamine receptor inhibits the [3H]-ACh release by increasing K(+) conductance, as high K(+) concentrations abolished the inhibitory control of 7-OH-DPAT on [3H]-ACh release. This effect could be mediated by potassium channels different from those sensitive to 4-AP, TEA and quinine. In addition, the presynaptic D(2) dopamine-receptor activation seems to not involve changes in intracellular Ca(2+).     

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.     

Different pathways by which extracellular Ca2+ promotes calcitonin gene-related peptide release from central terminals of capsaicin-sensitive afferents of guinea pigs: effect of capsaicin, high K+ and low pH media.

Different modes by which Ca2+, entering the nerve terminal, promotes transmitter secretion as well as the ability of protons to release neuropeptides, have been shown in peripheral endings of capsaicin-sensitive afferents. We have studied these two aspects in the central endings of these neurons by measuring the release of calcitonin-gene related peptide-like immunoreactivity (CGRP-LI) from slices of the dorsal half of the guinea pig spinal cord. Although capsaicin (1 microM) released both CGRP-LI and substance P-like immunoreactivity (SP-LI), CGRP-LI was chosen as the sole suitable marker of peptides released from central terminals of capsaicin-sensitive afferents, since after in vitro desensitization to capsaicin (1 microM capsaicin for 20 min), high K+ (80 mM) failed to evoke CGRP-LI release, whereas SP-LI release was still observed. The capsaicin (1 microM)-evoked CGRP-LI release was entirely dependent on extracellular Ca2+. It was unaffected by 0.3 microM tetrodotoxin (TTX), slightly reduced by 0.1 microM omega-conotoxin (CTX) and blocked by 10 microM Ruthenium red (RR). The Ca(2+)-dependent K+ (80 mM)-evoked CGRP-LI release was unaffected by TTX, markedly reduced by CTX and only moderately inhibited by RR. Low pH (pH 5) produced a remarkable increase in CGRP-LI outflow that was abolished after exposure to capsaicin, reduced by about 50% in Ca(2+)-free medium and unaffected by TTX (0.3 microM). The Ca(2+)-dependent component of the proton-evoked CGRP-LI release was abolished in the presence of RR (10 microM) and slightly inhibited by CTX (0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of serotonin uptake into mouse brain synaptosomes by ionophores and ion-channel agents.

[3H]Serotonin uptake into mouse cerebrocortical synaptosomes was decreased by the K+ ionophore valinomycin, the K+ and Na+ ionophore gramicidin, and the proton ionophore carbonylcyanide m-chlorophenylhydrazone. The Na+/H+ exchanger monensin reduced uptake at non-depolarizing concentrations. Uptake was also decreased by inhibition of the Na+, K(+)-ATPase with ouabain and by tetrodotoxin-sensitive activation of voltage-dependent sodium channels with veratridine, batrachotoxin and scorpion venom. In contrast, the Ca2+ channel agents BAY K8644 and nimodipine were ineffective. The effect of reducing the Na+ gradient depended upon whether the internal Na+ concentration was raised (i.e. by scorpion venom, monensin) or the external Na+ concentration was lowered (37 mM NaCl in the medium).     

Cobalt-sensitive and dihydropyridine-insensitive stimulation of dopamine release from tuberoinfundibular neurons by high extracellular concentrations of barium ions

Recently, it has been demonstrated that Ca2+ entrance into the neuronal cytoplasm can occur upon the activation of 3 different types of specific voltage-dependent channels which can be characterized according to the following criteria: (1) voltage threshold for activation; (2) tendency to inactivation; (3) bivalent cation permeability; and (4) drug sensitivity. In this study we investigated, in tuberoinfundibular dopaminergic (TIDA) hypothalamic neurons, the biochemical and pharmacological properties of Ca2+ channels, by comparing the effects of high extracellular concentrations of Ba2+ and Ca2+ ions on [3H]dopamine (DA) release from TIDA neurons. The results obtained show that extracellular Ba2+ ion concentrations dose-dependently (10-20 mM) stimulated [3H]DA release from superfused TIDA neurons and that this effect was prevented by Co2+ ions (2 mM). In addition, superfusion of TIDA neurons with a concentration of Ca2+ ions equimolar to that of Ba2+ ions (20 mM) failed to modify [3H]DA release. The fact that tetraethylammonium (10 mM), a blocker of K+ currents in excitable cells, did not mimick the stimulatory action of Ba2+ ions on [3H]DA release, seems to exclude that the effect of Ba2+ ions was dependent on the inhibition of K+ channels in TIDA neurons. The omission of Ca2+ ions from the extracellular medium did not prevent the stimulatory effect on [3H]DA release elicited by elevated concentrations of Ba2+ ions, but rather reinforced this effect. Finally, nitrendipine (50 microM) did not modify the stimulatory effect of high extracellular Ba2+ ions on [3H]DA release from TIDA neurons.     

Simultaneous action of MK-801 (dizclopine) on dopamine, glutamate, aspartate and GABA release from striatum isolated nerve endings

The simultaneous effect of MK-801 on the baseline- and depolarization (20 microM veratridine or 30 mM high K+)-evoked release of endogenous dopamine, glutamate (Glu), aspartate (Asp), and GABA is investigated in the same preparation of rat striatum isolated nerve endings. MK-801, in the microM range, selectively increases the baseline and high K+ depolarization-evoked release of dopamine, without causing any effect on the baseline or on the high K+-evoked release of Glu, Asp and GABA. In addition to this selective action on dopamine release, MK-801 inhibits the veratridine depolarization-evoked release of all the neurotransmitters tested, including dopamine. In SBFI and fura-2 preloaded striatal synaptosomes, MK-801 inhibits the elevation of internal Na+ (Na(i)) and the elevation of internal Ca2+ (Ca(i)) induced by veratridine depolarization. The elevation of Ca(i) induced by high K+ depolarization is unchanged by MK-801. This study reveals two separate MK-801 actions. (1) The voltage-independent action, which increases dopamine release selectively, and might contribute to the effects of MK-801 on motor coordination. (2) The voltage-dependent action, which inhibits all the veratridine-evoked responses including the evoked release of the excitatory amino acids (which are particularly concentrated in striatum nerve endings), and might contribute to the anticonvulsant and neuroprotective effects of MK-801.     

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.     

Effects of inhibitors of Na,K-ATPase on the membrane potentials and neurotransmitter efflux in rat brain slices

The potassium potential EK, of rat brain slices was estimated by determining the uptake of 86Rb+. The ERb was the same for slices prepared from five rostral brain regions, the average value being 66.4 mV. The ERb values in the presence of 20 microM ouabain were only slightly lower than the resting values; increasing concentrations of ouabain above 20 microM resulted in a graded depolarization in all five brain regions. High concentrations (1 mM) of two other inhibitors of Na+,K+-ATPase, dihydro-ouabain and strophanthidin, produced no more depolarization than did 20 microM ouabain. Competitive binding studies indicated that the differential effects were due to the relative binding to brain slices. Erythrosin B, an inhibitor of Na+,K+-ATPase, had no measurable effect on ERb. Intermediate concentrations of the Na+/H+ ionophore monensin slightly hyperpolarized striatal slices, whereas the same monensin concentrations plus 20 microM ouabain, 1 mM strophanthidin or 70 microM erythrosin B resulted in marked depolarization. Measurement of the membrane potential via uptake of methyltriphenylphosphonium cation indicated that ERb was indeed a valid estimation of the membrane potential. EK was measured directly by monitoring 42K+ uptake in striatal slices and was found to be essentially identical to ERb. Uptake of 22Na+ was consistent with the values for ERb or EK. Several conditions that resulted in little or no measurable depolarization of striatal slices did induce efflux of exogenously loaded GABA and dopamine; these conditions included 20 microM ouabain, 1 mM dihydro-ouabain or strophanthidin, and 70 microM erythrosin B. Neurotransmitter efflux in the absence of general cell depolarization was not accompanied by altered rates of respiration or decreased ATP levels.     

Actions of neomycin on neuronal L-, N-, and non-L/non-N-type voltage-sensitive calcium channel responses

The effects of neomycin on neuronal voltage-sensitive calcium channel (VSCC) responses were investigated by evaluating its effects on calcium-dependent neuronal responses that are sensitive and insensitive to the N-type voltage-sensitive calcium channel antagonist omega-conotoxin GVIA and the L-type VSCC antagonist nitrendipine. Chick synaptosomal 45Ca2+ influx and K(+)-evoked release of [3H]norepinephrine from chick cortical brain slices were omega-conotoxin GVIA sensitive and nitrendipine insensitive, suggesting that these responses are mediated predominantly by N-type VSCC. The K(+)-evoked increase of intracellular calcium in cortical neurons and the K(+)-evoked release of [3H]norepinephrine from rat brain cortical slices was partially sensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses are mediated by N-, L- and non-L/non-N-type VSCC. Rat synaptosomal 45Ca2+ influx and the K(+)-evoked release of [3H]D-aspartate from rat hippocampal slices were completely insensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses were mediated predominantly by non-L/non-N-type VSCC. Neomycin caused a concentration-dependent and virtually complete inhibition of all response parameters, with IC50 values ranging from 90 to 400 microM. The results suggest that neomycin is a nonselective inhibitor of neuronal responses mediated by L-, N-, and non-L/non-N-type VSCC.     

Intrinsic membrane potential oscillations in hippocampal neurons in vitro

Membrane potential oscillations (MPOs) of 2-10 Hz and up to 6 mV were found in almost all stable hippocampal CA1 and CA3 neurons in the in vitro slice preparation. MPOs were prominent for pyramidal cells but less pronounced in putative interneurons. MPOs were activated at threshold depolarizations that evoked a spike and the frequency of the MPOs increased with the level of depolarization. MPOs were distinct from and seemed to regulate spiking, with a spike often riding near the top of a depolarizing MPO wave. Analysis of the periodicity of the oscillations indicate that the period of MPOs did not depend on the afterhyperpolarization (AHP) following a single spike. MPOs persisted in low (0-0.1 mM) Ca2+ medium, with or without Cd2+ (0.2 mM), when synaptic transmission was blocked. Choline-substituted low-Na+ (0-26 mM) medium, 3 microM tetrodotoxin (TTX) or intracellular injection of QX-314 reduced or abolished the fast Na(+)-spike and reduced inward anomalous rectification. About 40% of CA1 neurons had no MPOs after Na+ currents were blocked, suggesting that these MPOs were Na(+)-dependent. In about 60% of the cells, a large depolarization activated Ca(2+)-dependent MPOs and slow spikes. MPOs were not critically affected by extracellular Ba2+ or Cs2+, or by 0.2 mM 4-aminopyridine, with or without 2 mM tetraethylammonium (TEA). However, in 5-10 mM TEA medium, MPOs were mostly replaced by 0.2-3 Hz spontaneous bursts of wide-duration spikes followed by large AHPs. Low Ca2+, Cd2+ medium greatly reduced the spike width but not the spike-bursts. In conclusion, each cycle of an MPO in normal medium probably consists of a depolarization phase mediated by Na+ currents, possibly mixed with Ca2+ currents activated at a higher depolarization. The repolarization/hyperpolarization phase may be mediated by Na+/Ca2+ current inactivation and partly by TEA-sensitive, possibly the delayed rectifier, K+ currents. The presence of prominent intrinsic, low-threshold MPOs in all hippocampal pyramidal neurons suggests that MPOs may play an important role in information processing in the hippocampus.     

Sodium overload through voltage-dependent Na(+) channels induces necrosis and apoptosis of rat superior cervical ganglion cells in vitro

Using the failure to exclude trypan blue as a criterion for cell death, we found that veratridine, the voltage-dependent Na(+) channel activator, exerted its toxicity to cultured sympathetic neurons in a dose-dependent manner (half-maximal toxicity occurred at 2 microM). The co-presence of tetrodotoxin completely reversed the toxicity only at concentrations of veratridine < 20 microM. Veratridine neurotoxicity was due to the influx of Na(+); a medium low in Na(+) (36 mM) completely abolished its neurotoxicity, whereas a Ca(2+)-free medium did not attenuate its neurotoxicity. Furthermore, the buffering action of 1, 2-Bis-(2-aminophenoxy)ethane-N,N,N',N',-tetraacetate (BAPTA) on veratridine-induced increase in intracellular Ca(2+) levels neither blocked veratridine neurotoxicity in normal medium, nor attenuated the low Na(+) effect. Elevated K(+) effectively blocked veratridine neurotoxicity in a Ca(2+)-dependent manner. Cytoplasmic pH measurements using a fluorescent pH indicator demonstrated that cellular acidification (from pH 7.0 to pH 6.5) occurred upon treatment with veratridine. Both veratridine-induced acidification and cell death were ameliorated by 5-(N-ethyl-N-isopropyl)amiloride, the specific inhibitor of the Na(+)/H(+) exchanger (IC(50) = 0.5 microM). Finally, necrosis occurred predominantly in veratridine neurotoxicity, but both staining with bis-benzimide and TUNEL analysis showed nuclear features of apoptosis in sympathetic neurons undergoing cell death.     

A novel toxicity-based assay for the identification of modulators of voltage-gated Na+ channels

Voltage-gated Na+ channels are promising drug targets. Screening of large numbers of putative modulators, however, can be demanding and expensive. In this study, a simple, cheap, and robust assay to test the pharmacological modulation of Na+ channel function is presented. The assay makes use of the fact that the intracellular accumulation of Na+ ions can be cytotoxic. The toxicity of the Na+ channel activator veratridine in the presence of an inhibitor of the Na+/K+ ATPase (ouabain) in a Nav1.2a (rat brain IIA alpha) expressing cell line is assessed. Na+ channel blockers should reduce toxicity in this model. CHO cells which recombinantly expressed rat Nav1.2a subunits were seeded in 96-well plates, and cell survival was tested after 24 h incubation in medium containing veratridine and ouabain in the presence or absence of Na+ channel blockers. Propidium iodide fluorescence was used as toxicity readout. Veratridine (100 microM) or ouabain alone (500 microM) were not toxic to the cells. In the presence of 500 microM ouabain, however, veratridine induced halfmaximal cell death with an EC50 value of 15.1 +/- 2.3 microM. Ouabain's EC50 was 215.3 +/- 16.7 microM (with 30 microM veratridine). The effects of a number of Na+ channel blockers were tested and compared with their Na+ channel blocking activity measured in voltage-clamp experiments. Blockers from various chemical classes reduced toxicity half maximally with IC50 values ranging from 11.7 +/- 1.4 nM (tetrodotoxin) to 280.5 +/- 48.0 microM (lamotrigine). There was a linear relationship between the log IC50 values obtained by the two methods (slope: 1.1 +/- 0.08; correlation coefficient: 0.93). In summary, these data show that this novel toxicity assay is well suited to test Na+ channel blockers.     

Galanin and Glibenclamide Modulate the Anoxic Release of Glutamate in Rat CA3 Hippocampal Neurons

The effects of brief anoxic episodes on intracellularly recorded CA3 pyramidal neurons have been studied in the hippocampal slice preparation. Anoxia induced a depolarization occasionally preceded by a transient hyperpolarization associated with a fall in input resistance. The anoxic depolarization was due to the release of glutamate from presynaptic terminals since it was blocked by tetrodotoxin (TTX) (1 microM) or by the broad spectrum excitatory amino acid antagonist kynurenate (1 mM). In the presence of TTX (1 microM) or kynurenate (1 mM), anoxia only induced a hyperpolarization which was due to activation of a K+ conductance. The anoxic depolarization was blocked by galanin, a hormone which activates ATP sensitive K+ (K+ATP) channels. Anoxic depolarization was increased by the potent sulfonylurea agent glibenclamide (GLIB) which blocks K+ATP channels. Bath applications of these agents had little effect when applied in oxygenated Krebs solution suggesting that their action may be mediated by K+ATP channels. Since excessive release of glutamate during anoxia is neurotoxic, agents such as galanin which activate K+ATP channels may provide tissue specific protection against anoxic damage.