The role of osmotic pressure and membrane potential in K(+)-stimulated taurine release from cultured astrocytes and LRM55 cells

The effects of [K+]o on taurine release from glial cells were studied with primary cultures of cerebellar astrocytes and with LRM55 cells, a continuous glial cell line. The characteristics of K(+)-stimulated taurine release were virtually identical in the 2 cell types. Both cerebellar astrocytes and LRM55 cells released taurine when stimulated with high-K+ medium prepared by isosmotically substituting KCl for NaCl, but neither cell type released taurine when stimulated with hyperosmotic high-K+ medium prepared by adding solid KCl to control medium. The membrane potential of LRM55 cells was measured by intracellular recording and was insensitive to changes in [K+]o below 20 mM. LRM55 cells released taurine when stimulated with nondepolarizing concentrations of K+ (13-22 mM) if the isosmotically prepared high-K+ medium was used, but the cells did not release taurine when treated with a depolarizing concentration of K+ (50 mM) if hyperosmotic high-K+ medium was used. The time course of K(+)-stimulated taurine release was quite slow, having a time to peak of 10-15 min. Small changes (2.5-10%) in the osmolarity of the medium strongly affected taurine release by cerebellar astrocytes and LRM55 cells. K(+)-stimulated taurine release from both cell types was inhibited when the osmolarity was increased with sucrose or NaCl and was enhanced when the osmolarity was reduced. Similarly, baseline taurine release was suppressed by small elevations in osmolarity and increased by reduced osmolarity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of taurine from astrocytes during potassium-evoked swelling

Cultured astrocytes superfused with isosmotic solutions containing high concentrations of potassium, i.e., 25, 56, 75, and 100 mM, showed a proportional increase in cell volume corresponding to 25, 36, 57, and 75% greater than the cell volume in physiological solutions. This volume increase was abolished in low chloride or hypertonic solutions. The release of 3H-taurine previously accumulated by astrocytes was stimulated by potassium at all concentrations examined. During 4-minute exposure to 25, 56, 75, or 100 mM of potassium, cells released 13.5, 15.6, 20.2, or 36.2%, respectively, of the total labeled taurine accumulated during the preloading period. The potassium-stimulated release of 3H-taurine was calcium-independent and insensitive to BaCl2 and bumetanide. Substitution of chloride by gluconate to concentrations necessary to maintain the K+ X Cl- product constant abolished the potassium-stimulated release of 3H-taurine. Superfusion with solutions made hypertonic with sucrose also decreased the potassium-elicited efflux of 3H-taurine. In both conditions, the depolarizing effect of potassium measured with 3H-TPP+ was unchanged. High potassium concentrations and hyposmotic solutions released 3H-taurine by a nonadditive mechanism. These results indicate that in cultured astrocytes high concentrations of potassium produce both swelling and depolarization, but only swelling elicits the release of taurine. These observations suggest an involvement of taurine in cell-volume regulation in astrocytes.     

The effect of phencyclidine on the basal and high potassium evoked extracellular GABA levels in the striatum of freely-moving rats: an in vivo microdialysis study

The effect of phencyclidine (PCP) on the γ-aminobutyric acid-ergic (GABAergic) transmission in the striatum of freely-moving rats was investigated using an in vivo microdialysis. The high potassium (100 mM) increased the extracellular GABA level to 4000% of the basal level. Although the basal GABA level in the striatal dialysate did not show either calcium dependency or tetrodotoxin (TTX) sensitivity, the high potassium evoked GABA level was reduced by 82% under calcium-free conditions (with 12.5 mM magnesium) and by 54% in the presence of 10 μM TTX. The systemic administration of PCP (7.5 mg/kg) or the local perfusion of PCP (100 μM and 1 mM) significantly inhibited the high potassium evoked GABA release in the rat striatum. The local perfusion of MK-801 (10 μM and 100 μM), a more potent and selective N-methyl- -aspartate (NMDA) receptor antagonist, also inhibited the high potassium evoked striatal GABA release. These drugs did not show any significant effect on the basal extracellular GABA level. NMDA (1 mM) either partly or completely blocked the effect of PCP (1 mM) or MK-801 (100 μM) on the high potassium evoked striatal GABA release. On the other hand, nomifensine (100 μM), a dopamine uptake blocker, did not show any effect on the high potassium evoked GABA release. These results suggest that PCP inhibited the striatal GABAergic neuronal transmission through its antagonism of the NMDA receptor.     

Effects of the anion channel blocker DIDS on ouabain- and high K(+)-induced release of amino acids from the rat cerebral cortex

Amino acid release from the rat cerebral cortex was analyzed using an in vivo cortical cup perfusion model. Topical applications of ouabain or high extracellular K(+) were used to mimic two dimensions of ischemic conditions which promote cell swelling and amino acid release. Ouabain (30 microM) induced significant releases of taurine, gamma-aminobutyric acid (GABA), aspartate, glutamate and phosphoethanolamine. The anion channel blocker, 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS; 1 mM), inhibited ouabain-induced release of all these amino acids except for glutamate. Exposure to high extracellular K(+) (75 mM) induced a delayed rise in the levels of taurine in the superfusates and an immediate increase in GABA levels. There were no significant releases of other amino acids. The release of taurine and GABA was sensitive to the blocking of anion channels with DIDS. Both ouabain- and high K(+)-induced taurine release is likely to be mediated by DIDS sensitive anion channels. The extracellular accumulation of the other amino acids, where insensitive to DIDS, may be mediated by mechanisms other than swelling-induced anion channels.     

Control by GABA and tachykinins of the evoked release of acetylcholine in striatal compartments under different modalities of NMDA receptor stimulation

The contribution of endogenously released dopamine, GABA and its co-transmitters, substance P (SP) and neurokinin A (NKA), to the control of the evoked release of acetylcholine was investigated in vitro in the striosomes and the matrix of the rat striatum under various modalities of NMDA receptor stimulation (NMDA 50 microM or 1 mM without or with 10 microM D-serine). Sulpiride, bicuculline, SR140333 and SR48968, the antagonists of D(2), GABA A, NK(1) and NK(2) tachykinin receptors, respectively, were used for this purpose. (1) In both striatal compartments, the dopamine-mediated inhibitory regulation of the evoked release of acetylcholine only occurred when D-serine was co-applied with 50 microM or 1 mM NMDA. (2) In striosomes, the dopamine-dependent inhibitory effects of SP and NKA on the evoked release of acetylcholine only occurred when D-serine was co-applied with 50 microM or 1 mM NMDA. (3) A similar inhibitory regulation by NKA, but not SP, was found in the matrix when 1 mM NMDA was co-applied with D-serine. (4) In contrast, the dopamine-dependent facilitatory effect of GABA on the evoked release of acetylcholine did not require added D-serine and was more important with 1 mM than 50 microM NMDA. In the presence of D-serine, and depending on the NMDA concentration, the facilitatory regulation of GABA was reduced (matrix) or suppressed (striosomes). This latter effect was partially restored in the presence of SR48968. Therefore, the dopamine-dependent inhibitory effects of tachykinins on the evoked release of acetylcholine only occurred when NMDA receptors were stimulated in the presence of saturating concentrations of D-serine.     

Transient increases in extracellular K+ produce two pharmacological distinct cytosolic Ca2+ transients

Transient increases in extracellular K+ are observed under various conditions, including repetitive neuronal firing, anoxia, ischemia and hypoglycemic coma. We studied changes in cytoplasmic Ca2+ ([Ca2+]cyt) evoked by pulses of KCl in human neuroblastoma SH-SY5Y cells and rat dorsal root ganglia (DRG) neurons at 37 degrees C. A "pulse" of KCl evoked two transient increases in [Ca2+]cyt, one upon addition of KCl (K+on) and the other upon removal of KCl (K+off). The K+on transient has been described in many cell types and is initiated by the activation of voltage-dependent Ca2+ channels followed by Ca2+-evoked Ca2+ release from intracellular Ca2+ stores. The level of KCl necessary to evoke the K+off transient depends on the type of neuron, in SH-SY5Y cells it required 100 mM KCl, in most (but not all) of dorsal root ganglia neurons it could be detected with 100-200 mM KCl and in a very few dorsal root ganglia neurons it was detectable at 20-50 mM KCl. In SH-SY5Y cells, reduction of extracellular Ca2+ inhibited the K+on more strongly than the K+off and slowed the decay of K+off. Isoflurane (1 mM) reduced the K+on)- but not the K+off-peak. However, isoflurane slowed the decay of K+off. The nonspecific cationic channel blocker La3+ (100 microM) had an effect similar to that of isoflurane. Treatment with thapsigargin (TG) at a concentration known to only deplete IP3-sensitive Ca2+ stores did not affect K+on or K+off, suggesting that Ca2+ release from the IP3-sensitive Ca2+ stores does not contribute to K+on and K+off transients and that the thapsigargin-sensitive Ca2+ ATPases do not contribute significantly to the rise or decay rates of these transients. These findings indicate that a pulse of extracellular K+ produces two distinct transient increases in [Ca2+]cyt.     

Role of brain-derived neurotrophic factor in the protective action of N-methyl-D-aspartate in the apoptotic death of cerebellar granule neurons induced by low potassium

Several neurotrophic factors, including brain-derived neurotrophic factor (BDNF), and neurotransmitters, such as glutamate, may influence neuronal apoptotic death. Rat cerebellar granule neurons (CGN) cultured in low potassium (5 or 10 mM KCl) for more than 5 days in vitro (DIV) die apoptotically. These cells survive in the presence of high potassium (25 mM KCl, K25) or N-methyl-D-aspartate (NMDA), an agonist of glutamatergic receptors. CGN transferred from high to low potassium die apoptotically. Here, we characterized the effect of BDNF and NMDA on the apoptotic death induced by low potassium in CGN. Cell death of CGN by culturing in low potassium for 6 DIV was inhibited by BDNF and NMDA. When CGN were cultured in K25 and transferred to a low-potassium medium, 65% of neurons died after 48 hr. Under these conditions, BDNF, NMDA, or BDNF + NMDA increased CGN survival. Both BDNF and NMDA decreased caspase-9 activity and mRNA caspase-3 levels and activity induced by low potassium. CGN survival induced by BDNF is mediated by TrkB activation, whereas that induced by NMDA is mediated by NMDA receptor and TrkB activation. NMDA, but not BDNF, raised [Ca(2+)](i), which was reduced by low-potassium treatment. These results suggest that NMDA receptor stimulation induces CGN survival through the influx of extracellular Ca(2+) that may evoke the release of BDNF and the activation of TrkB. Complementary mechanisms induced by depolarization and changes in Ca(2+) levels would also contribute to the neuroprotection exerted by NMDA and potassium.     

NMDA receptor mediates dopamine release in the striatum of unanesthetized rats as measured by brain microdialysis.

We have studied the characteristics associated with the activation of the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor on the release of dopamine (DA) in the striatum of awake rats as measured by brain microdialysis technique. NMDA dose-dependently stimulated the striatal DA release in Mg(2+)-free Ringer's solution. The stimulation was significant at 90 microM and the maximum observed effect was at the highest concentration tested (800 microM). The selective NMDA receptor antagonist, 2-amino-5-phosphonovalerate (AP5; 300 microM), blocked the stimulatory effect of NMDA. The NMDA-induced release of DA was reduced by 1.2 mM Mg2+ and totally blocked by 2.5 mM of the cation. Glycine (200 microM) potentiated the response evoked by 300 microM NMDA while 7-chloro-kynurenate (100 microM), an antagonist of the glycine site, reduced markedly this response. Neither atropine (100 microM) nor tetrodotoxin (TTX) (5 microM) prevented the stimulatory effect of NMDA. These results suggest that glutamate released from corticostriatal terminals presynaptically stimulates the release of DA via an NMDA receptor.     

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.     

Glutamate release and spreading depression in the fascia dentata in response to microdialysis with high K: role of glia

To see electrophysiological and neurochemical events during microdialysis with high [K+], direct current (DC) and excitatory postsynaptic field potentials (fEPSPs) due to perforant path stimulation were recorded in the granule cell layer of the fascia dentata, while 3, 25, 50 or 100 mM KCl was perfused through a microdialysis probe placed 1.5 mm from the recording electrode. Glutamate and glutamine content of the dialysate was measured by high performance liquid chromatography. Raising [K+] from 3 to 25 mM reduced the efflux of glutamine, without affecting that of glutamate or the electrical activity. In about 50% of experiments, 50 mM K+ induced large (20-30 mV) negative waves of spreading depression (SD), and a suppression of fEPSPs. In the other 50%, without SD, fEPSPs did not change. Glutamate efflux increased 3-fold in both groups. SD waves were produced in all experiments with 100 mM K+ which evoked a more than 10-fold increase in glutamate release. Glutamine efflux decreased equally, by about 50%, with the 3 concentrations of K+. Microdialysis with 20 mM fluoroacetate, a glial metabolic poison, decreased the spontaneous efflux of glutamine and glutamate and increased the incidence of SD waves. Results suggest that perfusion of 50 or 100 mM K+ through a microdialysis probe causes spreading depression which blocks surrounding electrical activity. The activity of glia partly protects against spreading depression caused by high [K+].     

Taurine Release Evoked by NMDA Receptor Activation is Largely Dependent on Calcium Mobilization from Intracellular Stores

It is known that the activation of N-methyl-d -aspartate (NMDA) receptors leads to an increase in extracellular taurine concentration in different brain regions. The mechanism that mediates this effect is not totally understood. In this study, rat hippocampal slices were used to determine the dependence of NMDA-induced taurine release on extracellular calcium and/or on calcium mobilization from intracellular stores. NMDA was administered through a microdialysis probe inserted into the slice, at the level of CA1 stratum radiatum, which was also used to collect amino acids from the extracellular space. Field potentials evoked by stimulation of the Schaffer collaterals and recorded in the stratum pyramidale of CA1 were used as a control of NMDA receptor activation. NMDA induced a marked increase in extracellular taurine levels and a decrease in field potential amplitude, and both effects were suppressed in the presence of MK-801, a blocker of the NMDA receptor-linked channel. Dantrolene, an inhibitor of calcium release from intracellular stores, partially inhibited the extracellular taurine increase, while 2-nitro-4-carboxyphenyl-N,N-diphenyl carbamate (NCDC), an inhibitor of phosphatidylinositol-specific phospholipase C activation, had no effect. Removal of extracellular calcium diminished, but did not abolish, the extracellular taurine increase caused by NMDA. The remaining taurine response was totally suppressed by dantrolene, and also by NCDC. These results demonstrate that the release of taurine induced by NMDA receptor activation is triggered by the increase in cytoplasmic calcium concentration. We suggest that, under physiological conditions, calcium influx provides the signal for NMDA-induced taurine release, which is amplified by calcium-dependent calcium mobilization from intracellular stores. In the absence of extracellular calcium, NMDA is still able to evoke taurine release through a mechanism that implies calcium release from inositol 1,4,5-trisphosphate-sensitive stores.     

Weak organic acids induce taurine release through an osmotic-sensitive process in in vivo rat hippocampus

Isotonic media containing sodium salts from weak organic acids induce cell swelling in several experimental preparations (Grinstein et al., 1984; Jakubovicz et al., 1987). In vivo perfusion of rat dentate gyrus, using a microdialysis probe, with modified Krebs-Ringer bicarbonate solutions in which 50 mM NaCl was isotonically substituted by the sodium salts from organic acids with a pKa value of greater than 2 (acetate, propionate, or pyruvate), induced a reversible increase in the extracellular taurine concentration. By contrast, similar NaCl substitutions with sodium salts from the stronger organic acids isethionate and methane-sulfonate did not change extracellular taurine levels. Extracellular taurine increases evoked by acetate, propionate, or pyruvate were almost completely abolished when the perfusion liquid was made hypertonic by adding sucrose (50 mM). A 30% reduction of the acetate-induced extracellular taurine increase was observed both when amiloride was present or when the [Na+]0 was lowered. Both conditions are known to inhibit Na+/H+ exchange. These results are compatible with the hypothesis that acid load-induced taurine release is stimulated by an osmotic sensitive mechanism, part of which is dependent on activation of the Na+/H+ exchange.     

Age-related changes in NMDA-induced [3H]acetylcholine release from brain slices of senescence-accelerated mouse.

From the brain slices of normal mice (ddY strain, subcloned from dd strain in National Institute of Health in Japan), N-methyl-D-aspartic acid (NMDA) at 0.01-1 mM evoked [3H]acetylcholine (ACh) release in a concentration dependent manner. [3H]ACh release evoked by 1 mM NMDA was significantly inhibited by 2-amino-5-phosphonovaleric acid (APV), phencyclidine (PCP) and 5-methyl-10,11-dihydroxy-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). The effects of NMDA were not seen in the Ca2+ free medium and were inhibited by physiological concentration (0.83 mM) of Mg2+. NMDA seems to cause ACh release from nerve terminals through the receptor-ion channel mediated mechanism in the mouse brain. Based upon these results, we determined the activity of a high K(+)- or NMDA-evoked [3H]ACh release using prone/8 strain of senescence-accelerated mouse (SAM-P/8) (a murine model of accelerated aging and memory dysfunction) and SAM-resistance/1 strain (SAM-R/1) (normal aging mice as the control) and these release activities were compared between both strains and during aging. [3H]ACh release evoked by 30 mM KCl was significantly lower than that of age-matched SAM-R/1 at 9 and 12 months. NMDA evoked the [3H]ACh release at 2, 6, 10 and 14 months in R/1 mice. In SAM-P/8 mice the activity of NMDA-evoked release was seen at 2 months, but markedly decreased afterwards. Nonsignificant difference was observed on the uptake of [3H]choline and on the spontaneous release of [3H]ACh between SAM-P/8 and SAM-R/1 strains, and during aging.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-independent release of [3H]spermine from chick retina

Spermine has been shown to influence NMDA receptor function through an interaction at the coagonist site for glycine in the central nervous system (CNS) and the retina. In order to support a role for spermine as neurotransmitter or neuromodulator in the chick retina, specific stimulated-release of spermine should be demonstrated. Isolated chick retinas, preloaded with [3H]spermine, were stimulated with 1 mM NMDA and other glutamate agonists at ionotropic receptors, in a continuous superfusion system. [3H]spermine was released from the retina by depolarization with 50 mM KCl, in a Ca2+-independent manner. Inhibition of Na+/K+-ATPase by ouabain or digitoxigenin also induced spermine release following 36 min in the presence of the drugs; such effect seems unrelated to changes in Na+ electrochemical gradients, since nigericin and veratrine did not induce release in Na+ containing medium. The lack of effect of glutamate, NMDA and kainate at 1 mM concentration, suggests that release of spermine in the retina is mediated by the reversal of uptake and not necessarily linked to EAA-receptor activation.     

Release of vesicular Zn2+ in a rat transient middle cerebral artery occlusion model

In the brain, Zn(2+) is stored in synaptic vesicles of a subgroup of glutamatergic nerve terminals. Although it has been reported that this Zn(2+) is released upon the excitation of nerves in vitro, there has been little study of the release of Zn(2+) during ischemia in vivo. Here, using brain microdialysis, the release of vesicular Zn(2+) was investigated in vivo. When the vesicular Zn(2+) was released into the synaptic cleft by a depolarizing stimulation achieved by perfusion with Ringer's solution containing high K(+) (100mM KCl), a significant increase in the extracellular concentration of Zn(2+) could be detected by microdialysis. Then, we investigated the release of vesicular Zn(2+) in a rat transient middle cerebral artery occlusion model using microdialysis. Consequently, the extracellular Zn(2+) level in the cortex increased within 15 min of the start of occlusion and reached a peak at 30 min, which was about twice the basal level. After 30 min, it declined with time returning to the basal level 15 min after reperfusion, which was performed after 60 min of occlusion. The results suggest that vesicular Zn(2+) would be released into the synaptic cleft during brain ischemia in vivo.     

Effects of taurine on the influx and efflux of calcium in brain slices of adult and developing mice

45Ca2+ influx was reduced by 10-mM taurine during a 5-min incubation in slices from the cerebral cortex, cerebellum and brainstem of adult mice. In both adult and developing cerebral cortex and cerebellar slices a 30-min exposure to 1.0-mM taurine was likewise effective on resting Ca2+ influx whereas in slices depolarized by 50 mM K+ the influx was not affected. Ca2+ efflux from adult mouse cerebral cortex slices was affected by extracellular Na+ (0-180 mM) but not by K+ (50 mM) or taurine (1 mM). In cerebral cortex slices of 3-day-old mice K+ depolarization stimulated Ca2+ efflux, which effect was antagonized by 1.0 mM taurine. The results suggest that taurine is able to modify Ca2+ influx and efflux in both adult and developing brain but only at relatively high concentrations.     

A chloride-dependent component of the release of labeled GABA and taurine from the chick retina

The characteristics and ionic dependence of the release of [3H]gamma-aminobutyric acid ([3H]GABA) and [3H]taurine from the chick retina, stimulated by kainic acid (KA) and by depolarizing concentrations of potassium was examined and compared to those of [3H]dopamine. KA (100 microM) highly stimulated the release of [3H]GABA (25-fold over resting efflux), induced a moderate increase in [3H]taurine and did not increase the efflux of [3H]dopamine. The efflux of [3H]GABA stimulated by KA was totally calcium-independent but it was markedly sodium and chloride dependent. Chloride dependence was assessed by replacing chloride with the impermeant anion gluconate, or by addition of the anion transport blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Depolarizing concentrations of KCl (56 mM) stimulated the release of [3H]GABA, [3H]taurine and [3H]dopamine to about the same extent. The release of [3H]GABA and [3H]taurine was only partially calcium dependent, in contrast to the highly calcium-dependent efflux of [3H]dopamine. A sodium-free medium increased the resting efflux and decreased the potassium-stimulated release of [3H]GABA and [3H]taurine; the resting efflux of [3H]dopamine was unaffected and the potassium-induced efflux was somewhat increased. The potassium-stimulated efflux of [3H]GABA and [3H]taurine showed a chloride-dependent component which was higher for taurine whereas the resting efflux was not modified. The stimulated release of [3H]dopamine was increased in a chloride-free medium. The ionic dependence of KA and potassium stimulated efflux of [3H]GABA and [3H]taurine showed properties similar to those of the homoexchange-activated efflux of amino acids which was also found sodium and chloride dependent and clearly different from the calcium-coupled neurotransmitter release process. Exposure of retinas to KA and potassium produced retinal cell swelling which is prevented in a chloride-free medium. Results are discussed in terms of a particular efflux mechanism for [3H]GABA and [3H]taurine in the retina in response to stimulation associated with changes in ionic gradients and retinal cell swelling.     

Separate neuronal and glial Na+,K+-ATPase isoforms regulate glucose utilization in response to membrane depolarization and elevated extracellular potassium.

The role of cell type-specific Na+,K+-ATPase isozymes in function-related glucose metabolism was studied using differentiated rat brain cell aggregate cultures. In mixed neuron-glia cultures, glucose utilization, determined by measuring the rate of radiolabeled 2-deoxyglucose accumulation, was markedly stimulated by the voltage-dependent sodium channel agonist veratridine (0.75 micromol/L), as well as by glutamate (100 micromol/L) and the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) (10 micromol/L). Significant stimulation also was elicited by elevated extracellular potassium (12 mmol/L KCl), which was even more pronounced at 30 mmol/L KCl. In neuron-enriched cultures, a similar stimulation of glucose utilization was obtained with veratridine, specific ionotropic glutamate receptor agonists, and 30 mmol/L but not 12 mmol/L KCl. The effects of veratridine, glutamate, and NMDA were blocked by specific antagonists (tetrodotoxin, CNQX, or MK801, respectively). Low concentrations of ouabain (10(-6) mol/L) prevented stimulation by the depolarizing agents but reduced only partially the response to 12 mmol/L KCl. Together with previous data showing cell type-specific expression of Na+,K+-ATPase subunit isoforms in these cultures, the current results support the view that distinct isoforms of Na+,K+-ATPase regulate glucose utilization in neurons in response to membrane depolarization, and in glial cells in response to elevated extracellular potassium.     

Glutamate release evoked by glutamate receptor agonists in cultured chick retina cells: Modulation by arachidonic acid

We studied the effect of ionotropic glutamate receptor agonists on the release of endogenous glutamate or of [³H]D -aspartate from reaggregate cultures (retinospheroids) or from monolayer cultures of chick retinal cells, respectively. Kainate increased the fluorescence ratio of the Na⁺ indicator SBFI and stimulated a dose-dependent release of glutamate in low (0.1 mM) Ca²⁺ medium, as measured using a fluorometric assay. Under the same experimental conditions, the release evoked by N-methyl-D -aspartate (NMDA; 400 μM) was about half of that evoked by the same kainate concentration; α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA; 400 μM) did not trigger a significant response. In the presence of 1 mM CaCl₂, all of the agonists increased the [Ca²⁺]_i, as determined with the fluorescence dye Indo-1, but the glutamate release evoked by NMDA and kainate was significantly lower than that measured in 0.1 mM CaCl₂ medium. Inhibition by Ca²⁺ of the kainate-stimulated release of glutamate was partially reversed by the phospholipase A₂ inhibitor oleiloxyethyl phosphorylcholine (OPC), suggesting that the effect was mediated by the release of arachidonic acid, which inhibits the glutamate carrier. Accordingly, kainate, NMDA, and AMPA stimulated a Ca²⁺-dependent release of [³H]arachidonic acid, and the direct addition of the exogenous fatty acid to the medium decreased the release of glutamate evoked by kainate in low (0.1 mM) CaCl₂ medium. In monolayer cultures, we showed that NMDA, kainate, and AMPA also stimulated the release of [³H]D -aspartate, but in this case release in the presence of 1 mM CaCl₂ was significantly higher than that evoked in media with no added Ca²⁺. The ranking order of efficacy for stimulation of Ca²⁺-dependent release of [³H]D -aspartate was NMDA ≪ kainate < AMPA. © 1996 Wiley-Liss, Inc.     

Metabotropic glutamate receptors modulate [(3)H]acetylcholine release from cultured amacrine-like neurons.

Retinal amacrine cells express metabotropic glutamate receptors (mGluRs), but their physiological role is unknown. We investigated the effect of mGluR on [(3)H]acetylcholine release ([(3)H]ACh) from cultured chick amacrine-like neurons. Activation of group III mGluR with the agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) inhibited [(3)H]ACh release evoked by 25 mM KCl in a dose-dependent manner, and this effect was sensitive to pertussis toxin. In contrast, activation of group I or II mGluR with (S)-3, 5-dihydroxyphenylglycine (DHPG) and (2S,2'R,3'R)-2-(2', 3'-dicarboxycyclopropyl)glycine (DCG-IV), respectively, did not affect significantly [(3)H]ACh release. The effect of L-AP4 on [(3)H]ACh release was sensitive to nitrendipine, suggesting that it is, at least in part, due to inhibition of L-type Ca(2+) channels. Activation of group III mGluR also partly inhibited omega-conotoxin GVIA-sensitive Ca(2+) channels, coupled to [(3)H]ACh release. The L-AP4 did not affect the cAMP levels measured in amacrine-like neurons depolarized with 25 mM KCl or stimulated with forskolin, indicating that the effect of group III mGluR on [(3)H]ACh release is not due to inhibition of adenylyl cyclase activity. Inhibition of protein kinase A with KT-5720 was without effect on [(3)H]ACh release evoked by 25 mM KCl, further indicating that the effect of group III mGluR on [(3)H]ACh release cannot be attributed to the inhibition of the kinase. The effect of L-AP4 on [(3)H]ACh release was reversed by DHPG or by DCG-IV, and activation of group II mGluR also partially inhibited cAMP production stimulated by forskolin. Taken together, our results show that the effect of group III mGluR on [(3)H]ACh release may be due to a direct inhibition of L- and N-type Ca(2+) channels and is modulated by group I and group II mGluR.