Two conventional protein kinase C isoforms, alpha and beta I, are involved in the ATP-induced activation of volume-regulated anion channel and glutamate release in cultured astrocytes

Volume-regulated anion channels (VRACs) are activated by cell swelling and are permeable to inorganic and small organic anions, including the excitatory amino acids glutamate and aspartate. In astrocytes, ATP potently enhances VRAC activity and glutamate release via a P2Y receptor-dependent mechanism. Our previous pharmacological study identified protein kinase C (PKC) as a major signaling enzyme in VRAC regulation by ATP. However, conflicting results obtained with potent PKC blockers prompted us to re-evaluate the involvement of PKC in regulation of astrocytic VRACs by using small interfering RNA (siRNA) and pharmacological inhibitors that selectively target individual PKC isoforms. In primary rat astrocyte cultures, application of hypoosmotic medium (30% reduction in osmolarity) and 20 μM ATP synergistically increased the release of excitatory amino acids, measured with a non-metabolized analog of l -glutamate, d -[³H]aspartate. Both Go6976, the selective inhibitor of Ca²⁺-sensitive PKCα, βI/II, and γ, and MP-20-28, a cell permeable pseudosubstrate inhibitory peptide of PKCα and βI/II, reduced the effects of ATP on d -[³H]aspartate release by ∼45–55%. Similar results were obtained with a mixture of siRNAs targeting rat PKCα and βI. Surprisingly, down-regulation of individual α and βI PKC isozymes by siRNA was completely ineffective. These data suggest that ATP regulates VRAC activity and volume-sensitive excitatory amino acid release via cooperative activation of PKCα and βI.     

ATP regulates anion channel-mediated organic osmolyte release from cultured rat astrocytes via multiple Ca2+-sensitive mechanisms

Ubiquitously expressed volume-regulated anion channels (VRACs) are activated in response to cell swelling but may also show limited activity in nonswollen cells. VRACs are permeable to inorganic anions and small organic osmolytes, including the amino acids aspartate, glutamate, and taurine. Several recent reports have demonstrated that neurotransmitters or hormones, such as ATP and vasopressin, induce or strongly potentiate astrocytic whole cell Cl^– currents and amino acid release, which are inhibited by VRAC blockers. In the present study, we explored the intracellular signaling mechanisms mediating the effects of ATP on D-[³H]aspartate release via the putative VRAC pathway in rat primary astrocyte cultures. Cells were exposed to moderate (5%) or substantial (30%) reductions in medium osmolarity. ATP strongly potentiated D-[³H]aspartate release in both moderately swollen and substantially swollen cells. These ATP effects were blocked (80% inhibition) by intracellular Ca²⁺ chelation with BAPTA-AM, calmodulin inhibitors, or a combination of the inhibitors of protein kinase C (PKC) and calmodulin-dependent kinase II (CaMK II). In contrast, control D-[³H]aspartate release activated by the substantial hyposmotic swelling showed little (25% inhibition) sensitivity to the same pharmacological agents. These data indicate that ATP regulates VRAC activity via two separate Ca²⁺-sensitive signaling cascades involving PKC and CaMK II and that cell swelling per se activates VRACs via a separate Ca²⁺/calmodulin-independent signaling mechanism. Ca²⁺-dependent organic osmolyte release via VRACs may contribute to the physiological functions of these channels in the brain, including astrocyte-to-neuron intercellular communication. volume-regulated anion channels; protein kinase C; calcium/calmodulin-dependent kinase II; glutamate release; neuron-glia communication     

Carbenoxolone inhibits volume-regulated anion conductance in cultured rat cortical astroglia

Accumulating evidence indicate that the gap-junction inhibitor carbenoxolone (CBX) regulates neuronal synchronization, depresses epileptiform activity and has a neuroprotective action. These CBX effects do not depend solely on its ability to inhibit gap junction channels formed by connexins (Cx), but the underlying mechanisms remain to be elucidated. Here we addressed the questions whether CBX modulates volume-regulated anion channels (VRAC) involved in the regulatory volume decrease and regulates the associated release of excitatory amino acids in cultured rat cortical astrocytes. We found that CBX inhibits VRAC conductance with potency comparable to that able to depress the activity of the most abundant astroglial gap junction protein connexin43 (Cx43). However, the knock down of Cx43 with small interfering RNA (siRNA) oligonucleotides and the use of various pharmacological tools revealed that VRAC inhibition was not mediated by interaction of CBX with astroglial Cx proteins. Comparative experiments in HEK293 cells stably expressing another putative target of CBX, the purinergic ionotropic receptor P2X7, indicate that the presence of this receptor was not necessary for CBX-mediated depression of VRAC. Finally, we show that in COS-7 cells, which are not endowed with pannexin-1 protein, another astroglial plasma membrane interactor of CBX, VRAC current retained its sensitivity to CBX. Complementary analyses indicate that the VRAC-mediated release of excitatory amino acid aspartate was decreased by CBX. Collectively, these findings support the notion that CBX could affect astroglial ability to modulate neuronal activity by suppressing excitatory amino acid release through VRAC. They also provide a possible mechanistic clue for the neuroprotective effect of CBX in vivo.     

Purinergic activation of anion conductance and osmolyte efflux in cultured rat hippocampal neurons

The majority of mammalian cells demonstrate regulatory volume decrease (RVD) following swelling caused by hyposmotic exposure. A critical signal initiating RVD is activation of nucleotide receptors by ATP. Elevated extracellular ATP in response to cytotoxic cell swelling during pathological conditions also may initiate loss of taurine and other intracellular osmolytes via anion channels. This study characterizes neuronal ATP-activated anion current and explores its role in net loss of amino acid osmolytes. To isolate anion currents, we used CsCl as the major electrolyte in patch electrode and bath solutions and blocked residual cation currents with NiCl(2) and tetraethylammonium. Anion currents were activated by extracellular ATP with a K(m) of 70 microM and increased over fourfold during several minutes of ATP exposure, reaching a maximum after 9.0 min (SD 4.2). The currents were blocked by inhibitors of nucleotide receptors and volume-regulated anion channels (VRAC). Currents showed outward rectification and inactivation at highly depolarizing membrane potentials, characteristics of swelling-activated anion currents. P2X agonists failed to activate the anion current, and an inhibitor of P2X receptors did not block the effect of ATP. Furthermore, current activation was observed with extracellular ADP and 2-(methylthio)adenosine 5'-diphosphate, a P2Y(1) receptor-specific agonist. Much less current activation was observed with extracellular UTP, suggesting the response is mediated predominantly by P2Y(1) receptors. ATP caused a dose-dependent loss of taurine and alanine that could be blocked by inhibitors of VRAC. ATP did not inhibit the taurine uptake transporter. Thus extracellular ATP triggers a loss of intracellular organic osmolytes via activation of anion channels. This mechanism may facilitate neuronal volume homeostasis during cytotoxic edema.     

Is the Volume-Regulated Anion Channel VRAC a “Water-Permeable” Channel?

The volume-regulated anion channel, VRAC, plays an important role in cell volume regulation. This channel is permeable for a wide variety of anions, amino acids, and organic osmolytes, including taurine. However, nothing is known about possible water permeability of this channel. Water permeability of endothelial cells is estimated from the initial rate of cell swelling because of a hypotonic challenge. As a result of simultaneous volume and current measurements, it will be shown that water permeability is decreased by inhibition of VRAC. It is concluded that water permeates VRAC and might be able to accelerate water transport by providing an additional permeation pathway for water. Therefore VRAC can be considered as a water-permeable, "wet" channel.     

Activation of microglia with zymosan promotes excitatory amino acid release via volume-regulated anion channels: the role of NADPH oxidases

Microglia are the resident immune cells of the CNS, which are important for preserving neural tissue functions, but may also contribute to neurodegeneration. Activation of these cells in infection, inflammation, or trauma leads to the release of various toxic molecules, including reactive oxygen species (ROS) and the excitatory amino acid glutamate. In this study, we used an electrophysiologic approach and a d‐[ ³ H] aspartate (glutamate) release assay to explore the ROS‐dependent regulation of glutamate‐permeable volume‐regulated anion channels (VRACs). Exposure of rat microglia to hypo‐osmotic media stimulated Cl^? currents and d ‐[³H]aspartate release, both of which were inhibited by the selective VRAC blocker, DCPIB. Exogenously applied H₂O₂ potently increased swelling‐activated glutamate release. Stimulation of microglia with zymosan triggered production of endogenous ROS and strongly enhanced glutamate release via VRAC in swollen cells. The effects of zymosan were attenuated by the ROS scavenger, MnTMPyP, and by two inhibitors of NADPH oxidase (NOX), diphenyliodonium and thioridazine. However, zymosan‐stimulated glutamate release was insensitive to other NOX blockers, apocynin and HEBSF. This pharmacologic profile pointed to the potential involvement of apocynin‐insensitive NOX4. Using RT‐PCR we confirmed that NOX4 is expressed in rat microglial cells along with NOX1 and NOX2. To check for potential involvement of phagocytic NOX2, we stimulated this isoform using protein kinase C (PKC) activator, phorbol 12‐myristate 13‐acetate or inhibited it with the broad spectrum PKC blocker, Gö6983. Both agents potently modulated endogenous ROS production by NOX2 but not VRAC activity. Taken together, these data suggest that the anion channel VRAC may contribute to microglial glutamate release and that its activity is regulated by endogenous ROS originating from NOX4.     

Osmotic swelling-provoked release of organic osmolytes in human intestinal epithelial cells

Human Intestine 407 cells respond to osmotic cell swelling by the activation of Cl(-)- and K(+)-selective ionic channels, as well as by stimulating an organic osmolyte release pathway readily permeable to taurine and phosphocholine. Unlike the activation of volume-regulated anion channels (VRAC), activation of the organic osmolyte release pathway shows a lag time of approximately 30-60 s, and its activity persists for at least 8-12 min. In contrast to VRAC activation, stimulation of organic osmolyte release did not require protein tyrosine phosphorylation, active p21(rho), or phosphatidylinositol 3-kinase activity and was insensitive to Cl(-) channel blockers. Treatment of the cells with putative organic anion transporter inhibitors reduced the release of taurine only partially or was found to be ineffective. The efflux was blocked by a subclass of organic cation transporter (OCT) inhibitors (cyanine-863 and decynium-22) but not by other OCT inhibitors (cimetidine, quinine, and verapamil). Brief treatment of the cells with phorbol esters potentiated the cell swelling-induced taurine efflux, whereas addition of the protein kinase C (PKC) inhibitor GF109203X largely inhibited the response, suggesting that PKC is involved. Increasing the level of intracellular Ca(2+) by using A-23187- or Ca(2+)-mobilizing hormones, however, did not affect the magnitude of the response. Taken together, the results indicate that the hypotonicity-induced efflux of organic osmolytes is independent of VRAC and involves a PKC-dependent step.     

Osmolyte contents of cultured astrocytes grown in hypoosmotic medium

Primary rat cerebral astrocyte cultures were grown for 2 weeks in isoosmotic medium (305 mosmol) and then placed in similar medium with a reduced NaCl concentration. During the first hour of growth in this moderately hypoosmotic medium (240 mosmol), the cells lose 88% of their taurine contents, 62% of their alanine contents, and 54% of their aspartate contents while regaining normal volume. Loss of these amino acids accounts for 43% of observed volume regulation. Contents of these amino acids remain decreased during 24 h of growth in hypoosmotic medium. In contrast, potassium, glutamate, glutamine, and asparagine contents are not changed, relative to cells in isoosmotic medium, at time points between 1 h and 24 h of hypoosmotic exposure. The data suggest astrocytes contribute to net loss of amino acids, but not potassium, from brains exposed to hypoosmotic conditions in situ.     

Effects of Anion Channel Blockers on Hyposmotically Induced Amino Acid Release From the In Vivo Rat Cerebral Cortex

A cortical cup model with continuous perfusion of artificial cerebrospinal fluid (containing 134 mM NaCl) was used to investigate the effects of anion channel blockers on the hyposmotically-induced release of amino acids from the in vivo rat cerebral cortex. The hyposmotic stimulus (25 mM NaCl) evoked a release of taurine, glutamate, aspartate, glycine, phosphoethanolamine and GABA. Topically applied anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (1 mM); 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (2 mM); 5-nitro-2-(3-phenylpropylamino) benzoic acid (350 M); niflumic acid (500 M); tamoxifen (20 M) and arachidonic acid (0.5 M) all significantly reduced the hyposmotically-induced release of taurine. The releases of glutamate, aspartate, glycine, phosphoethanolamine and GABA were variably susceptible to inhibition by these compounds. These results demonstrate that osmoregulatory processes in cortical cells, in vivo, involve amino acids, with taurine playing a dominant role. The efflux of taurine and, to a lesser extent, the other amino acids may be mediated by anion channels.     

Valproate doubles the anoxic survival time of normal developing mice: possible relevance to valproate-induced decreases in cerebral levels of glutamate and aspartate, and increases in taurine

We have previously reported that chronic administration of valproate in developing mice decreased brain aspartic and glutamic acid levels and increased the brain taurine content. The direction of the valproate-induced changes in the cerebral levels of these neurotransmitter amino acids - excitatory in the case of aspartate and glutamate, inhibitory in the case of taurine - appeared relevant to the mechanism of its anticonvulsant action. Since the neuropathology of hypoxia-ischemia also appears to be mediated by release of glutamate/aspartate at the synapse, the valproate-induced reduction of the levels of these neuroexcitatory/neurotoxic amino acids suggested that valproate might increase the tolerance of young mice to anoxia. A doubling of the length of survival of the intact animal in an atmosphere of pure nitrogen gas and a three-fold increase in the duration of respiratory activity (gasping) of the isolated head after chronic administration of valproate support the speculation.     

Release of Endogenous Glutamate,Aspartate, GABA,and Taurine from Hippocampal Slices from Adult and Developing Mice under Cell-Damaging Conditions

The releases of endogenous glutamate, aspartate, GABA and taurine from hippocampal slices from 7-day-, 3-, 12-, and 18-month-old mice were investigated under cell-damaging conditions using a superfusion system. The slices were superfused under hypoxic conditions in the presence and absence of glucose and exposed to hydrogen peroxide. In the adult hippocampus under normal conditions the basal release of taurine was highest, with a response only about 2-fold to potassium stimulation (50 mM). The low basal releases of glutamate, aspartate, and GABA were markedly potentiated by K⁺ ions. In general, the release of the four amino acids was enhanced under all above cell-damaging conditions. In hypoxia and ischemia (i.e., hypoxia in the absence of glucose) the release of glutamate, aspartate and GABA increased relatively more than that of taurine, and membrane depolarization by K⁺ markedly potentiated the release processes. Taurine release was doubled in hypoxia and tripled in ischemia but K⁺ stimulation was abolished. In both the mature and immature hippocampus the release of glutamate and aspartate was greatly enhanced in the presence of H₂O₂, that of aspartate particularly in developing mice. In the immature hippocampus the increase in taurine release was 10-fold in hypoxia and 30-fold in ischemia, and potassium stimulation was partly preserved. The release processes of the four amino acids in ischemia were all partially Ca²⁺-dependent. High concentrations of excitatory amino acids released under cell-damaging conditions are neurotoxic and contribute to neuronal death during ischemia. The substantial amounts of the inhibitory amino acids GABA and taurine released simultaneously may constitute an important protective mechanism against excitatory amino acids in excess, counteracting their harmful effects. In the immature hippocampus in particular, the massive release of taurine under cell-damaging conditions may have a significant function in protecting neural cells and aiding in preserving their viability.     

Effect of Impact Trauma on Neurotransmitter and Nonneurotransmitter Amino Acids in Rat Spinal Cord

N-Methyl-D-aspartate (NMDA) administration exacerbates neurological dysfunction after traumatic spinal cord injury in rats, whereas NMDA antagonists improve outcome in this model. These observations suggest that release of excitatory amino acids contributes to secondary tissue damage after traumatic spinal cord injury. To further examine this hypothesis, concentrations of free amino acids were measured in spinal cord samples from anesthetized rats subjected to various degrees of impact trauma to the T9 spinal segment. Levels of excitatory and inhibitory neurotransmitter amino acids [gamma-aminobutyric acid (GABA), glutamate, aspartate, glycine, taurine] and levels of nonneurotransmitter amino acids (asparagine, glutamine, alanine, threonine, serine) were determined at 5 min, 4 h, and 24 h posttrauma. Uninjured surgical (laminectomy) control animals showed modest but significant declines in aspartate and glutamate levels, but not in other amino acids, at all time points. In injured animals, the excitatory amino acids glutamate and aspartate were significantly decreased by 5 min posttrauma, and remained depressed at 4 h and 24 h as compared with corresponding laminectomy controls. In contrast, the inhibitory amino acids, glycine, GABA, and taurine, were decreased at 5 min postinjury, had partially recovered at 4 h, and were almost fully recovered at 24 h. The nonneurotransmitter amino acids were unchanged at 5 min posttrauma and significantly increased at 4 h, with partial recovery at 24 h. At 4 h postinjury, severe trauma caused significantly greater decreases in aspartate and glutamate than did either mild or moderate injury. These findings are consistent with the postulated role of excitatory amino acids in CNS trauma.     

Release of Endogenous Amino Acids from the Striatum from Developing and Adult Mice in Ischemia

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K⁺-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K⁺ stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K⁺ stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.     

Increased release of excitatory amino acids by the actions of ATP and peroxynitrite on volume-regulated anion channels (VRACs) in astrocytes

Rapid swelling of astrocytes in primary culture by exposure to hyposmotic medium (or slower swelling by exposure to high K+ medium) leads to release of the excitatory amino acids (EAAs) glutamate and aspartate. One question that arises is whether these phenomena are only relevant to pathological states such as ischemia and trauma where marked astrocytic swelling occurs or whether much smaller astrocytic volume changes, that might be encountered under physiological states, will cause such release. We have recently found that extracellular ATP strongly potentiated volume-regulated anion channels (VRACs)-mediated-excitatory amino acid release in non-swollen and osmotically swollen primary astrocyte cultures. However, ATP does not seem to directly activate but instead positively modulates VRACs and we postulate that a minor fraction of these are active under isoosmotic conditions based on the finding that in hyperosmotic media the ATP-induced increase was inhibited. Agonist and inhibitor analysis suggests that the effect of ATP is mediated by several subtypes of metabotropic P2Y receptors. Thus, the concept of volume transmission may be extended to volume-mediated transmission, whereby moderate cell swelling causes release of neurotransmitter substances. The product of the superoxide oxygen radical and nitric oxide, peroxynitrite, formed under pathological conditions such as cerebral ischemia, also potentiated the release of D-[3H]aspartate from astrocyte cultures exposed to limited or marked swelling via intracellular signaling mechanisms involving tyrosine kinases (TKs). Thus, the enhancement of cell volume-dependent release of excitatory amino acids from astrocytes can be physiological or pathological and its magnitude depends on the degree of the cell volume increase.     

Inhibition by cigarette smoke of lipid peroxidation-induced neurotransmitter release

Effects of water-soluble substance in cigarette smoke on neurotransmitter release were investigated using nerve terminals (synaptosomes) prepared from rat cerebral cortex. 2,2′-Azobis (2-amidinopropane) dihydrochloride (ABAP), a peroxyl radical-generator, enhanced the depolarization-evoked release of glutamate and aspartate from synaptosomes with concomitant increase in thiobarbituric acid-reactive substances (TBA-RS) levels in membrane lipids of synaptosomes. The trapped smoke-substance attenuated the lipid peroxidation-enhanced release of excitatory amino acids during the depolarization with reduction in TBA-RS, although it failed to affect the basal release of neurotransmitters. These data suggest that cigarette smoke may possess antioxidant properties to reduce oxidation-induced enhancement of transmitter release from nerve terminals.     

Differential effect of beta-N-oxalylamino-L-alanine, the Lathyrus sativus neurotoxin, and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate on the excitatory amino acid and taurine levels in the brain of freely moving rats

We studied the effect of beta-oxalylamino-L-alanine, a glutamate analog present in Lathyrus sativus seeds and implicated in the etiopathogenesis of neurolathyrism, and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate on the extracellular levels of aspartate, glutamate and taurine in the primary motor cortex of freely moving rats. We found that while both neurotoxins increase the level of aspartate and glutamate, only (+/-)-alpha(-amino-3-hydroxy-5-methylisoxazole-4-propionate is able to modulate the level of taurine. GYKI-52466, a non-competitive non-NMDA antagonist, inhibited beta-oxalylamino-L-alanine-induced increase of aspartate, but not that of glutamate. Conversely, this antagonist proved to be very efficient in blocking the stimulating effect of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate on all three amino acids. We suggest that beta-oxalylamino-L-alanine increases the level of glutamate in vivo by a mechanism not connected to its effect on the non-NMDA receptors, which might involve the inhibition of glutamate transport. This would allow the excitatory neurotransmitter to reach a concentration sufficient to stimulate the non-NMDA receptors, which in their turn mediate the specific release of aspartate. Although the role of aspartate as a neurotransmitter is still under discussion, it might indeed amplify the excitotoxic cascade through its action on NMDA receptors. We speculate that this sequence of events might represent an important step in the molecular cascade leading to the appearance of the selective motoneuron degeneration in neurolathyrism.     

Effects of Kainic Acid in Rat Brain Synaptosomes: The Involvement of Calcium

Abstract: The effects of kainic acid were investigated in preparations of rat brain synaptosomes. It was found that kainic acid inhibited competitively the uptake of d -[³H]aspartate, with a K _i of approximately 0.3 m m . Kainic acid also caused release of two excitatory amino acid neurotranstnitters, aspartate and glutamate, in a time- and concentration-dependent manner, but had no effect on the content of γ-aminobutyric acid. Concomitant with the release of aspartate and glutamate, depolarization of the synaptosomal membrane and an increase in intracellular calcium were observed, with no measurable change in the concentration of internal sodium ions. The increase in intrasynaptosomal calcium and decrease in transmem-brane electrical potential were prevented by the addition of glutamate, whereas the kainate-induced release of ra-dioactive aspartate was substantially inhibited by lowering the concentration of calcium in the external medium. It is postulated that kainic acid reacts with a class of glutamate receptors located in a subpopulation of synaptosomes, presumably derived from the glutamatergic and aspartatergic neuronal pathways, which possesses high-affinity uptake system(s) for glutamate and/or aspartate. Activation of these receptors causes opening of calcium channels, influx of calcium into the synaptosomes, and depolarization of the synaptosomal plasma membrane with consequent release of amino acid neurotransmitters.     

Disturbances of Amino Acids from Temporal Lobe Synaptosomes in Human Complex Partial Epilepsy

We have studied the levels of neuroactive amino acids in synaptosomes (P₂ fraction) isolated from brain tissue of ten patients with medically intractable epilepsy who were undergoing temporal lobectomy. First, lateral temporal tissue (nonfocal) was removed followed by medial temporal tissue (focal). A synaptosomal fraction (P₂) was immediately prepared from each tissue and analyzed for free amino acid concentrations. Statistically significant reductions were seen in glutamine and GABA concentrations in focal tissue compared to nonfocal tissue. The ratio of excitatory amino acids (aspartate and glutamate) to inhibitory amino acids (taurine and GABA) was significantly higher in focal tissue compared to nonfocal. The glutamine/glutamate ratio was significantly reduced. These data support the hypothesis that alterations in the balance between excitatory and inhibitory amino acids may be involved in the expression of epilepsy.     

Astrocyte Heterogeneity: Endogenous Amino Acid Levels and Release Evoked by Non-N-Methyl-D-Aspartate Receptor Agonists and by Potassium-Induced Swelling in Type-1 and Type-2 Astrocytes

The aim of the present study was to determine whether endogenous amino acids are released from type-1 and type-2 astrocytes following non-N-methyl-D-aspartate (NMDA) receptor activation and whether such release is related to cell swelling. Amino acid levels and release were measured by HPLC in secondary cultures from neonatal rat cortex, highly enriched in type-1 or type-2 astrocytes. The following observations were made. (a) The endogenous level of several amino acids (glutamate, alanine, glutamine, asparagine, taurine, serine, and threonine) was substantially higher in type-1 than in type-2 astrocytes. (b) The spontaneous release of glutamine and taurine was higher in type-1 than in type-2 astrocytes; that of other amino acids was similar. (c) Exposure of type-2 astrocyte cultures to 50 microM kainate or quisqualate doubled the release of glutamate and caused a lower, but significant increase in that of aspartate, glycine, taurine, alanine, serine (only in the case of kainate), and glutamine (only in the case of quisqualate). These effects were reversed by the antagonist CNQX. (d) Exposure of type-1 astrocyte cultures to 50-200 microM kainate or 50 microM quisqualate did not affect endogenous amino acid release, even after treating the cultures with dibutyryl cyclic AMP. (e) Exposure of type-1 or type-2 astrocyte cultures to 50 mM KCl (replacing an equimolar concentration of NaCl) enhanced the release of taurine greater than glutamate greater than aspartate. The effect was somewhat more pronounced in type-2 than in type-1 astrocytes. Veratridine (50 microM) did not cause any increase in amino acid release. (f) The release of amino acids induced by high [K+] appeared to be related to cell swelling, in both type-1 and type-2 astrocytes. Swelling and K(+)-induced release were somewhat higher in type-2 than in type-1 astrocytes. In contrast, neither kainate nor quisqualate caused any appreciable increase in cell volume. It is concluded that non-NMDA receptor agonists stimulate the release of several endogenous amino acids (some of which are neuroactive) from type-2 but not from type-1 astrocytes. The effect does not seem to be related to cell swelling, which causes a different release profile in both type-1 and type-2 astrocytes. The absence of kainate- and quisqualate-evoked release in type-1 astrocytes suggests that the density of non-NMDA receptors in this cell type is very low.     

Amino Acids as Osmolytes in the Retina

Amino acids play a role as osmolytes during the regulatory volume decrease subsequent to hyposmotic swelling, but less is known about its role when swelling occurs in isosmotic conditions. In this work we examined the efflux of labelled GABA, taurine and glutamate (traced as D-aspartate) from the chick retina, after isosmotic swelling evoked by KCl-containing solutions, and compared its features to those in hyposmotic swelling. In both conditions, GABA and taurine efflux were more sensitive to swelling than glutamate, as assessed by the activation threshold and the amount released. The amino acid efflux in hyposmotic media was decreased by DIDS, tamoxifen and NPPB, agents acting as Cl channels blockers, which also inhibit the osmosensitive Cl efflux. The component associated with swelling in the KCl-stimulated efflux was assessed by the reduction observed when Cl is replaced by an impermeant anion, or by the influence of hyperosmotic media. GABA and taurine efflux exhibited a large swelling-dependent component, which was lower for D-aspartate. This component was markedly decreased by NPPB, but this was due to an effect of the blocker preventing swelling. These results suggest that the influx of Cl, acting as K counterion, which is responsible for cell swelling, occurs through a pathway sensitive to NPPB, similarly to that activated by hyposmolarity. This finding may be of interest in studies aiming at preventing the cell edema which occurs in a number of pathologies.