Induced Release of γ-Aminobutyric Acid by a Carrier-Mediated, High-Affinity Uptake of L-Glutamate in Cultured Chick Retina Cells

[3H]gamma-aminobutyric acid (GABA) was taken up by cultured embryonic retina cells during the initial stages of cell differentiation. The accumulated GABA was released in the bathing medium and a transient increase in the efflux of GABA was observed when cultures were pulse-stimulated (2 min) with 0.1 mM L-glutamate but not with D-glutamate. The EC50 for L-glutamate to evoke [3H]GABA release was approximately 15 microM. This value is close to the Km for high-affinity uptake of L-glutamate by retina cells. When Na+ ions were replaced by Li+ ions, L-glutamate-induced release of GABA was abolished. Moreover, L-[14C]glutamate uptake by retina cells was significantly reduced when NaCl was replaced by LiCl in the incubation medium. L-Glutamate elicited release of GABA was Ca2+ independent, and was observed when Ca2+ was replaced by Co2+ or when Mg2+ ions were increased to 10 mM concentration. D-Aspartate, which is taken up by the same high-affinity uptake mechanism as L-glutamate, induced an increase in [3H]GABA efflux comparable to L-glutamate. The addition of unlabeled GABA to the medium also promoted the release of accumulated [3H]GABA. However, GABA was twofold less effective than L-glutamate in eliciting [3H]GABA release. The addition of both GABA and L-glutamate to the incubation medium indicated that [3H]GABA efflux due to L-glutamate and GABA was additive. L-Aspartate also promoted an increase in the efflux of [3H]GABA accumulated by retina cells. However, L-aspartate effect was significantly decreased in the absence of Ca2+ or when Na+ ions were replaced by Li+. Our results indicate that at least three releasable pools of GABA are present in the chick embryo retina cells: (a) a GABA-promoted GABA release-homoexchange, (b) a Ca2+-dependent L-aspartate-promoted release, and (c) a Ca2+-independent, Na+-dependent L-glutamate-evoked release. In addition, our data strongly suggest that the L-glutamate-promoted GABA release is due to a process of exchange of L-glutamate with GABA, which may play a fundamental role in the fine control of the excitability of local circuits in the retina.     

Oxalate accumulation in rat renal cortical slices

Tubular transport of oxalate is thought to be an energy-mediated process which may contribute to the renal deposition of calcium oxalate in a variety of pathologic states. In order to examine this possibility, the renal handling of oxalate was investigated in rat renal cortical slices in vitro. Slices incubated in vitro with 1 microM [14C]oxalate in Krebs-Ringer bicarbonate buffer at 25 degrees C for 180 min achieved a mean slice to medium ratio of 2.8 +/- 0.08 (SEM) and a mean tissue concentration of 7.7 +/- 0.2 mumol/kg dry wt (N = 64). Section freeze-dry autoradiographs demonstrated maximum uptake within proximal tubule cells but no crystals were evident. Substituting N2 for O2, adding KCN, or removing Ca2+ increased uptake of 14C-oxalate. Dinitrophenol (DNP) and iodoacetamide (IoAc), however, significantly decreased, and O degrees C eliminated slice uptake. Slices incubated with 100 microM [14C]oxalate showed a further increase in tissue accumulation and the appearance of [14C]oxalate crystals. Crystals formed in vitro were deposited throughout the tissue. Oxalic acid did not appear to share the organic acid by renal cortical slices in vitro is largely independent of energy-mediated mechanisms.     

The release of [3H]GABA formed from [3H]glutamate in rat hippocampal slices

to compare the storage and release of endogenous GABA, of [3H]GABA formed endogenously from glutamate, and of exogenous [14C]GABA, hippocampal slices were incubated with 5 microCi/ml [3,4-3H]1-glutamate and 0.5 microCi/ml [U-14C]GABA and then were superfused in the presence or absence of Ca+ with either 50 mM K+ or 50 microM veratridine. Endogenous GABA was determined by high performance liquid chromatography which separated labeled GABA from its precursors and metabolites. Exogenous [14C]GABA content of the slices declined spontaneously while endogenous GABA and endogenously formed [3H]GABA stayed constant over a 48 min period. In the presence of Ca+ 50 mM K+ and in the presence or absence of Ca2+ veratridine released exogenous [14C]GABA more rapidly than endogenous or endogenously formed [3H]GABA, the release of the latter two occurring always in parallel. The initial specific activity of released exogenous [14C]GABA was three times, while that of endogenously formed [3H]GABA was only 50% higher than that in the slices. There was an excess of endogenous GABA content following superfusion with 50 mM K+ and Ca2+, which did not occur in the absence of Ca2+ or after veratridine. The observation that endogenous GABA and [3H]GABA formed endogenously from glutamate are stored and released in parallel but differently from exogenous labelled GABA, suggests that exogenous [3H] glutamate can enter a glutamate pool that normally serves as precursor of GABA.     

Inhibitory effects of calcium channel blockers on thyroid hormone uptake in neonatal rat cardiomyocytes

The effects of the Ca2+ channel blockers verapamil, nifedipine, and diltiazem on triiodothyronine (T3) and thyroxine (T4) uptake were tested in cultured cardiomyocytes from 2-day-old rats. Experiments were performed at 37 degrees C in medium with 0.5% BSA for [125I]T3 (100 pM) or 0.1% BSA for [125I]T4 (350 pM). The 15-min uptake of [125I]T3 was 0.124 +/- 0.013 fmol/pM free T3 (n = 6); [125I]T4 uptake was 0.032 +/- 0.003 fmol/pM free T4 (n = 12). Neither T3 nor T4 uptake was affected by 1% DMSO (diluent for nifedipine and verapamil). Uptake of [125I]T3 but not of [125I]T4 was dose dependently reduced by incubation with 1-100 microM verapamil (49-87%, P < 0.05) or nifedipine (53-81%, P < 0.05). The relative decline in [125I]T3 uptake after 4 h of incubation with 10 microM verapamil or nifedipine was less than after 15 min or 1 h, indicating that the major inhibitory effect of the Ca2+ channel blockers occurred at the level of the plasma membrane. The reduction of nuclear [125I]T3 binding by 10 microM verapamil or nifedipine was proportional to the reduction of cellular [125I]T3 uptake. Diltiazem (1-100 microM) had no dose-dependent effect on [125I]T3 uptake but reduced [125I]T4 uptake by 45% (P < 0.05) at each concentration tested. Neither the presence of 20 mM K+ nor the presence of low Ca2+ in the medium affected [125I]T3 uptake. In conclusion, the inhibitory effects of Ca2+ channel blockers on T3 uptake in cardiomyocytes are not secondary to their effects on Ca2+ influx but, rather, reflect interference with the putative T3 carrier in the plasma membrane.     

Uptake and Release of Glycine in the Guinea Pig Cochlear Nucleus

This study attempts to determine if the cochlear nucleus (CN) contains glycinergic synaptic endings. The uptake and release of exogenous radiolabeled glycine were measured in vitro in the three major subdivisions of the guinea pig CN: anteroventral, posteroventral, and dorsal. A kinetic analysis of [3H]glycine uptake revealed the presence in each CN subdivision of a high- and a low-affinity uptake mechanism. The high-affinity mechanism had a Km of 25.2-30.5 microM and a Vmax of 3.8-4.8 nmol/10 mg of cell water/5 min, whereas the low-affinity mechanism had a Km of 633-718 microM and a Vmax of 26.6-37.1 nmol/10 mg of cell water/5 min. At steady state, the high-affinity mechanism accumulated 10 microM [3H]glycine from the medium, achieving tissue concentrations that were 13-24 times that in the medium. The high-affinity uptake was dependent on the temperature and on the concentrations of NaCl and glucose in the incubation medium. It exhibited a high degree of substrate specificity, as determined by the effects of structural analogues of glycine on the uptake of [3H]glycine. Each CN subdivision also contained two mechanisms mediating [14C]glycine release. One was activated by depolarizing electrical stimuli, produced a rapid transient release of [14C]glycine, and was dependent on the presence of extracellular Ca2+. The other was continuous, producing a slow spontaneous efflux of [14C]glycine. Released glycine could be removed primarily by uptake, because during release measurements, the amount of [14C]glycine detected in the medium decreased when glycine uptake activity was optimized. The electrically evoked, Ca2+-dependent release and the high-affinity uptake of glycine may mediate the synaptic release and inactivation of glycine, respectively. These findings, therefore, support the presence of glycinergic synaptic endings in each CN subdivision.     

Ca2+-dependence of the evoked release from guinea pig cerebral cortex slices of endogenous 14C-labelled amino acids, labelled via D-[U-14C]glucose.

Spontaneous and electrically evoked release of exogenous labelled amino acids and endogenous amino acids labelled from D-[U-14C]glucose were compared in control and Ca2+-free medium using guinea pig cerebral cortex slices. Spontaneous release of all labelled amino acids, except that of endogenous 14C-labelled threonine-serine-glutamine (unseparated) and exogenous [14C]aspartate, was doubled in Ca2+-free medium. The major portion of the electrically evoked release of endogenous [14C]glutamate, [14C]aspartate, gamma-amino[14C]butyrate (14C-labelled GABA) and exogenous 3H-labelled GABA was Ca2+-inpendent. More than half of the evoked release of the other labelled amino acids was Ca2+-independent. As the pattern of Ca2+-dependence of the evoked release concurred with the selectivity of the evoked release for endogenous [14C]-glutamate, [14C]aspartate, and 14C-labelled GABA, it was concluded that these labelled amino acids were probably released from the amino acid 'transmitter pool'.     

Comparison of synaptosomal and glial uptake of pipecolic acid and GABA in rat brain

The active uptake of [³H]pipecolic acid increased with incubation time and its uptake at 3 min was half of that at 20 min. [¹⁴C]GABA uptake rose earlier, and its uptake at 3 min was almost 80% of that at 20 min. On the other hand, a ratio (pellet/medium) of [³H]pipecolic acid uptake into glial cell-enriched fractions, was much less (0.4–0.6) than that of [¹⁴C]GABA (25.8–74.1). GABA, 10^–4 M, and pipecolic acid, 10^–4 M, produced a significant inhibition of [³H]pipecolic acid uptake into P₂ fractions. Pipecolic acid, 10^–4 M, significantly reduced the synaptosomal and glial uptake of [¹⁴C]GABA. GABA, 10^–4 M, affected neither spontaneous nor high K⁺-induced release of [³H]pipecolic acid from brain slices. It is suggested that pipecolic acid is involved in either synaptic transmission or in its modulation at GABA synapses in the central nervous system.     

Characterization of calcium transport by basal plasma membranes from human placental syncytiotrophoblast.

We have studied the mechanisms involved in calcium (Ca2+) transport through the basal plasma membranes (BPM) of the syncytiotrophoblast cells from full-term human placenta. These purified membranes were enriched 25-fold in Na+/K(+)-adenosine triphosphate (ATPase), 37-fold in [3H] dihydroalprenolol binding sites, and fivefold in alkaline phosphatase activity compared with the placenta homogenates. In the absence of ATP and Mg2+, a basal Ca2+ uptake was observed, which followed Michaelis-Menten kinetics, with a Km Ca2+ of 0.18 +/- 0.05 microM and Vmax of 0.93 +/- 0.11 nmol/mg/min. The addition of Mg2+ to the incubation medium significantly decreased this uptake in a concentration-dependent manner, with a maximal inhibition at 3 mM Mg2+ and above. The Lineweaver-Burk plots of Ca2+ uptake in the absence and in the presence of 1 mM Mg2+ suggest a noncompetitive type of inhibition. Preloading the BPM vesicles with 5 mM Mg2+ had no significant effect on Ca2+ uptake, eliminating the hypothesis of a Ca2+/Mg2+ exchange mechanism. This ATP-independent Ca2+ uptake was not sensitive to 10(-6) M nitrendipine nor to 10(-4) M verapamil. An ATP-dependent Ca2+ transport was also detected in these BPM, whose Km Ca2+ was 0.09 +/- 0.02 microM and Vmax 3.4 +/- 0.2 nmoles/mg/3 min. This Ca2+ transport requires Mg2+, the optimal concentration of Mg2+ being approximately 1 mM. Preincubation of the membrane with 10(-6) M calmodulin strongly enhanced the initial ATP-dependent Ca2+ uptake. Finally, no Na+/Ca2+ exchange process could be demonstrated.     

Uptake and release of [3H]gamma-aminobutyric acid by embryonic spinal cord neurons in dissociated cell culture

We have investigated the uptake and release of [3H]gamma-aminobutyric acid (GABA) by embryonic chick spinal cord cells maintained in culture. Cells dissociated from 4- or 7-d-old embryos were studied between 1 and 3 wk after plating. At 3 degrees C, [3H]GABA was accumulated by a high affinity (Km approximately equal to 4 microM) and a low affinity (Km approximately equal to 100 microM) mechanism. The high affinity transport was markedly inhibited in low Na+ media, by ouabain, at 0 degrees C, and by 2,4-diaminobutyric acid. Autoradiography, after incubation in 0.1 microM [3H]GABA, showed that approximately 50% (range = 30-70%) of the multipolar cells were labeled. These cells were neurons rather than glia; action potentials and/or synaptic potentials were recorded in cells subsequently found to be labeled. Non-neuronal, fibroblast-like cells and co-cultured myotubes were not labeled under the same conditions. The fact that not all of the neurons were labeled is consistent with the suggestion, based on studies of intact adult tissue, that high affinity transport of [3H]GABA may be unique to neurons that use GABA as a neurotransmitter. Our finding that none of fifteen physiologically identified cholinergic neurons, i.e., cells that innervated nearby myotubes, were heavily labeled after incubation in 0.1 microM [3H]GABA is significant in this regard. The newly taken up [3H]GABA was not metabolized in the short run. It was stored in a form that could be released when the neurons were depolarized in a high K+ (100 mM) medium. As expected for a neurotransmitter, the K+-evoked release was reversibly inhibited by reducing the extracellular Ca++/Mg++ ratio.     

Separation of carrier mediated and vesicular release of GABA from rat brain slices.

In this study the temperature dependence of [3H]GABA release from brain slices evoked by electrical field stimulation and the Na+/K+ ATPase inhibitor ouabain was investigated. [3H]GABA has been taken up and released from hippocampal slices at rest and in response to electrical field stimulation (20 V, 10 Hz, 3 msec, 180 pulses) at 37 degrees C. When the bath temperature was cooled to 7 degrees C, during the sample collection period, the tissue uptake and the resting outflow of [3H]GABA were not significantly changed. In contrast, the stimulation-induced tritium outflow increased both in absolute amount (Bq/g) and in fractional release and the S2/S1 ratio was also higher at 7 degrees C. Perfusion of the slices with tetrodotoxin (TTX, 1 microM) inhibited stimulation-induced [3H]GABA efflux indicating that exocytotic release of vesicular origin is maintained under these conditions. 15 min perfusion with ouabain (10-20 microM) induced massive tritium release both in hippocampal and in striatal slices. However, the fraction of [3H]GABA outflow evoked by ouabain was much higher in the hippocampus than in the striatum. Sequential lowering the bath temperature from 37 degrees C to 17 degrees C completely abolished ouabain-induced [3H]GABA release in both brain regions, indicating that it is a temperature-dependent, carrier-mediated process. When the same experiments were repeated under Ca2+ free conditions, cooling the bath temperature to 17 degrees C, although substantially decreased the release but failed to completely abolish the tritium outflow evoked by ouabain, a significant part was maintained. Our results show that vesicular (field stimulation-evoked) and carrier-mediated (ouabain-induced) release of GABA is differentially affected by low temperature: while vesicular release is unaffected, carrier-mediated release is abolished at low bath temperature. Therefore, lowering the temperature offers a reliable tool to separate these two kinds of release and makes possible to study exclusively the pure neuronal release of GABA of vesicular origin.     

Selective inhibition of human platelet phospholipase A2 by buffering cytoplasmic calcium with the fluorescent indicator quin 2. Evidence for different calcium sensitivities of phospholipases A2 and C

Human platelets labelled with either [14C]arachidonic acid or [32P]orthophosphate were loaded or not with the Ca2+ fluorescent indicator quin 2. They were then incubated in the presence or in the absence of human thrombin (1 U/ml) in a medium where Ca2+ concentration was adjusted near zero or to 1 mM. Under these conditions, phospholipase A2 activity, as detected by the release of [14C]arachidonate and of its metabolites, or by the hydrolysis of [14C]phosphatidylcholine, was severely impaired in quin 2-loaded platelets upon removal of external Ca2+. However, Ca2+ was not required in non-loaded platelets, where a maximal phospholipase A2 activity was detected in the absence of external Ca2+. In contrast, phospholipase C action, as determined from the amounts of [14C]diacylglycerol, [14C]- or [32P]phosphatidic acid formed, appeared to be much less sensitive to the effects of quin 2 loading and of Ca2+ omission. By using various concentrations of quin 2, it was found that the inhibitory effect exerted against phospholipase A2 could be overcome by external Ca2+ only when the intracellular concentration of the calcium chelator did not exceed 2 mM. At higher concentrations averaging 3.5 mM of quin 2, phospholipase A2 activity was fully suppressed even in the presence of external Ca2+, whereas phospholipase C was still active, although partly inhibited. It is concluded that platelet phospholipase A2 requires higher Ca2+ concentrations than phospholipase C to display a maximal activity. By comparing platelet phospholipase A2 activity under various conditions with the values of cytoplasmic free Ca2+ as detected by quin 2 fluorescence, it is proposed that cytoplasmic free Ca2+ in control platelets stimulated with thrombin can attain concentrations above 1 microM, probably close to 5-10 microM, as recently determined with the photoprotein aequorin (Johnson, P.C., Ware, J.A., Cliveden, P.B., Smith, M., Dvorak, A.M. and Salzman, E.W. (1985) J. Biol. Chem. 260, 2069-2076).     

Modulation of GABA Receptor Binding by Ca+2

In frozen-thawed repeatedly washed rat cortical synaptic membranes, Ca2+ (1-5 mM) decreased the binding of [3H]muscimol whereas it increased the binding of [3H]gamma-aminobutyric acid (GABA). However, the binding of [3H]GABA was decreased by the same extent as the binding of [3H]muscimol when the membranes were incubated with baclofen (a selective ligand for the GABAB binding site) and Ca2+. Scatchard analysis of [3H]muscimol binding revealed that Ca2+ reduced the density of GABA binding sites without affecting the dissociation constant. Ca2+ was more potent than Ba2+, Mg2+ was ineffective, and the Ca2+ antagonist La3+ stimulated [3H]muscimol binding. The inhibition of [3H]muscimol binding by Ca2+ was not influenced by calmodulin (50 micrograms/ml), trifluoperazine (10(-5) M), verapamil (10(-6) M), quinacrine (10(-4) M), cordycepin (0.1 mM), leupeptin (20 microM), or soybean trypsin inhibitor (0.1 mg/ml). Moreover, the effect of Ca2+ was additive to that of GABA-modulin. These results indicate that Ca2+ decreases the number of GABAA binding sites while unveiling GABAB binding sites.     

Glutamate as a Putative Transmitter in the Cerebellum: Stimulation by GABA of Glutamic Acid Release from Specific Pools

The aim of the present paper was to determine whether the release of glutamate from putative "glutamergic" terminals in the cerebellum is influenced by gamma-aminobutyric acid (GABA). In a group of preliminary experiments, we present biochemical evidence in favour of a neurotransmitter role of glutamate in the cerebellum: (1) endogenous glutamate was released from depolarized cerebellar synaptosomal preparations in a Ca2+-dependent away; (2) [14C]glutamate was synthesized from [14C]glutamine in cerebellar synaptosomes, and the newly synthesized [14C]glutamate was released released in a Ca2+-dependent way; (3) the elevation of cyclic GMP elicited by depolarization of cerebellar slices in the presence of Ca2+ was partly reversed by the glutamate antagonist glutamic acid diethyl ester, which probably prevented the interaction of endogenously released glutamate with postsynaptic receptors. GABA and muscimol at low concentrations (2--20 micrometers) potentiated the depolarization-induced release of D-[3H]aspartate (a glutamate analogue which labels the glutamate "reuptake pool") from cerebellar synaptosomes. The effect was concentration dependent and was largely prevented by two GABA antagonists, bicuculline and picrotoxin. The stimulation of D-[3H]aspartate release evoked by muscimol was linearly related to the logarithm of K+ concentration in the depolarizing medium. GABA did not affect the overall release of endogenous glutamate, but potentiated, in a picrotoxin-sensitive manner, the depolarization-evoked release of [14C]glutamate previously synthesized from [14C]glutamine. Since nerve endings are the major site of glutamate synthesis from glutamine, GABA and muscimol appear to exert their stimulatory effect at the level of "glutamergic" nerve terminals, probably after interacting with presynaptic GABA receptors. The possible functional significance of these findings is briefly discussed.     

γ-Aminobutyric Acid and Glycine Modulate Each Other''s Release Through Heterocarriers Sited on the Releasing Axon Terminals of Rat CNS

The ability of gamma-aminobutyric acid (GABA) and glycine (Gly) to modulate each other's release was studied in synaptosomes from rat spinal cord, cerebellum, cerebral cortex, or hippocampus, prelabeled with [3H]GABA or [3H]Gly and exposed in superfusion to Gly or to GABA, respectively. GABA increased the spontaneous outflow of [3H]Gly (EC50, 20.8 microM) from spinal cord synaptosomes. Neither muscimol nor (-)-baclofen, up to 300 microM, mimicked the effect of GABA, which was not antagonized by either bicuculline or picrotoxin. However, the effect of GABA was counteracted by the GABA uptake inhibitors nipecotic acid and N-(4,4-diphenyl-3-butenyl)nipecotic acid. Moreover, the GABA-induced [3H]Gly release was Na+ dependent and disappeared when the medium contained 23 mM Na+. The effect of GABA was Ca2+ independent and tetrodotoxin insensitive. Conversely, Gly enhanced the outflow of [3H]GABA from rat spinal cord synaptosomes (EC50, 100.9 microM). This effect was insensitive to both strychnine and 7-chlorokynurenic acid, antagonists at Gly receptors, but it was strongly Na+ dependent. Also, the Gly-evoked [3H]GABA release was Ca2+ independent and tetrodotoxin insensitive. GABA increased the outflow of [3H]Gly (EC50, 11.1 microM) from cerebellar synaptosomes; the effect was not mimicked by either muscimol or (-)-baclofen nor was it prevented by bicuculline or picrotoxin. The GABA effect was, however, blocked by GABA uptake inhibitors and was Na+ dependent. Gly increased [3H]GABA release from cerebellar synaptosomes (EC50, 110.7 microM) in a strychnine- and 7-chlorokynurenic acid-insensitive manner. This effect was Na+ dependent. The effects of GABA on [3H]Gly release seen in spinal cord and cerebellum could be reproduced also with cerebrocortical synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ATP on Phosphatidylinositol-Phospholipase C and Inositol 1-Phosphate Accumulation in Rat Brain Synaptosomes

Incubation of rat brain synaptosomes prelabeled with [2-3H]inositol resulted in a time-dependent release of labeled inositol 1-phosphate. This process was Ca2+ dependent, and ATP (1 mM) enhanced the inositol 1-phosphate formation three- to fivefold. Using [1-14C]arachidonoyl-phosphatidylinositol which was introduced into saponin-permeabilized synaptosomes, ATP (1 mM) and free Ca2+ (approximately 20 microM) enhanced the phospholipase C hydrolysis of this substrate to form labeled diacylglycerol. When the same permeabilized synaptosomal preparation was incubated with [2-3H]inositol-phosphatidylinositol, ATP not only enhanced the formation of labeled inositol 1-phosphate, but also inhibited the conversion of inositol 1-phosphate to inositol. Furthermore, ATP appeared to reduce the Ca2+ requirement of the phosphatidylinositol-phospholipase C. Inhibition of the conversion of inositol 1-phosphate to inositol could not be overcome by increasing the Mg2+ concentration in the incubation medium. Although the ATP effect is not viewed as a receptor-mediated event, it is possible that such an event may occur in synaptosomes under conditions in which intrasynaptic Ca2+ concentration becomes elevated.     

Release of Previously Incorporated γ-[3H]Aminobutyric Acid in Rabbit Caudate Nucleus Slices

The release of gamma-aminobutyric acid (GABA) was studied in slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused. Aminooxyacetic acid was present throughout. Both the tritium in the slices and that in the superfusate consisted practically entirely of [3H]GABA. Stimulation for 2 min by electrical field pulses of 3 ms width and 9 V/cm voltage drop (36 mA current strength) at 5 or 20 Hz elicited an overflow of [3H]GABA that amounted to 0.23 or 0.47% of the tritium content of the tissue, respectively, and was diminished by 85% in the presence of tetrodotoxin. At higher current strength, less of the stimulation-evoked overflow was tetrodotoxin-sensitive. cis-1,3-Aminocyclohexane carboxylic acid diminished the uptake of [3H]GABA into the tissue but did not change the percentage released by electrical stimulation. Ca2+ withdrawal greatly accelerated basal [3H]GABA efflux and almost abolished the response to stimulation. Nipecotic acid 10-1,000 microM enhanced both the basal and (up to eightfold) the stimulation-evoked overflow. The method described allows us to elicit electrically a quasiphysiological, i.e., Ca2+-dependent and tetrodotoxin-sensitive, neuronal release of [3H]GABA. Nipecotic acid diverts released [3H]GABA from reuptake to overflow.     

Depolarization Increases Chloride-Dependent Glutamate Sequestration in Synaptic Membranes of Rat Cerebral Cortex

To assess the functions of Cl- -dependent glutamate "binding" (Cl- -dependent glutamate uptake) in synaptic membranes, possible effects of depolarization on the uptake were examined. When rat cerebral cortical slices were preincubated with depolarizing agents such as veratrine (7 micrograms/ml), 10 microM aconitine, 56 mM K+, and 50 microM monensin, [3H]glutamate uptake by the crude synaptic membranes, which were subsequently prepared from the pretreated slices, was increased by 60-85%. Stimulation of the glutamate uptake by predepolarization was dependent on Na+ but not on Ca2+. The bindings of gamma-[3H]aminobutyric acid and 5-[3H]hydroxytryptamine were not significantly affected by the predepolarization. Veratrine pretreatment increased the maximal density of the glutamate uptake sites without affecting the affinity for glutamate. Several characteristics of the uptake sites increased by the veratrine pretreatment coincided with those of Cl- -dependent glutamate uptake sites. Na+-dependent glutamate binding (Na+-dependent glutamate uptake) to the membranes was not affected by pretreatment with veratrine. The content of endogenous glutamate and the noninulin space in the membrane fractions were not changed by the predepolarization. The increase in the glutamate uptake induced by pretreatment with high K+ was reversible: it returned to the control level after a second incubation of the slices in control medium. These results suggest that the Cl- -dependent glutamate sequestration system in synaptic membranes is regulated by the membrane potential.     

Phosphorylation of Cl-, HCO3--stimulated Mg2+-ATPase of plasma membranes of carp (Cyprinus carpio L.) brain sensitive to GABA(A)-ergic ligands

Phosphorylation of the sensitive to GABA(A)-ergic ligands Cl-, HCO3--stimulated Mg2+-ATPase of the plasma membranes from fish brain by [gamma-32P]ATP was investigated in the presence of Mg2+. It was established, that formation of the phosphoprotein at 0-1 degrees C is dependent on time incubation and concentration of Mg2+ in the incubation medium. Hydroxylamine (50 mM) and pH (10) completely inhibited formation of phosphorylated intermediate. Ions of Cl- (10 mM)+HCO3- (2 mM) and also GABA (1-100 microM) dephosphorylated the enzyme. The dephosphorylating effect of GABA on the membrane samples did not appear in the presence of bicuculline. o-Vanadate (10 microM) eliminates the dephosphorylating effect of anions and GABA on the phosphoprotein. It was established by SDS-PAAG electrophoresis and autoradiographia that investigated phosphorylation and GABA(A)-induced dephosphorylation is performed by the protein with molecular weight aproximately 56 kDa. Such molecular weight has a subunit which forms oligomer composition of the sensitive to GABA(A)-ergic ligands Cl-, HCO3--ATPase from fish brain. The obtained data demonstrated that Cl, HCO3- ATPase from fish brain can be directly phosphorylated by [gamma-32P]ATP in the presence of Mg2+ and forms the phosphorylation intermediate.     

Bidirectional Movement of γ-Aminobutyric Acid in Rat Spinal Cord Slices

Abstract: The bidirectional movement of GABA (γ-aminobutyric acid) was studied in slices of rat spinal cord which were incubated in small volumes of medium. The appearance in the medium of endogenous GABA and the disappearance from the medium of [¹⁴C]GABA were used to calculate the rates of unidirectional uptake and unidirectional release of GABA. Under these conditions, no net uptake of GABA was observed when slices were incubated in media containing concentrations of GABA as high as 25 μm . Elevated potassium (60 mm ) stimulated the unidirectional release of endogenous GABA from spinal cord slices by a calcium-dependent process. Ouabain (0.1 mm ) more than doubled the unidirectional release of endogenous GABA in a calcium-independent manner, while unidirectional uptake was inhibited by 44%. Nipecotic acid (1.0 mm ) stimulated unidirectional release and inhibited unidirectional uptake of GABA.     

Relationship between synaptosomal uptake and release of [14C]gaba, [14C]diaminobutyric acid and [14C]beta-alanine.

[¹⁴C]GABA is taken up by rat brain synaptosomes via a high affinity, Na⁺-dependent process. Subsequent addition of depolarizing levels of potassium (56.2 MM) or veratridine (100 μM) stimulates the release of synaptosomal [¹⁴C]GABA by a process which is sensitive to the external concentration of divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺. However, the relatively smaller amount of [¹⁴C]GABA taken up by synaptosomes in the absence of Na⁺ is not released from synaptosomes by Ca²⁺ -dependent, K ⁺-stimulation. [¹⁴C]DABA, a competitive inhibitor of synaptosomal uptake of GABA (Iversen & Johnson , 1971) is also taken up by synaptosomal fractions via a Na ⁺ -dependent process; and is subsequently released by Ca²⁺ -dependent, K⁺-stimulation. On the other hand, [¹⁴C]β-alanine, a purported blocker of glial uptake systems for GABA (Schon & Kelly , 1974) is a poor competitor of GABA uptake into synaptosomes. Comparatively small amounts of [¹⁴C] β-alanine are taken up by synaptosomes and no significant amount is released by Ca²⁺ -dependent, K⁺-stimulation. These data suggest that entry of [¹⁴C]GABA into a releasable pool requires external Na⁺ ions and maximal evoked release of [¹⁴C]GABA from the synaptosomal pool requires external Ca²⁺ ions. The GABA analogue, DABA, is apparently successful in entering the same or similar synaptosomal pool. The GABA analogue, β-alanine, is not. None of the compounds or conditions studied were found to simultaneously affect both uptake and release processes. Compounds which stimulated release (veratridine) or inhibited release (magnesium) were found to have minimal effect on synaptosomal uptake. Likewise compounds (DABA) or conditions (Na⁺-free medium) which inhibited uptake, had little effect on release.