Efflux of γ-aminobutyric acid caused by changes in ion concentrations and cell swelling simulating...

Acta Neurochir (Wien) (1997) 139:453463 Ac ta N e u r o c h i r u r g i c a

�9 Springer-Verlag 1997 Printed in Austria

Efflux of y-Aminobutyric Acid Caused by Changes in Ion Concentrations and Cell Swelling Simulating the Effect of Cerebral Ischaemia

T. S. Haugstad 1' 2, H. E. Karlsen 4, P. Krajt~i 3, B. Due-TOnnessen 2, M. Larsen 1' 2, C. Sandberg ~' 2, O. Sand 4, E Brandtzaeg 3, and I. A. Langmoen 1' 2

i Institute for Surgical Research, 2 Department of Neurosurgery and 3 Institute for Pathology, The National Hospital, Rikshospitalet, and 4 Institute of Biology, University of Oslo, Oslo, Norway

Summary The relationships among ischaemic GABA efflux from brain

tissue and extracellular and intracellular concentrations of sodium, chloride and potassium ions were investigated by means of 1) transverse hippocampal slices from rat and 2) functional expression of a high affinity GABA transporter in Xenopus oocytes. Brain slices were incubated for 20 min in medium where extracellular sodium and chloride were substituted with impermeant ions. Iseth- ionate (Iseth) substitution for chloride generated a 7-fold increase in GABA efflux. Choline (Chol) but not N-methyl-D-glucamine (NMDG) substitution for sodium likewise increased GABA efflux. Reducing the osmolarity of the medium by decreasing both sodium and chloride concentrations (Hyp) increased GABA efflux 3-fold. This release was blocked by mannitol (Man). Blocking sodium channels with 1 uM of tetrodotoxin (TTX) also increased the release 3-fold. Energy deprivation (ED) increased the GABA release 50-fold. ED/Iseth left the release unchanged, ED/Chol increased the GABA efflux by 23%, whereas ED/NMDG reduced the release by 41%. Adding mannitoI did not block the ED-evoked release, whereas TTX reduced it by 52%. Release of preloaded [3H]-GABA from oocytes expressing the GAT-1 GABA transporter was then examined. Depolarisation by current injection or 100 mM extracellular K + did not increase GABA release. Sodium chloride injection, however, caused membrane depolarisation and a 100-fold increased GABA efflux from the oocytes. This release was blocked when the osmolarity was increased extracellnlarly by adding mannitol. These results show that 1) TTX releases GABA from brain tissue but blocks release during ED, 2) the high affinity GABA carrier must be altered in order to reverse, 3) ischaemic GABA release is sodium independent, and is modulated by large cations, 4) mannitol blocks the reversal of high affinity carriers in oocytes, but the release from brain slices during ED is unaffected. Taken together, the results suggest that ischaemic release of GABA from brain tissue does not occur by means of reversed high affinity carriers alone, but rather that it is controlled by more complex mechanisms.

Keywords: Cerebral ischaemia; GABA release; brain slice; GAT- 1.

Introduction

The cell death that occurs during and after cerebral ischaemia has been closely linked to the massive inward flux of ions that is known to occur [25, 53]. Chloride influx is one of the factors that have been implicated in the excitotoxic insult to neurons, since it has been shown that glutamate analogues are toxic to hippocampal neurons only in the presence of extracellular chloride [50]. An important channel of chloride transport is coupled to the GABA receptor [9]. When this channel is blocked, or if chloride is omitted from the extracellular medium, neuron recovery after energy deprivation improves [21]. During cerebral ischaemia, a massive release of GABA and other amino acids takes place [6, 28]. In this way chloride channels are activated, and GABA-induced chloride influx may thus play a role in ischaemic brain damage.

Although amino-acid release during cerebral ischaemia has been extensively studied, the precise release mechanisms of GABA and other amino acids are still disputed. In early phases of ischaemia, the release seems to some extent to be calcium dependent [35], but the large release that occurs slightly after the rapid anoxic depolarisation [6], is calcium independent [21, 30, 32]. The rapid depolarisation is the result of a simultaneous flux of several types of ions across the cell membrane [26]. This results in a relative extracellular hyposmolarity of --~ 150 mOsm [28]. At the same time, the intercellular space is reduced by 50% [38], due to the osmotic swelling of the cells [37, 68].

The ionic composition of the extracellular fluid

454 T. S. Haugstad et al.: Ion Dependence of Brain GABA Efflux

resulting from the massive ion fluxes during anoxic depolarisation is thought to favour release of neurotransmitters by reversal of uptake mechanisms [40, 46]. Direct evidence for carrier mediated release of amino-acids has, however, been difficult to obtain, although etectrophysiological studies indicate that this may take place [44, 60].

The release pattern of amino acids during hyposmotic stress [28, 29], where GABA and other amino acids are released by mechanisms probably dependent on increased membrane tension [28, 55, 63, 67] is very different from the release pattern caused by brain energy deprivation. Patch clamp techniques have revealed that some membrane proteins alter their functional state when the cell membrane is stretched [45, 59]. In fact, cell response to changes in the environment is often mediated by sensitivity to mechanical forces [31]. Both glial cells and neurons are known to possess channels that are activated by such mechanisms [5, 33].

The present report focuses the dependence of reversed GABA transport on ion concentrations, membrane potential and membrane tension. Brain slices are suited for this type of study, since the slice technique allows exposure of an isolated brain seg- ment to a strictly controlled physical and chemical environment, and collection of released and contained endogenous amino acids for analysis [39]. In order to study the function of a specific type of amino-acid carrier, it is favourable to isolate the carrier and install it in an indolent biological membrane. Thus, mRNA coding for the GABA high affinity carrier GAT-1 [23], was injected into Xenopus oocytes [54], and oocytes expressing high affinity uptake were used to study the release of preloaded [3H]GABA. In both models, ionic concentrations were manipulated to simulate some of the changes that occur during cerebral ischaemia.

Methods

Preparation of Brain Slices

Hippocampal slices were prepared as described previously [39]. In brief, male Wistar rats (100-200 g) were decapitated, the brains cooled for 2 rain at 4 ~ in artificial cerebrospinal fluid (ACSF), the hippocampus carefully removed, and cut in 0.5 mm thick transverse slices. The slices were then transferred to a pre-incubation chamber where they were submerged in ACSF saturated by a gas mixture containing 95% 02 and 5% CO2. The ACSF consisted of (in raM) NaC1 123; KCI 3.75; KH2PO4 1.25; MgSO2 1; CaC12 2; NaHCO3 26; glucose (dextro-rotated) 10. The pH was 7.4. Follow- ing a 60-90 minutes rest in the pre-incubation chamber, they were transferred to the test chambers. The slices were placed on trans-

portable grids submerged in 5 separate wells containing 5 ml of ACSF (35 ~ saturated with 95% 02 and 5% CO> After 30 min, the grids supporting the slices were transferred to 5 separate test wells with the test solutions. Energy deprivation was induced by exposing the slices to ACSF with 0 mM glucose and oxygen substituted by 95% N> The slices were kept in these test wells for 20 rain, then removed into 8% tri-chloro-acetic acid (TCA) with 2 mM ethylene diamine tetra-acetic acid (EDTA), and stored at -70 ~ until analysis for amino acid and protein content. ACSF samples collected from the wells for amino-acid determination at the end of the experiment (representing total release from the test period), and the TCA cell extract (representing intracellular content of amino- acids), were cleaned and deproteinised by ultracentrifugation through a 5 • 103 Dalton cut-off filter (Nihon Millipore, Yone- zawa, Japan), and stored at -70 ~ until analysis. The total amino acid content was calculated for the individual well as the content of amino acids in the medium plus the amount extracted from the slices by TCA.

Measurement of Endogenous GABA

The content of GABA in the ACSF and the TCA extracts was determined by high performance liquid chromatography (HPLC, Perkin Elmer, Norwalk, Connecticut, USA) by a method modified from Lindroth and Mopper [41 ] and Godel et al. [20]. Care was taken to avoid contamination of amino acids in the laboratory. In brief, the amino acids were automatically pre-column derivatised by autosampIer (Perkin Elmer ISS 200) with 40 mM o-phthalaldehyde (Fluka Chemie AG, Buchs, Switzerland) and 3-mercapto propionic acid (Sigma, St. Louis, MO, USA) at pH 10.5. The amino-acids were separated on a reversed phase Hypersit ODS column (3.1 • 200 ram, Chrompak International B.W., Middelburg, The Nether- lands) at 42 ~ by gradient elution (Perkin Elmer Binary LC pump 250). Mobil phases were: A: 0.08 M sodium acetate (Sigma) buffer, pH 6.0, and B: acetonitrile (Rathburn Chemicals Limited, Walker- burn, Scotland)/0.4 M sodium acetate buffer/methanol (Rathburn) (14/4/1). Fluorescence peaks (Perkin Elmer LC 240, excitation wavelength 340 nm, emission wavelength 460 nm) were integrated by Perkin Elmer 1020X software on a PE Nelson computer.

Protein Measurements

The protein content of the slices was determined according to the method of Lowry [43], after homogenisation in 1 M NaOH.

Statistical Analysis

The results were tested statistically using the Wilcoxon rank sum test and are presented as mean _+ standard error of the mean (SEM).

Expression of the GABA Transporter in Oocytes

A 4.054-bp GAT-I-cDNA [23] in pBluescript (SK+), linearised with Not I served as template for in vitro transcription of GAT-1 RNA. The transcription reaction contained approximately 1 ug of template, 40 mM Tris (pH 7.5), 50 mM NaCI, 8 mM MgC12, 2 mM Spernfidine, 1 mM each of rATP, rCTP and rUTP, 0.2 mM rGTP, 1 mM of m7-rGTP, and 80 U of T7 RNA polymerase in a total volume of 100 ]ul. After an incubation period of 2 h at 37 ~ the reaction mixture was treated with 40 U of de-oxyribonuclease I for 30 rain at 37 ~ phenol/chloroform extracted and spun through Sephadex G-50 column. Correct size of GAT 1 RNA, recovered by

T. S. Haugstad et al.: Ion Dependence of Brain GABA Efflux 455

ethanol precipitation, was ascertained by gel electrophoresis. Oocytes were bar-vested from mature Xenopus females, injected with 50 ng mRNA synthesised from the GAT-1 clone, and sample oocytes tested after 2 4 ~ 8 h incubation for [3H]GABA uptake. The oocytes were incubated for 15 min at room temperature in a medium containing (in mmol) NaC1 88, KC1 1, NaHCO3 2.4, Ca(NO3)2 0.33, CaC12 0.41, MgSO4 0.82, buffered to pH 7.4 with HEPES 10, and the transport reaction initiated by the addition of [3H]GABA (100 TBq/mol; Amersham) to a final concentration of 0.2 ~M. After a 60 rain incubation period, the oocytes were rapidly washed and solubilised in 10% SDS, and the radioactivity was measured by liquid scintillation counting. Oocytes from batches that showed expression of high affinity GABA carrier were preloaded by micro- injection with [3H]GABA to an estimated intra-oval concentration of 10 mM (100 GBq/mol). After I5 rain rest, an oocyte was placed in a perfusion chamber, and two micro-electrodes (and, for some experiments, an injection capillary), installed. A resting period of 5-10 min was allowed for the oocyte to regain its membrane potential (> -55 mV). The perfusion fluid (containing (in mM): NaC1 115, KC1 2.5, CaC12 1.8, buffered with HEPES 10 to pH 7.2) was sampled in 1 or 2 rain fractions, and the radioactivity measured by liquid scintillation counting.

Results

Calcium Dependent Release of GABA From Rat Hip- pocampal Slices

The exocytotic release of neurotransmitters from synaptic vesicles is dependent on the presence of extracelMar calcium [ 1, 36]. In order to evaluate the depolarisation induced calcium dependent release of GABA, slices were incubated for 2 rain (n = 10) under the following conditions: 1) normal ACSF, 2) ACSF with 50 mM K + (substituted for Na +, to depolarise the cell membrane), and 3) ACSF with 50 mM

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Fig. L Release of GABA from rat hippocampal slices (n = 10) was measured during 2 min incubation under control conditions (Con- trol), during membrane depolarisation with 50 mM K + stimulation/0 mM Ca++/4 mM Mg ++ present in the medium (in order to block synaptic transmission and thus vesicular release, 50 mM K+/BST). Values are presented as mean _ standard error. Signifi- cant difference from control (bar 2) and from 50 mM K + (bar 3) is denoted by asterisks (**p < 0.01 )

K § 0 mM Ca ++ and 4 mM Mg ++ (to depolarise the membrane and block synaptic transmission, BST). Depolarising the cell membrane caused GABA release to increase from 39 + 6 to 237 + 29 pmol/mg protein/rain. Seventy percent of this release was calcium dependent (Fig. 1).

The Effect of Reduced Ion Gradients and Membrane Depolarisation on GABA Efflux

If high affinity uptake of GABA follows Michae- lis-Menten kinetics [18], the thermodynamics of the proposed stochiometry predicts that alterations in extracellular sodium or chloride concentrations and the membrane potential will alter the extracellular concentration of GABA [2, 23]. In order to test this prediction, extracellular chloride was substituted with isethionate (Iseth, 4 mM of chloride remaining). This caused an increase in GABA efflux from slices during 20 rain of incubation by 766%, from a control value of 149 + 69 to 1290 + 394 pmol/mg protein/min (n = 9, p < 0.01, see Fig. 2). When sodium ions were substituted by choline (Chol, 5 mM of sodium remaining), there was an increase in GABA efflux by 630% (n = 8, p < 0.01). However, when sodium was substituted in the same manner by N-methyl-D-glucamine (NMDG, 9.2 mM sodium remaining), GABA effiux was unchanged (n = 8). Sodium and chloride

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Fig. 2. GABA release was measured after 20 minutes incubation of brain slices (n = 9) under the following conditions: 1) normal conditions (Control), 2) chloride ions substituted with isethionate (lseth), 3) sodium ions substituted with choline (Chol), 4) sodium ions substituted with N-methyl-D-glucamine (NMDG), 5) hypo- somlar medium with 75 mM sodium and chloride ions removed (Hyp), 6) medium with 75 mM sodium and chloride ions removed, osmotically conected by 150 mM mannitol (Hyp/Man), 7) normal medium with 1 mM tetrodotoxin added to block sodium channels (TTX). Values are presented as mean _+ standard error. Significant difference from control is denoted by asterisks (*p<0.05; **p < 0.0t)


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Fig. 3. Tissue contained (hatched part of bar) and released (open part of bar) amount of GABA was measured from slices incubated for 20 min under following conditions: 1) normal medium (n = 7,

Control), 2) hyposmolar medium with 75 mM sodium and chloride ions removed (n = 10, Hyp), 3) membrane depolarisation with potassium (n = 10, 50 mM K+), 4) membrane depolarisation with veratridine (n = 7, 50/~M Ver), and 5) combined oxygen and glucose deprivation (energy deprivation, ED). For variability data see Results

concentrations were then reduced each by 75 mM (without substitution), creating a relative extracellular hyposmolarity of 150 mOsm (Hyp). This increased the effiux by 321% (n -- 10, p < 0.01). Adding mannitol to the sodium chloride reduced medium (Hyp/Man), to substitute for the missing osmoles, blocked this increase (n = 10). Finally, when 1 pM of tetrodotoxin (TTX) was added to the incubation, to block sodium channels [61], the GABA efflux increased by 292% (n = 9, p < 0.05%).

Experiments were then performed to measure the relative effect on the GABA release and tissue content (Fig. 3). Under control conditions (n = 7), the slices released 272 + 55 pmol/mg protein of GABA, and the content at the end of the incubation period was 13140 + 1320 pmol/mg protein. Slices that were exposed to hyposmolar conditions (n = 10) released 628 + 57 pmol/mg protein of GABA (p < 0.01), and contained 14168 + 1584 pmol/mg protein (not significant). When slices were exposed to 50 mM K + for 20 min, without Ca ++ (n = 10, depolarising neurons and glia, and blocking synaptic release [36]), 503 + 123 pmol/mg protein of GABA was released to the medium (p < 0.05), and the content increased to 15246_+ 971 pmol/mg protein (p < 0.05). When 50 pM of veratridine was added to the ACSF with 0 mM Ca ++ (n = 7, depolarising neurons by opening sodium channels, while synaptic mechanisms were blocked) increased the release of GABA to 2670 + 562 pmol/mg protein (p < 0.05), and reduced the con-

tent to 12083 + 2489 pmol/mg protein (n.s.). Energy deprivation (removing both glucose and oxygen from the medium, ED) caused a large increase in release to 13559 + 819 pmol/mg protein (p < 0.01), whereas the content was unchanged, 13375 + 802 pmol/mg protein.

Effect of Changes in Ionic Composition and Osmolar- it)/on the Efflux of GABA During Energy Deprivation

A series of experiments was performed in order to evaluate the dependence of the massive GABA release caused by energy deprivation on 1) the presence of specific ions in the extracellular compartment, 2) increased membrane tension, and 3) membrane permeability of Na + through voltage gated channels.

Slices that were exposed to energy deprivation for 20 rain (n = 9, Fig. 4), released 8353 + 636pmol GABA per mg protein, compared to 149 _+ 69 pmol/ mg protein when oxygen and glucose was present. When chloride was substituted with isethionate, the release was unchanged (5% increase). However, when most of the sodium ions were substituted by choline, the GABA effiux increased by 23% (p < 0.05), and when sodium was substituted by N- methyl-D-glucamine, the release was reduced by 41% (p < 0.05). Decreasing the calcium concentration to

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Fig. 4. The release of GABA from brain slices (n = 9) incubated for 20 minutes was measured under the following conditions: 1) normal medium (Con), 2) combined oxygen and glucose deprivation (energy deprivation, ED), 3) ED with chloride substituted by isethionate (ED/Iseth), 4) ED with sodium ions substituted with

choline (ED/Chol), 5) ED with sodium ions substituted with N-

methyl -D-glucamine (ED/NMDG), 6) ED/O mM Ca ++, 7) ED with the relative extracellular hyposmolarity generated during ED corrected by adding 150 rnM mannitol (ED/Man), and 8) ED with 1 m M tetrodotoxin added to block sodium channels (ED/TTX). Values are presented as mean _+ standard error. Significant difference from ED is denoted by asterisks (*p < 0.05; **p < 0.01)

T. S. Haugstad et aI.: Ion Dependence of Brain GABA Efflux 457

0.1 raM, and increasing magnesium to 4 mM (in order to block exocytotic release of GABA), left the release during energy deprivation unchanged (reduced by 5%). When 150 mM mannitol was added during ED to dehydrate the tissue and prevent increase in cell volume and membrane tension, GABA release was unchanged. Finally, 1 ~M of TTX added during ED, reduced the GABA efflux by 52% (p < 0.01).

GABA Release from Xenopus Oocytes Expressing a High Affinity Transporter

Oocytes injected with 50rig of GAT-1 mRNA showed an uptake of up to 100 times higher than water injected oocytes. In order to demonstrate a voltage dependent reversal of GABA transport, the membranes of individual oocytes expressing the GAT protein were preloaded with 10 mM [3H]-GABA and depolarised by current injection and by high extracellular potassium. In addition, the intracellular sodium chloride concentration was increased by injection into the oocyte.

In the first set of experiments, the membrane was initially hyperpolarised to -80 mV, then depolarised to 0 mV in one step by current injection for 60 sec- onds, and then again hyperpolarised (Fig. 5). This procedure (n = 8) did not change the GABA release. In the second set of experiments, the perfusion medium was altered to contain 100 mM K + (at the expense of Na +) for 5 rain, and then again changed back to 5 mM (Fig. 6). This depolarised the oocyte membrane

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Fig. 5. Release of preloaded [3H]-GABA from Xenopus oocytes (n = 8) expressing the GAT-1 rat brain high affinity GABA carrier was measured by liquid scintillation counting. Depolarising the membrane by current injection to 0 mV for 60 sec did not release GABA. Values are presented as mean + standard error

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Fig. 6. Release of preloaded [3H]-GABA from Xenopus oocytes (n = 6) expressing the GAT-1 rat brain high affinity GABA carrier was measured by liquid scintillation couting. The perfusion medium was altered to contain 100 mM K + for 5 min (black horizontal bar) in order to depolarise the oocyte. Circles denote [3H]-GABA release in fmol/sec, squares denote membrane potential in mV. Depolarising the membrane by high extracellular potassium did not release GABA. Values are presented as mean +_ standard error

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Fig. 7. Release of preloaded [3H]-GABA from Xenopus oocytes was measured by liquid scintillation counting. NaCI injection (arrow) elevating the intra-oval concentration to - 1 0 0 mM generated a massive [3H]-GABA efflux in oocytes expressing the GAT-1 rat brain GABA transporter (circles). Substituting NaC1 injection with KC1 generated a similar [3H]-GABA release (squares). NaC1 injection into control oocytes that did not express the transporter protein did not generate measurable changes in release of [3H]- GABA (triangles). The plotted values are from three representative experiments

to ~ -5 mV (n = 6). It did not, however, cause release of detectable amounts of GABA.

In the third set of experiments, a sodium chloride solution was injected into oocytes preloaded with [3H]GABA by means of a pre-installed micro-elec- trode, elevating the intracellular sodium concentra-


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Fig. 8. Release of preloaded [3H]-GABA from Xenopus oocytes was measured by liquid scintillation counting. NaCI injection (arrow) (-100mM intracellularly) generated a massive [3H]- GABA efflux in oocytes expressing the GAT-1 rat brain GABA transporter (circles). When 135 mM (squares) and 270 mM (triangles) mannitol was added to the perfusing medium, [3H]-GABA release was attenuated or blocked. The plotted values are from three representative experiments

tion to ~ 100 mM (n = 8). This caused rapid depolarisation to 0 mV and a delayed (3-6 min) massive efflux of GABA, amounting to 100 times of the control release (Fig. 7). In contrast, no significant release occurred when water injected oocytes (i. e., without carrier) were injected with sodium chloride (~100 mM). When sodium chloride injection was exchanged with potassium chloride ( ~ 100 mM), the membrane was depolarised to -5 mV, and [3H]GABA released to the same extent as with NaC1 (Fig. 7). Sodium chloride injection was also performed in oocytes which were superfused by medium containing mannitol, to balance the increase in intracellular osmotic pressure. [3H]GABA release was attenuated by 135 mM and completely blocked by 270 mM mannitol (Fig. 8). Extracellular mannitol also improved recovery of the membrane potential.

Discuss ion

Release and uptake of amino acids in the nervous system are controlled by complex sets of mechanisms. Five different modes of amino acid release

have been postulated [7]: 1) exocytosis (synaptic vesicles), 2) carrier reversal, 3) channel diffusion, 4) spontaneous release, and 5) bleb formation. The for- mer three are controlled by the membrane gradient of ions, charge and solutes, the fourth occurs by an unknown, temperature insensitive mechanism, and the fifth is caused by severe energy depletion and cer- tain toxins.

The calcium dependent release of GABA which

occurred during brief high potassium stimulation, probably represents vesicular release from presynap- tic compartments [27, 46]. A calcium dependent release of GABA from synaptosomes of comparable magnitude was also seen (M. Larsen, T. S. Haugstad and I. A. Langmoen, unpublished results). The remaining 30% of GABA release that was not affect- ed when Ca ++ was removed from the medium, probably represents carrier mediated [46] or swelling induced [28, 51] release.

Carrier mediated release is considered to be evoked by one or more of three basic conditions [40]: 1) increased cytoplasmic concentration of the neurotransmitter (caused by inhibiting vesicular uptake, or by increasing the synthesis), 2) reduced Na + gradient or 3) extracellular presence of neurotransmitter analogues that are transported by the same carrier. Reducing extracellular Na + to the level of intracellular concentration by substitution with choline, increased the GABA efflux, in keeping with carrier mediated release. However, substituting sodium with the larger cation NMDG, did not affect the release of GABA.

High affinity transport of GABA has been extensively studied, and the carrier protein has been cloned [23]. It is known to depend on the presence of both sodium and chloride ions. According to the proposed stochiometry of the carrier, the extracellular concentration of GABA is predicated by the following equation [2]:

[GABA]o = [GABA]i([Na+]][Na+]o)2([C1-]][C1-]o)exp{VF/(RT) } (1)

where the subscripts "i" and "o" denote "intracellular" and "extracellular" concentrations of GABA, Na + and C1-, respectively, V is the membrane potential and T the temperature (in Kelvin). F and R denote the physical constants of Faraday and the gas con- stant, respectively. Thus, not only reduced sodium gradient, but also reduced chloride gradient should cause a carrier mediated release of GABA. According to this equation, choline and NMDG substitution for

T. S. Haugstad et al.: Ion Dependence of Brain GABA Efflux

sodium should cause a 850 and 250 times increase in extracellular GABA (smaller release with NMDG, since the sodium concentration was somewhat higher), whereas the observed value was a seven-fold and an unchanged release, respectively. Substituting chloride with the impermeant anion isethionate should increase GABA 30 times, whereas an eight-fold increase was observed.

In preliminary experiments we have observed that during conditions of ample energy supply, equilibrium between release and uptake is reached already by 10 minutes after the brain slices have been submerged in amino acid free ACSF. In situations where a large release is predicted, one may not obtain equilibrium within the 20 rain incubation time, due to limitations in the capacity of the release mechanism, or due to limitations to extracellular diffusion in the slice tissue. Still, if a single release mechanism is responsible, one should suspect that the observed values vary proportional to predictions. The predicted extracellular GABA values are different from the ones observed both in absolute and in relative terms. Thus, the observations question the validity of the concept of GABA release by carrier reversal in these situations. Since GABA transport by the high affinity carrier depends on the sodium ratio to the second power, sodium substitution should be a more potent stimulus for reversal of the carrier than chloride substitution, whereas the observed values were contrary to this notion. Even if a possible slight hyperpolarising effect of sodium substitution is taken into account [17], a large efflux is predicted. The different effect of different substituting cations is also difficult to explain in terms of release by carrier reversal, unless NMDG in some way should inhibit the carrier. One possible explanation is that low sodium medium inhibits re-uptake of released GABA. Assuming that there is a continuous basal release in the slice, and a continuous re-uptake, inhibition of re-uptake caused by low sodium concentrations will result in a net efflux from the slice. However, this does not explain why sodium substitution with NMDG does not increase the overflow of GABA from the slice. High affinity uptake occurs by cotransport of GABA and Na +, and it is quite unlikely that the large, membrane impermeant ion NMDG + is transported along with GABA instead of sodium, while the smaller choline is not. One possible solution to this question may be sought in the surface charge theory, since ion substitution may exert strong forces on membrane proteins [48]. This effect is caused by interaction of cations in

459

the medium with the negatively charged phospholipid groups on the membrane surface.

Veratridine is a plant toxin from species of the lily family (Veratrum) which opens sodium channels and causes intracellular sodium accumulation and membrane depolarisation [16, 31, 47]. Exposing slices to 50 pM veratridine for 20 min with synaptic release blocked, caused a --15 times increase in extracellular GABA. Assuming that the intracellular sodium concentration rises to 70 mM [47], and the membrane is depolarised to - 0 mV, equation 1) predicts carrier reversal and 150-fold increase in extracellular GABA. TTX, on the other hand, is a fish poison which blocks sodium channels (both inactivating and non-activating) and prevents sodium influx [31]. Neurons are unaffected or slightly hyperpolarised by TTX [17, 66], whereas glial cells depolarise [56]. (The latter effect is caused by blocking voltage dependent sodium channels, which in gliai cells seem to serve the purpose of regulating the intracellular sodium concentration, and prevent that sodium is depleted via the membrane Na*/K%ATPase [56, 57, 61].) According to equation 1), lowering intracellular sodium would lead to increased GABA uptake. Assuming that TTX leads membrane depolarisation even to 0 mV [56], the extracellular GABA concentration still would be reduced by 20%. Instead, TTX caused a four-fold increase in GABA release.

The release evoked during energy deprivation showed the same puzzling difference in results with different substituting ions. Energy deprivation causes membrane depolarisation [6], increase in extracellular potassium, inward flow of sodium [26], and cell swelling [38, 68]. Nerve cells and glia react different- ly to these conditions, since they possess different sets of ion channels. Substituting sodium with choline at the same time as the slices were exposed to ED, modestly increased the GABA release, while substitution with NMDG caused a marked reduction. Again, the changes observed were not in proportion with predictions from thermodynamic principles, since the choline and NMDG-containing media would be expected to increase extracellular GABA during energy deprivation considerably (ten-fold to a thousand fold, depending on the estimates made for intracellular sodium concentration). However, TTX attenuated the GABA release to an extent comparable with predictions.

The effects of TTX on energy deprivation leaves the possibility that GABA is released by carrier reversal an open option. TTX prevents increase in intracel-

460

lular sodium concentrations which would be necessary for reversal of the GABA carrier. This is in keeping with the results of other authors, demonstrating that TTX and drugs that block sodium channels, improve neuronal recovery, and attenuate ischaemic glutamate release [12, 47, 52, 61, 62]. However, the effect of choline and of NMDG substitutions are difficult to explain by this model. Again, the effect of choline could be explained as increased overflow due to decreased uptake, but if this was the case, one would expect the more impermeant NMDG ion to cause the same increase in release. Another option would be that decreased extracellular concentration of sodium depletes intracellular sodium when cation channels open during ischaemia. This could possibly lead to reduced sodium dependent GABA binding to an intracellular site on the GABA carrier. But then again choline substitution should lead to a more pro- found depletion, and less GABA binding and release than with NMDG substitution (due to lower sodium concentrations). One possible explanation would be that the substituting cations and sodium channel blockers influence the release of GABA by acting on structures on the outside of the plasma membrane different from the voltage gated sodium channels, such as a cation recognition site on carriers or channels associated with amino acid transport.

When the intracellular GABA was measured (see ref. [29] for discussion of the relationship between tissue content and intracellular amount), it was found that energy deprivation left the content unchanged, whereas the total amount of GABA was doubled. Increase in the intracellular content of neurotransmitters is considered a main cause of release by carrier reversal [40]. Thus, this result is consistent with the theory of carrier mediated ischaemic amino acid release. However, we have shown earlier that when mannitol was added during energy deprivation, the released amount of GABA was unchanged, whereas the intracellular content was reduced by 32% [29]. Mannitol also increased the permeability of several other amino acids, including aspartate and glutamate. Taken together, the results from the brain slice experiments favour the opinion that GABA is released from brain tissue during energy deprivation by a different pathway, and not solely by a mechanism following Michaelis-Menten kinetics.

Since Guastella and collaborators first cloned the GAT- 1 high affinity GABA transporter [23], a number of other carriers of GABA and other amino acids have been cloned [19, 22, 34, 42, 49, 58, 65]. Whereas high

T. S. Haugstad er aL: Ion Dependence of Brain GABA Effiux

affinity glutamate carriers cotransport sodium ions [3, 46], GABA transporters are sodium and chloride dependent [23, 44]. For some time a fixed stochiometry has been assumed for the amino acid transport [4, 8, 10, 11, 60], but recent reports challenge this concept [13, 191.

The oocyte experiments demonstrate that membrane depolarisation and high extracellular potassium does not reverse high affinity GABA transport. According to equation 1), membrane depolarisation to 0 mV by either K + or current injection should increase extracellular GABA twenty-fold. Chloride injection should increase efflux 400-fold, and injection of sodium and chloride (to an estimated intracellular concentration of 100 mM) 6000-fold. Injection of potassium should not affect the release according to theory. The observations show that potassium injection has the same effect as sodium injection, as both sodium and potassium injected together with chloride to an estimated intracellular concentration of 100 mM of each ion species generate a 100-fold increase in release.

Moreover, when osmotic forces were corrected for by adding mannitol to the extracellular medium, the release was blocked. This means that during conditions of membrane depotarisation, a relative intracellular hyperosmotarity must be present in order to generate outward GABA transport. Apparently, the functional state of the GAT-1 transporter is altered by the osmotic imbalance or factors related to it. At present, the mechanism leading to altered function is open to speculation, The membrane stretch caused by increased interior osmotic pressure could generate mechanical forces that act on the protein structure. In fact, mechanosensitive channels were first discovered in excitable cells [24], and are known to exist in neurons [5] and glial cells [33]. Another possibility is that second messenger systems activated in the oocyte by the osmotic stress alter the function of the carrier protein by chemical means, for example through phosphorylation [14, 15] or that the protein is altered by changes in the oxidative state of thiol groups [64].

The deviation of the present results from the enzyme kinetic model for carrier function supports notions espoused by other authors. Fairman et al. [19] have demonstrated uncoupled anion currents linked to high affinity glutamate transport. Another perspec- tive on amino acid transport has been presented by Cammack e t al . [13], who have investigated the currents generated by GAT-1 GABA carrier expressed in a cell line while changing the extracellular and intra-


cel lular concentra t ions of ions and GABA. They f ind

the GAT-1 G A B A carrier may best be described as a

channel or pore with compl ica ted gating mechan i sms

on either side of the membrane . M e m b r a n e depolari-

sation keeps the inside gate closed, but Na + and C1-

ions may act together to keep it open. Their interpre-

tat ion implies that G A B A transport does not fol low

Michae l i s -Menten kinetics. Rather, it must be consid-

ered as a complexly control led process of ion cotrans-

port. Under pathological condi t ions, when the intra-

cel lular concent ra t ion of Na + rises, G A B A efflux is

facilitated. This model explains some of the discrep-

ancies f rom predict ions made by enzyme kinet ics

observed in the present study. However, it does not

satisfactorily account for the b locking effect of extra-

cel lular mann i to l on G A B A release.

Taken together, the present results suggest that the

high affinity G A B A carrier, in addit ion to proper ion-

ic condi t ions, requires an osmotic imbalance in order

to reverse G A B A transport. However, together with

the observat ion that t issue dehydra t ion by manni to l

does not prevent G A B A efflux dur ing energy depriva-

tion, reversal of this carrier is apparent ly not the only

factor responsible for ischaemic G A B A efflux. Rath-

er, G A B A release from ischaemic bra in seems to be

control led by more complex mechanisms . The

present results also ques t ion the val idi ty of a pure

Michae l i s -Menten model of brain amino acid trans-

port.

Acknowledgement This work was supported by The Norwegian Council on Cardi-

ovascular Diseases. The GAT-1 clone [23] was supplied by J. Guastella, Pasadena, USA. We are thankful to J. Berg-Johnsen, Oslo, and professors Jon Storm-Mathisen, Oslo, John Hablitz, Birmingham, USA, and David Attwell, London, UK for useful comments on the manuscript. Professors A. O. Aasen and H. Nornes have provided excellent working facilities.

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C o m m e n t s

The authors put forward the hypothesis that cell injury in the brain from, e. g., ischaemia is attributable to a massive influx of ions, in this case of chloride. The concept was developed with regard to observations that glutamate analogues which are injuring hippocampal neurons are active only in the presence of C1- -ions in the exn-acellular space. Since Cl--influx is coupled to activation of GABA receptors, a relationship can be conceived for respective investigations. In other words, if the GABA-dependent ion channel is blocked, or if CI- -ions are omitted from the medium, ischaemia may not induce neuronal damage. This is, indeed, an interesting alternative to the classical concept that onset of ischaemia induces terminal depolarisation of neuronal cells (probably also of glial cells), thereby leading to a massive efflux of excitatory transmitters

and K+-ions, whereby Na +- (and CF)-ions are shifted into the intracellular compartment. Currently, there is more evidence that the glutamate (or excitatory transmitter)-dependant influx of Na § -ions under these circumstances is the leading step followed by influx of e l - -ions for maintenance of electroneutrality. The hypothesis of the article favours instead glutamate- and Na+-ion independent mechanisms, emphasising an involvement of GABA and CI -ions - so far, so good.

The authors have established a neat in vitro technology for measurement of release of GABA as end point. The preparation was modified and exposed to various conditions to find out the mechansims enhancing, or inhibiting efflux of GABA into the medium. In addition, the high affinity GABA-carrier GAT-1 was injected into Xenopus oocytes also for analysis of the release of radioactively labelled GABA. Altogether, the article is a compre- hensive report on a great number of experiments, with exchange of C1 - or Na+-ions against non-permeable substitutes (isethionate or choline), which all were enhancing release of GABA. Even lowering of the osmolarity was raising efflux of GABA, which could be blocked by normalising the osmoconcentration by mannitol. The same is true for energy failure (omission of glucose and O2-free atmosphere). In essence, the authors can largely reject their working hypothesis that the ischaemia-induced release of GABA must be attributed to a reversal of the high affinity GABA transporter alone. Rather, other complex mechanisms seem to be involved. I found it difficult to understand, why replacement of C1-- or Na § ions, or induction of energy failure all were enhancing efflux of GABA. Assuming that a major step for the influx of Na+-iens following induction of ischaemia is the terminal depolarisation with massive release of glutamate, I wonder, whether the GABA hypothesis of cell swelling by massive influx of C1- -ions is really necessary, as the latter ions have to follow the Na+-ions for reasons of electroneutrality (cf. above).

A. Baethmann

Correspondence: Tor S. Haugstad, M.D., Ph.D., Institute for Surgical Research, The National Hospital, Rikshospitalet, N-0027 Oslo, Norway.

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