Inhibition of y-aminobutyric acid uptake into astrocytes by pentobarbital

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Dc7partment of Aizatomy, University o f Saskatchewan, Suskatoon, Susk., Canada S7N OW0 AND

B. R. SASTRY~ Departmetzt of Physiology, C o k l ~ g eo f Medicine, Ur~iversityo f S a s k a t c h e ~ ~ a n , Sask~itoon,Sask., Canada S7N OMfO Received August 11, 1978

HERTZ, L., and SASTRY, B. R. 1978. Inhibition of y-aminobutyric acid uptake into astrocytes by pentobarbital. Can. J. Physiol. Pharmacol. 56, 1083-1087. Pentobarbital (0.5-2 m M ) , but not phenobarbital, was found to inhibit the uptake of y-aminob~~tyric acid into mouse astrocytes in primary cultures by up to 45%. This inhibition was additive to a reduction in uptake rate caused by excess potassium. Its possible role in the pharmacological action of pentobarbital is discussed.

Introduction That uptake of GABA into glial cells may play a major role in termination of the transmitter activity of this amino acid was first suggested on the basis of experiments with bulk-prepared glial cells (Henn and Hamberger 197 1; SelIstrom and Wamberger 1975). Later, this concept has been supported by studies on peripheral ganglia (Bowery and Brown 1972; Youiig et 01. 1973; Roberts 1976), rat retina (Neal and Tversen 1972; Marshall and Voaden 1974), glioma cell lints (Hutchison e f al. 1974; Schrier and Thompson 1974; Schubert 1975 ) , spinal cord explant cultures (Hosli et al. 1972), and primary cultures of astrocytes from the cerebral hemispheres (Schousboe, Hertz et al. 1977). The Tatter preparation is outstanding in showing an intense accumulation of GABA, which is inhibited by high concentrations of potassium as well as by certain drugs, e.g., nipecotic acid (Schousboe, Hertz et al. 1977; Schousboe et al. 1978) and represents a net uptake, not a homoexchange (Hertz, Wu et al. 1978). Presynaptic inhibition in the spinal cord and postsynaptic inhibition of mitral cells in the olfactory bulb are supposed to be mediated by GABA (Schmidt 1964; Curtis et al. 1971; Nicoll 197 l ) , and the duration of both these responses is prolonged by barbiturates (Eccles e f al. 1963; Nicoll 1972). This led Cutler and co-workers (Cutler and Bodzinski 1974; Cutler et al. 1974) to study the ABBREVIATIONS: GABA, y-aminobutyric acid; MEM, minimum essential medium, dBcAMP, dibutyryl cyclic AMP. 'Author to whom correspondence should be addressed. 'Canadian Medical Research Council Scholar.

effect of barbiturates on GABA uptake into brain slices. They reported that pentobarbital competitively inhibits the uptake of GABA and concluded that this might be due to an illhibition of the uptake of GABA into presynaptic terminals (Cutler et al. 1974). In view of the intense net uptake of GABA into astrocytes, the effect of certain other drugs on this uptake, and an effect of barbiturates on astrocytic metabolism (Roth-Schechter and MandeB 1976; Roth-Schechter et al. 1976; Hertz, Mukerji et al. 1978) it seems, however, likely that an inhibition of GABA uptake into astrocytes could, at least partly, explain the prolongation of GABA-induced inhibition. To test this possibility, the effect of pentobarbital on GABA uptake into astrocytes in primary cultures was studied in the present work.

Methods and Materials Cult14re Primary cultures of astrocytes were prepared essentia!ly as described by Booher and Sensenbrenner (1972) but with some minor modifications. The areas superficial to the lateral ventricles were carefully dissected out from brains s f newborn DBA mice and, after the meninges had been cleaned off, placed in a modified Eagle's MEMhwith 20% horse selum4 (7.5 ml/ brain), cut into small pieces, disintegrated by the vortex method (Rullaro and Brookman 19761, and passed twice through sterile nylon nitex sieves with pore sizes of 80 km (first sieving) and 10 pm (second sieving). Two and one-half millimetres of this cell suspension was planted in "agle's (1959) MEM with twice the original concenti-ations of amino acids, except glutamine ( 2 m M ) , and four times the original concentrations sf vitamins. No GABA is added to this medium, but a low concentration of GABA is present in the serum added (Wu ct ak. 1978). 'A few cultures, included in Fig. 1 but not in Table 1, were grown with 20% fetal calf serum as previsusly described (Hertz, Mukerji et al. 1978).



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a 60-mm Falcon plastic petri dish and incubated a t 37°C in a 95%-5% (v/v) mixture of atmospheric air and COz. The culture meditrln was changed after 3 days and subsequently two times per week. After 1 week the concentration of serum was reduced to l o % , and after 2 weeks the cultures reached confl~aency. They were thereafter in general cultured for 2-3 more weeks in a MEM medium with 0.25 m M dBcAMP and 5% horse serum. Treatment with dBcAA4P has previously been found to induce a morphological and biochemical differentiation of cultured astrocytes (e.g., Schousboe, Bock rt crl. 1977).

t i p take Thirty minutes before the uptake experiment, the medium was replaced with a serum-free MEM containing the desired concentration of unlabelled GABA and of pentobarbital o r phenobarbital. After 30 minutes of preincubation (at 37°C) this medium was replaced with 3 rnl of an identical medium (same concentrations of GABA and the barbiturate) containing '4C-labelled GABA ( [ I -'T]GABA, 49.4 mCi/mmol ( I C i 1- 37 G B q ) ) and the uptake of ''C during 5 min was measured at 37°C. Previous experience (Schousboe, Hertz et ak. 1977) has shown that the GABA uptake is rectilinear during this length of time, i.e., that initial uptake rates are measured. Thereafter. the cultures were rapidly washed twice with nonradioactive, cold medium, and 1.5 in1 1.0 !\/I NaOH was added. Radioactivity was counted in the dissolved cultures and in the incubation media by aid of a scintillation counter (Nuclear-Chicago Isocapi300). and the protein content was determined using a conventional Lowry technique (Lowry et ul. 1951). Uptake rates were calculated from the uptakes of "C into the tissue and the specific activities in the media. Slip plies Falcon plastic tissue culture dishes (No. 3002) were purchased from Falcon (Oxnard, CA). and nylon nitex from 'Fobler, Ernst and Traber (New York, NY); horse serum was supplied from the Department of Anatomy, all amino acids and vitamiins from Sigma Chemicals Company (St. Louis, M O ) , and the radioisotopes from New England Nrnclear Corp. (Boston, M A ) . All other chemicals were of purest grade available from regular commercial sources.

Results Table 1 shows the uptake of GABA (nanomoles per minute per milligram protein) into astrocytes incubated in a medium containing 20 p M GABA and either 5 or 25 mlLa potassium as well as either 0 or 2.0 m!W pentobarbital. The medium with the low concentratio~zof potassi~lmand without pentobarbital is identical with that previously used to determine the kinetics of GABA uptake into astrocytes grown with fetal calf serum5 (Schousboc, Hertz et a" 1977' Hertz' Wu 1978)' a'1d the present protein at of Oa074 nmO1/min per a GABA concentration of 20 p M is in reasonably


'In contrast with the present work, no preincubation with unlabelled GABA was used by Schousboe, Hertz ~ ' 6ul. (1977). Little, if any, alteration o f the uptake kinetics is, however, provoked &y this difference.

Pentobarbital concentration, mM

FIG.1. Inhibition of GABA uptake as a function of the pentobarbital concentration at GABA coilcentrations of 5 20 (Q), o r 50 ( @ ) /M.The inhibition is expressed as a percentage of the uninhibited value. i.e., the G A n A uptake without any pentobarbital in the medium, and the inhibitions observed in the individual experiments were calculated in relation to the uninhibited valaies of the same batch. Results shown are mean rk SEM and the aver;iges of three to seven individual experiments. At the last point ( 2 mM) the SEM for the 20 ,uM values is shown in a clownward direction and that for the 5 ,LLMvalues is an upward direction.


good agreement with the previous determination o f a K,,, of 40-45 p M and a V of 0.35-0.40 nimol/rnin per milligram p r o t e i ~ This ~ . uptake was inhibited ky about 35 5% in the presence of 2 nlM pentobarbital (Table 1, left column). The inhibition was at least as pronounced (e.g., 47.1 7;at a GABA concentration of 20 p!W and a pentobarbital concentration of 2 m M ) when cultures were used that had not been exposed to dBcAMP during the culturing. The effect of 0.5-2.0 mM pentobarbital in the presence of different GABA concentrations is shown in Fig. 1. It can be seen that 2 mh/l pentobarbital exerted approximately the same degree of inhibition regardless whether the GABA concentration was 5, 20, or 50 pM,%hereas lower concentrations of pentobarbital (0.5-1 mM) seemed to lead to a rela"The is even closer than that shown in Fig. 1 sirice no attempt was made to sllbtract the nonsaturable part of the GABA uptake, which is negligible at 5 and 20 il~l GABA but not quite negligible (about 15% of the total uptake) at 50 p:M (Schousboe, Hertz tJf rrl. 1977). The inhibition of 30% of the total uptake with 50 p M GABA shown in Fig. 1 thus corresponds to an inhibition of the saturable uptake of ahout 35%.


TABLE 1. Uptake of GABA (nanomoles per minute per milligram protein) ints cultured GABA in the presence or absence of astrocytes exposet1 to 5 or 25 m M potassium and 20 2 m M pentobarbital

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Potassium, m M

Pentobarbital. mM 0

2 .0 Perltobarbital inhibition, p$ ---



0.0742 + 0 -0054 (7) 0.0470.+0.0013 (3)

0 .0557 i0.0005 (3) 0.03691fr0.0035(9)

37 (I" -


< 0.01)

34 ( P

Potasslunl inhibition, C, 25 ( P 21 ( P

< 0.02) < 0.05)

< 0.01)


Nure: Initial uptake rates for [I-'4C]CABA were measured as described i n the text. Inhibition by 25 m M potassiulrm (compared with 5 m h d ) o r 2 mMpentobarbital (compared with no pentobarbital) is expressed as 3 perce~ltage of the uninhibited uptake rate. 'The statistical significance of the inhibition (but not its nu~nericalvalue), calculated by Student's t test, is given in parentheses.

tively more pronounced inhibition at the lower concentrations of GABA. This suggests that part of, but probably not the entire pentobarbital action is due to a competitive inhibition. Even 0.5 mil4 pentobarbital did, however, inhibit the GABA uptake significantly at all three GABA concentrations tested (Fig. 1). Phenobarbital did not share the inhibitory effect of pentobarbital cince the uptake of GABA was not inhibited by 1 m1V of this drug. A rise of the potassium concentration in the incubation medium from 5 to 10 in!V had little, if any. effect (results not presented), but an increase to 25 m!V led. in accordance with previous results, t c ~ an inhibition of the uptake of 25% (Table 1. upper line). Pentobarbital, at a concentration of 2 mM, caused approximately the same inhibition in the potassium-rich medium as in the medium with a normal potassium concentration (Table 1, lower line). Thus, the effects by pentobarbital and by potassium seem to be additive. as confirmed by the fact that the difference between the GABA uptake with and without excess potassium in a medium containing 2 mM pentobarbital (Table 1, middle line) was statistically significant.

Discussion The present experiments have shown that pentobarbital causes an inhibition of the uptake of GABA into astrocytes in primary cultures. This raises two questions, i.e., is this effect limited to astrocytes, and does it play any role in the phartnacological action of pentobarbital. The first of these questions can be at least partly answered by comparing the GABA uptake. and the effect of pentobarbital on this uptake, in astrocytes and in more complex preparations containing both neurons and glial cells, e.g., brain slices. As previously discussed (Schousboe. Hertz et a?. 1977; Hertz 1977). the GABA uptake ints astrocytes is an

intense net uptake, but is not quite as intense as the uptake into brain slices or synaptosomes. This suggests that a substantial amount of GABA is accumulated also into neuroniil constituents, and it is in keeping with this concept that nerve terminals also become labelled after exposure to [WIGABA (Storm-Mathisen 1976). An additional neuronal uptake does, however, not preclude that pentobarbital might affect exclusively the uptake into astrocytes, and the pentobarbital :GABA ratio required to exert an inhibition of 30-40% is, if anything, lower for astrocytes than for brain slices (compare present results with those of Cutler et al. 1974). The fact that the inhibition observed in the present study (30-45 % ) is approximately similar to that (4045% ) observed in brain slices (Cutler et a?. 8974) suggests, nevertheless, that pentobarbital affects both neuronal and astrocytic uptake. Such an effect on both cell types is typical for several drugs affecting GABA uptake, e.g., nipecotic acid and guvacine (Schousboe 1979). and it inight be of importance that pentobarbital resembles these compounds in being a heterocyclic six-membered ring. In contrast with pentobarbital, phenobarbital did not inhibit the GABA uptake. This distinction between pentobarbital and phenobarbital effects is at variance with the barbiturate action on energy metabolism since the potassium-induced stimulation of respiration in astrocytes (and brain slices) is affected by both pentobarbital and phenobarbital (Hertz, Mukerji et al. 1978; Hertz, E., and Hertz, L.: unpublished experiments). Also, the observation that the inhibitory action of pentobarbital and potassium are independent of each other suggests that there is no direct correlation between the effect of pentobarbital on GABA accumulatioil and on potassium-stimulated oxygen uptake. It seems likely that the pentobarbital effect on astrocytic uptake of GABA is of importance for the in v i ~ waction of the barbiturate since postsynaptic



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inhibition in the cat hippocampus and the depolarizing action of GABA on sympathetic frog ganglia are enhanced by pentobarbital concentrations of 0.1-0.2 m/M (Nicoll et al. 1975; Nicoll 1978). These conceiltrations are similar to those expected under anaesthesia (Richards 1972) and oilly slightly lower that that of 0.5 rnA4 found in the present study to have a distinct effect on astrocytic uptake of GABA. Nipecotic acid, which inhibits astrocytic as well as neuronal uptake of GABA (Schousboe 19791, causes analogously a prolongation of the depolarizing response to a low concentration of GABA in isolated rat superior ganglia (Galvan 1 9771, and this drug resembles pentobarbital in potentiating the action of GABA on afferent excitability in the feline spinal cord (Sastry 1978). The concept that the effect by either pentobarbital or nipecotic acid on spinal cord at least partly is due to interference with the termination of GABA activity is further supported by the observation that it is reduced by pretreatment with semicarbazide (Sastry 19781, a drug known to inhibit the in vivs formation of GABA and deplete the content of GABA in the spinal cord (Bell and Anderson 1972). Since phenobarbital does not inhibit the uptake of GABA, the electrophysiological response to GABA should not be affected if phenobarbital is used instead of pentobarbital. It is, therefore, also indicative of a functional role of the inhibition by phenobarbital that MacBonald and Barker (1978) recently have reported that pentobarbital, but not phenobarbital, markedly prolongs the response of cultured spinal cord ileurons to added GABA. It is, however, uncertain to what extent alteration of GABA uptake could contribute to this alteration (Ransom and Barker 1976).

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Inhibition of gamma-aminobutyric acid uptake into astrocytes by pentobarbital.

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