Relationships between ethylenediamine and GABA transport systems in rat brain slices

Neurochemistry blternational, Vol. 5. No. 1. pp. 57 to 64. 1983. Printed in Great Britain.

0197-0186/83/010057-08503.00/0 © 1983 Pergamon Press Ltd.

RELATIONSHIPS BETWEEN ETHYLENEDIAMINE A N D GABA T R A N S P O R T SYSTEMS IN RAT BRAIN SLICES L. P. DAVIES, C. A. DREW, S. CHEN CHOW, J. H. SKERRITT and G. A. R. JOHNSTON Department of Pharmacology, University of Sydney, N.S.W. 2006, Australia (Received 1 June 1982; aecepted 22 June 1982)

Abstraet--[14C]EDA was accumulated by slices of adult rat cerebral cortex, although the tissue:medium ratios achieved were very much lower than those for GABA. EDA uptake was temperature dependent and appeared to take place by both sodium dependent and sodium independent mechanisms. Kinetic analysis of the uptake revealed a major low affinity component with an apparent K m of 1.11 + 0.05 mM and a Vmax of 9.8 + 0.2/~mol,,' h/g wet wt, with a second site of K m about 20/~M but a 50 fold lower Vm~~. Inhibition studies indicate that EDA may be transported in part by the 'small basic" amino acid transport system and in part by polyamine systems shown to be present in CNS tissue. High levels of displaceable binding of radioactive EDA to glass-fibre filters were observed; studies using [~4C]EDA may be complicated by binding to tissue macromolecules. Potassium stimulated, calcium dependent release of radioactivity from brain slices labelled with [~4C]EDA in the presence of sodium ions was observed. Extracellular EDA stimulated the release of [3H]GABA and [3H]beta-alanine from preloaded slices, although GABA and beta-alanine did not stimulate [~4C]EDA release. It appears that extracellular EDA can counterexchange with intracellular GABA or beta-alanine, but that EDA which is accumulated by the tissue may then be bound or move to pools not directly accessible to these amino acids. Ouabain released radioactivity from slices labelled by [~4C]EDA in the presence of sodium but not from slices labelled in the absence of sodium. These results suggests that EDA is not acting simply as a substrate for GABA transport sites.

W h e n applied by iontophoresis to single cells in the cerebral cortex or globus pallidus of rats, ethylenediamine (EDA) is an inhibitor of neuronal firing with a potency comparable to that of g a m m a - a m i n o b u tyric acid (GABA) (Forster, Lloyd, Morgan, Parker, Perkins and Stone, 1981). The observation that this action is blocked by bicuculline, a c o m p o u n d widely regarded as a relatively specific G A B A antagonist (e.g. Curtis, Duggan, Felix and Johnston, 1971) prompted a more detailed examination of EDA and various diamine analogues as possible GABA-mimetics (Perkins, Bowery, Hill and Stone, 1981). Of a series of compounds, only EDA, piperazine and 1,3-diaminop r o p r a n e had significantly inhibitory effects on cell firing (Perkins and Stone, 1982). In other systems, EDA has been reported to cause an increase in the rate of basal [14C]GABA release from prelabelled, perfused slices of mouse brain (Forster et al., 1981), to weakly inhibit radioactive GABA uptake in rat and mouse brain slices (Forster et al., 1981; Davies, Hambley and Johnston, 1982), to weakly inhibit [ 3 H ] G A B A binding to rat brain G A B A receptors and to act as a GABA agonist, albeit with low potency, in

enhancing [3H]diazepam binding to well-washed rat brain m e m b r a n e s (Davies et al., 1982). We have investigated the uptake and release of [14C]EDA, to determine whether E D A is a substrate at GABA uptake and release sites.

EXPERIMENTAL PROCEDURES Uptake studies

Sprague Dawley rats were killed by decapitation and their brains rapidly removed on ice. Cerebral cortices were dissected flee of white matter and chopped into small prisms (0.1 x 0.1 x ca. l mm) using a McIlwain tissue chopper. Slices were suspended in Krebs bicarbonate Ringer and washed 4-5 times by decantation. Assays were carried out in a final volume of 2.5 ml and contained 0.8/~M [14C]EDA (0.05 ~Ci)and 100 ~1 of slice suspension (approx. 10 mg wet wt of tissue). Slices were preincubated with shaking for 8 min before addition of radioactivity and incubation for a further period. Incubations were either carried out at 37°C or on ice. Blanks, containing an excess of EDA (10 mM), were incubated on ice. Assays were terminated by the addition of 2.5 ml ice-cold Ringer, rapid filtration on glass-fibre filters (Whatman GF/A or GF/C) and tubes and filters washed by a further 2 × 3 ml washes 57

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with ice-cold saline. Radioactivity on discs was determined b 3, liquid scintillation counting.

R cteasc st udic,, The procedure for the release studies was as described previously (Davies, Johnston and Stephanson, 1975). Cortical slices (50rag wet wt of tissue in 500/d of bufferi were added to 5 ml of gassed incubation medium and after a 10 min preincubation at 37 C, the appropriate radioisotope ([*~'(']EDA (0.375 p('il, [3H]GABA (0.5 HCi) or [3H]betaalaninc (l.25ttCil) was added and the slices incubated lot a further 20 rain. The slices were then collected by ~acuum tiltratiou on W h a t m a n GF/A filter discs (2.5cm diameterL washed with medium (10 ml tit 37 ('1 and transferred to filter holders (Swinnex 25, Millipore Corp. I. These lissuc "beds' were then perfuscd with medium (37 C. 0.5 ml, rain) and fractions of perfusatc collected every 3 ram. [~H]GABA and [-~H]beta-alanine prelabelling and release were carried out in the presence of amino-ox',acetic acid i20ttM linal conccntrationL to reducc metabolism of the radioactivity. The incubation and perfusion media used were either a Krcbs bicarbonate buffcr, a sodium-free modilication of this buffer, or a phosphate buffered solution. The Krebs bicarbonate buffer (carbogenated) contained lin mM): NaCI 1120), KCI (3.0i. CaC1 e (I.51, MgCIe (1.2), l)-glucose II0), NaHPO.~ (1.2) and NaHCO3 1251. In "sodium-free" buffers. NaCI. NaHCO3 and N a H P O 4 were omitted and

iso-osmotic sucrose and J'ris HCI buffer 125mM) used instead. The phosphate buffer (oxygenated) contained Na('l (I 18,5), K('I i4,75), ('a('l e (1.33), MgCIe (1.18), b-glucose (5.8t and sodium phosphate, pH 7.4 (15.4). The a m o u n t of each isotope released per minute ~as expressed as the 'fractional rate constant', which represents the radioactivity released in each fraction determined as a proportion of that present in the tissue tit the time of release.

,~ldleria/s 1"~C]EDA (25 mCi, mmoli and [ 3H ]beta-alanine 14[ .5 ( ' i rmllol) were from Amersham Internatiomd Ltd ( U . K . ) a n d [q-I]GABA ( 9 7 . 0 C i m m o l ) were from New England Nuclear (Boston). EDA (A.R. grade) was from Merck (l)armstadl); all other chemicals wcrc from Sigma Chemical Corp. RESULTS

Uptake of EDA Slices i n c u b a t e d with [ 1 4 C ] E D A at 3 7 C s h o w e d a significant time d e p e n d e n t a c c u m u l a t i o n of radioactivity {Figs IA a n d 3A). At 0 ' C there was little or no time d e p e n d e n t a c c u m u l a t i o n of radioactivity: however, even at "zero-time" (0-2 s between a d d i t i o n of

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Fig. I(A). Time course of [~*C]EDA accumulation in rat brain cortex slices. Uptake was measured at 37°C (@), at 0°C (11) and at 0°C in the presence of excess (10raM) E D A (O). The difference between accumulation at 0°C and blanks was shown to be due to displaceable binding to the glass-fibre filters used in the assay. (B). [14C]EDA uptake measured after a 30 min incubation in buffers in which sodium-ion concentration was varied from 0 to 146.2 mM. Other details as for (A). Each point is the mean ( _+S.E.M.) of quadruplicate determinations.

Ethylenediamine uptake and release

10(

1

radioactivity and filtration), there was significant radioactive accumulation in the slices relative to blanks (samples incubated at 0°C with excess unlabelled EDA), both at 0°C and at 37°C (Figs 1A and 3A). This contrasts with [ 3 H ] G A B A uptake where no difference between zero-time accumulation of radioactivity at 0~C and zero-time blanks (incubated at 0 C and containing an excess of unlabelled GABA) was observed (see Fig. 2). At comparable times (20min; see Fig. 2) the tissue to medium ratio for EDA was very much lower than for GABA. In experiments performed without slices, excess EDA significantly displaced [14C]EDA ' b o u n d ' to filters, sufficient to explain an apparent zero-time accumulation of EDA.

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Sodium dependence of EDA accumulation

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Fig. 2, 'Time course of [3H]GABA uptake in rat brain slices at 37'C (O), 0c'C (11) and at 0'C in the presence of excess (1 mM) GABA (©), measured as tissue:medium ratios (calculated assuming tissues are 80%, waterl. For comparison the tissue:medium ratios achieved for EDA after 20 min incubation are given (&). Each point is the mean of quadruplicate determinations.

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In view of the known sodium-dependence of GABA uptake, the effect of sodium depletion on [ ' 4 C ] E D A accumulation into slices of rat brain was tested (Fig. 1B). While changing the sodium concentration from 146 to 0 r a M had no effect on blanks (samples incubated at 0 C with excess unlabelled EDA) there was actually an increase rather than a decrease in [14C]EDA accumulation in slices incubated in the absence of sodium (Fig. I B). It should be noted that the results obtained in Fig. I(B) were obtained after a 30 rain incubation period. To clarify this effect further

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Fig. 3(A). Time course of [~'~C]EDA uptake in rat brain slices (details as for Fig. 1). In the same experiment slices were also incubated in sodium free medium. (B). The time course of uptake followed. Each point is the mean of quadruplicate determinations.

60

L.P. DAVlI:Set al.

a time-course of accumulation was carried out in sodium-free buffer (see Fig. 3B). A time-dependent increase in radioactivity was observed both at 0 and 3 7 C . The amount of [~4C]EDA found in slices at zero time was significantly higher than those levels found in slices incubated in normal, sodium-containing buffer at zero-time. It was not until longer incubation times l > 3 0 m i n ) that the accumulation of [~'~C]EDA was greater in normal than in sodium-free buffer (Fig. 3A and B). Thus the apparent sodiumdependence of EDA uptake will depend on the time at which uptake is measured. Ouabain t l raM) inhibited [~4C]EDA uptake by 77".;I after 20 min incubation (Table 11.

Table 1. Inhibition of temperature-dependent EDA accumulation by rat brain slices Compound

Per cent inhibition

Monoamino compounds GABA Beta-alanine Glycine Glutamate Taurine L-Histidine cis-3-Aminocyclohexanecarboxylic acid 3-Aminopropane sulphonic acid O-Phosphoethanolamine Nipecotic acid Isonipecotic acid Piperidine Piperazine

29.1 ± 5.8 9.4 + 3.7 I3.5 ± 4.8 N.S. 11.3 _+ 4.2 26.0 ± 5.2 29.7 ± 2.7 N.S. 21.0 +_ 4.0 36.0 ± 3.3 N.S. N.S. N.S.

Diamino compounds Ethylenediamine 1.2-Diaminoethylphosphonic acid 2,4-Diaminobutyric acid 1,3-Diaminopropylphosphonic acid DL-Diaminopropionic acid

61.7 31.6 66.7 I9.0 47.4

_+ 3.7 + 7.1 ± 5.7 _+ 3.6 ± 3.2

Polyamino compounds Spermine Spermidine phosphate Putrescine dihydrochloride

46.5 + 3.4 50.4 ± 3.9 22.5 ± 2.0

Drugs Bicuculline methochloride p-Chloromercuriphenylsulphonic acid Ouabain (l.0mM)

N.S. 89.3 ± 1.4 77.5 ± 5.9

Unless otherwise stated, the above compounds were tested at 100/~M against the uptake of [~4C]EDA into rat brain slices. Inhibitors were preincubated with the slices for 8 rain prior to addition of radioactivity and incubation continued for a further 20 rain. Incubations were carried oat at 3T'C. Blanks, containing radioactivity were incubated for 20 rain on ice.

The effbct q]buffers on EDA uptake If uptake experiments were carried out in a phosphate-buffered medium instead of Krebs bicarbonate buffer, there was virtually no temperature dependent uptake over 60 rain. However, the addition of 10 mM phosphate ion to incubations carried out in Krebs bicarbonate buffers inhibited uptake by only 15 20!~,, suggesting that the lack of uptake in the phosphate containing buffer was not explained simply by the presence of phosphate.

Inhibition o[ EDA uptake A number of amino acids, diamino compounds and GABA analogues were tested at 100/~M as inhibitors of EDA accumulation. The inhibition of [J4C]EDA uptake (0.8 ,uM/by 100/~M unlabelled EDA was 62'I; (control uptake measured as total accumulation at 3 7 C mim,s accumulation measured at 0C). The most potent inhibitors were the di- and polyamino compounds, namely L-2,4-diaminobutyric acid, spermidine, DL-diaminopropionate and spermine. The other diamino compounds tested (viz. 1,2-diaminoethylphosphonic acid, 1,3-diaminoethylphosphonic acid) gave quite significant inhibitions. At 100,aM, p-chloromercuriphenylsulphonate inhibited uptake by 89";,.

Kinetics (ff EDA uptake Slices were incubated with [~4C]EDA and increasing concentrations of unlabelled EDA in the range 100/~M--20 raM. Results indicated an uptake site with a Km of 1.11 + 0.05 mM and a 1/ma x of 9.79 + 0.20 l~mot/h/g wet wt of tissue lweighted means of three experiments, analysed by Eadie--Hofstee plotsl. These plots suggested a second uptake site of higher affinity but much lower 1/~ x. A kinetic assay using a wider range of concentrations of EDA (20/~M-20 raM) gave a clear biphasic Eadie- Hofstee plot (Fig. 4) with a K m of 20.6 #M and a 1~,~ of 0.20 pmol/h/g wet wt for the high affinity site (cf. a Km of 0.96 mM and a l/m~~ of 9.61 ,umol/h/g for the major, low-affinity site as determined from this experiment).

Release of EDA Radioactivity from cerebral cortical slices preloaded with [~4C]EDA could be released by increasing the potassium concentration in the perfusing medium to 30 mM (Fig. 5). This potassium-stimulated release was calcium dependent: calcium-dependent and calcium-independent potassium-stimulated release were 592 + 5Y~o (N = 6) and 92 + 5~°~;i (N = 6) of basal efftux respectively (Fig. 5A; difference significant at p < 0.001). When pretabelling of the

Ethylenediamine uptake and release

(296 _+ 14~°;,; N = 4) (Fig. 7A; P < 0.001 for both), whereas in the absence of sodium, no increase in release was observed at either concentration of ouabain (Fig. 7B).

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S (in raM) Fig. 4. Slices were incubated with [I'*C]EDA (0.8 #M) and increasing amounts of unlabelled EDA at 9 concentrations in the range of 0.02 20 mM, both at 37 and 0°C. Temperature dependent uptake (calculated as the difference between uptake-at the two temperatures) was examined by EadieHofstee plot and data analysed by a computer assisted iterative curve fitting procedure (Rosenthal, 1967). Each point represents the mean of quadruplicate determinations at each concentration. Two other experiments gave similar results (see text).

It is apparent that there is a temperature-dependent accumulation of EDA in rat brain slices which continues for at least 60 min. On the basis of tissue to medium ratios however, the accumulation of [14C]EDA is very much lower than for GABA. The apparently instantaneous accumulation at zero-time in samples incubated at either 0 or 37°C (relative to blanks containing excess EDA and incubated at 0°C) is largely due to binding of EDA to the glass-fibre filters. In contrast to GABA high affinity uptake which is sodium-dependent and completely abolished in the absence of sodium (Johnston, 1978a), the uptake of [14C]EDA is only partially sodium-dependent. In zero-sodium medium there is still a time and temperature dependent EDA accumulation. Accumulation at early times was actually greater in sodium-free than in normal medium, but at longer times uptake in sodium-free medium levelled off whereas accumu-

~4C E .DA /~% slices and the subsequent release were carried out in the absence of sodium, the potassium stimulated release in the presence of calcium was 367 + 36Vo (N -- 10) of basal release, compared with 435 + 62~o (N = 10) in the absence of calcium (difference not significant; Fig. 5B).

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The effect of G A B A and beta-alanine on EDA release EDA (1 raM) stimulated the efflux of [3H]GABA by 480 +_ 76~o (N = 8), [3H]beta-alanine by 53 -t- 9~o (N = 8) and [I'*C]EDA by 72 + 12~o (N = 9) over basal efftux from preloaded tissue slices (Figs 6A, B, C respectively; differences significant at P <0.005). However, neither GABA nor beta-alanine at 1 mM had any effect on basal release of radioactivity from slices labelled with [14C]EDA (Fig. 6D).

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Fig. 5(A). Slices were prelabelled with [14C]EDA in normal Krebs bicarbonate buffer and perfused in the same buffer with (B) or without (r-I) CaCl 2. After 70 min perfusion the KCI concentration was increased from 3 to 30 mM for 10 min. Both experiments are mean values from 6 perfusion channels. (B). Details as for experiment as in (A) except that the prelabelling and release buffer did not contain sodium ions. In both experiments N = 10.

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Fig. 6(A). Slices were prelabelled with [3H]GABA and pcrfused with Krebs bicarbonate Ringer. Scventy minutes after the start of perfusion, slices were exposed to solution containing 1 mM EDA (N = 8). (BI. Details as for {A) except that slices were prelabelled with [3H]beta-alanine (n = 8). (('). Slices prelabelled with [E'~C]EDA and perfused with normal Krebs bicarbonate Ringer were exposed for 10min to mM EDA {N = 9). [D). As for (CI except that the perfused slices were exposed for I0 rain to either 1 mM GABA (O1 IN = I(I} or 1 mM beta-alanine (@) (N = 7).

lation In normal medium continued for at least as tong as 60 min (the longest time point measured). The differences in these time-course curves (Fig. 3A and B) suggest that there may be separate sodium dependent and sodium independent uptake processes. In relation

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Fig. 7(A). Using normal Krebs bicarbonate Ringer, slices were prelabelled and perfused. After 70 min perfusion, slices were exposed either to 0.1 mM ouabain (O) (N = 8) or to 1 mM ouabain (O) (N = 4) for 10 min. (B). Details as for (A) except that prelabelling of the slices and the release perfusion were carried out in sodium-free medium (N = 4 for both 0.1 and 1 mM ouabain).

to this, high concentrations of ouabain (1 mM) only partially inhibited temperature-dependent EDA accumulation (77.50;i). This should not be taken as a measure of the relative activities of the sodium dependent and sodium independent "uptake' systems however, since Nomura, Schmidt-Glenewinkel and Giacobini (1980) showed that the apparently sodium-independent uptake of piperidine was inhibited by ouabain. Results (Figs IB and 3B) show that there are some changes in 0 C 'accumulation' with sodium ion concentration and with time; although most of the 0 C c o m p o n e n t of EDA accumulation was due to lilter binding, the effect of sodium depletion on this binding was not examined further. Neither GABA nor beta-alanine had much effect on EDA uptake, although GABA appeared to be marginally more potent of the two. This is in contrast to our previous results in which EDA was shown to be a better inhibitor of [3H]beta-alanine uptake than it was of [ 3 H ] G A B A uptake (Davies e t al., 1982). Neither piperidine nor piperazine, cyclic "analogues" of EDA, had any inhibitory effect on EDA uptake. whereas the polyamines spermine and spermidine gave significant inhibitions. A qow-affinity" active transport system for piperidine in synaptosomes prepared from mouse brain has been reported (Nomura et al., 1980). The lack of inhibition of EDA uptake by piperidine suggests that the accumulation of [I'*]EDA is not taking place via this piperidine system.

Ethylenediamine uptake and release Low affinity uptake systems for spermine and spermidine have been described in mouse cerebral cortex slices (Picolli and Lajtha, 1971), and more recently two sodium dependent high affinity uptake sites for spermine (3.8 nM and 0.44 #M) have been noted in rat brain cortical slices (Harman and Shaw, 1981). Since spermine and spermidine inhibited EDA accumulation it is possible that some of the sodium dependent EDA uptake may be taking place at these rather than GABA-related sites. Kinetic analysis of the temperature dependent component of EDA uptake revealed a site with an apparent K m of 1.11 + 0.05 mM, which is similar to the value of 1.36 mM reported for EDA uptake by Lloyd and Stone (1982). In addition to this low affinity site, results suggest a further higher affinity site with a K m of 20.6 #M and a 50-fold lower Vmax. It is possible that this site represents EDA uptake at the sodium dependent high affinity GABA uptake site (Johnston, 1978a), although further experiments would be needed to confirm this. Some of the high affinity GABA uptake inhibitors such as nipecotic acid, cis-3-amino cyclohexane carboxylic acid and L-2,4-diaminobutyric acid (Johnston, 1978b) significantly inhibited temperature-dependent EDA accumulation whereas isonipecotic acid (4-amino piperidine) (inactive vs GABA uptake) was inactive. Of further interest was the inhibition of EDA accumulation by L-histidine and the particularly potent inhibition by L-2,4-diaminobutyric acid. These two compounds are inhibitors of low affinity uptake of GABA (Blasberg and Lajtha, 1966; Kennedy and Neal, 1978), suggesting that the 'small basic~ amino acid transport system (Cohen and Lajtha, 1972) which transports L-histidine and L-2,4-diaminobutyric acid (and GABA, with low affinity) may be involved in part in EDA accumulation. This low affinity uptake site may be important in the inactivation of GABA since it has been shown (Lodge, 1979) that L-histidine potentiates the depressant effects of GABA on cat spinal neurons. Potassium-stimulated, calcium-dependent release of EDA radioactivity is from pools labelled by sodiumdependent uptake, since there was no calcium-dependent, stimulated release if the prelabelling and perfusion of the slices was carried out in sodium-free buffers. Surprisingly neither GABA nor beta-alanine were able to stimulate the release of radioactivity from slices labelled with [14C]EDA, although unlabelled EDA did so. It is possible that EDA taken up by the tissue is sequestered in subcellular pools separate from those containing GABA or beta-alanine, although if this is the case EDA must still be available

63

for depolarization-induced release. Despite the fact that GABA or beta-alanine could not release [14C]EDA or [14C]EDA-derived radioactivity, unlabelled EDA was able to significantly stimulate release of [3H]GABA and [3H]beta-alanine from prelabelled slices. It appears that extracellular EDA can counterexchange with intracellular GABA and beta-alanine, but that EDA which is accumulated by the tissue may then be bound or move to pools not directly accessible to GABA or beta-alanine. The differing actions of ouabain on the release of ['4C]EDA accumulated in the presence or absence of sodium suggest that there are sodium dependent and sodium independent uptake sites and that these label separate intracellular pools. This supports those conclusions made from the uptake studies. The uptake and release patterns for [14]EDA are thus quite complicated. Studies suggest the presence of at least two uptake sites which are sodium dependent and sodium independent (with associated intracellular storage pools). Kinetic analysis of temperature dependent EDA uptake suggest the presence of a major low-affinity uptake (Km approx. 1 mM) and a less significant but higher affinity site (Km approx. 20 pM). This latter site may represent the high affinity GABA carrier. Inhibitor studies give some indication that EDA may also be taken up by the low-affinity GABA transporter (the 'small basic' amino acid transport system which is largely but not completely dependent upon the presence of sodium ions). Radioactive EDA which is accumulated by tissues can be released by potassium depolarization of the tissue in a calcium dependent manner, in a similar way to transmitter release. We have shown that radioactive EDA undergoes very significant displaceable binding to glass-fibre filters. This suggests that EDA may also undergo binding to tissue macromolecules which could further complicate analysis of results from uptake and release studies. Furthermore, the ability of EDA to chelate some metal ions (e.g. Zn 2+, Mn 2+, Mg 2+) may need to be taken into account, given our findings on the influence of different incubation media on EDA transport.

REFERENCES Blasberg, R. J. and Lajtha, A. (19661. Heterogeneity of the mediated transport systems of amino acid uptake in brain. Brain Res. 1, 86 104. Cohen, S. R. and Lajtha, A. (1972). Amino acid transport. In: Handbook of Neuroehemistry (Lajtha, A. ed.), Vol. 7, pp. 177-210. Plenum Press, New York. Curtis, D. R., Duggan, A. W., Felix, D. and Johnston, G. A. R. (1971). Bicuculline, an antagonist of GABA and

(~4

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