The differential effects of excitatory amino acids on uptake of 45CaCl2 by slices from mouse striatum

Neurophcrrmacolog,r Vol. 23, No. I, pp. 89-94, 1984 Printed in Great Britain

0028-3908/84 $3.00 + 0.00 Pergamon Press Ltd

THE DIFFERENTIAL EFFECTS OF EXCITATORY AMINO ACIDS ON UPTAKE OF 45CaC12BY SLICES FROM MOUSE STRIATUM K. C. RETZ and J. T. COYLE* Division of Child Psychiatry, Departments of Psychiatry, Neuroscience, Pharmacology and Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A. (Accepted 26 April 1983)

Summary-Exposure to L-glutamate (10 mM) or 60 mM K + for 1min significantly stimulated the uptake of %a2 + in slices from mouse striatum. Glutamate-induced stimulation was antagonized by 30 mM MgZ+ and by 5 or 10 mM t_-glutamic acid diethyl ester, but not by 5 p M tetrodotoxin. Under these I-min incubation conditions, neither kainate nor N-methyl-D,L-aspartate significantly affected the uptake of ‘%a’+ ion. By contrast, following preincubation for IOmin, glutamate and the conformationally restricted analogues, ibotenate, quisqualate, and kainate significantly inhibited the 60 mM K + -induced stimulation of the uptake of 45Ca2+ These effects of glutamate and kainate were not significantly atTected by the presence of 1 mM Ca*+ in the preincubation medium. These results suggest that glutamate may activate a receptor directly linked to CaZ+ channels, whereas kainate may indirectly modulate the intracellular disposition of Ca* + Key words: glutamic acid, kainic acid, excitotoxins, calcium, depolarization.

The growing evidence that L-glutamic acid serves as a major excitatory neurotransmitter in the CNS has been recently reviewed (Coyle, 1980; Cotman, Foster and Lanthorn, 1981; Watkins, 1981; Watkins and Evans, 1981). In particular, the corticostriatal gluamatergic projection has been well characterized. It appears that glutamate not only has direct excitatory effects on neurons intrinsic to the striatum, but also stimulates the release of dopamine (Nieoullon, Cherany and Glowinski, 1978) presumably at presynaptic receptor sites (Gieoguieff, Kenel and Glowinski, 1977; Roberts and Anderson, 1979). Although the mechanisms for this latter effect remain obscure, a coupling of the glutamate receptor to a Ca2+ ion channel could be involved as the influx of Ca* + is thought to be a prerequisite for the release of neurotransmitter (see, Blaustein, 1975). Because of the involvement of the striatum in neuronal projections regulating motor function (see, Graybiel and Ragsdale, 1979) elucidation of the role of excitatory amino acids in neurotransmission in this region could greatly aid our understanding of the regulation of motor activity in both normal and pathological states. Indeed, it has been demonstrated that the intrastriatal injection of kainic acid, a conformationally restricted analogue of glutamate, does produce in rodents a neuropathology that is biochemically similar to that observed in the terminal stages of Huntington’s Disease (Coyle, McGeer, Mc-

Geer and Schwartz, 1978). The mechanism responsible for the perikaryal-specific neuronal degeneration induced by kainate and related excitotoxins remains unclear (Retz and Coyle, 1982). Notably, chohnergic agonist-induced myopathy shares the acute histopathological features with those of the excitotoxins (Salpeter, Leonard and Kasprzak, 1982). Since the cause of the cytotoxic effects on the myocytes appears to involve an increased intracellular concentration of Ca’+ and activation of Ca2 + -dependent proteases (Leonard and Salpeter, 1979; Salpeter, Kasprazak, Feng and Fertuck, 1979) the effects of excitatory amino acids and their analogues on the intracellular disposition of Ca’+ might be involved in their neurotoxic mechanisms. Using unpurified synaptosome preparations from rat striatum to examine the effects of glutamate and related excitatory amino acids on the uptake of 45Ca2 + , a specific, tetrodotoxin-insensitive stimulation by glutamate has been observed (Retz, Young and Coyle, 1982). In the present report, an examination was made of the effects of glutamate and conformationally restricted analogues on the uptake of 4sCa2 + in slices prepared from mouse striatum and the LaCI, post-incubation technique (Van Breeman, Farinas, Casteels, Berba, Wytuck and Deth, 1973) was employed to enhance the sensitivity of the measurements. METHODS

*To

whom correspondence should be addressed: Department of Psychiatry, Meyer 4-163, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore. MD 21205, U.S.A.

Adult male mice (Ha:ICR), 253Og, purchased from Blue Spruce Farms (Albany, New York) were used in all studies. Kainic acid, t-glutamate mono89

K. C. RETZ and J. T. CQYLE

90

sodium salt, N-methyl-D,L-aspartic acid (NMDLA), L-glutamic acid diethyl ester-HCl (GDEE), tetrodotoxin (TTX), LaCl, and N-2_hydroxyethylpiperazineN’-2’ethane sulfonic acid (HEPES) were purchased from Sigma Chemical Co. (St Louis, Missouri). All other chemicals were obtained from common laboratory suppliers. Animals were killed by cervical dislocation. Striata were dissected on a 0°C cold plate and placed in ice-cold modified Krebs buffer with the following composition (mM): 132 NaCl, 4.8 KCl, 2.4 MgSO,, 20 HEPES and 10 dextrose. Tissue slices of 100 p thickness were prepared using a McIlwain-type tissue chopper and they were immediately suspended in 20ml buffer equilibrated with bubbling 100% OZ. After washing three times with 10 ml buffer, the slices were preincubated for 60min at 37°C under continuous bubbling OZ. Buffers were changed every 20min. Preincubation in a Ca-free buffer would be expected to deplete the content of intracellular Ca2 + ion by approx. 50% (Lolley, 1963). In those experiments where the effects of preincubation with amino acids were examined, the amino acids were added after 50 min of preincubation. After 10 min, the drug solutions were removed by aspiration and the slices were rinsed with two 10 ml portions of buffer. Following preincubation, most of the buffer was withdrawn from the slices and the uptake of 4SCaCl, was measured in aliquots. The reaction, conducted in 20ml glass scintillation vials, was initiated by the addition of a 0.3 ml aliquot of slices (approx. 0.4-2.5 mg protein) to pre-equilibrated 3.0 ml aliquots of buffer containing the compounds studied and 1.OmM 45CaC12(1 mCi/mmol). In studies with 60 mM K + ion, the concentration of Na + ion was adjusted to maintain osmolarity. The reaction was terminated by the addition of lOm1 of ice-cold Cafree buffer and all vials were placed in an ice-water bath. After the removal of the buffer, the slices were then post-incubated for 60 min as described in a buffer free of Ca containing 10 mM LaCl,; this procedure displaces extracellular bound Ca2+ and entraps intracellular Ca2+ (Van Breeman et al., 1973). After this post-incubation, all buffer was removed by aspiration; the tissues were dispersed in 1.0 ml of 7% trichloroacetic acid using sonication; the radioactivity of 45CaCl, was measured by liquid scintillation spectrometry. Protein was determined on separate aliquots with bovine serum albumin as a standard (Lowry, Rosebrough, Farr and Randall, 1951). The radioactivity of 45Ca2f, taken up under basal conditions (4.8 mM K+ buffer), was linearly proportional to the total protein (0.4-2.5 mg) present in each assay. RESULTS

The ability of the La3+ ion to entrap intracellular Ca* + while displacing extracellular Ca* + was verified

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40

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Fig. 1. Loss of 45CaCl, from striatal slices from mouse brain in the presence of LaCl,. Striatal slices were preincubated for 60min prior to a 1 min incubation with 1 mM “jCaC1, (1 mCi/mm$. Immediately after terminating the uptake with 3.3 vol of ice-cold Ca-free buffer. the tissue slices were post-incubated for up to 60 min in da-free buffer with or without 1OmM LaCl,. At the time periods indicated, all buffer was removed by aspiration and the tissue slices were analyzed for 45CaZ+and protein as described under Methods. The data are presented as the mean + SEM (N = 4). The mean 45CaZ+content of tissues without post-incubation post-incubation was 8.9 nmol/mg protein. *LaCl, significantly different from post-incubation in Ca-free buffer by t-test, P < 0.01; by **P < 0.001.

using two approaches. First, slices were incubated with 45CaC12 as described in Methods, and postincubated for up to 60min in buffer free of Ca but containing 10 mM LaCl,. The results (Fig. 1) demonstrate greater retention of 45CaC12in the presence of the La3+ ion at all times examined. Based on these findings and similar observations in cortical slices (Bull and Trevor, 1972) and hippocampal slices (Bairnbridge and Miller, 1981), a 60min postincubation time was used in all further studies. In the second approach, the ability of La3 + (0.5-10.0 mM) to compete directly with the 45Ca2C ion for uptake and extracellular binding sites was examined. As can be seen in Fig. 2, the La3 + ion decreased the observed basal rates of the uptake of 45Ca2+ at all concentrations, reaching nearly 80% inhibition at the highest concentration. With the La3+ post-incubation technique, the uptake of “?a2 + was time-dependent from 0.5-5.0min of incubation under both basal and 60 mM K + conditions (Fig. 3). However, the apparent rate decreased markedly between 0.5 and 2.0 min

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Fig. 2. Inhibition of 45CaC1, uptake by LaC1,. Mouse striatal slices were preincubated for 60 min prior to a 1min incubation with 1mM 45CaC1,(1 mCi/mmoi) in the presence of 0.5-10.0 mM LaCI,. Uptake was terminated, the tissue slices were post-incubated with IOmM LaCl,, and were processed as described under Methods. The data are expressed as mean percentage of control k SEM (N = 4-6). All data were significantly different from control using ANOVA and the Student-Newman-Keul’s test, P -c0.05.

I

of incubation, consistent with a biphasic process of uptake. These findings are similar to those observed in cortical slices from rat (Ichida, Osugi, Noguchi and Yoshida, 1981). With the 1 min incubation conditions, 60 mM K+ ion produced an 81% stimulation of the uptake of 4sCa2+ (Table 1). Thirty millimolar Mg2+ ion, a physiological Ca2 + antagonist, markedly attenuated this stimulation, whereas 5pM tetrodotoxin, a blocker of voltage-dependent Na + channels, had no significant effect. A statistically significant 24% simulation of the uptake of 45Ca2C was observed with 5.0mM glutamate, but not with kainate (up to

1.0 mM) or N-methyl-D,L-aspartic acid (up to 2.0 mM). The stimulation of the uptake of 45Ca2+ by glutamate was antagonized by 30 mM Mg2 + ion and

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Fig. 3. Time course of the uptake of 45CaCl,. Mouse striatal slices were preincubated for 60 min prior to incubation for 0.5-5.0 min with 1mM CaCl, (1 mCi/mmol) in the presence of buffer containing 4.8 mM KC1 (basal conditions) or 60 mM KCl. Uptake was terminated and the slices were post-incubated with 10 mM LaCI, as described under Methods. The data presented with the solid line are expressed as nmol %aC12 uptake/mg protein per time indicated. The data presented with the broken line are expressed as “apparent rate” in nmol 45CaC1, uptake/mg protein per min (mean k SEM, N = 7). The symbols “m” denote 4.8 mM K + buffer and “A” denote 60 mM K + buffer. by the glutamate antagonist L-glutamic acid diethyl ester-HCl (5, 10 mM), but not by 5 PM tetrodotoxin. In experiments not shown, the glutamate-induced

Table I. Effects of 6OmM K+ depolarization and acidic amino acids on the uptake of “Ca2+ bv striatal slices from mouse brain Condition 4.8mM K+ buffer plus l.OmM kainate plus 2.0mM NMDLA plus 30mM Mg2+ plus 5mM GDEE plus IOmM GDEE plus 5pM TTX 60mM K+ buffer plus 30mM Mg”+ plus 5pM TTX 4.8mM K + buffer plus 5.0 mM glutamate plus 30mM Mg’+ plus 5mM GDEE plus lOmM GDEE PIUS 5uM TTX

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45Ca2+ uptake (nmol/mg protein per min)

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2.02 + 0.07 2.15 kO.10 2.17~0.12 1.63 +O.lO 1.86&0.13 2.00 * 0.10 2.08 + 0.14 3.66 + 0.24 2.59 + 0.10 3.84 + 0.40

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Slices were prepared and preincubated in Ca*+-free buffer for 60 min, exposed to drugs and 1.0 mM %aCI, for 1.0 min, and post-incubated for 60 min with buffer containing 10 mM LaCI,. Results are expressed as the mean f SEM; N = 5-8. *Significantly different from 4.8 mM K+ buffer by ANOVA and Student-Newman-Keul’s test, P < 0.05. (See text for definitions of abbreviations.)

K. C. RITZ and J. T. COVLE

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Fig. 4. Net stimulation of the uptake of JSCaClz by 60 mM KCI buffer in the presence or absence of glutamate during preincubation. Tissue slices were exposed to L-glutamate during the last IOmin of the 60min preincubation period. The slices were then washed twice with IOmi of oxygenated 4.8 mM K + buffer and incubated for 1.0 min in the presence of 1.OmM JSCaCll (1 mCi/mmol) in buffer containing either 4.8 mM K+ or 60mM K + ion. incubations were terminated and the slices were processed as described in Methods. Preincubation with any of the concentrations of glutamate examined was without significant effect on the ratebf uptake of 45Ca” under 4.8 mM K + basal conditions (not shown). The net stimuiation produced by 60mM K‘” foliowin~ preincubation under basal conditions and preincubation with glutamate are presented for each concentration examined. These data are expressed as net stimulation of uptake of %a’ ’ by 60 mM KC1 (mean f SEM, N = 4-8). *Drug preincubation is statistically different from 4.8 mM K + preincubation using r-test, P i 0.05; by **P < 0.02; by ***p

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stimulation of the uptake of “Ca*+ was not affected by the presence of I .O mM kainate and vice versu. Several other neurotransmitter agonists were without significant effect on the rate of the uptake of 4’Ca2 + including: 0.2 mM dopamine; 0.2 mM bethanechol and two GABAergic agonists, I.0 mM muscimoi and 10.0 mM THIP (4,5,6,7-tetrahydroisoxazolo[5,4-clpyrindin-3-01). Because of the known neurotoxic effects of excitatory acidic amino acids, the effects of preincubation were examined (10 min, see Methods). Pre~ncubation with any of the amino acids examined was without significant effect on the rate of the uptake of 4sCaZ + under 4.8 mM K + basal conditions (not shown). However, with preincubation with glutamate. inhibition of the net stimulation produced by 60 mM K + ion reached statistical significance following the 1.0, 2.0, 5.0 and IO.OmM preincubations

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Fig. 5. Net stimulation of uptake of 45CaCI, by 60 mM KCI buffer in the presence or absence of kainate, ibotenate or quisqualate during preincubation. Tissue slices were exposed to kainate (O.Oi-2.OmM). 2.0mM ibotenate. or 2.0mM quisquaiate during the last IOmin of the 60min preincubation period. For experimental details. see the legend to Fig. 4. Preincubation with any of the acidic amino acids examined was without significant erect on the rates of uptake of ‘%Ya + under 4.8 mM K + basal conditions (not shown). *Drug preincuhation is statistically different from 4.8 mM K + preincubation using t-test. by P < 0.05: by **P ~0.02; by ***P
(Fig. 4). With preincubation with kainate significant inhibition was observed with concentrations as small as 0.2mM. In a few preliminary experiments, the effect of preincubation with 1.O mM Ca’ + was examined and found not to alter significantly the effects of either 1.0 or 10.0 mM glutamate or 0.2-2.0 mM kainate in the preincubation studies (not shown). Preincubation with two glutamate congeners. quisqualate and ibotenate also significantly depressed the net 60 mM K +-induced stimulation of the uptake of 4’Ca’ + (Fig. 5). DISCUSSION

The present studies have shown differential effects of glutamate and kainate on the rate of the uptake of ‘%?a’+ by striatal slices from the mouse. The results obtained with a 1 min incubation in the presence 4SCaC12 are in good agreement with previous obsetvations in striatal synaptosomes from the rat where glutamate but not kainate nor N-methyl-I>,t_-aspartic acid stimulated the uptake of “Ca” (Retz et rtl., 1982). Although earlier studies reported a greater

Excitatory

amino acids and Ca’+

degree of stimulation by glutamate of the uptake of 45Ca2t in slices from cortex of guinea pig (Ramsey and Mcllwain, 1970) and cortex of rat (Cooke and Robinson, 1971), recently Ichida, Tokunaga, Moriyama, Oda, Yanake and Kita (1982) using cortical slices of rat, found that lower concentrations of glutamate gave percentage increases of a degree similar to those found in the present study. Moreover, previous studies using calcium-sensitive electrodes for intracellular recording also observed a glutamate-stimulated influx or Ca” (Sonnhof and Buhrle, I98 I ). The specificity of the glutamate-induced uptake or “‘Cal +, demonstrated by the inhibition by L-glutamic acid diethyl ester-HCl and the lack of effect of agonists for other neurotransmitter receptors in the striatum, is in agreement with some recent studies in cortical slices from rat (Ichida it ul., 1982). The current observations that stimulation of the uptake of “Ca’ + by glutamate was sensitive to the Mg’ ’ ion, but not to tetrodotoxin, is also in agreement with earlier studies in striatal synaptosomes from the rat (Retz o/ (I/., 1982). The insensitivity to tetrodotoxin is not surprising in that stimulation by glutamate of the synthesis of cGMP in cerebellar slices (Foster and Roberts, 1980) is also insensitive to tetrodotoxin (Ferkany et (I/., unpublished). However, it must be noted that in cortical slices from guinea pig and rat. glutamate-stimulated uptake of Ca” is apparently sensitive to tetrodotoxin (Ramsey and Mcllwain. 1970; Cooke and Robinson. 1971). The present findings in the I min incubation studies support the evidence from pharmacological studies that glutamate, kainate and N-methyl-I,.L-aspartic acid each interact with unique excitatory receptors (Coyle, 1980: Cotman (‘/ u/.. 1981; Watkins, 1981; Watkins and Evans, 1981). Moreover. the present study on slices and previous findings with synaptosomes (Retz (~1u/., 1982) are consistent with the hypothesis that glutamate-stimulated uptake of Ca’+ could account for the tetrodotoxin-insensitive mechanism responsible for the presynaptic effects of glutamate in releasing dopamine and norepinephrine from nerve terminals (Giorguieffet u/., 1977; Roberts and Anderson. 1979; Nelson. Zacek and Coyle, 1980).

In the IOmin preincubation experiments, glutamate, kainate, ibotenate and quisqualate were all effective in reducing 60 mM K ’ -stimulated uptake of “Ca”. From the I min incubation studies, it would appear that the inhibition seen with the glutamate receptor agonists could result from prolonged direct stimulation of glutamate receptors directly linked to Ca’ ’ channels, accompanied by ionic and energy imbalances interfering with the ability of the neurons to maintain their osmolar integrity (Bradford and Mcllwain, 1966; Harvey and Mcllwain, 1968; Okamoto and Quastel. 1970; Biziere and Coyle, 1979). An explanation obvious.

of the inhibition

However.

seen with

kainate

is less

it has been shown that prolonged

uptake

93

exposure of striatal slices to kainate and in the striatum in uiuo produces ionic and energy imbalances similar to those observed following glutamate, these alterations being enhanced by the presence of glutamate at the synapse (Biziere and Coyle, 1978, 1979; Retz and Coyle, 1982). Further elucidation of this question will require the availability of selective kainate antagonists and knowledge of the endogenous ligands for the kainate receptor and their physiological function. It is possible that prolonged agonist occupation of glutamate and kainate receptors produces neurotoxicity as a result of a Ca’+ channel-mediated process, directly in the case of glutamate, and probably indirectly through a concerted mechanism in the case of kainate. Because the presence of Cal ’ in the preincubation medium was not obligatory for the inhibition of 60 mM K +-induced stimulation of the with uptake of “‘Ca’+, seen following preincubation either glutamate or kainate, mobilization of intracellular Ca’ ’ stores may be more important than the uptake of Ca’ + per .w. especially so for kainate. Consistent with this, increased free levels of intracellular Ca’ + would inhibit the K ’ -induced uptake of Ca’ ’ as observed with all four excitotoxins. Elevated intracellular levels of free Ca’ + could activate proteases, which might account for the acute and chronic neurotoxic effects of these as agents, as suggested on the basis of the myopathic model by Salpeter c)t cd. (I 982).

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