Suppressive Effects of Various Amino Acids Against Ouabain-Induced Seizures in Rats

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Suppressive Effects of Various Amino Acids against Ouabain-Induced Seizures in Rats

Y. TSUKADA, N. INOUE, J. DONALDSON, and A. BARBEAU

SUMMARY: The suppressive effect of thionic acid showed increases in inci INTRODUCTION various amino acids against ouabain- dence of running and leaping seizures induced seizures was investigated in while L-arginine in high concentrations Recent investigations on the young female rats. The amino acids caused aggravation in clonic-tonic biochemistry of convulsive seizures were injected into the left lateral ven seizures. L-cysteine, even in low concen have revealed the existence of ab tricle 10 minutes prior to the intra trations, also brought about an increase normalities in the metabolism or ventricular administration of 5 jj.g. of in the occurrence and incidence of brain content of various amino ouabain. Animals receiving 1.9 x 10A M clonic-tonic seizures. The ED^of hypo acids. For example, an accumulation solutions of hypotaurine and of fir taurine was 10.11 x 10~2 M for running 3, of alanine and decreased citrate alanine were almost completely protected seizures and 4.63 x 10- M for clonic- levels were observed in the precon from the ouabain seizures. Administra tonic seizures; that of fi -alanine was tion of L-alanine and of glycine was also 14.01 x 10~2 M for running seizures and vulsive stage; on the other hand, effective, although running and leaping 5.50 x 10-2 M for clonic-tonic seizures. GABA increased in rat brain during seizures still occurred to some extent. However, hypotaurine and fi-alanine, tonic seizures as well as during Betaine reduced only clonic-tonic and the most effective compounds tested in post-convulsive stages (Nahorski et whole body flexion and extension the present studies, provided less protec al, 1970). According to some au seizures. In contrast, L-proline exclu tion than taurine previously examined thors, electroconvulsive shocks sively suppressed clonic-tonic and focal by us under similar conditions (Izumi markedly inhibit the conversion of clonic seizures. Rats injected with ise- et al., 1973). 14C-glucose to amino acids and the incorporation of 14C into pro teins in mouse brain (Dunn and RESUME: Nous avons etudie I'effet ayant regu I'acide isethionique mont- Giudetta, 1971) and in rat neo- suppresseur de plusieurs acides amines rerent une augmentation de Yincidence cortical slices (Jones and Mcllwain, contre les convulsions induites par la des convulsions cursives et des sauts. 1971). These findings were chal ouabaine chez lajeune rate. Les acides La L-arginine, en forte concentration, lenged by others (Barkulis et al., amines furent injectes dans le ventricule aggravait les convulsion tonico- 1960; Orrego and Lipmann, 1967). lateral gauche 10 minutes avant Vad cloniques. II enfut egalement ainsi pour ministration intraventriculaire de 5/j.g. la L-cysteine, meme en faibles concen Recently, van Gelder et al. (1972) de ouabaine. Les animaux recevant des trations. La DEgo de I'hypotaurine est reported lower levels of GABA and solutions de concentration 1.9 x 10-1 M de 10.11 x 10~2 M pour les convulsions aspartic acid in all regions of epilep d'hypotaurine et de fi -alanine etaient cursives et de 4.63 x 10-2 M pour les tic human cortex sampled and lower presque completement proteges contre convulsions tonico-cloniques; celles de concentrations of glutamic acid and les convulsions induites. L'administra la fi-alanine sont de 14.01 x 10-2 M et taurine together with very high tion de L-alanine et de glycine etait de 5.50 x 10-2 M, respectivement. Ce- glycine levels in human cortical egalement efficace, meme si les de- pendant il faut noter que Yhypotaurine epileptic foci. Koyama (1972) ob charges cursives et les sauts etaient et la fi -alanine, les composes les plus served similar decreased concentra encore presents. La betaine ne reduisait efficaces dans la presente etude, offrent tions of taurine, glutamate, asparate que les convulsions tonico-cloniques ou tous deux une protection moindre contre celles se manifestant par une flexion et les convulsions induites par la ouabaine and GABA during the period pre une extension du corps. Par contraste, que la taurine que nous avions pre- ceding cobalt induced convulsions la L-proline supprimait exclusivement alablement etudie en detail (Izumi et al., in cats. However, Reynolds and Gal les convulsions tonico-cloniques oupure- 1973). lagher (1973) found neither changes ment cloniques localisees. Les rates in amino acid levels nor alterations of U-14C-glucose incorporation into amino acids during any of the stages of the seizure induced by Flurothyl.

Department of Neurobiology, Clinical Research Some amino acids have been Institute of Montreal, 110 Pine Avenue West, shown to be neurotransmitter candi Montreal, Quebec, Canada. dates with inhibitory or excitatory Reprint address: Dr. Andre Barbeau, Clinical Research Institute of Montreal, 110 Pine Avenue functions. Micro-electrophoretic West, Montreal, Quebec, Canada H2W 1R7. techniques to substantiate this

214 - NOVEMBER 1974

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nypothesis were reviewed by Curtis administration of amino acid and sons of seizure incidence and of la and Crawford (1969). Uptake studies ouabain solutions which were in tency in onset of the first seizure. have been carried out in central jected through the cannula at a rate nervous system tissue or retina (Bal- of 25nl./min. Two different doses of RESULTS car and Johnston, 1973; Ehinger, each amino acid (9.5 x 10-2 M or 1.9 J Occurrence and incidence 1972; Kaczmarek and Davison, x 10 M) were administered 10 mi Pretreatment of the animals with 1972; Snyder, Young et al., 1973; nutes prior to the injection of 5^g. in hypotaurine (the main precursor of Snyder, Yamamura et al., 1973). 50 p.1. ouabain. Moreover, various taurine), /3-alanine, L-alanine and Finally, some authors have ap concentrations of hypotaurine and glycine caused a dose dependent de proached the problem with the help j3-alanine were used to investigate crease in seizure occurrence and in of autoradiography (Ehinger and median effective doses (ED50) for cidence (Tables 1 and 2). Test rats Falek, 1971; Iversen and Schon, running and clonic-tonic seizures. receiving hypotaurine at 10 minutes 1973). These investigations con The control animals received an in prior to the ouabain injection were cluded that 7-aminobutyric acid jection volume of 50 1. of buffered kept free from all seizure activities at (GABA), glycine, 0 -alanine and saline. The effects of amino acids high concentrations (1.9 x 10-1 M). taurine may be inhibitory neuro against the different types of Moreover, even in low concentra transmitters, while glutamic acid and ouabain-induced seizures previously tions (9.5 x 10-2 M), the compound aspartic acid have excitatory func described in rats (Izumi et al., 1973) completely suppressed clonic-tonic, tions. were observed for fifteen minutes whole body flexion and extension after the administration of ouabain. On the other hand, once recom seizures (WBFE), and to a lesser mended trials of treatment with Preparation of solutions extent, focal clonic seizures. This amino acids in human epilepsy no Ouabain solutions were prepared concentration of hypotaurine also longer appear useful today, except at a concentration of 5ju,g./50/il. in decreased the incidence of running possibly with gamma-amino-beta- warm 0.85% saline. The amino acids and leaping seizures. hydroxybutyric acid (GABOB) were dissolved in a NaCl solution Intraventricular administration of (Hahashi, 1972). Recently, however, containing 5 mM phosphate buffer, 1.9 x 10*1 Mf3 -alanine showed sig some antiseizure effects of taurine to obtain an osmotic pressure of 290 nificant protective effects against all have been reported (Barbeau and to 300 mosm. and the solutions were types of ouabain-induced seizures. Donaldson, 1974; Izumi et al, 1973; checked by osmometry. The pH of At low concentrations (9.5 x 10-2 M) van Gelder, 1972). van Gelder (1972) the amino acid solutions was ad the compound completely abolished first demonstrated the suppressive justed to 7.0-7.2. The control solu focal clonic and WBFE seizures and effect of taurine administered sys- tion contained 5mM. phosphate buf significantly decreased clonic-tonic temically against cobalt-induced fer in a NaCl solution. seizures. seizures in cat and mouse. Izumi et al. (1973) then found that the in Ouabain, betaine and isethionic Animals injected with high con traventricular injection of taurine acid were purchased from Sigma centrations of either L-alanine or revealed more marked antiseizure Chemical Co., glycine, L-alanine glycine were protected from WBFE, effects than GABA against ouabain- and L-arginine from California clonic-tonic and focal clonic seizures induced seizures in rats. Moreover, Biochemical Research, hypotaurine and to a lesser extent from running Barbeau and Donaldson (1974) re and L-cysteine from Calbiochem, and leaping seizures. Low concen ported that taurine also possessed L-proline from Nutritional Biochem trations of these two amino acids also anticonvulsant activity against cer ical Corporation and /3-alanine from completely suppressed WBFE and tain types of human epilepsy. Eastman Kodak Co. clonic-tonic seizures. Furthermore, glycine had some suppressive effect The present studies were carried STATISTICS against leaping and running seizures out to investigate the activity against at a low concentration. Administra Median effective doses (EDso ± 2 1 ouabain-induced seizures in rats of 95% confidence limits) of tion of 9.5 x 10- M or 1.9 x 10- M various amino acids, including some hypotaurine and /3 -alanine against betaine protected rats from WBFE precursors, a metabolite and various clonic-tonic and running seizures and clonic-tonic seizures only. High structural analogues of taurine in were obtained by the graphic doses of L-proline intensified run order to delineate the specificity of method of Litchfield and Wilcoxon ning seizures but clonic-tonic sei this amino acid. zures were decreased in incidence. (1949). Statistical analysis for differ 2 ences in seizure occurrence between Isethionic acid (9.5 x 10- M) showed MATERIALS AND METHODS control and test groups was carried no significant protective action. Female Sprague-Dawley rats out by the "difference between At the other end of the spectrum, weighing approximately 180 g. were proportions of two small samples the animals receiving 50/xl. of 1.5 x implanted with a cannula into the left method" (Winer, 1971). Student's 10-1 M L-arginine had some en lateral ventricle as described previ t-test (Goldstein, 1964) was applied hancement of clonic-tonic seizures. ously (Donaldson et al., 1971). A to analysis of differences between Similarly intraventricular injections volume of 50 fx\. was used for the control and test groups in compari- of L-cysteine markedly increased

Tsukada et al NOVEMBER 1974 - 215

TABLE 1-1 Suppressive Effects of Ai"nino Acids against Ouaba in-Induced Seizures. Occurrence (1)

TAURINE GABA HYPOTAURINE Concentration (M) Control 4.75x10-" 9.5x10-" 9.5x10-" 1.9xlOJ Control J.5xl0-» 1.9x10-' Number of Rats 9 9 6 9 6 7 8 6

Running 8 4 0** j** 0** 7 5 0** Leaping 8 4 0** i** 0** 7 4 0** Focal clonic 4 0* 0 4 0 4 1 0* Clonic-tonic 8 Q** 0** 0** 0** 4 0* 0* Whole body flexion and extension 7 3 0** 0** 0** 5 0** 0* Occurrence: Number of rats showing each type of seizures Differences between control and test animals indicated by **P<0.01 and *P<0.05. Data on taurine and GABA were quoted from the study by Izumi et al. (1973) (9.5 x 10-3 M L-leucine was used as a control for taurine and GABA in that study).

TABLE 1-2 Suppressive Effects of Amino Acids against Ouabain-lnduced Seizures: Occurrence (2)

B -ALANINE L-ALANINE GLYCINE 2 J Concentration (M) Control 9.5x10-" 1.9x10-' Control 9.5xl0- 1.9xlO Control 9.5x10-" 1.9x10-' Number of Rats 6 6 6 6 6 6 7 6 6

Running 6 5 i ** 6 6 3 7 5 4 Leaping 6 5 0** 6 6 2* 7 2* 2 Focal clonic 4 0* 0* 4 2 0* 4 2 0" Clonic-tonic 3 1 0 3 0 0 3 0 0 Whole body flexion and extension 4 0* 0* 3 0 0 3 0 0 Occurrence: Number of rats showing each type of seizures. Differences between control and test animals indicated by **P<0.01 and *P<0.05. Data on taurine and GABA were quoted from the study by Izumi et al. (1973) (9.5 x 10-3 M L-leucine was used as a control for taurine and GABA in that study).

TABLE 1-3 Suppressive Effects of A •nino Acids against Ouabain-lnduced Seizures: Occurrence (3)

BETAINE L-PROLINE L-ARGININE Concentration (M) Control 9.5x10-" 1.9x10-' Control 9.5x10-" 1.9xlOJ Control 9.5x10-" 1.5x10-' Number of Rats 6 6 6 6 6 6 6 6 6

Running 6 6 6 6 6 6 6 6 6 Leaping 6 6 5 6 6 6 6 5 6 Focal clonic 3 2 2 4 3 1 5 4 3 Clonic-tonic 3 0 0 3 1 0 3 3 6 Whole body flexion and extension 4 0* 1 4 2 3 4 2 1 Occurrence: Number of rats showing each type of seizures Differences between control and test animals indicated by **P<0.01 and *P<0.05. Data on taurine and GABA were quoted from the study by Izumi et al. (1973) (9.5 x 10 3 M L-leucine was used as a control for taurine and GABA in that study).

TABLE 1-4 Suppressive Effects of Amino A cids against Ouabain-lnduced Seizures Occurrence (4)

ISETHIONIC ACID L-CYSTEINE Concentration (M) Control 9.5x10-" Control 6.3xl0«" 9.5x10-" Number of Rats 5 5 6 6 6 Running 5 5 6 6 6 Leaping 5 5 6 6 5 Focal clonic 3 2 3 3 4 Clonic-tonic 3 3 3 6 6 Whole body flexion and extension 4 5 3 5 4

216 - NOVEMBER 1974 Suppressive Effects of Various Amino Acids

TABLE 2-1 Suppressive Effects of Amino Acids against Ouabain-Induced Seizures • Incidence (1)

TAURINE GABA HYPOTAURINE CONCENTRATION (M) CONTROL 4.75 x 10-2 9.5 x 10-2 9.5 x 10-2 1.9 x 10-> CONTROL 9.5 x 10-2 1.9 x 10-1 NUMBER OF RATS 9 9 6 9 6 7 8 6 Running 1.9 ±0.4 1.0 ±0.4 o** 0.1 ± 0.1** o** 2.1 ± 0.3 1.0 ± 0.4* Leaping 1.3 +0.3 0.7 + 0.3 o** 0.1 ± 0.1** o** 2.1 ± 0.4 0.5 ± 0.2** Q** Focal clonic 1.4 +0.6 0* 0 0.8 ± 0.4 0 0.7 ± 0.3 0.1 + 0.1 0* Clonic-tonic 2.9 ±0.8 o** o** 0.7 ± 0.3 0* 0* Whole body flexion Q** o** and extension 1.3 ±0.3 0.3 ±0.2* g** 0.9 ± 0.5 0 0 0** o** Incidence: Number of seizures/animal/observation period (mean ± S.E.). Differences between control and test animals indicated by **P<0.01 and *P<0.05 (**) means increased incidence, significant toP<0.001. Data on taurine and GABA were quoted from the study by Izumi et al. (1973) (9.5 x 10-3 M L-leucine was used as a control for taurine and GABA in that study).

TABLE 2-2 Suppressive Effects of A mino Acids against Ouabain-Induced Seizures: Incidence (2)

/3-ALANINE L-ALANINE GLYCINE CONCENTRATION (M) CONTROL 9.5 x 10-2 1.9 x 10-1 CONTROL 9.5 x 10-2 1.9 x 10-1 CONTROL 9.5 x 10-2 1.9 x 10-1 NUMBER OF RATS 6 6 6 6 6 6 7 6 6 Running 2.0 ± 0.4 1.0 ±0.3 0.2 ±0.2** 1.5 ±0.3 1.0 ± 0 0.5 ± 0.2* 1.9 ±0.3 0.8±0.2* 0.7 ±0.2* Leaping 1.8 +0.5 1.0 ± 0.3 Q** 1.3 ± 0.3 1.0 ±0 0.3 ± 0.2* 2.0 ± 0.4 0.3 ±0.2** 0.3±0.2** Focal Clonic 1.5 + 0.7 0 0 1.5 ±0.6 0.3 ± 0.2 0* 0.9 ± 0.6 0.3±0.2 0 Clonic-tonic 0.7 ±0.3 0.2 ± 0.2 0 0.7 ± 0.3 0 0 1.4 ±0.7 0 0 Whole body flexion and extension 0.8 ± 0.3 0* 0* 0.5 ± 0.2 0 0* 0.7 ± 0.4 0 0

TABLE 2-3 Suppressive Effects of A mino Acids against Ouabain-Induced Seizures: Incidence '3)

BETAINE L-PROLINE L-ARGININE CONCENTRATION (M) CONTROL 9.5 x 10-2 1.9 x 10-' CONTROL 9.5 x 10-» 1.9 x 10-1 CONTROL 9.5 x 10-2 1.5 x 10-1 NUMBER OF RATS 6 6 6 6 6 6 6 6 6 Running 2.2 ± 0.8 1.5 ± 0.3 1.3 ±0.3 1.2 ± 0.2 1.7 + 0.2 1.8 ± 0.2* 1.0 ±0 1.0 ±0 1.2 ±0.1 Leaping 2.0 + 0.7 1.3 ± 0.2 1.3 ± 0.4 1.2 + 0.2 1.5 ± 0.2 1.5 ±0.2 1.0 ± 0 0.8±0.1 1.2 ±0.1 Focal clonic 0.8 + 0.5 0.3+ 0.2 0.3 ± 0.2 1.0 ± 0.4 0.5 ± 0.2 0.5 ±0.5 1.0 ± 0.2 1.2 ±0.6 0.7 ± 0.3 Clonic-tonic 0.5 ±0.2 0* 0* 0.5 + 0.2 0.2 ± 0.2 0* 0.5 ± 0.2 0.5 ± 0.2 1.8 ± 0.5* Whole body flexion and extension 0.8 ± 0.5 0 0.3 ±0.2 0.7 ± 0.3 0.3 ± 0.2 0.5 ±0.2 0.7 + 0.3 0.3 + 0.2 0.2 ± 0.1

TABLE 2-4 Suppressive Effects of A mino Acids against Ouabain-Induced Seizures: Incidence (4)

ISETHIONIC ACID L-CYSTEINE 2 CONCENTRATION (M) CONTROL 9.5 x 10--2 CONTROL 6.3 x 10- 9.5 x 10-2 NUMBER OF RATS 5 5 6 6 6 Running 1.6 ± 0.4 2.4 ± 0.6 1.8 ± 0.5 1.7 ± 0.4 1.5 ± 0.2 Leaping 1.0+0.4 1.6 ± 0.6 1.3 ± 0.2 1.3 ± 0.2 1.8 ± 0.9 Focal clonic 1.0+0.4 0.8 ± 0.6 0.7 ± 0.3 1.7 + 0.8 2.2 ± 1.0 Clonic-tonic 0.8 ±,0.4 0.8 ± 0.4 0.3 ± 0.2 5.5 ±1.1** 5.2 ± 1.4** Whole body flexion and extension 1.0 ± 0.5 1.2 ± 0.5 0.7 ± 0.3 0.8 + 0.2 1.0 ± 0.5 Incidence: Number of seizures/animal/observation period (mean + S.E.). Differences between control and test animals indicated by **P<0.01 and *P<0.05 (**) means increased incidence, significant to P< 0.001. Data On taurine and GABA were quoted from the study by Izumi et al. (1973) (9.5 x 10-3 M L-leucine was used as a control for taurine and GABA in that study).

ITsukada et al NOVEMBER 1974 - 217

the incidence of clonic-tonic seiz On the other hand, betaine, for clonic-tonic and running seizures ures at the concentrations of 6.3 x L-proline, isethionic acid and were obtained by the graphic 10-2 M and 9.5 x 10-2 M. L-arginine did not exhibit any sig method (Litchfield and Wilcoxon, nificant increases in latency, even at 1949). As shown in Table 3, there Latency of the first seizure the high concentrations. Finally, was a significant difference between L-cysteine-treated rats (6.3 x 10-2 M) the potency of hypotaurine and of Taui Intraventricular administration of displayed a shorter latency to the ^-alanine against running seizures, hypotaurine, L-alanine, (3 -alanine first seizure than that of the control but not against clonic-tonic sei Hyp. and glycine caused significant animals. zures. Furthermore, the potency of dose-dependent increases in the la hypotaurine was significantly differ fi-Al tency of the first seizure between the Median effective dose (ED50) ent between running and clonic- Medi test (at both 9.5 x 10-2 M or 1.9 x 10-1 The ED50 (with 95% confidence tonic seizures. Diffe M) and control rats (Fig. 1). limits) of hypotaurine and |3 -alanine n.s.: DISCUSSION Difife LATENCY OF SEIZURE ONSET Hypotaurine, 0 -alanine, L-alanine calcu AFTER VARIOUS AMINO ACIDS and glycine displayed stronger and EDM more widely ranged potency against 100% TIME ouabain-induced seizures than any §: F (min.) other amino acids tested, except taurine. However, even with the Kacz 15 **p Taurine ** PGABA more effective amino acids, running and leaping seizures were apparently the pi •. mark / more difficult to suppress than other types of seizures. L-cys Hypotaurine the ii / ' / Hypotaurine, L-cysteine and isethionic ures. acid Cy / /[ SHgi / ' / ** »L- Alanine Recently, taurine has been shown 10 — **< to have antiseizure activity in human stronj / epileptic patients (Barbeau and EDT, / T t / ' '/ ' Donaldson, 1974) and in experimen also 9 tal seizures (Izumi et al., 1973; van with 1 : » -1 **; ' / <'' Gelder, 1972). Therefore, it was of amine / / / interest to study the effectiveness of 1961). / - / / some precursors (hypotaurine and ents s / ' »0-Alanine L-cysteine) and metabolites can pi / ' *; (isethionic acid) of taurine. and C 5 Hypotaurine has been identified in crease I / _L tion o / / adult rat brains (Perry and Han sen, 1973). Oja et al. (1973) reported chelat >' / I / ' ' X that no measurable conversion of calciu hypotaurine to taurine occurred in Oni brain homogenates from adult rats incres rl **• t/ y E Glycine although some conversion was CSF1 L*+ found in the brain of 10-year-old fects rats. These authors suggested that Glasei i 1 1 the oxidation of hypotaurine to GABJ« 2 2 COHTROL 4.75)< I0' H 9.5xlO- M 1.9)tlO-'M taurine might be the limiting step in creme the biosynthesis of taurine in the centra CONCENTRATIONS OF AMINO ACID SOLUTIONS adult rat brain. If this is true, the cortex suppressive effect of hypotaurine Figure 1—Latency indicated as the time (minutes) between termination of ouabain Isetl against ouabain-induced seizures uptake injection and the first appearance of any seizure activity. When the seizure activity may be due to hypotaurine itself was abolished during the 15 min. observation period, latency was tentatively plotted lesser rather than to its conversion into as 15 min. Marin taurine. Lahdesmaki and Oja (1973) Welty Difference between control and two doses of each amino acid indicated by reported that hypotaurine competi ** P<0.01 and * P< 0.05. that th tively inhibited the transport of Isethionic acid, L-arginine, betaine, L-cysteine and L-proline graphically give flat potassi curves below the 1 min. mark and have not been reproduced here. Data of taurine taurine in rat brain slices, while heart s and GABA were quoted from the study by Izumi et al. (1973) (9.5 x 10-3 M L-leucine isethionic acid and L-cysteine had isethi< no action. This was confirmed by was used as a control for taurine and GABA). taurine

218 - NOVEMBER 1974 Suppressive Effects of Various Amino Acids Jsukad,

TABLE 3 Median Effectice Dose (ED so)

RUNNING CLONIC-TONIC

Taurine § 3.89 (2.36- 6.42) x 10-2 M 2.80 (1.76-4.51) x 10-2 M Hypotaurine 10.11 (8.57-11.91) x 10-2 M ^ * +. 4.63 (3.16,6.78) x 10-2 M J * J n.s. J3-Alanine . 14.01 (11.62-16.88) x 10-2 M 5.50 (5.00-6.06) x 10-2 M Median effective dose (EDso) is indicated by ED50 (with 95% confidence limits). Difference of potency between two substances or between two types of seizures indicated by *: Significant (P<0.05) and n.s.: not significant. Differences between taurine and the other two subgroups and between two types of seizures in /3 -alanine could not be calculated because of the differences in slope functions in these plotted lines. EDso for clonic-tonic seizure was calculated at half the value of control occurrence, because the occurrence did not reach 100% even in control animals. §: From the previous study by Izumi et al. (1973).

Kaczmarek and Davison (1972). In showed no antiseizure activity in the glycine in cat spinal cord slices was the present studies, hypotaurine had present studies. We did not investi not inhibited by L-alanine, marked antiseizure activity while gate cysteic acid, another precursor ^-alanine, GABA or taurine. Con L-cysteine significantly increased of taurine, but the role of cysteic versely, glycine did not inhibit the incidence of clonic-tonic seiz acid as a precursor is known to be GABA uptake (Balcar and Johnston, ures. less than that of hypotaurine. Furth 1973). Obata and Yoshida (1973) re Cysteine like penicillamine has an ermore , the inhibitory effect of cys ported that electrophoretic administ SH group in its structure which more teic acid on taurine uptake is known ration of picrotoxin or bicuculline strongly chelates Ca2+and Mg2+than to be weaker than that of hypo depressed the inhibitory action of EDTA (Boineau, 1973). Cysteine taurine (Lahdesmaki and Oja, 1973). GABA, but not that of glycine; by also has the strongest complexes contrast, strychnine blocked only with bivalent ions among numerous ^-alanine, L-alanine, glycine and the action of glycine in cat en- amino acids (Greenstein and Winitz, L-proline topeduncular cells. The same effect 1961). It is known that chelating ag of picrotoxin on the actions of 0-alanine and glycine and less so, ents such as EDTA and citric acid GABA and glycine was also exhi L-proline and L-alanine, are thought can play a role as excitants (Curtis bited in frog spinal neurones (Barker to be inhibitory neurotransmitter and Crawford, 1969). Thus the in and Nicoll, 1973). From such candidates in the brain or spinal cord crease in seizures with administra studies. Snyder, Young et al. (1973) (Curtis and Crawford, 1969). tion of L-cysteine may result from have asserted that proline should also /3-alanine inhibits the transport of chelation of bivalent ions especially be considered as a potential central GABA and of taurine from the CSF calcium, neurotransmitter since it behaved of cat, while glycine and L-alanine similarly to glutamic and aspartic On the other hand, it is known that has no significant effects (Snodgrass acids and glycine upon uptake and increased Ca+t" concentrations in and Lorenzo, 1973). The uptake of release. CSF have strong anticonvulsant ef taurine into rat cortical slices is also fects in cats (Zuckermann and inhibited by /3-alanine, but is unaf In the present studies of the four Glaser, 1973). Similarly release of fected by glycine (Kaczmarek and amino acids listed,|3-alanine was the GABA can be increased through in Davison, 1972; Lahdesmaki and Oja, most effective against ouabain- crements in the extracellular con 1973). Snyder, Young et al. (1973) induced seizures. L-alanine and centration of Ca-^ in cat cerebral found that the displacement of glycine also displayed considerably cortex (Kaczmarek and Adey, 1973). strychnine binding in the synaptic potent antiseizure activities. Isethionic acid increases calcium membrane by glycine and glycine L-proline suppressed only clonic- uptake in rat liver mitochondria to a analogs paralleled glycine-like tonic seizures, but permitted an in lesser extent than taurine (Dolara, neurophysiological activity. Displac crease in the incidence of running Marino et al., 1973). Moreover, ing potency was in the following seizures at high concentrations. Welty and Read (1964) postulated order: glycine, L-/3-alanine, taurine, that the intracellular accumulation of L-ce-alanine and finally proline. Betaine and L-arginine potassium caused by taurine in dog Alanine and glycine have been found Betaine (glycine betaine) was in heart slices, might actually be due to in synaptic vesicle from rat cerebral vestigated because of its structural isethionic acid converted from cortex (De Belleroche and Bredford, similarity to the inhibitory neuro taurine. Isethionic acid, however, 1973). The high affinity uptake of transmitter candidate glycine. Ar-

Tsukada et al NOVEMBER 1974 - 219

ginine was studied because of its Isethionic acid, L-cysteine and similarities to taurine. Finally protective effects against hepatic glycine displayed no inhibitory hypotaurine and (3 -alanine block the coma, insulin shock and epilepsy in property. This order corresponds transport sites of taurine. Hence the rat, probably through its action upon almost with that of the antiseizure antiseizure activity of some of these tissue ammonia levels (Roberge and activity recorded in the present taurine analogs might relate to the Charbonneau, 1969). However, be- studies, except for glycine which mediating role of calcium or at least taine and L-arginine had no suppres was found to be almost as effective to that of some membrane stabilizing sive effects against ouabain-induced against ouabain induced seizures as ions. However, isethionic acid seizures. Moreover, L-arginine /3-alanine. which showed no suppressive effect caused an increase in the incidence Electrolytes are intimately related against ouabain-induced seizures of clonic-tonic seizures at a dose of to seizure phenomena: ouabain in can also favor calcium accumulation 1.5 x 10-1 M. This aggravation may creases the extracellular concentra (Dolara, Marino et al., 1973), thus be due to increases in extra cellular tion of potassium, probably con the calcium mobilization theory potassium concentration caused by tributing to the seizure inducing cannot be the only explanation of some basic amino acids, including mechanism by its inhibitory action this activity. Because of this, Bar- arginine (Alberti et al., 1971). upon Na+-K+-ATP'ase. Perfusion of beau and Donaldson (1974) favor a the brain with high concentrations of role for taurine as stabilizer of mem Comparison with taurine and GABA potassium can cause epileptic seiz brane excitability rather than as a Comparison of the effect of ures (Zuckermann and Glaser, neurotransmitter. Huxtable and hypotaurine, the most effective 1968). Conversely, a high calcium Bressler (1973) also suggest that compound in this study, and that of concentration in the cerebrospinal taurine may function as a membrane taurine or GABA examined previ fluid can have anticonvulsant effects stabilizer. ously (Izumi et al., 1973), revealed in cat (Zuckermann and Glaser, Finally it should be recalled that that taurine is the most potent in 1973). Krnjevic and Lisiewicz (1972) 2 no amino acids examined in the pre hibitor of the occurrence and inci reported that Ca +Hnjected into spi sent series were more effective dence of ouabain-induced seizures. nal motoneurones caused a reduc 2 against ouabain-induced seizures At a concentration of 9.5 x 10* M, tion in excitability and a fall in mem than taurine, previously tested under taurine completely suppressed brane resistance and that the most the same conditions. Even with ef clonic-tonic and running seizures. common change in membrane poten fective amino acids, running and Significant suppressive effects were tial was a hyper-polarization. They 1 leaping seizures were difficult to also obtained with 4.75 x 10- M postulated that the increase in per 1 44 suppress compared with other types taurine. In high doses (1.9 x 10- M), meability caused by Ca " would ex of seizures. hypotaurine as well as GABA com plain the Ca-H- dependent release of pletely abolished all seizure activity. transmitter (Kasczmarek and Adey, At low concentrations (9.5 x 10-2 M) 1973; Rubin, 1970). Recent reports REFERENCES hypotaurine diminished clonic-tonic revealed that ATP-dependent cal ALBERTI, K. G., SHAHRIARI, A. A., LEVINE, H. D., and LAULER, D. P. seizures better than GABA. On the cium uptake, in conditions of high (1971). Reversal of ouabain-induced ar other hand, running seizures were external calcium (lOmM), is in rhythmias in the dog by intravenous ar more clearly suppressed by GABA creased significantly by taurine and ginine hydrochloride. Cardiovascular Re than by hypotaurine. isethionic acid (Dolara, Marino et search, 5, 226-235. BALCAR, V. J., and JOHNSTON, A. R. The difference of efficacy bet al., 1973) and that taurine increases the affinity of some cell structures (1973). 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