Seizure-susceptibility and decreased taurine transport in the genetically epileptic rat

Neurochem. Int. Vol. 6, No. 3, pp. 365-368, 1984

0197-0186/84 $3.00 + 0.00 Copyright © 1984 Pergamon Press Ltd

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SEIZURE-SUSCEPTIBILITY A N D DECREASED TAURINE TRANSPORT IN THE GENETICALLY EPILEPTIC RAT DOUGLAS W. BONHAUS and RYAN J. HUXTABLE* University of Arizona, Department of Pharmacology, Health Sciences Center, Tucson, Arizona 85724, U.S.A. (Received 31 August 1983; accepted 1 November 1983)

Abstraet--P z fractions from brains of genetically seizure-susceptible (SS) rats as compared to seizureresistant (SR) rats show decreased high affinity uptake of taurine. Uptakes of GABA and glutamate into P2 fractions did not differ between the substrains. In neonatal SS rats that had never had a seizure, the uptake of taurine is decreased both into the whole brain in vivo and into P2 fraction in vitro, as compared to age-matched SR rats. This indicates that decreased uptake is not a consequence of seizure activity per se. In non-seizure susceptible progeny of SS rats, the uptake of taurine into P2 fraction did not differ significantly from that of SR rats. In kidney cortex slices from SS rats, taurine uptake is slightly greater than in slices from SR rats. We propose that the decreased taurine transport in the P2 fraction of the brains of SS rats may reflect a defect in transport in vivo that contributes to seizure-susceptibility.

Taurine is a sulfur-containing fl-amino acid present in high concentrations in the central nervous system (CNS). When taurine is applied to brain slices, spinal cord preparations or intact brains, it has an inhibitory effect on neuronal firing. This inhibitory action has led many investigators to suggest that one physiological function of taurine may be that of modulating neuronal excitability (for reviews see Barbeau and Huxtable, 1978; Huxtable, 1981; Kuriyama et al., 1983). In some human epileptics and in certain experimental epilepsies there are alterations in the high affinity active transport of taurine. Airaksinen (1979) has reported decreased uptake of taurine into platelets of human epileptics. G o o d m a n et al. (1980) have found that human epileptics are disproportionately high renal reabsorbers of taurine. In the cerebellar P2 fraction of rats made epileptic by kindling there is an increased taurine transport (Fabisiak and Schwark, 1982) and in the genetically seizure-susceptible (SS) rat, we have found a decreased taurine transport in platelets and in P2 fractions derived from brain homogenates (Bonhaus and Huxtable, 1983). However, it has not been determined whether the alterations in taurine transport found in human and animal epilepsies are specific for this amino acid, or whether the alterations are a cause or consequence of, or are unrelated to, the seizure-susceptibility. To address these questions and to gain further insight

into the role of taurine in the brain, we investigated the relationship between decreased transport of taurine and seizure-susceptibility in the SS rat. EXPERIMENTAL PROCEDURES

Chemicals

[3H]Taurine (0.31TBq/mol), [14C]glutamic acid (2.18 TBq/mol) and [3H]GABA (9.25 TBq/mol) were obtained from New England Nuclear, Boston, Massachusetts. Other reagents were purchased from Sigma Chemical Co., St Louis, Missouri. Animals

Seizure-susceptible Sprague-Dawley rats, designated UAz; AGS (SD), (Consroe et al., 1981), were from the University of Arizona colony. Control, seizure-resistant (SR), Sprague-Dawley rats were obtained from Hilltop Lab Animals, Chatsworth, California. Animals designated SS in this study were from the substrain of maximally susceptible rats (Jobe et al., 1973). Non-susceptible progeny from SS rats were designated NSP. NSP rats, which comprise less than 5% of the progeny of SS rats, show no seizure response to the sound stimulus used to determine seizure susceptibility. Transport experiments

P2 fractions were prepared from brain homogenates by discontinuous sucrose gradient centrifugation as described by Gray and Whittaker (1962). The identifiable cellular constituents in this fraction are myelin, glial cell membranes, synaptosomes and mitochondria. Taurine transport into the P2 fractions of rat brains was determined as described by Hruska et al. (1978) and Bonhaus and Huxtable (1983). In brief, 4.0 #M [3H]taurine was incubated at 37°C with 0.5rag protein/ml in Krebsphosphate buffer for 5 rain. Incubation was terminated by the addition of cold buffer, and the protein collected for

*To whom correspondence should be addressed. 365

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DOUGLAS W. BONHAUS a n d RYAN J. HUXTABLE

scintillation counting by centrifugation. The uptakes of glutamate and gamma-aminobutyric acid (GABA) were determined in an identical manner except that incubations were carried out at 27"C and the concentration of substrate was 10 #M. These conditions are similar to those described by Roskoski (1978). Incubations with glutamate and GABA were terminated after 1 and 2 min, respectively. The specific activities of GABA and glutamate were 1.17 and 0.176 TBq/mol, respectively. Taurine uptake into brains of SS and SR rats in vivo was determined as previously described (Bonhaus and Huxtable, 1983). In brief, this procedure consisted of administering 370 KBq [3H]taurine/100 g body weight by intraperitoneal injection and measuring the brain to plasma ratio of radioactivity 4 h later. Taurine transport into kidney cortex slices was determined. Renal cortical slices were cut by hand to an approximate thickness of 0.5 mm. Slices, 5 15 mg dry weight, were incubated for 40min at 37°C with [3H]taurine (4k~M, 0.267 TBq/mol) in 6 ml of a continuously oxygenated Krebs phosphate buffer identical to that used in the P2 transport experiments. Incubations were terminated by removal of the slices, which were placed on gauze blotters and then transferred to glass vials for drying. Slices were dried at 80°C for 6 h, weighed, and solubilized in 1 ml of Protosot prior to scintillation counting. Taurine efflux from P2 fractions of SS and SR rats was determined by incubating the P2 fractions in a taurine-free buffer, otherwise identical to the buffer used for the uptake experiments, and measuring the change with time in concentration of endogenous taurine in the P: fraction. Statistics. Single comparisons to a control were conducted with the unpaired Student t-test. Multiple comparisons to control were conducted with Dunnetrs t-test (Dunnett, 1964). RESULTS The rate o f taurine uptake into P2 fractions o f brain homogenates was constant for l0 min and was directly proportional to protein concentration up to 1.2 mg protein/ml. T r a n s p o r t did not occur in incubations carried out in ice water baths. Taurine uptake into P2 fractions o f SS rats was 50% o f that in SR rats (Fig. 1). The rate o f G A B A uptake in P2 fractions o f brain homogenates was constant for at least 3 rain and was directly proportional to protein concentrations up to at least 0.24 mg/ml. Uptake did not occur in incubations carried out in ice water baths (Fig. 2). Net temperature-sensitive uptakes o f G A B A into Pz fractions of SR, NSP, and SS rats were (mean_+ SD) 0.32_+0.10, 0 . 2 6 + 0 . 0 4 and 0 . 2 2 + 0 . 0 9 p m o l / m g protein/min, respectively. There were no statistically significant differences between these values at the P < 0.05 confidence limit. The rate o f glutamate uptake into P2 fractions from SR rats was constant for 2 min, and was proportional to protein concentration up to at least 0.24mg/ml (Fig. 3). Transport did not occur in incubations carried out in ice water baths. The rate o f glutamate

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Fig. 1. Net temperature-sensitive uptake of taurine into P2 fractions of seizure-resistant rats (SR), seizure-susceptible rats (SS), and nonsusceptible progeny of SS rats (NSP). Values are shown as means + SD. In the SR, SS and NSP groups there were 11, 9 and 4 animals, respectively. Rates of uptake into SR, SS and NSP rat P fractions were 5.99 + 2.10, 3.05 _+ 1.50 and 4.36 _+0.39 pmol/mg protein/ min, respectively. Uptake in SS rats was statistically lower than that of SR rats (P < 0.05), Dunnett, 1955).

uptake into P2 fractions of SR and SS rats was 2.23 _+ 0.59 and 2.15 _+ 0.77 n m o l / m g protein/min, respectively. The rate o f taurine uptake into kidney cortex slices from SR rats was constant for at least 4 0 m i n (Fig. 4). Uptake did not occur in incubations carried out in ice water baths. The net temperature-sensitive uptake o f taurine was (mean + S D ) 3.66_+0.34 /

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Fig. 2. GABA uptake into P2 fractions of SR rats; linearity with time. Values are the mean + SD for 3 preparations at each time. O-uptake at 37~C; (D-uptake in an ice water bath.

Taurine transport and seizure-susceptibility $O

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Time (min) Fig. 3. Glutamate uptake into P2 fractions of SR rats; linearity with time. Values are the mean + SD of ast least 3 determinations at each time. pmol/mg dry wt/min in kidney slices of SR rats, and 4.19 + 0.21 in kidney slices of SS rats. The difference is statistically significant (P < 0.05). On incubation, there was no detectable effiux of taurine from P2 fractions of SS and SR rats. At the beginning of incubation, taurine concentrations in P2 fractions of SR and SS rats were 11.7 + 1.8 and 16.0 + 2.9 nmol/mg protein, respectively. After 10 min incubation, the concentrations of endogenous taurine in P2 fractions of SR and SS rats were 14.0 + 2.9 and 15.6 + 3 . 4 n m o l / m g protein, respectively. The transport of taurine in the neonatal (14-18 day old) SS rat was less than that of age-matched SR rat pups. In P2 fractions of SS rat pups the uptake was 57~o of that in SR rat pups. In rive uptake into brains of SS rats, expressed as the brain to blood ratio of [3H]taurine 4 h after intraperitoneal injection, was 4 8 ~ of that found in the SR neonate rat (Fig. 5). The corresponding values for adult rats are shown for comparison (Bonhaus and Huxtable, 1983). 0.2

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Fig. 4. Uptake of taurine into kidney cortex slices of SR rats; linearity with time. Each value represents the mean ___SD of 3 determinations at each time.

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Fig. 5. Taurine uptake into P2 fractions and whole brains of adult and neonatal SS rats. Values are the mean + SD expressed as a percentage of the corresponding values for SR rats. At least 7 animals were used in each group for the in vitro experiments and 4 animals per group for the in vivo experiments. In all cases the uptake into SS rats was lower than that in SR rats (P < 0.05). The uptake in adult rats has been reported previously (Bonhaus and Huxtable, 1983).

DISCUSSION

The P2 fraction isolated from brain homogenate contains mitochondria, synaptosomes and glial fragments (Gray and Whittaker, 1962). Based on studies of synaptosomally enriched subfractions of the P2 fraction, it appears that the decrease in taurine transport of the P2 fraction in the SS rat is due to a decrease in glial rather than synaptosomal transport (Bonhaus and Huxtable, 1983). The P2 transport of taurine is decreased in both 14-18-day old and adult SS rats. Decreased transport is not a generalized phenomenon affecting all amino acids, as no alteration is seen in glutamate or G A B A transport. The decrease in taurine transport in the P2 fraction of the SS rat is not due to a difference in efflux of endogenous taurine into the incubation medium as the amount of taurine released, if any, from the P2 fraction is not sufficient to alter the concentration of taurine in the incubation medium. The limited availability of NSP rats ( < 5 per 100 SS rats) made it necessary to use animals from the same litter for the data reported in Fig. 1. O f the three substrains tested, the rate of transport of taurine was lowest in those animals demonstrating audiogenic seizure-susceptibility (Fig. 1). Decreased taurine transport may be associated with seizure-susceptibility, but not with the seizure

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DOUGLASW. BONHAUSand RYAN J. HUXI'ABLE

process. Decreased transport is present in neonatal animals that have never had an audiogenic convulsion, and that are, in fact, recalcitrant to sound stimulation. Furthermore, electroencephalographically verified seizures in unrestrained SS rats have always been accompanied by convulsions (Laird H. E., personal communication). Thus, we are confident that the neonatal rats used in this study have never experienced seizure activity. Further support for the hypothesis that decreased taurine transport may contribute to seizuresusceptibility comes from the observation that the taurine transport antagonist, guanidinoethane sulfonic acid, is a potent convulsant (Mori et al., 1981; Huxtable, 1981). In P2 fractions of the cerebellum of rats made seizure-susceptible by amygdaloid kindling, the net temperature-sensitive transport of taurine is increased (Fabisiak and Schwark, 1982). One could hypothesize that the increased taurine transport in the genetically normal but experimentally epileptic rat is a compensatory response either to the kindling process or to subsequent seizures, whereas the decreased taurine transport in the genetically epileptic rat is a defect contributing to the seizuresusceptibility. If this reasoning is valid, then this in turn would suggest that the anticonvulsant action of taurine involves an intracellular mechanism, or a mechanism which requires the transport of taurine. Baba et al. (1983) have shown that the inhibitory action of taurine on cyclic A M P formation is a transport dependent process, indicating that this action of taurine is by an intracellular rather than extracellular process. The increased taurine transport in the kidney slices of SS rats indicates that there is not a global deficit in the transport of taurine. This finding is consistent with the observation of G o o d m a n et al. (1980) that human epileptics are disproportionally high renal reabsorbers of taurine. In summary, our findings suggest that decreased taurine transport in P2 fractions of SS rat brains is not a consequence of the seizure-susceptibility but rather may be contributing to the increased neuronal excitability. This in turn supports the hypothesis that the neuroinhibitory action of taurine involves an intracellular or transport-related mechanism of action.

Acknowledgements--Supported by USPHS HL 19394 and l'Association Canadienne de.l'ataxie de Friedreich. REFERENCES

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