Glutamate uptake in blood platelets from epileptic patients

Neurochemistry International 43 (2003) 389–392

Glutamate uptake in blood platelets from epileptic patients Sirpa Rainesalo a,b,∗ , Tapani Keränen c,d , Jukka Peltola b , Pirjo Saransaari a b

a Brain Research Center, University of Tampere, Tampere FIN-33014, Finland. Department of Neurology and Rehabilitation, Tampere University Hospital, P.O. Box 2000, Tampere FIN-33521, Finland c Department of Neurology, University of Turku, Turku FIN-20014, Finland d Department of Pharmacological Sciences, Medical School, University of Tampere, Tampere FIN-33014, Finland

Received 30 September 2002; received in revised form 19 November 2002; accepted 21 November 2002

Abstract Glutamate, the major excitatory amino acid neurotransmitter, is involved in epileptogenesis and initiation and spread of seizures. We studied glutamate uptake into blood platelets from patients with distinct epileptic syndromes: included were 20 patients with temporal lobe epilepsy and hippocampal sclerosis (TLE+HS), 20 with juvenile myoclonus epilepsy (JME) and 20 healthy volunteers matched for age and sex. The affinity of glutamate for the transporters was highest in patients with TLE + HS, but the maximal velocity of transport was highest in controls. There were no differences in the plasma levels of glutamate. Carbamazepine (CBZ), valproate (VPA) and lamotrigine (LTG) did not affect the uptake in vitro. The alterations observed in the uptake of glutamate in TLE + HS patients may reflect an up-regulated uptake of glutamate in the brain. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Glutamate uptake; Blood platelets; Neurotransmitters; Juvenile myoclonus epilepsy; Temporal lobe epilepsy; Hippocampal sclerosis

1. Introduction Glutamate is the major excitatory amino acid transmitter in the human central nervous system. It is involved in epileptogenesis, the initiation and spread of seizures, and seizure-related neuronal damage. Glutamate effects are mediated by ionotropic and metabotropic receptors (Chapman, 2000). The uptake into neurons and glial cells is the most important mechanism in terminating its actions by neuronal (EAAC-1/EAAT3/EAAT4) and glial (GLT-1 and GLAST/ EAAT1/EAAT2) transporters (Chapman, 2000). Excitotoxicity results from overstimulation of glutamate receptors due to high extracellular glutamate concentrations resulting from excessive release and/or inhibition of uptake. To date, the mRNA and protein levels of EAAT3 transporter have been shown to be increased in hippocampal and neocortical neurons in human epilepsy (Crino et al., 2002), whereas the expressions of EAAT1 and EAAT2 show either no change in epileptic patients (Tessler et al., 1999; Crino et al., 2002) or changes only in specific regions of the hippocampus in temporal lobe epilepsy (Mathern et al., 1999; Proper et al., 2002).

∗

Corresponding author. Tel.: +358-3-247-5111; fax: +358-3-247-4351. E-mail address: [email protected] (S. Rainesalo).

Human platelets have been shown to accumulate glutamate in a manner similar to that in as synaptosomal preparations (Mangano and Schwarcz, 1981b). The platelet model has been used in the investigation of a number of neurodegenerative disorders, such as Alzheimer’s disease (Ferrarese et al., 2000), amyotrophic lateral sclerosis (Ferrarese et al., 2001). Huntington’s disease (Mangano and Schwarcz, 1981a), and Parkinson’s disease (Ferrarese et al., 1999). To our knowledge, this platelet approach has not previously been used in the investigation of human epilepsy. In the present preliminary study, we investigated the uptake of glutamate in blood platelets isolated from patients with juvenile myoclonic epilepsy (JME) and patients with temporal lobe epilepsy and hippocampal sclerosis (TLE + HS). The plasma concentrations of selected neurotransmitter amino acids were also measured.

2. Experimental procedure 2.1. Patients We studied 20 patients with JME, 20 patients with TLE + HS and 20 healthy volunteers. The patients were recruited from among outpatients of the Department of Neurology, Tampere University Hospital, Finland. All were

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Table 1 Demographic data on patients JME Age (years, mean ± S.D.) 27 ± 8 Duration of epilepsy 14 ± 9 (years, mean ± S.D.) Sex ratio (male/female) 5/15 Etiology Idiopathic 20 Seizure frequency

Antiepileptic drugs (no mean range)

TLE + HS 43 ± 11 27 ± 12 9/11 Cryptogenic 13 Remote symptomatic 7

GTC 0 (0–1) per month Myoclonic seizures 0 (0–30) per month

All seizure types

1.55 (1–3) Valproate 16 Lamotrigine 7 Clonazepam 2 Topiramate 1 Carbamazepine 1

1.8 (1–3) Carbamazepine 13 Gabapentin 5 Lamotrigine 4 Vigabatrin 3 Oxcarbazepine 3 Valproate 3 Benzodiazepines 2 Topiramate 2 Phenytoin 1

2.9 (0–10) per month

JME: juvenile myoclonic epilepsy, TLE+HS: temporal lobe epilepsy with hippocampal sclerosis and GTC: generalized tonic-clonic seizures.

on antiepileptic drugs. The patients and volunteers were matched for age and sex. Demographic data are presented in Table 1. The study was approved by the Ethics Committee of Tampere University Hospital and all participating subjects gave written informed consent. 2.2. Blood samples Venous blood samples were collected into tubes containing ethylenediaminetetra-acetic acid (EDTA) as anticoagulant. The samples were processed as described by Mangano and Schwarcz (1981a) with slight modifications. Cooled tubes were centrifuged for 10 min at 200 × g at 4 ◦ C. Platelet-rich plasma was then collected and centrifuged for 10 min at 7000 × g at 4 ◦ C and the pellets then suspended in 2.0 ml of ice-cold 0.32 M sucrose. After centrifugation for 5 min at 7000 × g at 4 ◦ C, the platelets were resuspended in 0.32 M sucrose to one-fifth of the original blood volume.

lets were washed twice, extracted with H2 O and counted for radioactivity. The assays were done within 6 h after taking blood samples. The results were corrected by subtracting the radioactivity in unincubated blank samples. To study the effects of valproate (VPA), carbamazepine (CBZ) and lamotrigine (LTG) on glutamate uptake in platelets from healthy volunteers, 150–1200 ␮M VPA, 10–80 ␮M CBZ and 1–100 ␮M LTG were added to incubation mixtures 5 min prior to the addition of platelets. The assays were done in triplicate. To evaluate the breakdown of glutamate during the uptake experiments, a number of samples were investigated by thin-layer chromatography using n-butanol–acetic acid– water (80:20:20) as solvent. The breakdown of l-[3 H]glutamate was negligible during the experiments, since no significant amount of radioactivity was detected outside the glutamate spot. 2.4. Amino acid assays The amino acid concentrations in plasma were measured from blood samples taken into heparin-containing tubes. The cells were centrifuged down and the 500 ␮l plasma samples deproteinized with 50 ␮l of 50% sulfosalicylic acid containing 50 ␮l of 5 mM dl-diamino-n-butyric acid as internal standard. They were left to stand for an hour at +4◦ , whereafter they were centrifuged for 10 min at 16,000 × g. An aliquot of 300 ␮l of the supernatant was taken and 175 ␮l of 0.2 M lithium citrate buffer (pH 2.2) and 25 ␮l of saturated LiOH added. The mixtures were subjected to ion-exchange chromatography using an automatic Pharmacia LKB Alpha Plus amino acid analyzer with o-phthalaldehyde derivatization and fluorescence detection. Protein measurements were done by the method of Lowry et al. (1951). 2.5. Data analysis Comparisons of different experimental groups were performed by unpaired Student’s t-test. The data are presented as mean values ± S.D.

3. Results 2.3. Uptake assays Glutamate uptake by the platelet preparations was measured with l-[3 H]glutamate (Amersham, Bristol, UK, specific activity 1.55 PBq/mol) at concentrations from 5 to 500 ␮M (Mangano and Schwarcz, 1981a). Briefly, the samples were first preincubated with Tris-citrate buffer, pH 7.4, for 15 min under oxygen in a shaking water bath. Then 0.7 ␮M [3 H]glutamate was added and the incubation stopped after 10 min by adding 500 ␮l of cold saline followed by immediate centrifugation for 10 min at 10,000 × g. The pel-

The uptake of glutamate was linear within the protein content 50–500 ␮g for at least 20 min. It was enhanced at low glutamate concentrations in patients with TLE + HS compared to controls, the transport constant Km being 118.7 ␮M in controls and 49.5 ␮M in patients with TLE + HS (P = 0.002). The maximal velocity of transport, Vmax was 0.50 mmol/min/kg protein in volunteers and 0.36 mmol/min/kg protein in TLE + HS patients (P = 0.145). In patients with JME, the transport parameters [Km 80.3 ␮M (P = 0.09) and Vm 0.35 mmol/min/kg pro-

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Fig. 1. Concentration dependence of glutamate uptake in platelets from patients with JME (䊐), and TLE + HS (䊊) and from healthy volunteers (). Number of subjects in each group 20. Mean values ± S.E.M. are shown.

Table 2 Plasma concentrations (␮mol/l±S.D.) of aspartate, glutamate and glycine in different epileptic syndromes and in control subjects

JME TLE + HS Controls

Aspartate

Glutamate

Glycine

4.4 ± 1.2 4.1 ± 1.1 4.4 ± 1.0

36.9 ± 18.6 30.6 ± 13.2 30.7 ± 13.8

300 ± 165 218 ± 44 219 ± 84

JME: juvenile myoclonic epilepsy, TLE+HS: temporal lobe epilepsy with hippocampal sclerosis.

tein (P = 0.08)] did not significantly differ from the corresponding parameters in controls (Fig. 1). In vitro experiments, VPA, CBZ and LTG did not alter glutamate uptake by platelets at clinically relevant concentrations (data not shown). There were no significant differences in the plasma levels of glutamate, glycine or aspartate between any of the study groups (Table 2).

4. Discussion Both experimental and clinical studies suggest seizurerelated changes in the glutamate levels in the brain. In the hippocampus, the levels are already increased 15 min prior to seizures (Janhua et al., 1992; Wilson et al., 1996). The glutamate/GABA ratio increases in the epileptic hippocampus, possibly causing hippocampal sclerosis (During and Spencer, 1993). The uptake is the most important mechanism removing glutamate from the synaptic cleft. Lewis et al. (1997) have shown that the ATP-dependent uptake of glutamate increases as a response to seizures in epileptic mice. However, Ortiz et al. (1996) report that glutamate up-

take is elevated prior to the appearance of manifest seizures, pointing to the possibility of regulation of glutamate uptake by second messengers. In keeping with these findings in human epilepsy is the elevation of the mRNA and protein levels of EAAT3 in the hippocampus and neocortex (Crino et al., 2002). Platelets have been to shown to possess uptake mechanisms for several amino acid transmitters, among them GABA, glutamate, aspartate and glycine (Zieve and Solomon, 1968; Airaksinen, 1979; Hambley and Johnston, 1985). A statistically significant difference was now obtained only between the control subjects and patients with TLE + HS. These patients, with an up-regulated platelet glutamate uptake, had refractory epilepsy and more frequent seizures than JME patients. There is previous evidence that changes in the platelet uptake of glutamate reflect corresponding changes in the brain (Mangano and Schwarcz, 1981b). It is not known, however, which glutamate transporters are expressed in human platelets. This matter is now the subject of our on-going studies. It is likely that in the present study a part of glutamate penetrated into platelets by diffusion. Since this non-saturable component was not subtracted, the estimates of the kinetic parameters for the saturable uptake are correspondingly higher. It could be hypothesized that an up-regulated glutamate uptake may be a phenomenon compensatory to the increase in glutamate release caused by high seizure activity. This mechanism may serve as a means to prevent glutamate excitotoxicity. On the other hand, we found no evidence of general alterations in glutamate metabolism, since the plasma levels of glutamate in both patient groups did not differ from those in the control subjects. Contradictory results have been reported regarding plasma levels of amino acids in patients with epilepsy. The levels of glutamate have been seen to be

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increased in patients with idiopathic epilepsies exhibiting generalized spike-and-wave discharges in the EEG (Van Gelder et al., 1980; Janhua et al., 1992) and in patients with partial epilepsies (Janhua et al., 1992). On the other hand, glutamate levels are not altered in cases with familial idiopathic epilepsies (Haines et al., 1985) and in a small group of patients with both generalized and partial epilepsies (Monaco et al., 1975). Differences in the patient populations may be the main factor explaining the inconsistencies in the results reported. To our knowledge, there is no firm evidence that either conventional or novel antiepileptic drugs could interact with the expression and function of transporter systems of glutamate. In one experimental study valproate had no direct effect on glutamate transport (Hassel et al., 2001) and in another valproate reduced GLT-1, had no effect on EAAC-1 and increased GLAST in the hippocampi during epileptogenesis (Ueda and Willmore, 2000). Our in vitro experiments revealed no effects of carbamazepine, lamotrigine or valproate, the most common antiepileptic drugs in our patient population, on the uptake of glutamate into the platelets. It is thus, unlikely that antiepileptic drugs could underlie the differences in glutamate uptake between our patient and control subjects. In conclusion, our preliminary studies on the platelet model suggest a compensatory alteration in the uptake of glutamate in patients with refractory localization-related epilepsy and seizure-related focal destructive changes (hippocampal sclerosis). The alterations in glutamate uptake may not, however, be enough to prevent glutamate excitotoxicity.

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