Neurotoxic effect of acamprosate, N-Acetyl-Homotaurine, in cultured neurons

Abstract

Acamprosate (AC), N-acetyl-homotaurine, has recently been introduced for treating alcohol craving and reducing relapses in weaned alcoholics. AC may exert its action through the taurine system rather than the glutamatergic or GABAergic system. This conclusion is based on the observations that AC strongly inhibits the binding of taurine to taurine receptors while it has little effect on the binding of glutamate to glutamate receptors or muscimol to GABAA receptors. In addition, AC was found to be neurotoxic, at least in neuronal cultures, triggering neuronal damage at 1 mM. The underlying mechanism of AC-induced neuronal injury appears to be due to its action in increasing the intracellular calcium level, [Ca2+]i. Both AC-induced neurotoxicity and elevation of [Ca2+]i can be prevented by taurine suggesting that AC may exert its effect through its antagonistic interaction with taurine receptors.

Introduction

Addiction to alcohol produces numerous deleterious effects. Untreated alcohol abuse results in a variety of social, economic and medical consequences, all of which contribute to health care costs in the US in excess of 100 billion dollars annually [14]. Excessive alcohol use not only complicates treatment of general medical diseases, but can result in severe fatal outcomes. Hence, an effective therapeutic intervention of alcohol abuse is highly desirable.

Among various therapeutic agents that have been proposed for treating alcohol craving, naltrexone and acamprosate (AC) have received the most attention. Several preclinical studies have demonstrated that naltrexone, an opioid antagonist, reduced alcohol intake [15, 21, 22]. Unfortunately, a more recent follow-up study showed that the ability ofnaltrexone to facilitate abstinence compared to placebo diminishes over time [ 15].

Perhaps, the most promising therapeutic agent for reducing or preventing relapses in weaned alcoholics is AC, a homotaurine analog (N-acetyl-homotaurine). Numerous reports have shown that AC is effective in reducing alcohol craving and alcohol consumption and maintaining the abstinence, [11, 17, 19, 20, 23]. In addition, it has also been shown to be effective in reducing excitatory amino acids (EAA)-induced neurotoxicity [ 1 ] and alcohol withdrawal-induced seizures [3]. Although AC has already been used clinically in Europe [6, 17-20, 23] the mechanism(s) of action for its central effects remain elusive. In this communication, evidence is presented to support the conclusion that AC has a neurotoxic effect, at least in the cultured neuronal system and it may exert its action through its antagonistic interaction with the taurine receptors.

Materials and Methods

Materials Female Sprague-Dawley rats were obtained from Sasco (Wilmington, Mass.). Basal Medium Eagle (BME) and sodium bicarbonate were purchased from Life Technologies (Grand Island, N.Y.). Glutamine, poly-L-lysine (MW >300 kd), monosodium glutamate, ~-NADH, sodium pyruvate, taurine and heat-inactivated fetal bovine serum were obtained from Sigma (St. Louis, Mo.). [3H]-gtutamate (Glu; 1 mCi/ml) and [3H]-taurine (1 mCi/ml) were purchased from American Radiolabeled Chemicals (St. Louis, Mo.). [3H]-muscimol (1 mCi/ml) was obtained from Dupont NEN Research Products (Boston, Mass.). Fluorescent dyes (Fura 2-AM and Calcium Green-AM) were purchased from Molecular Probes (Eugene, Oreg.). Synthesis of A C AC was synthesized by acetylation of homotaurine with acetic anhydride. AC thus obtained was characterized as N-acetyl-homotaurine primarily by NMR analysis. Preparation of Neuronal Cultures Primary neuronal cultures were prepared from fetal rat brains as previously described [9]. Procedures for all rats used in this study were in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Care Unit and Use Committee of the University of Kansas. The rats were housed in the animal care facility at the University of Kansas and bred weekly. Briefly, brains dissected from fetal rats were mechanically dissociated in BME (Gibco BRL) supplemented with 7.6 mM sodium bicarbonate, 26.8 mM glucose, 2 mM glutamine, and 20% heat-inactivated fetal bovine serum. This medium was referred to as GME. The dissociated cells were plated in either 24-well plates (1 rnl/well), 35-mm tissue culture dishes or on 20-ram circular glass coverslip dishes (2 ml/dish), precoated with 5 mg/ml poly-L-Iysine (MW >300 kd). After incubation in a humidified incubator (37°C, 5% CO2) for 1-2 h, the incubation medium was then replaced with serum-free GME. Previously, it has been shown that neurons grown under similar conditions are morphologically and physiologically mature after 14 days in vitro (DIV) [9]. Furthermore, these cultures contain about 80-85% neurons as estimated by immunohistochemical staining using antibodies against neurofilament protein [9]. Treatment of Neuronal Cultures with Glutamate or AC L-Glu-induced neurotoxicity was studied in neuronal cultures at 14 DIV as described previously [5]. Prior to GIu treatment, the original culture media were replaced with fresh serum-free GME and the cultures were equilibrated in the incubator for 2 h. The experiments were carried out at room temperature in an environmental hood. For Glu treatment, cultured neurons were stimulated with 0.25 rm~i Glu for 5 rain. The stimulation was terminated by removal of the Glucontaining medium. Cultures were further washed twice and incubated with serum-free GME for 20-24 h to allow the process of neuronal injury to be completed. For AC treatment, the conditions were the same as those described above for the Glu treatment except Glu was replaced with various concentrations of AC. When AC was tested on Glu-induced neurotoxicity, cultured neurons were preincubated with AC for 10 rain before Glu treatment and AC was present throughout the Glu treatment at concentrations indicated. Determination of Neuronal Damage by Lactate Dehydrogenase Assay Neuronal damage was determined based on the release of lactate dehydrogenase (LDH) due to neuronal injury [8]. The preparation of cell suspensions and the measurement of LDH were the same as described [5]. Change of absorbance at 340 nm upon addition of [3-NADH was used for the determination of LDH activities. Release of LDH was expressed as the percentage of the amount of LDH activity in the medium to the total LDH activity. Total LDH activity is the sum of the LDH activity in the medium and the activity remaining in the cell. The percentage of LDH released was expressed as follows: LDH media LDH release (%) = x 100% LDH media + LDH cells Glutamate Receptor Binding Assay The preparation of synaptosomal membranes and the condition for Glu receptor binding assays were conducted according to Lee et al. [10] using [3H]-Glu as the ligand. Briefly, male Swiss Webster mice were decapitated and the whole brain was removed and homogenized in a 10 mM Tris-HC1, pH 7.4 buffer containing 0.32M sucrose and protease inhibitors (0.1 mM benzamidine HC1; 0.3 mM PMSF; 10 mM ACA; 0.1 mM EGTA). Synaptosomal fraction was obtained by differential centrifugation, followed by hypoosmotic 15/- sis and high speed centrifugation to obtain the synaptosomat membranes (P2 pellet). Prior to the binding assay, the P2 pellet was thawed and washed twice with 0.05% Triton X-100 in water and three more times with 50 rm~/Tris-HC1 buffer, pH 7.6. For the total binding (TB), the reaction mixture contained about 100-150 mg protein of washed membranes, 15-20 nM [3H]-Glu in I ml of 50 mM Tris-HC1 buffer, pH 7.2. After 45 rain incubation at room temperature, the reaction was terminated by brief centrifugation. The pellet was briefly washed twice before counting for radioactivity. For nonspecific binding (NB), the conditions were the same as those described above except that the membranes were preincubated with 1 mMunlabeled Glu for 10 rain prior to the addition of [3H]-Glu. In a typical assay, the ratio of TB to NB is about 5. The specific binding is the difference between TB and NB.