Yu R et al / Acta Pharmacol Sin 2002 Aug; 23 (8): 680-684
YU Rong1, XU Xin-Hong, SHENG Mei-Ping
Department of Pharmacology, Medical Center, Fudan University, Shanghai 200032, China
1 Correspondence to Assoc Prof YU Rong. Phn 86-21-6404-1900, ext 2228. Fax 86-21-6404-1761. E-mail ryu@shmu.edu.cn
Received 2002-01-16 Accepted 2002-05-27
KEY WORDS steroids; allopregnanolone; cell culture; lactate dehydrogenase; GABAA receptors
ABSTRACT
AIM: To exam the effects of allopregnanolone and ¦Ã-amino-butyric acid (GABA) on the excitotoxicity. METHODS: The excitotoxicity was evoked by kainate (KA) in the primary culture of rat cerebral cortical cells. Effect of allopregnanolone or GABA on the excitotoxicity was examined by the measurement of lactate dehydrogenase (LDH) activity released in the culture medium. RESULTS: Either acute (3 h) or chronic (24 h) treatment with KA (0.01-1 mmol/L) produced a concentration-dependent increase in LDH activity released. The EC50 values were (0.16±0.03) mmol/L and (0.257±0.015) mmol/L, respectively. Acute treatment with allopregnanolone (10-1000 nmol/L) for 3 h did not significantly affect the 0.2 ¦Ìmol/L KA-induced LDH activity. On the other hand, chronic treatment with allopregnanolone (10-1000 nmol/L) for 24 h, produced inhibition on the KA-induced LDH activity in a concentration-dependent manner. The EC50 value was (436±19) nmol/L. Acute treatment with GABA (0.1-100 ¦Ìmol/L) exacerbate the 0.2 mmol/L KA-induced LDH activity in a concentration-dependent manner, with an EC50 value of (2.7±1.0) ¦Ìmol/L; while chronic treatment with GABA had no significant effect. CONCLUSION: There were differential patterns between the effects of allopregnanolone and GABA on the KA-induced excitotoxicity.
INTRODUCTION
Kainate mediate excitatory synaptic transmission in the mammalian central nervous system (CNS) through ligand-induced opening of transmembrane ion channels. Over-activation of kainate receptors has been shown to play an important role in the mechanisms underlying several neurodegenerative disorders[1]. Generally, the effect of kainate is thought to be indirect through innervations of glutamatergic neurons. It is the release of glutamate that leads to neuronal death through activation of post synaptic N-methyl-aspartic acid (NMDA) receptors[2]. However, conflicting observations regarding the roles of presynaptic kainate- and postsynaptic NMDA-receptors have also been reported[3].
The neuroprotective potential of neurosteroid has gained increasing attention during the last years. The neurosteroid allopregnanolone is synthesized de novo in the brain from cholesterol[4]. It is a potent ¦Ã-amino-butyric acidA (GABAA) receptor modulator with anticonvulsant[5] and anxiolytic[6] effects. Recently, we noticed a protective effect of allopregnanolone against kainate-induced lethality in mice as observed in an earlier report[7]. Base upon the fact that allopregnanolone is the most efficacious endogenous compound to enhance GABAA receptor function in vitro[8], the role GABAA receptor in this potential protective pathway needs to be clarified. In this study, therefore, we examined the effects of allopregnanolone and GABA on the kainate (KA)-induced excitotoxicity by the measurement of lactate dehydrogenase (LDH) activity released in the culture medium of rat cerebral cortical cells.
MATERIALS AND METHODS
Animals Embryos were obtained from Sprague-Dawley rats (Experimental Animal Resource, Medical Center of Fudan University, Grade II, Certificate ¡í 2-2-22-2) between gestational d16 and 18.
Cultures The embryonic cerebral cortex was removed from the skull and placed into minimum essential medium (MEM) containing glucose 5 g/L and NaHCO3 1.2 g/L. Cells were mechanically dispersed by repeated triturations and filtered through a 200 mm metal mesh. Two mL aliquots of the cell suspension were plated at a final density of 1.5¡Á106 cells per Petri dish (35 mm) previously coated with poly-D-lysine 0.25 g/L. Cells were then cultivated at 37 ¡æ in a 5 % CO2 atmosphere.
Drugs Allopregnanolone, GABA, and kainic acid were purchased from Sigma Chemical Co (St Louis, MO, USA).
LDH assay As a quantitative measure of cellular toxicity, the LDH activity was estimated in 0.1 mL aliquots of culture medium. The activity of LDH was determined spectrophotometrically[9].
Data analysis To characterize the excitotoxic effect of KA, the LDH activities released into the culture medium were expressed in percent of total LDH activities detected in culture medium and the cellular fraction. From the concentration-response curve, a suitable concentration was derived for the subsequent study. In the study of modulations of KA toxicity, changes in LDH activities were expressed as percent of the value obtained with KA treatment alone. Allopre-gnanolone or GABA was tested at 5 doses spanning the doses producing 50 % attenuation or exacerbation of KA toxicity. EC50 values and Emax values were the averages from three separate experiments, each in triplicate. Data were expressed as mean±SD.
RESULTS
Characterization of KA-induced LDH release in cultured rat cerebral cortical cells Either acute (3 h) or chronic (24 h) treatment with KA (0.01, 0.05, 0.1, 0.5, 1 mmol/L) produced a concentration-dependent increase LDH activity released (Fig 1). The EC50 values were (0.16±0.03) mmol/L and (0.257±0.015) mmol/L, respectively. The Emax values were (16.0±1.8) % and (71±13) %, respectively. Apparently, the concentration of 0.20 mmol/L was suitable for KA in the subsequent modulating studies, either acute or chronic.
Fig 1. Representative concentration-effect curve of KA on the excitotoxic release of LDH in the culture medium of rat cerebral cortical cells. Similar results were repeated three times, each in triplicate.
Effect of allopregnanolone on KA-induced LDH release Acute (3 h) treatment with allopregnanolone (10-1000 nmol/L) and KA (0.20 mmol/L) produced no significant changes in the KA-induced LDH release. Chronic treatment (24 h) with allopregnanolone (10-1000 nmol/L) inhibited KA-induced LDH activity in a concentration-dependent manner (Fig 2). The EC50 value was (436±19) nmol/L. The Emax values were (73.6±0.8) %.
Fig 2. Representative concentration-effect curve of allopregnanolone on LDH activity in the culture medium of rat cerebral cortical cells induced by KA (0.2 mmol/L). Similar results were repeated three times, each in triplicate.
Effect of GABA on KA-induced LDH release Acute (3 h) treatment with GABA (0.1-100 mmol/L) for 3 h exacerbate the kainate (0.2 mmol/L)-induced LDH activity in a concentration-dependent manner, with an EC50 value of (2.7±1.0) ¦Ìmol/L (Fig 3). The Emax values were (75±15) %. Chronic (24 h) treatment with GABA did not significantly affect the KA-induced LDH activity.
Fig 3. Representative concentration-effect curves of GABA on LDH activity in the culture medium of rat cerebral cortical cells induced by KA (0.2 mmol/L). Similar results were repeated three times, each in triplicate.
DISCUSSION
Several recent investigations have shown that the neurosteroid allopregnanolone is neuroprotective in a variety of experimental paradigms. In kainate model of epilepsy, allopregnanolone was found to have a protective effect[10]. We also noticed a protective effect of allopregnanolone against kainate-induced lethality in mice. Since allopregnanolone allosterically modulate GABAergic transmission through a unique binding site on the GABAA receptor Cl- channel complex, it is possible that allopregnanolone protect the KA-induced excitotoxcity via the enhancement of GABAA receptor responses. In the acute study, we found that GABA exacerbated the KA-induced LDH activity release in a concentration-dependent manner. This result contradicted to the fact that GABA is protective against excitototoxic brain damage in vivo[11]. Since the excitotoxic effect of kainate is thought to be mediated indirectly through activation of post synaptic NMDA receptors[2], this seeming conflicting result could be due to an indirect trans-synaptic action, which is negligible in this culture setting.
On the other hand, allopregnanolone exhibited a neuroprotective
effect in a later stage. So far, there has been no report regarding the chronic
effect of allopreg-nanolone on the KA-induced excitotoxicity in the cultured
cortical cells. There was a report showing both positive and negative effects
of allopregnanolone on neuronal survival following anoxia[12].
It has been proposed that glutamate excitotoxicity can contribute to the neurodegeneration
associated with cerebral
hypoxia-ischemia[13]. Therefore, their data represent
the situation in glutamate excitotoxicity to certain extent. Our results of
the chronic effect of allopregnanolone on the KA-induced excitotoxicity were
consistent with their findings of the positive effect. The negative effect of
allopregnanolone was only observed at lower concentration (1×10-10
mol/L) and could be due to a more complicated effect of allopregnanolone on
the GABAA receptor. Our results, as discussed above, were more likely
produced through sites other than the GABAA receptor. Furthermore,
we noticed insignificant effect of allopregnanolone after acute treatment, indicating
the decreased excitatory neurotransmission could be developed only after chronic
treatment of allopregna-nolone.
Further support for the speculation of non-GABA mechanism involved was from the observed difference between allopregnanolone and GABA. Different time profile indicated different mechanism involved in their effects on KA-induced excitotoxicity. Allopregnanolone and pregnanolone sulfate manifested opposite effects of on the GABAA receptor complex, and pregnanolone sulfate can exacerbate NMDA-induced death of hippocampal neurons[14]. It was reasonable to expect the protective property of allopregnanolone. However, the difference of these nuerosteroids on the excitotoxicity can not simply explained by their bidirectional effects on the GABAA receptor, since the GABA itself did not protect the KA-induced excitotoxicity.
Therefore, the differential pattern between the effect of allopregnanolone and GABA on the KA-induced excitotoxicity revealed a non-GABA mechanism involved in the allopregnanolone protection along the pathway of KA-induced excitotoxicity.
ACKNOWLEDGEMENT To Professor XU Duan-Zheng for his assistance in statistical analysis.