European Journal of Pharmacology, 194 (1991) 131-132 © 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 001429999100206D EJP 0292R Rapid communication DL-threo-3-hydroxyaspartate reduces NMDA receptor activation by glutamate in cultured neurons Ann Marini 1 and Antonello Novelli 2 I Clinical Neuroscience Branch, National Institute for Neurological Disorders and Stroke (NINDS), Bethesda, MD 20892, U.S.A. and 2 Laboratory of Developmental Neurobiology, National Institute for ChiM Health and Development (NICHD), Bethesda, MD 20892, U.S.A. Received 23 January 1991, accepted 25 January 1991 131 DL-threo-3-hydroxyaspartate (THA) is a well known glutamate uptake blocker (Balcar and Johnston, 1972) and significantly decreases glutamate uptake in cerebel- lar neurons in primary culture (Marini et al., in prepara- tion). Little is known about the effect of THA on excitatory amino acid (EAA) receptors (McBean and Roberts, 1985). Based on their affinity for the synthetic glutamate analogue N-methyl-D-aspartate (NMDA), EAA receptors may be divided into two major subtypes, NMDA and non-NMDA receptors. Primary cultures of rat cerebellar neurons, mostly glutamatergic granule cells, have proved to be a useful system to study the physiology, biochemistry and toxicology of EAAs. In this system, NMDA receptor activation by glutamate leads to cGMP formation and ultimately, to neuronal death (Novelli et al., 1988). We now report that THA selectively reduces NMDA receptor activation by glutamate. Primary cultures of rat cerebellar neurons were pre- pared as previously described (Novelli et al. 1988) and were used after 8-10 days in vitro, cGMP studies were performed as previously described (Novelli et al., 1988). Culture dishes were washed twice with a prewarmed (37°C) incubation buffer containing (in mM): 154 NaC1; 5.6 KC1; 1 MgC12; 2.3 CaC12; 5.6 glucose; 8.6 HEPES; pH 7.4. MgC12 was omitted where indicated. Dishes were then incubated at 37°C for 30 min, and drugs were added prior to the end of this period for the amount of time indicated. The incubation was stopped by aspiration of the incubation buffer and addition of 1 ml 0.4 M HC104. cGMP levels were determined by radioimmunoassay. As shown in table 1, the excitatory amino acids glutamate (GA) and kainate (KA) potently stimulated cGMP formation in cerebellar neuronal cultures. Correspondence to: A. Novelli, Departamento de Biologia Funcional, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain. Glutamate stimulation of cGMP synthesis was selec- tively blocked by either the presence of 1 mM mag- nesium in the incubation buffer (89% inhibition), or by the specific NMDA receptor antagonist D(- )-2-amino- 5-phosphonovalerate (APV) (90% inhibition), thus prov- ing specificity of glutamate stimulation of cGMP for- mation via the NMDA receptor. Addition of up to 1 mM THA for 1 min before exposure to EAAs, did not reduce the stimulation of cGMP formation by either glutamate or kainate. However, a longer pre-incubation with 100 #M THA produced a time-dependent, selec- tive inhibition of glutamate-mediated cGMP synthesis, maximal after 10 min (81% inhibition). THA produced only a slight but statistically non-significant inhibition of kainate-mediated stimulation of cGMP formation. THA did not reduce cGMP increase following direct stimulation of particulate guanylate cyclase via the atrial natriuretic peptide (ANP) receptor (Chinkers et al., 1989). Similarly, THA did not reduce cGMP increase following direct stimulation of soluble guanylate cyclase by sodium nitroprusside (SNP) (Bohme et al., 1977). TABLE 1 THA effect on the stimulation of cGMP intracellular accumulation in cerebellar neurons in primary culture i. Drug concentration and ex- posure time: APV = 100/xM; SNP = 1 mM × 2 min; ANP = 10 nM x 2 min; KA =100 #MX1 min; GA =100 #Mxl min; THA =1 mMxl min or 100 #Mxl0 rain Antagonist Stimulant none 2 Ga 2 KA ANP SNP None 2 6 44 50 - - Mg2+ (1 mM) 1.7 5 4 59 8.3 380 APV (1 min) 3 0.8 4.4 4 41 8 370 THA (1 rain) 3 _ 56 77 - - THA (10 min) 3 1.8 8.5 4 46 7.8 360 1 Values (pmol cGMP/mg protein) are expressed as the means of two to six determinations, assayed in duplicate. S.D. was 10-20% of the mean. 2 Magnesium was omitted from the incubation buffer. 3 Time of addition before the cGMP stimulant. 4p < 0.01 vs. glutamate stimulation, by Student's t-test. (-) = not determined.