Pharmacological characterization of the N-methyl-D-aspartate (NMDA) receptor recognition site in porcine cerebral cortical membranes using [3H]-CGP 39653.

The high-affinity NMDA receptor competitive antagonist [3H]-CGP 39653 binds to Triton X-100 (0.04%) treated porcine cerebral cortex membranes in a saturable and reversible manner, with a KD of 6.1 +/- 0.97 nM and a Bmax of 944 +/- 55 fmol/mg protein. Association of ligand with the recognition site was rapid (estimated k1 = 1.1 x 10(7) M-1 min-1), and a steady state was reached within 30 min of incubation at 4 degrees C. Dissociation was also rapid (estimated k-1 = 0.2 min-1). The pharmacology of the binding site was similar to that for the rat brain, with mean pIC50 values (Hill slopes in parentheses, *indicating significant difference from unity) of 7.54 (0.51*), 6.99 (0.68*), 6.98 (0.71), 6.63 (0.80*), 6.31 (0.62*) and 5.17 (0.78) for R-CPP, L-glutamate, CGS 19755, cis-2,4-methanoglutamate, L-aspartate and NMDA, respectively. Other compounds (glycine, MK-801, kainate, S-AMPA and magnesium ions), previously observed not to interact competitively with the NMDA binding recognition site, showed a low affinity for the porcine cerebral cortex [3H]-CGP 39653 binding site. It is concluded that the pharmacological properties of the NMDA receptor recognition site labelled by [3H]-CGP 39653 are similar in the pig and rat cerebral cortices.

The effects of two inositol (1,4,5)-trisphosphate 5-phosphatase inhibitors on thyrotropin releasing hormone stimulated inositol (1,4,5)-trisphosphate production in GH3 cells.

The effects of FLA99 and EWP840, two disulfiram analogs which potently inhibit Ins(1,4,5)P3 5-phosphatase, upon basal and thyrotropin-releasing hormone (TRH)-stimulated inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) levels were investigated. Neither test compound affected the characteristics of the [3H]Ins(1,4,5)P3 binding site used in the competitive protein binding assay of Ins(1,4,5)P3 levels. In rat GH3 pituitary cell suspensions, TRH (100 nM) produced a large and time-dependent increase in Ins(1,4,5)P3 concentration, the maximum response being obtained within 5 seconds of stimulation in these cells. Neither FLA99 (100, 300 and 1000 microM) nor EWP840 (100 microM) produced obvious effects on the Ins(1,4,5)P3 response to TRH stimulation. Higher concentrations of EWP840 (300 and 1000 microM) abolished the Ins(1,4,5)P3 response to TRH stimulation. The lack of effect of the 5-phosphatase inhibition in the cells may indicate that 5-phosphatase is not the major metabolic pathway of this second messenger in this cell line under the assay conditions used.

Visualization of the NMDA recognition site in rat and mouse spinal cord by [(3)H]CGS 19755in vitro autoradiography.

The possibility to visualize the NMDA recognition site with [(3)H]CGS 19755in vitro autoradiography was evaluated in rat brain and spinal cord sections and thereafter used to study the distribution of the NMDA recognition site in rat and mouse spinal cord. The [(3)H]CGS 19755 binding was concentrated to the dorsal horn, in particular to the substantia gelatinosa. Notable binding was also seen in the intermediate area and ventral horn, while some binding was observed in the funiculi. No major differences were seen in [(3)H]CGS 19755 binding at various levels of the rat or mouse spinal cord, although a more dense binding was seen in the mouse. A similar distribution of the [(3)H]CGS 19755 specific binding and the NMDA receptor associated ion-channel site, labeled with [(3)H]TCP, was found in the mouse spinal cord. Taken together, our data indicate that the NMDA recognition site can be visualized in rat and mouse spinal cord byin vitro [(3)H]CGS 19755 autoradiography.

Characterization of [3H]CGS 19755 binding sites in the rat spinal cord.

[3H]Cis-4-phosphonomethyl-2-piperidine carboxylic acid ([3H]CGS 19755) was used to investigate the pharmacology and characteristics of the N-methyl-D-aspartate (NMDA) receptor recognition site from Triton X-100-treated membranes of rat spinal cord and cerebral cortex. The association of [3H]CGS 19755 was biphasic in both spinal cord and cerebral cortical membranes reaching a maximum after 5 min of incubation then decreasing to a steady level after an additional 10 min, suggesting that a proportion of the binding is unstable. The dissociation of the stable binding component was biphasic with rate constants at 4 degrees C of 1.55 and 0.020 min-1 for the spinal cord and 1.48 and 0.051 min-1 for the cerebral cortex. These multiple sites could not be captured in the saturation studies which were best fitted to a one-site model using non-linear regression analysis. Depending on the time of incubation with [3H]CGS 19755, KD and Bmax values differed; 9.9-26.1 nM and 25-96 fmol/mg protein vs 14.0-26.5 nM and 449-900 fmol/mg protein for spinal cord and cerebral cortex, respectively. The rank order of potency of inhibiting [3H]CGS 19755 binding was similar in both tissues: L-glutamate > CGS 19755 = CPP > NMDA. The specific NMDA agonist cis-2,4-methanoglutamate potently inhibited [3H]CGS 19755 binding as did MDL 100,925, although the latter was one order of magnitude less potent in the spinal cord than in the brain. The Hill coefficients were significantly lower than unity. In both tissues, AMPA, kainate and glycine competed poorly with [3H]CGS 19755.(ABSTRACT TRUNCATED AT 250 WORDS)