The quaking mouse: an epileptic mutant with alterations affecting the modulatory mechanisms of the NMDA receptor complex.

The binding of [3H]glutamate and of [3H]1-(1-(2-thienyl)cyclohexyl)piperidine [( 3H]TCP) has been examined in the genetically epileptic mutant mouse, quaking. The density of [3H]glutamate binding sites did not differ between the quaking mice and their controls of the same strain. In the absence of exogenous glutamate or glycine, the density of [3H]TCP binding sites was also similar in the two strains. In both the mutants and their controls, exogenously added glutamate, glycine and glutamate plus glycine dose-dependently increased the binding of [3H]TCP. In the 3 conditions, the modulation of [3H]TCP binding was significantly more efficient in the quaking mice than in the controls. Furthermore, in the presence of glutamate (10(-5) M), the increase of the affinity of the ligand for the ion channel binding site was higher in the mutants than in the controls. These results suggest that the modulatory mechanisms of the N-methyl-D-aspartate/ionophore receptor complex might be altered in these mutants. These alterations might be related to the previously observed anticonvulsant properties of NMDA receptor antagonists in the quaking mouse model of inherited epilepsy.

Purified rat brain microvessels exhibit both acid and neutral sphingomyelinase activities.

Purified rat brain microvessels have been shown to hydrolyze radiolabeled sphingomyelin by means of two different enzyme systems. Enzymatic activity was detected at pH 7.4 and was strongly stimulated by magnesium or manganese and inhibited by calcium. Activity at pH 5.1 could also be found and was not dependent on any of these cations. At neutral pH and in the presence of magnesium, the rate of sphingomyelin hydrolysis did not exhibit a linear relationship with protein concentration. In contrast, increasing the protein concentration from 0.05 to 0.5 mg/ml resulted in a constant increase of sphingomyelin hydrolysis at pH 5.1. Kinetic parameters of both neutral and acid activities have been determined and were similar in magnitude to values reported previously for neural sphingomyelinases. This work demonstrates the occurrence of a neutral sphingomyelinase activity in purified rat brain microvessels, an observation raising the question of its role at the level of the blood-brain interface.

Acidic sphingomyelinase: relationship with antidepressant-induced desensitization of beta-adrenoceptors.

Previous results indicate a dose-dependent decrease of lysosomal sphingomyelinase activity induced by tricyclic antidepressants in cell cultures. A possible association of this effect with the antidepressant-induced down-regulation of beta-adrenoceptors was postulated. We report here the determination of beta-adrenoceptor binding sites and lysosomal sphingomyelinase activity in the cerebral cortex of rats treated chronically with desipramine or with the potential antidepressant drug midalcipran (which is devoid of effect on beta-adrenoceptors). The effect of midalcipran on lysosomal sphingomyelinase activity was also determined on C6 glioma cells. In C6 glioma cells, midalcipran did not decrease sphingomyelinase activity, at variance with the enzymatic inhibition induced by desipramine (DMI). In the rat cerebral cortex, neither DMI nor midalcipran modified sphingomyelinase activity. In agreement with previously reported effects, DMI induced beta-adrenoceptor desensitization in the rat cerebral cortex, while midalcipran remained ineffective. Our results indicate that in the rat cerebral cortex, the activity of lysosomal sphingomyelinase is not modulated by chronic treatment with antidepressant drugs, whatever their effect on beta-adrenoceptor sites. Our results suggest that sphingomyelinase activity is not associated with the desensitization of beta-adrenoceptors, taken as an index of the therapeutic action of antidepressants. The results indicate that care should be taken when extrapolating to in vivo situations the conclusions derived from cell culture conditions.

Arachidonoyl-coenzyme A synthetase and nonspecific acyl-coenzyme A synthetase activities in purified rat brain microvessels.

Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl-CoA (EC 6.2.1.3) synthesis activity in the microvasculature of rat brain. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl-CoA synthesis by purified rat brain microvessels were 4.0 microM and 5.8 microM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol X min-1 X mg protein-1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 microM unlabelled arachidonic acid, with a Ki of 7.1 microM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 microM. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl-CoA synthesis. In comparison, radiolabelled palmitoyl-CoA formation was inhibited competitively by 15 microM unlabelled palmitic acid, with a Ki of 5.0 microM and to a much lesser extent by arachidonic acid (Ki, 23 microM). The Vmax of palmitoyl-CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl-CoA synthesis. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities were thermolabile. Arachidonoyl-CoA formation was inhibited by 75% after 7 min at 40 degrees C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)