Relationship Between Susceptibility to DMCM-Induced Generalized Motor Convulsions and Low-Affinity [3H]-Ouabain Binding in Membranes in Rat Brain.

BACKGROUND AND OBJECTIVE: Epilepsy is one of the most prevalent neurological disorders worldwide, but its underlying mechanisms have not yet been clarified. Among the possible molecular mechanisms that underlie its occurrence are those that are responsible for the neuronal ionic gradient, including the transmembrane enzyme Na+,K+;-adenosine triphosphatase (ATPase). Na+,K+-ATPase plays an important role in controlling neuronal excitability, and it is believed to be related to the pathophysiology of epilepsy. However, the specific isozymes that may be related to this neurological disorder remain to be determined. The α3 subunit-containing Na+,K+-ATPase isozyme has high affinity for ouabain and appears to play a major role in the pathogenesis of epilepsies. However, more studies are needed to evaluate the possible participation of Na+,K+- ATPase isozymes with lower affinity for ouabain (i.e., those that contain the α1 and α2 subunits). METHODS: The present study investigated whether rats with high (HTR) and low (LTR) thresholds for clonic convulsions that are induced by a benzodiazepine inverse agonist differ in the binding of [3;H]- ouabain to Na+,K+-ATPase isozymes with lower affinity to ouabain in discrete brain regions. RESULTS: Compared with the HTR group, the LTR group exhibited lower binding of [3H]-ouabain in the brainstem and frontal cortex. CONCLUSION: This finding supports the hypothesis that epilepsy is associated with impairments in Na+,K+- ATPase activity. The results also suggest that Na+,K+;-ATPase isozymes that contain the α1/α2 subunits in these brain regions may underlie the susceptibility to methyl 6,7-dimethoxy-4-ethyl-ß-carboline-3-carboxylate-induced convulsions.

Relationship between thresholds to convulsions induced by a benzodiazepine inverse agonist and [³H]-L-glutamate binding in the membranes of brain regions.

Although some studies have investigated the influence of kindling model of epilepsy on the glutamatergic neurotransmission, the relation between glutamatergic receptors and seizure susceptibility remains unclear. The present study sought to determine if rats with high (HTR) and low (LTR) thresholds to clonic convulsions induced by the benzodiazepine inverse agonist DMCM differed in the [(3)H]-L-glutamate binding to membranes from discrete brain regions. Compared to the HTR subgroup, the LTR subgroup presented a lower binding of [(3)H]-L-glutamate in the hippocampus, frontal cortex and amygdala plus limbic cortex, suggesting that glutamatergic receptors in these brain regions may underlie the susceptibility to DMCM-induced convulsions.

In vitro studies of the influence of glutamatergic agonists on the Na+,K(+)-ATPase and K(+)-p-nitrophenylphosphatase activities in the hippocampus and frontal cortex of rats.

BACKGROUND: The overstimulation of excitatory glutamatergic neurotransmission and the inhibition of Na(+),K(+)-ATPase enzymatic activity have both been implicated in neurotoxicity and are possibly related to the pathogenesis of epilepsy and neurodegenerative disorders. In the present study, we investigated whether glutamatergic stimulation by the glutamatergic agonists glutamate, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), kainate and N-methyl-d-aspartate (NMDA) modulates the Na(+),K(+)-ATPase and the K(+)-p-nitrophenylphosphatase activities in the crude synaptosomal fraction of the hippocampus and the frontal cortex of rats. RESULTS: Our results demonstrated that these glutamatergic agonists did not influence the activities of Na(+),K(+)-ATPase or K(+)-p-nitrophenylphosphatase in the brain structures analyzed. Assays with lower concentrations of ATP to analyze the preferential activity of the Na(+),K(+)-ATPase isoform with high affinity for ATP did not show any influence either. CONCLUSIONS: These findings suggest that under our experimental conditions, the stimulation of glutamatergic receptors does not influence the kinetics of the Na(+),K(+)-ATPase enzyme in the hippocampus and frontal cortex.

Rats with different thresholds to clonic convulsions induced by DMCM differ in the binding of [3H]-MK-801 and [3H]-ouabain in the membranes of brain regions.

Considering the putative participation of N-methyl-D-aspartate (NMDA) receptors and the Na(+), K(+)-ATPase enzymes in the susceptibility to convulsions induced by the benzodiazepine inverse agonist methyl 6,7-dimethoxy-4-ethyl-ß-carboline-3-carboxylate (DMCM), the present study sought to determine if rats with high (HTR) and low (LTR) thresholds to clonic convulsions induced by DMCM differed in the following aspects: the binding of NMDA receptors by [(3)H]-MK-801, Na(+), K(+)-ATPase activity (K(+)-stimulated p-nitrophenylphosphatase) and high-affinity [(3)H]-ouabain binding to membranes from discrete brain regions. Compared to the HTR subgroup, the LTR subgroup presented a lower binding of [(3)H]-MK-801 in the hippocampus, frontal cortex and striatum. The subgroups did not differ in K(+)-p-nitrophenylphosphatase activity, but the LTR subgroup had a lower density of isozymes with a high-affinity to ouabain in the brainstem and in the frontal cortex and a lower affinity to ouabain in the hippocampus than the HTR subgroup. These results suggest that NMDA receptors and ouabain-sensitive Na(+), K(+)-ATPase isozymes may underlie the susceptibility to DMCM-induced convulsions.

Rats with different thresholds for DMCM-induced clonic convulsions differ in the sleep-time of diazepam and [(3)H]-Ro 15-4513 binding.

The current study investigated the possible inherent relationship between convulsions and sleep involving the GABA(A)/benzodiazepine site complex. The aim of this study was to determine if rats with high (HTR) and low (LTR) thresholds for clonic convulsions induced by DMCM, a benzodiazepine inverse agonist, differ in the following aspects: (1) sensitivity to the hypnotic effects of the GABA(A) positive allosteric modulators diazepam, pentobarbital and ethanol and (2) in the binding of [(3)H]-flunitrazepam, a benzodiazepine agonist, measured by autoradiography, and [(3)H]-Ro 15-4513, a benzodiazepine partial inverse agonist, to membranes from discrete brain regions. The LTR subgroup presented a shorter diazepam-induced sleeping time compared to that of the HTR subgroup. Biochemical assays revealed that the LTR subgroup did not differ in [(3)H]-flunitrazepam binding compared to the HTR subgroup. With respect to the binding of [(3)H]-Ro 15-4513, the LTR subgroup had higher binding in the brainstem and lower binding in the striatum compared to the HTR subgroup. These results suggest that differences in the benzodiazepine site on the GABA(A) receptor may underlie the susceptibility to DMCM-induced convulsions and sensitivity to the hypnotic effect of diazepam.