RESUMO
Tolerance to the anticonvulsant effects of carbamazepine (CBZ) in the amygdala kindling paradigm is a contingent process, since it only develops in rats treated with CBZ before the kindling stimulation and not in those animals treated after the stimulation. The present study was designed to investigate the GABAA receptor system in CBZ contingent tolerance. Receptor autoradiography utilizing various radioligands that bind to different components of the GABAA receptor system and in situ hybridization with oligonucleotides that recognize different subunits of the GABAA receptor were performed. Kindling increased binding to benzodiazepine, picrotoxin, and GABA recognition sites selectively in the dentate gyrus of the hippocampus. Kindling also increased levels of mRNA for the alpha 4, beta 1, and beta 3 subunits but did not change alpha 1, alpha 2, or gamma 2 subunit levels. Rats tolerant to CBZ showed decreased [3H]muscimol binding, diazepam-insensitive [3H]Ro 15-4513 binding, and decreased alpha 4 subunit mRNA content compared to non-tolerant rats, whereas [3H]flunitrazepam binding, [35S]TBPS binding, and the levels of beta 1, and beta 3 subunit mRNAs remained elevated. The data suggest an indirect interaction of CBZ with the GABAA receptor system, since CBZ reportedly does not bind to this receptor system.
Assuntos
Tonsila do Cerebelo/fisiologia , Compostos Bicíclicos Heterocíclicos com Pontes , Carbamazepina/farmacologia , Expressão Gênica , Hipocampo/metabolismo , Excitação Neurológica , Receptores de GABA-A/biossíntese , Marcadores de Afinidade , Animais , Autorradiografia , Azidas/metabolismo , Benzodiazepinas/metabolismo , Compostos Bicíclicos com Pontes/metabolismo , Convulsivantes/metabolismo , Tolerância a Medicamentos , Estimulação Elétrica , Eletroencefalografia , Flunitrazepam/metabolismo , Lateralidade Funcional , Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Substâncias Macromoleculares , Muscimol/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/análise , Receptores de GABA-A/metabolismo , Convulsões/metabolismo , TrítioRESUMO
The presence of poorly oxygenated cells in solid tumors may account for clinical resistance to ionizing radiation and some chemotherapy in many cancers. Studies of the presence and spatial distribution, sensitivity to cancer therapies, and other physiological characteristics of hypoxic cells are hindered by the lack of markers specific for hypoxia and a relevant yet easily manipulated model system. We have chosen to use multicellular spheroids composed of murine EMT6 (fibrosarcoma) tumor cells as a model system and have applied an immunohistochemical marker specific for hypoxic cells with the ultimate goal of determining how cell populations change in response to radiation and/or chemotherapy. Large spheroids (500-700 microns in diameter) were selected and incubated in the presence of a hexafluorinated 2-nitroimidazole derivative, designated CCI-103F, which undergoes reductive metabolism and irreversibly binds to cellular macromolecules only under low oxygen tensions. A rabbit polyclonal antibody raised against a CCI-103F/protein adduct was used to visualize hypoxic cells using standard streptavidin-biotin-peroxidase immunohistochemical methods. Investigations using this spheroid model system promise to further our understanding of hypoxic cell resistance to cytotoxic therapies and of hypoxic cell biology in general.
