DIAZEPAM-INDUCED DOWN-REGULATION OF THE GABAA RECEPTOR α1 SUBUNIT MEDIATED BY A CALCIUM SIGNALING PATHWAY

NELSY BEATRIZ MEDINA; MICAELA PIETKO; MARIA CLARA GRAVIELLE

virtual

Congreso; Reunión Anual de la Society for Neuroscience; 2021

Society for Neuroscience

Benzodiazepines are widely prescribed drugs for the treatment of anxiety, sleep disorders, and epilepsy; however, their long-term use is limited by the development of tolerance. In previous studies we reported that prolonged exposure of rat cortical neurons to diacepam, a classic benzodiazepine, induced transcriptional repression of the GABAA receptor α1 subunit gene. This regulatory process was prevented in the presence of nifedipine, an inhibitor of L-type voltage-gated calcium channels. The aim of this work was to investigate the signaling cascade triggered by the persistent treatment with diazepam that leads to the regulation of GABAA receptor expression. Results from this study indicated that a 48 h treatment of rat cortical neurons with diazepam (1 μM) produced an increase in the intracellular calcium concentration measured in a multi-well plate reader using the calcium-sensitive fluorescent dye Fura 2-AM and a high potassium concentration (60 mM KCl) as a stimulus. This increase was abolished in the presence of nifedipine, suggesting that diazepam exposure induced an increase in the calcium influx through L-type voltage-gated calcium channels. Quantitative real-time PCR and western blots experiments showed that the diazepam-induced down-regulation of the α1 subunit was blocked by picrotoxin, a GABAA receptor channel blocker, and H-89, a protein kinase A inhibitor. Taken together, our results suggest that sustained stimulation of GABAA receptors by benzodiazepines activates an intracellular signaling cascade that involves the stimulation of calcium influx through L-type voltage-gated calcium channels which results in the activation of a protein kinase A cascade, finally leading to the transcriptional repression of α1 subunit gene. This regulatory mechanism depends on the activation of GABAA receptors. Elucidation of the signaling pathway activated by the sustained exposure to benzodiazepines will contribute to understand the molecular bases of tolerance.