N Miao1, K Nagao, C Lynch. 1. Department of Anesthesiology, University of Virginia Health Sciences Center, Charlottesville 22906-0010, USA.
Abstract
BACKGROUND: Although barbiturates activate alpha-aminobutyric acid type A receptors as part of their hypnotic effect, these drugs also inhibit voltage-gated calcium channels. The authors determined if barbiturates could decrease neuronal intracellular Ca2+ transients and the resulting glutamate release. METHODS: Neonatal rat cerebellar granule neurons were isolated and cultured on coverslips and studied at 37 degrees C. Spectrofluorometric assays were used during identical conditions to monitor intracellular Ca2+ with the Ca2+ -sensitive fluorophore fura-2 and glutamate release by a glutamate dehydrogenase-coupled assay, which produced the reduced form of nicotinamide-adenine dinucleotide phosphate in proportion to the amount of glutamate released. Neurons were depolarized by a rapid increase in external [K+] from 5 to 55 mM. Control responses were compared with those in the presence of 10, 30, and 100 microM thiopental; 3, 10, and 30 microM methohexital; decreased external [Ca2+]; or voltage-gated calcium channel blockers. RESULTS: Thiopental and methohexital depressed the intracellular Ca2+ transient peak and plateau in a dose-dependent manner, as did decreased Ca2+. The intermediate dose of either drug caused approximately 50% decrease in peak intracellular Ca2+ and 60% decrease in glutamate release. In the presence of specific L- and/or N-type voltage-gated calcium channel blockade by nicardipine or omega-conotoxin-GVIA, respectively, 30 microM thiopental further decreased the intracellular Ca2+ transient. Thiopental caused a dose-dependent decrease in glutamate release, which was proportional to the decreased peak intracellular Ca2+. CONCLUSIONS: Thiopental and methohexital depress the depolarization-induced increase in intracellular Ca2+ and the accompanying glutamate release, actions which can contribute to the anesthetic and neuronal protective effects of these drugs.
BACKGROUND: Although barbiturates activate alpha-aminobutyric acid type A receptors as part of their hypnotic effect, these drugs also inhibit voltage-gated calcium channels. The authors determined if barbiturates could decrease neuronal intracellular Ca2+ transients and the resulting glutamate release. METHODS: Neonatal rat cerebellar granule neurons were isolated and cultured on coverslips and studied at 37 degrees C. Spectrofluorometric assays were used during identical conditions to monitor intracellular Ca2+ with the Ca2+ -sensitive fluorophore fura-2 and glutamate release by a glutamate dehydrogenase-coupled assay, which produced the reduced form of nicotinamide-adenine dinucleotide phosphate in proportion to the amount of glutamate released. Neurons were depolarized by a rapid increase in external [K+] from 5 to 55 mM. Control responses were compared with those in the presence of 10, 30, and 100 microM thiopental; 3, 10, and 30 microM methohexital; decreased external [Ca2+]; or voltage-gated calcium channel blockers. RESULTS:Thiopental and methohexital depressed the intracellular Ca2+ transient peak and plateau in a dose-dependent manner, as did decreased Ca2+. The intermediate dose of either drug caused approximately 50% decrease in peak intracellular Ca2+ and 60% decrease in glutamate release. In the presence of specific L- and/or N-type voltage-gated calcium channel blockade by nicardipine or omega-conotoxin-GVIA, respectively, 30 microM thiopental further decreased the intracellular Ca2+ transient. Thiopental caused a dose-dependent decrease in glutamate release, which was proportional to the decreased peak intracellular Ca2+. CONCLUSIONS:Thiopental and methohexital depress the depolarization-induced increase in intracellular Ca2+ and the accompanying glutamate release, actions which can contribute to the anesthetic and neuronal protective effects of these drugs.