GABAergic mechanism of propofol toxicity in immature neurons.
Certain anesthetics exhibit neurotoxicity in the brains of immature but not mature animals. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, is excitatory on immature neurons via its action at the GABAA receptor, due to a reversed transmembrane chloride gradient. GABAA receptor activation in immature neurons is sufficient to open L-type voltage-gated calcium channels. As propofol is a GABAA agonist, we hypothesized that it and more specific GABAA modulators would increase intracellular free calcium ([Ca2+]i), resulting in the death of neonatal rat hippocampal neurons. Neuronal [Ca2+]i was monitored using Fura2-AM fluorescence imaging. Cell death was assessed by double staining with propidium iodide and Hoechst 33258 at 1 hour (acute) and 48 hours (delayed) after 5 hours exposure of neurons to propofol or the GABAA receptor agonist, muscimol, in the presence and absence of the GABA receptor antagonist, bicuculline, or the L-type Ca2+ channel blocker, nifedipine. Fluorescent measurements of caspase-3,-7 activities were performed at 1 hour after exposure. Both muscimol and propofol induced a rapid increase in [Ca2+]i in days in vitro (DIV) 4, but not in DIV 8 neurons, that was inhibited by nifedipine and bicuculline. Caspase-3,-7 activities and cell death increased significantly in DIV 4 but not DIV 8 hippocampal neuronal cultures 1 hour after 5 hours exposure to propofol, but not muscimol, and were inhibited by the presence of bicuculline or nifedipine. We conclude that an increase in [Ca2+]i, due to activation of GABAA receptors and opening of L-type calcium channels, is necessary for propofol-induced death of immature rat hippocampal neurons but that additional mechanisms not elicited by GABAA activation alone also contribute to cell death.
Kahraman
Sibel
S
Department of Anesthesiology, University of Maryland, Baltimore, MD 21201, USA.
Zup
Susan L
SL
McCarthy
Margaret M
MM
Fiskum
Gary
G
D000375:Aging / Q000502:physiology; D018686:Anesthetics, Intravenous / Q000633:toxicity*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D004594:Electrophysiology; D018755:GABA Agonists / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects; D008856:Microscopy, Fluorescence; D009118:Muscimol / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects*; D015742:Propofol / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000187:drug effects; D005680:gamma-Aminobutyric Acid / Q000502:physiology*
D018686:Anesthetics, Intravenous; D002120:Calcium Channel Agonists; D020746:Calcium Channels, L-Type; D018755:GABA Agonists; D011963:Receptors, GABA-A; D009118:Muscimol; D005680:gamma-Aminobutyric Acid; D053148:Caspase 3; D053179:Caspase 7; D002118:Calcium; D015742:Propofol