| Despite their importance, the mechanism of action of general anesthetics is still poorly understood. Facilitation of inhibitory GABA A receptors plays an important role in anesthesia but other targets have also been linked to anesthetic actions. Anesthetics are known to suppress excitatory synaptic transmission, but it has been difficult to determine whether they act on the neurotransmitter release machinery itself. The data in the present work suggest that clinical concentrations of both intravenous and volatile general anesthetics are able to inhibit neurotransmitter release through one or more direct interactions with the mammalian neurotransmitter release machinery. Three main findings in this work point to this conclusion. First, the commonly used volatile anesthetic, isoflurane, as well as two commonly used intravenous anesthetics, propofol and etomidate, were found to significantly inhibit neurotransmitter release from digitonin-permeablized mammalian neurosecretory cells stimulated with a 100 muM Ca2+ solution. Dose-dependent inhibition of neurotransmitter release was observed with all three anesthetics and significant amounts of inhibition occurred throughout each anesthetic's clinically relevant concentration range. Furthermore, inhibition of neurotransmitter release in permeablized neurosecretory cells was specific to anesthetic producing compounds and was not observed in the presence of the nonimmobilizer, F6, the inactive propofol isomer, 2,4-diisopropophenol, or diazepam. Second, clinically relevant concentrations of isoflurane, propofol and etomidate inhibited vesicle fusion in cultured hippocampal neurons as evidenced by the fact that release of the styryl dye RH414, loaded into synaptic vesicles, was markedly reduced in the presence of these anesthetics following ionomycin treatment. Thus, both inhalational and intravenous anesthetics appear to inhibit the neurotransmitter release machinery in neurons found within the mammalian CNS. Finally, overexpression of a truncated form of the T-SNARE, syntaxin 1A, completely blocked isoflurane and propofol's ability to inhibit neurotransmitter release from permeablized PC12 cells. This mutant form of syntaxin, md130A, lacks its C-terminal transmembrane domain as well as a portion of its SNARE motif. Overexpession of md130A alone had no affect on evoked neurotransmitter release. Furthermore, overexpression of wild-type syntaxin had no effect on isoflurane or propofol's ability to inhibit neurotransmitter release from permeablized cells. This finding suggests, but does not prove, that syntaxin 1A may be an intermediary in isoflurane's and propofol's ability to inhibit neurotransmitter release in mammalian cells. Interestingly, overexpression of md130A was found to have no effect on etomidate's ability to inhibit neurotransmitter release from permeablized cells. While md130A overexpression did not block etomidate's inhibition of the neurotransmitter release machinery this does not necessarily exclude syntaxin as a candidate effector of etomidate. Nevertheless, etomidate's site of action on the neurotransmitter release machinery appears to be distinct from that of isoflurane and propofol. |
