The broad, long-term objective of this proposal is to understand at a molecular level the mechanisms of action of general anesthetics on synaptic transmission in the central nervous system. The hypothesis to be evaluated is that general anesthetics effect neurotransmitter release by specific presynaptic mechanisms.
The Specific Aims are to 1) Determine the effects of general anesthetics on basal and depolarization- or ionophore-evoked neurotransmitter release from synaptosomes; 2) Determine the biochemical mechanisms of general anesthetic effects on neurotransmitter release from synaptosomes; and 3) Determine the effects of general anesthetics on purified brain protein kinase C and on Ca2+ dependent protein phosphorylation systems in synaptosomes. Identification of the mechanisms involved in the therapeutic and toxic effects of existing anesthetic agents will facilitate the development of more specific agents with fewer adverse effects. The experimental design is to identify the effects of general anesthetics on neurotransmitter release in a subcellular preparation (synaptosomes) that is free of cellular interactions and amenable to biochemical analysis, and then to characterize the mechanism(s) of the anesthetic effects by analyzing associated changes in presynaptic ion channel activity, synaptosomal membrane potential, Ca2+ concentration, Na+ concentration and flux, presynaptic receptor function and protein phosphorylation systems. The methods to be used include fluorometric and HPLC assays of anesthetic effects on basal and evoked glutamate, gamma-aminobutyric acid and dopamine release from rat brain synaptosomes, fluorometric assays of synaptosomal Ca2+ concentration (with fura-2), Na+ concentration (with SBFI) and membrane potential (with potential-sensitive probes); pharmacologic analysis of the roles of specific ion channels and of protein kinase C in the effects of general anesthetics on neurotransmitter release from synaptosomes; and biochemical analysis of general anesthetic effects on protein phosphorylation systems in intact synaptosomes and on purified protein kinase C in vitro. Determination of the presynaptic effects of general anesthetics on neurotransmitter release, and the specific mechanisms of these effects, is essential in understanding the mechanisms of general anesthetic action.
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| Ratnakumari, L; Hemmings Jr, H C (1998) Inhibition of presynaptic sodium channels by halothane. Anesthesiology 88:1043-54 |
| Murugaiah, K D; Hemmings Jr, H C (1998) Effects of intravenous general anesthetics on [3H]GABA release from rat cortical synaptosomes. Anesthesiology 89:919-28 |
| Ratnakumari, L; Hemmings Jr, H C (1997) Effects of propofol on sodium channel-dependent sodium influx and glutamate release in rat cerebrocortical synaptosomes. Anesthesiology 86:428-39 |
| Hemmings Jr, H C; Adamo, A I (1997) Effect of halothane on conventional protein kinase C translocation and down-regulation in rat cerebrocortical synaptosomes. Br J Anaesth 78:189-96 |
| Ratnakumari, L; Hemmings Jr, H C (1996) Inhibition by propofol of [3H]-batrachotoxinin-A 20-alpha-benzoate binding to voltage-dependent sodium channels in rat cortical synaptosomes. Br J Pharmacol 119:1498-504 |
