Neurons translate electrical and chemical stimuli into physiologically meaningful information by Ca2+ translocation and redistribution. The hypothesis the investigators will test in the proposed studies is that the volatile anesthetics (VA), halothane and isoflurane, interfere with normal extra- and intracellular Ca2+ translocation and distribution, and that the VA effect on Ca2+ homeostasis (translocation and redistribution) depends on the type of stimuli. Studies will be performed in a human neuroblastoma cell line, SH-SY5Y cells, that has many characteristics of adrenergic neurons, in rat hippocampal neurons in primary culture, and in HEK293 cells transfected with alpha and beta subunits of N-type Ca2+ channels. Using intracellular Ca2+ fluorescence, single channel and whole cell patch-clamp, radioisotopic measurement of noradrenaline (NA) secretion, and molecular biologic techniques to generate N-type Ca2+ channels, we will accomplish the following specific aims: 1) identify the mechanisms mediating the VA-induced increase in the K+-evoked [Ca2+]cyt transient and determine if VA also enhance [Ca2+]cyt transients resulting from electrical stimulation; 2) identify the mechanisms that lead to the VA-induced depression of the Carbachol-evoked [Ca2+]cyt; transient; 3) investigate the source of Ca2+ that modulates the VA action on neuronal L-type Ca2+ channels; 4) investigate the VA action on N-type Ca2+ channels, and the involvement of G-proteins; and 5) investigate which sources of Ca2+ are important for mediating NA release, whether VA exposure alters the relationship between [Ca2+]cyt and NA release, and determine whether the VA action on NA release is dependent on the exposure time to Vas. The results of these investigations will help to establish that modifications of Ca2+ homeostasis are a fundamental effect of the Vas and that they are directly related to the process of anesthesia. This study will lead to the identification of organelles and molecules that are targets of VA's that will assist in the future development of more specific anesthetic drugs.
NIH ExPorter
NIH ExPorter