Nuclear localization of the 82-kDa form of human choline acetyltransferase.

Choline acetyltransferase is the enzyme catalyzing synthesis of the neurotransmitter acetylcholine in cholinergic neurons. In human, transcripts encoding two forms of the enzyme with apparent molecular masses of 69 and 82 kDa are found in brain and spinal cord; the 82-kDa form differs from the 69-kDa enzyme only in terms of a 118-amino acid extension on its amino terminus. Using green fluorescent protein-tagged choline acetyltransferase, we show that the 82-kDa enzyme is targeted to nuclei of cells, whereas the 69-kDa protein is found in cytoplasm. Expression of site-directed and deletion mutants of the 82-kDa isoform reveals that the extended amino terminus contains a nuclear localization signal in the first nine amino acids which targets the protein to nucleus. This represents the first report of a neurotransmitter-synthesizing enzyme that is localized to the cell nucleus.

Nitrous oxide enhances Na+/Ca++ exchange in the neuroblastoma cell line SK-N-SH.

Changes in the concentration of cytosolic free calcium ([Ca++]i) play fundamental roles in the initiation and regulation of many neuronal processes. Altered regulation of [Ca++]i has been implicated in the action of some anesthetics. We investigated the effects of nitrous oxide (N2O) on Ca++ mobilization and membrane potential in the human neuroblastoma cell line SK-N-SH. [Ca++]i was monitored by fluorescence spectrophotometry of cells loaded with fura-2 or fluo-3. N2O reversibly suppressed carbachol-stimulated increases in [Ca++]i. N2O also inhibited increases in [Ca++]i induced by calcium ionophore or depolarization suggesting a mechanism involving enhanced efflux or sequestration of cytosolic Ca++. The inhibitory effect of N2O was attenuated when the transmembrane Na+ gradient was altered either by suspending cells in nominally Na(+)-free buffer or by pretreating cells with ouabain. The inhibitory effect of N2O was also attenuated by the Na+/Ca++ exchange inhibitor 3,4-dichlorobenzamil. The effects of N2O on membrane potential were measured fluorimetrically using bis(1,3-dibutylthiobarbituric acid)-trimethine oxonol. In the presence of N2O, resting membrane potential was hyperpolarized, a condition that would favor Ca++ efflux mediated by the electrogenic Na+/Ca++ exchanger. Taken together, these findings indicate that N2O suppresses carbachol-stimulated increases in [Ca++]i by enhancing Na+/Ca++ exchange activity. Enhancement of neuronal Na+/Ca++ exchange may contribute to the anesthetic action of N2O.

Pressure antagonism of nitrous oxide depression of intracellular calcium in a neuroblastoma cell line.

While the ability of increased pressure to reverse anaesthesia has been well documented, the cellular or molecular mechanism(s) responsible for their mutual antagonism have remained elusive. Previous work in our laboratory, using diverse cell types, has indicated several processes requiring Ca2+ are affected in opposite directions by hydrostatic pressure [as represented by helium (He)], narcotic gases, and some anesthetics. Here we report on the effects of elevated pressures of He, and of 1 atm abs of the anesthetic gas nitrous oxide (N2O), when present alone and in combination, on calcium mobilization in the human neuroblastoma cell line SK-N-SH. Cytosolic-free Ca2+ ([Ca2+]i) was monitored by fluorescence spectrophotometry in cell suspensions loaded with the intracellular Ca2+ indicator fura-2. N2O reversibly depressed the carbachol-stimulated increase in [Ca2+]i (P < 0.01). The application of both 18 and 35 atm abs He attenuated this N2O-induced depression of carbachol-stimulated increase in [Ca2+]i. These findings support the hypothesis that pressure/anesthetic antagonism may be due in part to effects on neuronal [Ca2+]i and its regulation.