The previous studies have supplied a fair amount of new data and prolific publications. The investigators have shown that volatile anesthetics inhibit NO dependent cGMP accumulation. This inhibition is independent of guanylyl cyclase activation or an interaction with NO. Further, volatile anesthetics do not affect the activity of guanylyl cyclases or of NOS, nor do volatile anesthetics cause an inactivation of NO directly. The data from this lab and from others indicate that the volatile anesthetics are likely to act at a proximal step in the pathway leading to NO. This may correspond to the receptor or binding site of volatile anesthetics and be represented by a signal transduction pathway or channel protein. The investigators will determine whether anesthetics inhibit NO production by limiting the availability of calcium for NO synthase activation, by inhibiting calmodulin or its interaction with NOS, by activating protein kinase C leading to phosphorylation and decreased activity of NOS, or by limiting the availability of L-arginine, the substrate for NO production. Brain slice and cultured neuronal cell pharmacology studies will be performed to define the specific neurotransmitter pathways which stimulate NO production and are inhibited by anesthetics, and to investigate the molecular mechanisms of neuronalNO signaling inhibition by inhalational anesthetics. A novel combination of cerebral autoradiography with cGMP immunohistochemistry will be used to quantitate and localize anesthetic effects of NO stimulated cGMP in specific neuronal pathways related to the anesthetic state using both isolated brain slices and whole animal preparations. To further define the mechanisms of upregulation of NOS mRNA and protein expression by inhalational anesthetics, studies will be performed to determine the time course and dose-response for upregulation of NOS mRNA and protein by inhalational anesthetics in isolated cells and in whole animals. The role of enhanced transcription versus mRNA degradation and molecular mechanisms of transcriptional regulation will be investigated. In situ hybridization will be used to assess structural location of changes in NOS expression as clues to functional correlates.
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