The long term objectives of this proposal are to increase knowledge concerning the cellular and molecular actions of anesthetics with the aim that this knowledge will aid the development of anesthetics which are more specific in their actions and having fewer unwanted side effects. The specific aims of this proposal are to study the pre and post-synaptic actions of (1) inhalation and intravenous anesthetics, (2) protein kinase C, (3) cAMP dependent protein kinase and (4) G proteins at the neuromuscular junction of the rat diaphragm. Interactions of these "second messengers" with anesthetics at pre- and post-synaptic sites will also be investigated. This information will be gained by using compounds known to either block or facilitate the actions of the "second messenger" being studied. The proposed studies will be performed in the rat diaphragm muscle using the 2 microelectrode voltage clamp. With this technique the amplitude and decay phase of the transmembrane current are analyzed. Monoexponential decay phases will be compared by observing changes in the amplitude and time constant of decay of the MEPC and EPC. Biexponential decay phases will be evaluated with the Marquardt-Levenberg algorithm which provides values for the amplitude and time constant of decay of both phases of the biexponential decay. The sum of the products (time constant of decay times the amplitude) of each component yields the total current flow across the membrane. This value can be determined for both mono and biexponential decay phases, and allows their comparison. Alteration of nerve terminal function will be evaluated by tetanic stimulation of the cut muscle preparation and recording the endplate current. Endplate currents will be analyzed for amplitude, quantum content, mobilization rate of transmitter, and rundown. Rundown of tetanically elicited endplate current of nerve terminal origin will be separated from that due to blockade of the receptor associated ion channel block or receptor desensitization by microiontophoresis of acetylcholine onto the endplate region at the same frequency used for tetanic stimulation. Currents induced by iontophoretic application of acetylcholine should not produce rundown if origin of the rundown is at the nerve terminal. Rundown which occurs as a result of open ion channel block or desensitization of the receptor will occur. Desensitization can be separated from open ion channel block by determining time constant of decay over a range of clamped membrane potential. Control measurements will be made followed by application of the test compound which will be applied for sufficient time to permit its action. Pertussis toxin will be administered 2-9 days before the planned study. Alteration in the actions of anesthetics as a result of the action or inhibition of these "second messengers" will also be evaluated.