In preliminary experiments of the applicant, general and local anesthetics have been found to influence the activity and the oligomeric state of the Ca-ATPase of sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. These results are consistent with the interpretation that tetrameres and larger oligomers are much less active than monomers and dimers of Ca-ATPase. The long term objective is to elucidate the molecular mechanisms of these effects of anesthetics on the Ca-ATPase and to understand their relevance to the myocardial depressive effects of general anesthetics and the antiarrhythmic effects of local anesthetics. Ca-ATPase will be labeled with a covalent phosphorescent probe, and time resolved phosphorence spectroscopy will be used to estimate the oligomeric state of the Ca-ATPase in SR membranes of cardiac and skeletal muscle. The influences of general and local anesthetics on the oligomeric state and activity of Ca-ATPase of cardiac and skeletal SR will be determined. Experiments with SR from AT-1 cells, an atrial tumor cell line deficient in phospholamban, will help to determine whether phospholamban is responsible for the very different responses of cardiac and skeletal SR Ca-ATPase to general anesthetics. The applicants will determine the influence of anesthetics on the responses of the oligomeric state and activity of cardiac Ca-ATPase to its two major physiologic regulators, Ca and phosphorylation of phospholamban, and vice versa. In both skeletal and cardiac SR, they will inquire whether the effect of local anesthetics to promote the formation of large oligomers of Ca-ATPase depends upon the positively charged quaternary ammonium moiety of most local anesthetics. The influence of anesthetics on the response of cardiac and skeletal Ca-ATPase to altered pH, and vice versa, will be investigated. The literature documents profound and poorly understood influences of pH on the responses of cardiac SR Ca-ATPase to inhalational anesthetics that the proposed experiments are designed to explain. Atomic force microscopy will provide an independent method to confirm or refute the anesthetic induced changes in oligomeric state of Ca-ATPase that are indicated by the previous results with time resolved phosphorescence spectroscopy.
