The introduction of inhalational anesthetics a century and a half ago marks a major medical and pharmacologic achievement. Neuronal membranes are favored as a likely site of anesthetic effect, either by biophysical interaction of anesthetics with membrane lipids or by induction of conformational changes in membrane proteins. Clinical anesthesia probably results from interference with information transfer at the synaptic level of brain organization. The overall objective of this proposal is to test the hypothesis that anesthetics interfere with synaptic transmission by altering lipid modulation of the Ca++-ATPase pump. There is good evidence that enzymatic methylation of membrane phospholipids does modulate transduction of biologic signals across cell membranes. Studies from our laboratory show that halothane and isoflurane stimulate phospholipid methylation (PLM) as much as two fold in rat brain synaptosomes. More recent work shows a reduction in Ca++-ATPase pump activity in synaptosomal plasma membrane (SPM) obtained from cerebra, cerebella, midbrains and medullae of rats anesthetized with halothane. This reduction varies from 25% to 75%, with the greatest inhibition seen in the medulla. Pump activity returns to control levels in SPM from rats allowed to recover from anesthesia.
The first aim of this proposal is to define characteristics of the calcium pump response to halothane. Dose response of Ca++ pump activity in synaptosomal plasma membranes will be evaluated at halothane concentrations ranging from 0.5 to 2.0%. We recently observed significant PLM stimulation over a wide range of halothane dosage. Possible concomitant halothane effects on SPM gated Ca++ channels and pump activity will also be tested. Linkage of PLM to the calcium pump response to halothane will be examined in experiments in which PLM can be inhibited or stimulated. A large effort will be directed toward testing for calmodulin, lipid, Ca++-ATPase interaction in the anesthetic response. We propose to assess anesthetic effects on Ca++ pump activity in calmodulin depleted and repleted SPM and to study anesthetic effects on purified and reconstituted rat brain Ca++-ATPase.
A second aim i s to determine where and how inhalational anesthetics affect the PLM pathway. Selective enzyme assays and kinetic studies will be used to identify putative, irreversible changes in methylating enzymes resulting from anesthetic exposure.
A third aim i s to evaluate the generality of calcium pump and PLM responses to anesthetics.
