These experiments will better define how inhalational anesthetics may mediate their actions by alterations in cellular Ca influx, internal Ca release and uptake. In the heart, such definition may provide insights into dysrhythmia-producing effects and contractile depressant actions of anesthetics. These studies will also determine if these anesthetic-induced alterations in cellular Ca metabolism occur in neural tissue, contributing to the anesthetic state. Knowledge of anesthetic mechanisms will permit more rational drug combinations to induce and maintain anesthesia, while defining hormonal and neural changes which accompany anesthesia. In addition to inducing general anesthesia, the inhalational anesthetics depress cardiac contractility largely by decreasing the delivery of Ca which activates myofibrillar activation. Halothane and enflurane depress both Ca entry through the Ca channel and decrease the rapidly-released Ca from the intracellular store (the sarcoplasmic reticulum or SR). In contrast, isoflurane causes less depression of inward Ca currents, while it and enflurane markedly depress a local anesthetic-sensitive, late component of tension which appears to be due a separate type of Ca release from the SR. In addition, isoflurane and enflurane markedly depress contractures caused by rapid cooling, while halothane has little effect. These differential effects of anesthetics will be assessed in the following ways: 1. Isolated vesicles of the SR will be separated into the Ca pumping portion (longitudinal SR) and the Ca release portion (junctional SR) to determine if Ca uptake and release from these different regions is specifically altered by anesthetics. 2. Study of anesthetic effects upon Ca currents in isolated heart cells will be completed using the whole cell voltage clamp technique, specifically examining effects on the different types of Ca channels. 3. Intracellular Ca measurements employing Ca-sensitive fluorescent dyes (FURA-2) in isolated myocytes will determine if anesthetics increase baseline intracellular Ca levels, thereby decreasing Ca available for myofibrillar activation, as well as depressing Ca entry. Release of hormones or neurotransmitters when stimulated (stimulation-secretion (S-S) coupling) from neuronal and endocrine cells is similar to excitation-contraction (E-C) in heart in that it requires Ca, which may be derived from extracellular sources, but may also have an intracellular source. To assess the interference by anesthetics of Ca-dependent processes in neural and endocrine cells, cultured dorsal root ganglion (DRG) cells and pituitary tumor cells will be studied as follows: 1. Anesthetic effects upon Ca and K currents in these cells will be studied using the whole cell patch voltage clamp technique, as well as single ion channel recording. Narcotic drugs and endorphins have already been shown to depress Ca uptake and Ca-dependent slow action potentials in DRG and other neuronal cells. 2. Anesthetic effects on Ca uptake and release will be measured by radioisotopic flux methods, with simultaneous measurement of neurotransmitter and hormone release (determined by HP liquid chromatographic and RIA techniques). 3. Changes in intracellular Ca levels caused by anesthetics will be determined employing Ca-sensitive fluorescent dyes. Potential differential effects of the inhalational anesthetics, comparable to those in heart, will be explored. Possible mechanisms involving guanine-nucleotide (G) protein will also be tested.
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