To understand the mechanisms by which volatile anesthetic agents produce many of their associated side effects it is important to understand how control of the functional state of cells is altered by these agents. Cell Ca2+ is an important effector of many cell functions, and the proposed studies will examine the mechanisms by which halothane, enflurane, and isoflurane affect Ca2+ mobilization, phosphoinositide generation, and function in three types of cells: ventricular myocytes from rat and guinea pig, arterial and venous vascular smooth muscle cells, and renal epithelial cells. These cells were selected because they represent organs in which anesthetic induced changes in function are clinically important, and their mechanisms of signal transduction have been well characterized.
In Specific Aim One, we will quantify changes in cytosolic Ca2+ (Ca2+i) availability in cells exposed to halothane, enflurane, or isoflurane.
In Specific Aim Two, we will examine these changes in comparison with muscle cell contraction.
In Specific Aim Three, we will characterize the mechanisms of anesthetic induced alterations in Ca2+ homeostasis by (a) examination of plasma membrane Ca2+ and K+ current, alone and with simultaneous direct measurements of [Ca2+]i; (b) study of the generation and metabolism of intracellular phosphoinositides (important mediators of Ca2+ homeostasis) in cells exposed to the volatile anesthetic agents; and (c) determination of changes in internal Ca2+ stores, release of Ca2+ from these stores by phosphoinositides in cells exposed to the volatile anesthetic agents. Finally, the interactions of hypoxia and anesthesia on Ca2+ mobilization will also be examined. The following techniques will be used to accomplish these aims: Single cell spectrofluorometry in a controlled atmosphere chamber using light microscope techniques, computer enhanced video image analysis with edge and motion detection, whole cell voltage clamp with intracellular dialysis, and phosphoinositide determination by HPLC and competitive binding assay. The goal of these basic cellular studies is to examine and compare (a) changes in Ca2+ homeostasis produced by anesthetic exposure in normal and hypoxic cells; (b) functional changes associated with alterations in Ca2+i homeostasis; and (c) possible mechanisms by which these changes are mediated. In the long term, these investigations will provide further information into the cellular mechanisms of anesthetic action, thereby enhancing patient care, and aiding the development of improved volatile anesthetic agents.
| Knight, P R; Smith, M D; Davidson, B A (1994) Effects hypoxia/reoxygenation on intracellular calcium ion homeostasis in ventricular myocytes during halothane exposure. Adv Pharmacol 31:109-23 |
| Wilde, D W; Davidson, B A; Smith, M D et al. (1993) Effects of isoflurane and enflurane on intracellular Ca2+ mobilization in isolated cardiac myocytes. Anesthesiology 79:73-82 |
| Wilde, D W; Knight, P R; Sheth, N et al. (1991) Halothane alters control of intracellular Ca2+ mobilization in single rat ventricular myocytes. Anesthesiology 75:1075-86 |
