The complex effects of anesthetics on airway smooth muscle (ASM) have the potential for both benefit (e g , relief of bronchospasm in patients with asthma) and harm (e.g., interference with normal mechanisms matching lung ventilation and perfusion). The overall goal of this research program is to continue exploration of how anesthetics affect the airways. Some anesthetics relax ASM in part by decreasing the force produced for a given concentration of cytosolic calcium (i.e., decreasing calcium sensitivity), a novel mechanism of anesthetic action. This proposal will focus on the mechanisms responsible for this effect, which is significant at clinically relevant anesthetic concentrations, by pursuing two specific aims. We have found that some (but not all) anesthetics reduce calcium sensitivity during stimulation of ASM by inhibiting receptor activation of GTP-binding proteins (G proteins).
AIM A will explore mechanisms responsible for this action by first defining the specific receptor-G protein pathways that regulate calcium sensitivity in porcine airways, using techniques such as immunoprecipitation in membrane preparations from homogenized smooth muscle and permeabilized smooth muscle preparations. The sites of anesthetic action on these pathways, which preliminary data suggest is specific both to anesthetic agent and to pathway, will then be determined, with emphasis on the role of G proteins and their associated receptors.
AIM B will determine if the anesthetics which affect calcium sensitivity also affect the function of purified G proteins, examining guanine nucleotide binding, GTPase activity, and biophysical parameters of protein association in heterotrimeric G protein subunits with and without associated receptors. These results will be compared with those obtained in AIM A to ensure that any effects observed in isolated proteins are relevant to actions in tissues. Because these G protein-coupled receptor signaling pathways are common to many types of cells, investigation of these anesthetic mechanisms in ASM may yield insight into anesthetic actions in other organ systems. Also, pathways that control calcium sensitivity are a potential target for therapeutic manipulation, and exploration of these systems and anesthetic effects may facilitate the development of novel bronchodilators.
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