EXCEED THE SPACE PROVIDED. Volatile anesthetics such as halothane, isoflurane and sevoflurane are potent bronchodilators. The long-term goal of the proposed research is to understand the mechanisms by which volatile anesthetics relax airway smooth muscle (ASM). During the previous funding period, we found that anesthetics inhibit ACh-induced intraceUular calcium ([Ca2 ]i) oscillations. Paradoxically, anesthetics enhance both baseline and ACh-induced IP3 production. Accordingly, the first major hypothesis of the present proposal is that volatile anesthetics reduce agonist-induced [Ca2+]l responses in ASM by depleting sarcoplasmic reticulum (SR) Ca2+ stores. In ASM, agonist-induced elevation of [Ca2+]_involves Ca2+ influx and SR Ca2+ release via IP3 and ryanodine receptor (RyR) channels. SR Ca2+ release through RyR channels is mediated via Ca2+ itself as well as the second messenger, cyclic ADP ribose (cADPR). SR Ca 2 stores are maintained by active Ca2 reuptake and by plasma membrane Ca2+ influx triggered by SR depletion (Ca 2. release activated Ca2+ influx; Icrae).In the proposed studies, we will examine volatile anesthetic effects on these specific mechanisms of SR Ca2+ regulation. In addition to affecting [Ca2 ]_ regulation, volatile anesthetics may also decrease ASM contractility by interfering with mechanisms distal to Ca2+. Accordingly, our second major hypothesis is that volatile anesthetics decrease ASM contractility by interfering with EC coupling.
Specific Aims : 1) To determine the mechanisms by which volatile anesthetics affect regulation of IP_ and cADPR levels in ASM; 2) To determine the mechanisms by which volatile anesthetics affect SR Ca z release in ASM; 3) To determine the mechanisms by which volatile anesthetics affect SR Ca z reuptake in ASM; 4) To determine the effect of volatile anesthetics on Ca z release activated Ca z+influx (Icrac)in ASM; and 5) To determine the effect of volatile anesthetics on EC coupling in ASM. With reference to [Ca2+]i, we specifically hypothesize that anesthetics deplete SR Ca2+ stores by: increasing IP3 levels, increasing SR Ca 2 'leak' through both IP3 and RyR channels, decreasing SR Ca 2+ reuptake and decreasing Ca 2+ influx via Ierac. Furthermore, we hypothesize that anesthetics inhibit EC coupling by interfering with Ca2+-calmodulin interactions and myosin light chain activation. The proposed studies will use real-time confocal microscopy of [Ca2*]i and biochemical techniques to examine the effects of volatile anesthetics on these mechanisms of [Ca2 ]i and force regulation in freshly dissociated porcine ASM ceils. These interactions will be examined in the context of unstimulated (baseline) conditions as well as with ACh stimulation. The studies will initially focus on the effects of halothane; however, when significant effects are observed, the effects of isoflurane and sevoflurane will also be explored. PERFORMANCE SITE ========================================Section End===========================================
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