The Essential Role of Calcineurin, Calcium Sparks, and Ryanodine Receptor Type-1 in Asthmatic Airway Hyperresponsiveness

Airway smooth muscle cells (ASMCs) encircle the respiratory passageways below the two main bronchi functioning to regulate bronchomotor tone. In healthy individuals ASMCs seem quite vestigial, however, potentially fatal "asthma attacks" which pose a life or death risk to hundreds of millions of people worldwide are the result of ASMC mediated airway hyperresponsiveness (AHR), which is the excessive contraction to normally non-harmful non-specific stimuli. A better understanding of ASMC contraction is needed to advance asthma therapeutics.;These current studies have investigated the novel role of protein phosphatase 2B (calcineurin, CaN) in ASMC contraction and asthma using pharmacological and genetic approaches in mice and in freshly isolated ASMCs imaged by the confocal line-scan technique. The results of this work show that CaN promotes mouse ASMC contraction by up-regulating the frequency of spontaneous Ca 2+ sparks through ryanodine receptor type-1 (RyR1) not type 2 or 3, independent of extracellular Ca2+ and inositol 1,4,5-triphosphate receptors (IP3Rs). Genetic knockout of the CaN catalytic subunit Aalpha (CaN Aalpha KO) resulted in a significantly lower spontaneous frequency of Ca2+ sparks versus wild type. Equally fascinating, RyR1 heterozygous gene deletion decreases the frequency of spontaneous Ca 2+ sparks. As was previously published, inhibition and activation of protein kinase C-epsilon (PKC&egr;) increases and decreases Ca2+ sparks respectively, however, inhibition of CaN is now known to block these effects. Likewise, the effects of CaN inhibition and activation are lost in PKC&egr; KO mouse ASMCs. In an ovalbumin challenged murine model of asthma, both spontaneous Ca2+ spark signaling is increased in accord with an in-vivo measure of AHR. RyR1 heterozygous gene deletion prevents the in-vivo increase in AHR and blocks the increase in Ca 2+ sparks. Moreover, in asthma CaN expression and activation are increased, while PKC&egr; expression is reduced. Most importantly, CaN inhibition during the development of asthma in a mouse model, through treatment with calcineurin-auto inhibitory peptide or heterozygous gene deletion, prevents in-vivo methacholine induced AHR.;This thesis shows that CaN coordinates opposite PKC&egr; to up-regulate Ca2+ sparks and ASMC contraction through RyR1. Calcineurin, Ca2+ sparks and the RyR1 play an essential role in asthmatic airway hyperesponsiveness extending our understanding of the cellular and molecular mechanisms responsible for asthma attacks while identifying Calcineurin as a therapeutic drug target for the prevention of asthmatic AHR.