Regulation of Normal and Asthmatic Lung Function by GCPR

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Abstract

Asthma, a pathological condition of reversible airways obstruction, comprises both inflammation of the lung as well as hyper-contractility of the bronchiolar smooth muscle. Such airway hyperresponsiveness (AHR) can exist in the absence of frank inflammatory infiltrates, however, suggesting that primary abnormalities in airway smooth muscle (ASM) contractility may exist in this disease. The primary substances that induce bronchial smooth muscle contraction are agonists of G protein coupled receptors (GPCRs). Allergic reactions are initiated by allergen crosslinking of of high affinity IgE receptors on lung mast cells MCs sensitized by IgE, and the allergic mechanism is probably the most common inciter of the pathophysiological cascade in asthma. Many of the compounds contained in mast cell granules or synthesized by mast cells act on procontractile GPCRs to induce bronchoconstriction. Examples include histamine, cysteinyl leukotrienes (LTD4), endothelin 1, adenosine, and bradykinin. In general, these agonists induce activation of the heterotrimeric G protein G-alpha q, which increases the concentration of intracellular calcium in smooth muscle cells, promoting actin-myosin interactions. A large family of Regulators of G protein signaling (RGS) proteins inactivate G-alpha q by accentuating the intrinsic GTPase activity of the G protein and by blocking downstream effector interactions. However, the physiological function of RGS proteins in the lung is unknown. The principal objective of this project is to determine which RGS proteins are expressed in specific cell types in the lung and to enumerate their functions in this tissue. The first objective will be accomplished primarily by immunohistochemistry using specific antibodies. Preliminary data suggest expression of RGS1 in cells with a morphology similar to alveolar macrophages and of RGS9 in alveolar pneumocytes. Immunofluorescence using marker antibodies for these cell types will be performed to identify cells expressing these RGS proteins. RGS5 was found to be abundantly expressed in human and mouse bronchial smooth muscle. Moreover, RGS5 expression is induced or diminished by physiological regulators of ASM function such as endothelin-1, acetylcholine, bradykinin, and isoproterenol. To elucidate how RGS5 regulates the ASM function in normal lung and in asthma, we have obtained RGS5 knockout mice. The tracheas of these mice will be excised and their contraction studied in a physiological salt solution bath connected to a pressure transducer at baseline and after agonist stimulation or electric field stimulation. Airway responses in these mice after allergen exposure will be measured by whole body plethysmography. Contractility of cultured human airway smooth muscle cells will be studied in vitro by measuring distensibility of collagen gel matrices connected to the pressure transducer. RGS5 expression will be knocked-down using siRNA, and the proliferation and synthetic function (e.g. cytokine secretion) of transfected cells will be cells measured directly.