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 major substances that induce bronchial smooth muscle contraction are natural ligands of G protein coupled receptors (GPCRs). Allergic reactions are initiated by allergen crosslinking of of high affinity IgE receptors on lung mast cells sensitized by IgE, and this allergic mechanism is considered 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 actomyosin interactions. In contrast, ligands acting on G-alpha-s-coupled receptors, such as isoproterenol, increase intracellular levels of cyclic AMP (cAMP), facilitating ASM relaxation. ? A large family of Regulators of G protein signaling (RGS) proteins binds to the G protein alpha subunits Gi and Gq (but not Gs) through a conserved RGS domain and inactivates them by accentuating their intrinsic GTPase activity and by blocking downstream effector interactions. 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 organ. The first objective is accomplished primarily by immunohistochemistry and immunoblotting using specific antibodies. RGS5 was shown to be expressed by PCR and immunoblotting in human and mouse bronchial smooth muscle cells. In human cultured airway smooth muscle (ASM) cells, RGS5 expression appears to be regulated by interleukin-13 (IL-13), which is a cytokine central to the AHR found in asthma. ? We are examining the function of RGS5 in ASM using Rgs5 knockout mice and RNA interference to knock down gene expression in cultured human ASM cells. To elucidate how RGS5 regulates ASM contraction in mice, we are using the slice contraction technique. The small diameter bronchi of the mice are excised, and thin lung sections are prepared and cultured in a tissue culture dish. A bronchoalveolar unit tethered to the dish by alveolar tension is obtained after several days, from which airway diameter (contraction) can be evaluated by microscopy before and after agonist stimulation. Preliminary data indicate cultured tracheal smooth muscle cells from these mice, the mobilization of calcium in knockout cells to the muscarinic receptor agonist carbachol and prostaglandin E2 (PGE2) were reduced compared to wild-type. Future studies will examine bronchial contractility in these mice after allergen sensitization and challenge in a model of allergic airway inflammation. Airway responsiveness of whole animals will be measured by plethysmography before and after allergen exposure. We have also established siRNA duplexes that effectively reduce expression of endogenous RGS5 in cultured human ASM cells. We are currently examining intracellular calcium concentration in siRNA-transfected human ASM cells and cultured mouse ASM from wild-type and knockout mice. We also plan to study the proliferation and synthetic function (e.g. cytokine secretion) of cultured ASM cells directly.
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