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 2009, we found that beta adrenergic agonists, a primary therapeutic agent for asthma, induced a marked reduction in transcription of several RGS mRNAs in human ASM, including RGS5. Human ASM with siRNA-induced extinction of RGS5 expression showed enhanced activity of signaling pathways leading to contraction like intracellular calcium flux and myosin light chain phosphorylation in response to physiological ligands such as bradykinin and acetylcholine. Contraction of precision-cut lung slices (PCLS) treated with RGS5-encoding lentivirus was reduced compared to control while the responses of slices infected with RGS5 shRNA lentivirus were increased. Contraction of PCLS from Rgs5 knockout mice in response to acetylcholine were markedly enhanced. These studies indicate that RGS5 is a physiological regulator of ASM contractility. Future studies will examine bronchial contraction in vivo using Rgs5 knockout mice after allergen sensitization and challenge in a model of allergic airway inflammation. Airway responsiveness of whole animals will be measured by invasive plethysmography before and after allergen exposure. An outgrowth of this project was to examine the function of RGS5 in parathyroid in collaboration with Dr. Olson. We found that (1)RGS5 is highly expressed in parathyroid cells;(2) parathyroid adenomas express elevated levels of RGS5 compared to matched pair normal tissue;(3) RGS5 can inhibit calcium-induced IP3 production in response to Calcium sensing receptor (CaSR) stimulation; (4) mice nullizygous for RGS5 have abnormally depressed plasma PTH levels but normal serum calcium. Future studies will examine the phenotype of Rgs5-/- mice backcrossed onto strains mimicking primary hyper- and hypo-parathyroidism.
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