Asthma, a pathological condition of reversible airway obstruction, is comprised of both inflammation of the lung and 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) number or contraction 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 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 actin-myosin interactions and muscle fiber shortening. 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. Although short-acting and long-acting inhaled beta2-adrenergic receptor agonists (SABA and LABA, respectively) relieve asthma symptoms, use of either agent alone without concomitant anti-inflammatory drugs (corticosteroids) may increase the risk of disease exacerbation in some patients. We found previously that pretreatment of human precision-cut lung slices (PCLS) with SABA impaired subsequent beta2-agonist-induced bronchodilation, which occurred independently of changes in receptor quantities. In current work, we provided evidence that prolonged exposure of cultured human airway smooth muscle (HuASM) cells to beta2-agonists directly augments procontractile signaling pathways elicited by several compounds including thrombin, bradykinin, and histamine. Such treatment did not affect expression of surface receptor, G protein, or downstream effector (phospholipase C-beta and myosin light chain) pathway components, but rather induced a dramatic reduction in RGS4 and RGS5 proteins, which are inhibitors of G-protein-coupled receptors (GPCR). Knockdown of RGS5 in HuASM increased intracellular calcium flux and myosin light chain (MLC) phosphorylation in response to these ligands, which are prerequisites for contraction, while overexpression of RGS5 inhibited these responses. Precision-cut lung slices from RGS5-deficient mice contracted more to carbachol than WT slices. These results indicated that RGS5 controls GPCR-evoked G-alpha-q-dependent signaling pathways in ASM. Repetitive beta2-agonist use may not only lead to reduced bronchoprotection but also to sensitization of excitation-contraction pathways as a result of reduced RGS5 expression. A second project in collaboration with Dr. Panettieri identified the expression of RGS4 in human and mouse bronchial smooth muscle. A population of RGS4+ smooth muscle cells was found to be increased in bronchioles of asthmatics compared to non-asthmatics. The degree of RGS4 immunostaining correlated positively with the severity of asthma symptoms and inversely with the FEV1 and bronchodilator response. RGS4 expression was increased by smooth muscle growth factors such as platelet-derived growth factor, which was linked to increased PI3-kinase dependent proliferation and reduced excitation-contraction. These results suggested the presence of a highly proliferative, poorly contractile RGS4+ bronchial myocyte population in severe asthmatics, which could contribute to the fixed airway obstruction observed in this population. Future studies will examine in vivo bronchial responsiveness of RGS4 and RGS5 knockout 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 also plan to examine contraction of human precision-cut lung slices overexpressing RGS proteins or RGS siRNA. We also examined 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;and (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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