RGS2 is a novel mediator of hypoxia-induced pulmonary artery constriction.

摘要

Pulmonary hypertension (PH) is characterized by an increase in blood pressure in the pulmonary vasculature associated with excessive vasoconstriction of pulmonary arteries and vascular remodeling. Pulmonary vasoconstriction is controlled by endogenous mediators, including those that act by stimulating G protein-coupled receptors (GPCRs). Vasoconstrictors such as serotonin (5-HT), endothelin-1 (ET-1) and thromboxane signal via GPCRs coupled to the G protein Gq. Gq activates signaling cascades in vascular smooth muscle that cause vasoconstriction. Regulators of G Protein Signaling 2 (RGS2) proteins turn off Gq signaling and thus inhibit the downstream pathways that cause vasoconstriction. Inhibition of RGS2 increases Gq-coupled GPCR vasoconstriction caused by many endogenous vasoconstrictors, including those implicated in PH.;Hypoxia is a key factor in the development of PH. Acute hypoxia causes hypoxic pulmonary vasoconstriction (HPV), which regulates ventilation by diverting blood flow away from poorly oxygenated regions of the lung. HPV is associated with an increase in calcium (Ca2+) mobilization, initially due to Ca2+ release from intracellular stores and followed by a sustained increase in Ca2+ influx through voltage-gated Ca2+ channels. Chronic hypoxia causes PH, in part, by enhanced Ca2+ mobilization contributing to HPV. Thus I hypothesized that hypoxia reduces RGS2 in pulmonary arteries, causing increased calcium mobilization and excessive vasoconstriction that may contribute to the development of PH. My objectives were:;1. To characterize the effects of RGS2 on Gq-coupled GPCR agonistmediated constriction and Ca2+ signaling in human pulmonary arterial smooth muscle (HPASM) cells. RGS2 protein was knocked-down (KD) in HPASM cells using RGS2 siRNA. I measured Gq-coupled GPCRmediated Ca2+ mobilization and contraction in RGS2 KD and scrambled HPASM cells.;2. To determine the effects of RGS2 on mouse pulmonary artery vasoconstriction activated by Gq-coupled GPCR agonists. I prepared precision cut lung slices (lung slices) from RGS2 KO and WT mice and assessed Gq-coupled GPCR agonist-mediated constriction concentrationresponse curves.;3. To determine the effects of hypoxia on mouse pulmonary artery vasoconstriction activated by Gq-coupled GPCR agonists. RGS2 KO and WT lung slices were incubated in hypoxia for 48 h and concentrationresponse curves for 5-HT and U46619 (thromboxane A2 mimetic) were analyzed.;4. To determine whether hypoxia enhancement of Gq-coupled GPCR agonist-mediated Ca2+ signaling and pulmonary arterial constriction is caused by down-regulation of RGS2 expression, HPASM cells were incubated in hypoxia (1% O2) or normoxia (21% O2) for 1-48 h and RGS2 expression was measured by western blotting. HPASM cells were also cultured for 48 h in hypoxia and Ca2+ mobilization and contraction of HPASM cells measured. Additionally, I incubated RGS2 KD HPASM cells in hypoxia and measured RGS2 expression along with Ca 2+ mobilization and contraction.;5. To determine whether RGS2 KO mice develop right ventricular hypertrophy. I measured the heart wt /body wt (HW/BW) and Fulton Index of RGS2 KO and WT mice.;U46619- and ET-1-mediated Ca2+ mobilization and contraction were significantly increased in RGS2 KD HPASM cells compared to scrambled control cells. Pulmonary arteries from RGS2 KO lung slices also exhibited enhanced constriction induced by 5-HT and U46619 compared to WT mice. Hypoxia enhanced the Ca2+ mobilization and contraction induced by U46619 and ET-1 in HPASM cells. Hypoxia did not significantly down-regulate RGS2 expression in RGS2 KD HPASM cells but did increase the Ca2+ mobilization and contraction of RGS2 KD HPASM cells. Hypoxia enhanced the pulmonary arterial constriction of WT mouse lung slices; however, it did not increase constriction of RGS2 KO lung slices. HW/BW and Fulton Index were significantly increased in RGS2 KO mice in comparison to WT mice.;In conclusion, RGS2 plays a role in GPCR-mediated contraction and Ca 2+ mobilization of HPASM cells and mouse pulmonary arteries. Hypoxia downregulates RGS2, which is associated with augmented Ca2+ signaling and constriction that may contribute to the development of PH.