Hypoxia increases pulmonary arterial thromboxane receptor internalization independent of receptor sensitization.

Persistent Pulmonary Hypertension of the Newborn (PPHN) is characterized by sustained vasospasm and an increased thromboxane:prostacyclin ratio. Thromboxane (TP) receptors signal via Gαq to mobilize IP3 and Ca(2+), causing pulmonary arterial constriction. We have previously reported increased TP internalization in hypoxic pulmonary arterial (PA) myocytes. Serum-deprived PA myocytes were grown in normoxia (NM) or hypoxia (HM) for 72 h. TP localization was visualized in agonist-naïve and -challenged NM and HM by immunocytochemistry. Pathways for agonist-induced TP receptor internalization were determined by inhibiting caveolin- or clathrin-mediated endocytosis, and caveolar fractionation. Roles of actin and tubulin in TP receptor internalization were assessed using inhibitors of tubulin, actin-stabilizing or -destabilizing agents. PKA, PKC or GRK activation and inhibition were used to determine the kinase responsible for post-agonist receptor internalization. Agonist-naïve HM had decreased cell surface TP, and greater TP internalization after agonist challenge. TP protein did not sort with caveolin-rich fractions. Inhibition of clathrin prevented TP internalization. Both actin-stabilizing and -destabilizing agents prevented TP endocytosis in NM, while normalizing TP internalization in HM. Velocity of TP internalization was unaffected by PKA activity, but PKC activation normalized TP receptor internalization in HM. GRK inhibition had no effect. We conclude that in hypoxic myocytes, TP is internalized faster and to a greater extent than in normoxic controls. Internalization of the agonist-challenged TP requires clathrin, dynamic actin and is sensitive to PKC activity. TP receptor trafficking and signaling in hypoxia are pivotal to understanding increased vasoconstrictor sensitivity.

Hypoxia and nitric oxide exposure promote apoptotic signaling in contractile pulmonary arterial smooth muscle but not in pulmonary epithelium.

RATIONALE: Neonatal pulmonary hypertension is characterized by hypoxia, abnormal vascular remodeling, and impaired alveolarization. Nitric oxide (NO) regulates cell replication and activation of apoptosis. Our objective was to examine cell phenotype-specific effects of hypoxia and NO exposure on cumulative apoptotic signal in neonatal pulmonary epithelial cells and arterial smooth muscle. DESIGN/METHODS: Primary cultured newborn porcine pulmonary arterial myocytes and epithelial cells were grown in normoxic (21% O2) or hypoxic conditions (10% O2). Myocyte phenotype was predetermined by serum-supplementation or -deprivation. Cells were exposed to sodium nitroprusside (10(-7) -10(-4) M) or diluent for 3 days. Cell survival was estimated by MTT assay; BAX, Bcl-2, and cleaved caspase-3 by Western blot; cell cycle entry by laser scanning cytometry. RESULTS: Hypoxic epithelial cells exhibited a small increase in anti-apoptotic Bcl2, and decrease in BAX. Cell survival and active caspase-3 were unchanged. Exposure to NO had no impact on epithelial apoptosis, but initiated necrosis. In contractile myocytes, pro-apoptotic BAX abundance and caspase-3 activation were increased by hypoxia, augmented by NO exposure promoting apoptosis. Hypoxia decreased BAX/Bcl-2 ratio and promoted survival of synthetic myocytes; NO increased apoptosis of normoxic synthetic myocytes, but decreased apoptosis of hypoxic synthetic myocytes. CONCLUSION: The effect of NO on pulmonary apoptosis is phenotype-dependent. A cumulative apoptotic effect of hypoxia and NO in vitro exerted on contractile myocytes may lead to contraction of this subpopulation, while synthetic myocyte survival and proliferation is enhanced by hypoxia and NO. Epithelial survival is unaffected. We speculate that alveolar rarefaction reported after neonatal hypoxia may arise from growth arrest in the vascular rather than the epithelial compartment.

Hypoxic neonatal pulmonary arterial myocytes are sensitized to ROS-generated 8-isoprostane.

8-Isoprostane, a ROS-derived prostanoid that acts via the thromboxane receptor (TP), is implicated in neonatal pulmonary hypertension. The purpose of this study was to examine the effect of hypoxia on vascular smooth muscle ROS generation, 8-isoprostane activity, and TP binding. First-passage neonatal porcine pulmonary artery myocytes were exposed to 10% O(2) (hypoxic myocytes; HM) or 21% O(2) (normoxic myocytes) for 72 h. Hypoxia increased in vitro generation of ROS, superoxide, and 8-isoprostane. ROS generation was ablated by inhibition of mitochondrial complex III. SOD1 and 3 activities were increased, but SOD2 activity decreased by 45% in HM. 8-Isoprostane generation was driven by the addition of peroxide and nitric oxide; incubation with permeative PEG-SOD, but not PEG-catalase or impermeative SOD, attenuated hypoxia-induced 8-isoprostane generation. 8-Isoprostane affinity for TP was markedly increased in HM. Myocyte 8-isoprostane challenge caused TP internalization and calcium release only in HM; this was sensitive to TP blockade and was normalized by activation of adenylyl cyclase. We propose that hypoxia induces superoxide accumulation in pulmonary artery myocytes through inhibition of mitochondrial SOD2 activity, promoting peroxynitrite-induced generation of 8-isoprostane. 8-Isoprostane binds to sensitized TP receptors, causing receptor internalization and signaling to calcium release in hypoxic myocytes. 8-Isoprostane may be an important pulmonary vasoconstrictor during neonatal hypoxia.