Abstract-The excitability of pulmonary artery smooth muscle cells (PASMC) is regulated by potassium (K ϩ ) conductances. Although studies suggest that background K ϩ currents carried by 2-pore domain K ϩ channels are important regulators of resting membrane potential in PASMC, their role in human PASMC is unknown. Our study tested the hypothesis that TASK-1 leak K ϩ channels contribute to the K ϩ current and resting membrane potential in human PASMC. We used the whole-cell patch-clamp technique and TASK-1 small interfering RNA (siRNA). Noninactivating K ϩ current performed by TASK-1 K ϩ channels were identified by current characteristics and inhibition by anandamide and acidosis (pH 6.3), each resulting in significant membrane depolarization. Moreover, we showed that TASK-1 is blocked by moderate hypoxia and activated by treprostinil at clinically relevant concentrations. This is mediated via protein kinase A (PKA)-dependent phosphorylation of TASK-1. To further confirm the role of TASK-1 channels in regulation of resting membrane potential, we knocked down TASK-1 expression using TASK-1 siRNA. The knockdown of TASK-1 was reflected by a significant depolarization of resting membrane potential. Treatment of human PASMC with TASK-1 siRNA resulted in loss of sensitivity to anandamide, acidosis, alkalosis, hypoxia, and treprostinil. These results suggest that (1) TASK-1 is expressed in human PASMC; (2) TASK-1 is hypoxia-sensitive and controls the resting membrane potential, thus implicating an important role for TASK-1 K ϩ channels in the regulation of pulmonary vascular tone; and (3) treprostinil activates TASK-1 at clinically relevant concentrations via PKA, which might represent an important mechanism underlying the vasorelaxing properties of prostanoids and their beneficial effect in vivo. Key Words: pulmonary circulation Ⅲ potassium channels Ⅲ TASK-1 Ⅲ treprostinil Ⅲ hypoxic pulmonary vasoconstriction T he membrane potential of pulmonary artery smooth muscle cells (PASMC) is an important regulator of arterial tone. These cells have a resting membrane potential of approximately Ϫ65 to Ϫ50 mV in vitro, close to the predicted equilibrium potential for potassium (K ϩ ) ions. The opening of K ϩ channels in the PASMC membrane increases K ϩ efflux, which causes membrane hyperpolarization. This closes voltage-dependent Ca 2ϩ channels, decreasing Ca 2ϩ entry and leading to vasodilatation. Conversely, inhibition of K ϩ channels causes membrane depolarization, Ca 2ϩ entry, cell contraction, and vasoconstriction.Background or leak K ϩ -selective channels, as defined by a lack of time and voltage dependency, play an essential role in setting the resting membrane potential and input resistance in excitable cells. Two-pore domain K ϩ (2-PK) channels have been shown to conduct several leak K ϩ currents. The activity of 2-PK channels is strongly regulated by protons, protein kinases, and hypoxia. Alteration of K ϩ conductance can influence cellular activity via membrane potential changes.Both RT-PCR and Northern blot analyses p...

-Prematurely born infants who require oxygen therapy often develop bronchopulmonary dysplasia (BPD), a debilitating disorder characterized by pronounced alveolar hypoplasia. Hyperoxic injury is believed to disrupt critical signaling pathways that direct lung development, causing BPD. We investigated the effects of normobaric hyperoxia on transforming growth factor (TGF)-␤ and bone morphogenetic protein (BMP) signaling in neonatal C57BL/6J mice exposed to 21% or 85% O2 between postnatal days P1 and P28. Growth and respiratory compliance were significantly impaired in pups exposed to 85% O2, and these pups also exhibited a pronounced arrest of alveolarization, accompanied by dysregulated expression and localization of both receptor (ALK-1, ALK-3, ALK-6, and the TGF-␤ type II receptor) and Smad (Smads 1, 3, and 4) proteins. TGF-␤ signaling was potentiated, whereas BMP signaling was impaired both in the lungs of pups exposed to 85% O2 as well as in MLE-12 mouse lung epithelial cells and NIH/3T3 and primary lung fibroblasts cultured in 85% O2. After exposure to 85% O2, primary alveolar type II cells were more susceptible to TGF-␤-induced apoptosis, whereas primary pulmonary artery smooth muscle cells were unaffected. Exposure of primary lung fibroblasts to 85% O2 significantly enhanced the TGF-␤-stimulated production of the ␣1 subunit of type I collagen (I␣1), tissue inhibitor of metalloproteinase-1, tropoelastin, and tenascin-C. These data demonstrated that hyperoxia significantly affects TGF-␤/BMP signaling in the lung, including processes central to septation and, hence, alveolarization. The amenability of these pathways to genetic and pharmacological manipulation may provide alternative avenues for the management of BPD.

The NADPH oxidases are involved in vascular remodeling processes and oxygen sensing. Hypoxia-induced pulmonary arterial remodeling results in thickening of the vessel wall and reduction of the area of vessel lumen, leading to pulmonary hypertension and cor pulmonale. The proliferation of pulmonary artery adventitial fibroblasts (PAFB) is critically involved in this process. In this study, we analyzed the role of the non-phagocytic NADPH oxidase subunits NOX1 and NOX4 in PAFB. NOX4 was predominantly expressed in comparison to NOX1 at mRNA levels. Under hypoxic conditions, NOX4 was significantly upregulated at mRNA and protein levels. Silencing of NOX4 by siRNA caused reduction of ROS levels under both normoxic and hypoxic (24 h) conditions and suppressed the significant hypoxic-induced ROS increase. PAFB proliferation was significantly decreased in cells transfected with NOX4 siRNA, whereas apoptosis was enhanced. Also, the expression of NOX4 was studied in PAFB isolated from the lungs of patients with idiopathic pulmonary arterial hypertension (IPAH). Interestingly, a significant increase of NOX4 mRNA expression was observed under hypoxic conditions in PAFB from the lungs with IPAH compared to healthy donors. In conclusion, NOX4 maintains ROS levels under normoxic and hypoxic conditions and enhances proliferation and inhibits apoptosis of PAFB.