Jernigan NL, Walker BR, Resta TC. Reactive oxygen species mediate RhoA/Rho kinase-induced Ca 2ϩ sensitization in pulmonary vascular smooth muscle following chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 295: L515-L529, 2008. First published July 11, 2008 doi:10.1152/ajplung.00355.2007.-Recent evidence supports a prominent role for Rho kinase (ROK)-mediated pulmonary vasoconstriction in the development and maintenance of chronic hypoxia (CH)-induced pulmonary hypertension. Endothelin (ET)-1 contributes to the pulmonary hypertensive response to CH, and recent studies by our laboratory and others indicate that pulmonary vascular reactivity following CH is largely independent of changes in vascular smooth muscle (VSM) intracellular free calcium concentration ([Ca 2ϩ ]i). In addition, CH increases generation of reactive oxygen species (ROS) in pulmonary arteries, which may underlie the shift toward ROK-dependent Ca 2ϩ sensitization. Therefore, we hypothesized that ROS-dependent RhoA/ROK signaling mediates ET-1-induced Ca 2ϩ sensitization in pulmonary VSM following CH. To test this hypothesis, we determined the effect of pharmacological inhibitors of ROK, myosin light chain kinase (MLCK), tyrosine kinase (TK), and PKC on ET-1-induced vasoconstriction in endotheliumdenuded, Ca 2ϩ -permeabilized small pulmonary arteries from control and CH (4 wk at 0.5 atm) rats. Further experiments examined ET-1-mediated, ROK-dependent phosphorylation of the regulatory subunit of myosin light chain phosphatase (MLCP), MYPT1. Finally, we measured ET-1-induced ROS generation in dihydroethidiumloaded small pulmonary arteries and investigated the role of ROS in mediating ET-1-induced, RhoA/ROK-dependent Ca 2ϩ sensitization using the superoxide anion scavenger, tiron. We found that CH increases ET-1-induced Ca 2ϩ sensitization that is sensitive to inhibition of ROK and MLCK, but not PKC or TK, and correlates with ROK-dependent MYPT1 Thr696 phosphorylation. Furthermore, tiron inhibited basal and ET-1-stimulated ROS generation, RhoA activation, and VSM Ca 2ϩ sensitization following CH. We conclude that CH augments ET-1-induced Ca 2ϩ sensitization through ROS-dependent activation of RhoA/ROK signaling in pulmonary VSM.endothelin-1; fura 2; pulmonary hypertension; superoxide anion; protein kinase C; tyrosine kinase; myosin light chain phosphatase PULMONARY HYPERTENSION, ASSOCIATED with high altitude exposure and chronic obstructive pulmonary disease, is attributed to both structural remodeling as well as enhanced vasoconstriction in the pulmonary vasculature. Although it has been widely believed that vascular remodeling due to medial hypertrophy of pulmonary vascular smooth muscle (VSM) is the key mediator of increased vascular resistance following chronic hypoxia (CH), it is now evident that vasoconstriction may play a more prominent role than remodeling in the elevation of pulmonary vascular resistance and arterial pressure in rats (51). Consistent with this possibility, CH-induced pulmonary hypertension can be acutely reversed by in...

(VSMCs) to contract in response to increases in intraluminal pressure. Although mechanosensitive ion channels are thought to initiate VSMC stretch-induced contraction, their molecular identity is unknown. Recent reports suggest degenerin/epithelial Na ϩ channels (DEG/ENaC) may form mechanotransducers in sensory neurons and VSMCs; however, the role of DEG/ENaC proteins in myogenic constriction of mouse renal arteries has not been established. To test the hypothesis that DEG/ ENaC proteins are required for myogenic constriction in renal vessels, we first determined expression of ENaC transcripts and proteins in mouse renal VSMCs. Then, we determined pressure-and agonistinduced constriction and changes in vascular smooth muscle cytosolic Ca 2ϩ and Na ϩ in isolated mouse renal interlobar arteries following DEG/ENaC inhibition with amiloride and benzamil. We detect ␣-, ␤-, and ␥ENaC transcript and protein expression in cultured mouse renal VSMC. In contrast, we detect only ␤-and ␥-but not ␣ENaC protein in freshly dispersed mrVMSC. Selective DEG/ENaC inhibition, with low doses of amiloride and benzamil, abolishes pressure-induced constriction and increases in cytosolic Ca 2ϩ and Na ϩ without diminishing agonist-induced responses in isolated mouse interlobar arteries. Our findings indicate that DEG/ENaC proteins are required for myogenic constriction in mouse interlobar arteries and are consistent with our hypothesis that DEG/ENaC proteins may be components of mechanosensitive ion channel complexes required for myogenic vasoconstriction. mechanotransduction; renal blood flow autoregulation; amiloride; benzamil; isolated renal vessel; stretch-activated cation channel; calcium; sodium MYOGENIC VASOCONSTRICTION is an intrinsic property of most resistance vessels characterized by a decrease in luminal diameter in response to an increase in intraluminal pressure. The response is important in establishing basal vascular tone and maintaining blood flow autoregulation. It is well established that vascular smooth muscle (VSM) membrane depolarization and subsequent Ca 2ϩ influx via voltage-gated Ca 2ϩ channels mediate myogenic constriction (23,32,36). However, the mechanism that transduces mechanical stimuli (pressure-induced stretch) into a cellular event (depolarization) is less understood. Although mechanosensitive nonselective cation channels are thought to initiate pressure-induced depolarization (9,29,38,46), the molecule(s) involved has not been identified.Members of the degenerin/epithelial Na ϩ channel (DEG/ ENaC) family have recently been identified as mechanosensors in a variety of species and cell types (2,17,20,28,33). In mammals, DEG/ENaC proteins are found at several important sites of mechanotransduction, including dorsal root ganglion, arterial baroreflex sensory neurons, osteoblasts, keratinocytes, and vascular smooth muscle cells (VSMCs) (5, 12-14, 18, 30, 34). Two subfamilies of proteins are expressed in mammals: ENaC and acid-sensing ion channel. Acid-sensing ion channel proteins are activated by protons ...

Chronic hypoxia (CH) associated with respiratory disease results in elevated pulmonary vascular intracellular Ca(2+) concentration, which elicits enhanced vasoconstriction and promotes vascular arterial remodeling and thus has important implications in the development of pulmonary hypertension (PH). Store-operated Ca(2+) entry (SOCE) contributes to this elevated intracellular Ca(2+) concentration and has also been linked to acute hypoxic pulmonary vasoconstriction (HPV). Since our laboratory has recently demonstrated an important role for acid-sensing ion channel 1 (ASIC1) in mediating SOCE, we hypothesized that ASIC1 contributes to both HPV and the development of CH-induced PH. To test this hypothesis, we examined responses to acute hypoxia in isolated lungs and assessed the effects of CH on indexes of PH, arterial remodeling, and vasoconstrictor reactivity in wild-type (ASIC1(+/+)) and ASIC1 knockout (ASIC1(-/-)) mice. Restoration of ASIC1 expression in pulmonary arterial smooth muscle cells from ASIC1(-/-) mice rescued SOCE, confirming the requirement for ASIC1 in this response. HPV responses were blunted in lungs from ASIC1(-/-) mice. Both SOCE and receptor-mediated Ca(2+) entry, along with agonist-dependent vasoconstrictor responses, were diminished in small pulmonary arteries from control ASIC(-/-) mice compared with ASIC(+/+) mice. The effects of CH to augment receptor-mediated vasoconstrictor and SOCE responses in vessels from ASIC1(+/+) mice were not observed after CH in ASIC1(-/-) mice. In addition, ASIC1(-/-) mice exhibited diminished right ventricular systolic pressure, right ventricular hypertrophy, and arterial remodeling in response to CH compared with ASIC1(+/+) mice. Taken together, these data demonstrate an important role for ASIC1 in both HPV and the development of CH-induced PH.

Ϫ -specific spin trap tiron on vasoconstrictor reactivity to depolarizing concentrations of KCl in isolated lungs and Ca 2ϩ -permeabilized, pressurized small pulmonary arteries from control and CH (4 wk at 0.5 atm) rats. Using the same vessel preparation, we examined effects of CH on KCl-dependent VSM membrane depolarization and O 2 Ϫ generation using sharp electrodes and the fluorescent indicator dihydroethidium, respectively. Finally, using a RhoA-GTP pull-down assay, we investigated the contribution of O 2 Ϫ to depolarization-induced RhoA activation. We found that CH augmented KCl-dependent vasoconstriction through a Ca 2ϩ sensitization mechanism that was inhibited by HA-1077 and tiron. Furthermore, CH caused VSM membrane depolarization that persisted with increasing concentrations of KCl, enhanced KClinduced O 2 Ϫ generation, and augmented depolarization-dependent RhoA activation in a O 2 Ϫ -dependent manner. These findings reveal a novel mechanistic link between VSM membrane depolarization, O 2 Ϫ generation, and RhoA activation that mediates enhanced myofilament Ca 2ϩ sensitization and pulmonary vasoconstriction following CH. pulmonary hypertension; Rho kinase; membrane potential CHRONIC HYPOXIA (CH) associated with high-altitude exposure and chronic obstructive pulmonary disease leads to elevated pulmonary vascular resistance and pulmonary hypertension. The vasoconstrictor component of CH-induced pulmonary hypertension is mediated, in part, by generalized hypoxic pulmonary vasoconstriction resulting from global airway hypoxia. However, hypoxic vasoreactivity is largely blunted following CH (31), suggesting that additional mechanisms provide a major contribution to the development of pulmonary hypertension. Findings that basal tone is elevated in pulmonary arterial rings from CH rats (34, 54) and that vasodilators substantially lower pulmonary vascular resistance in CH rats acutely returned to a normoxic environment (34) provide further evidence that the vasoconstrictor response to CH is multifaceted and a primary determinant of pulmonary hypertension. Consistent with these findings, recent studies from our laboratory and others have identified an effect of CH on induction of myogenic tone in small pulmonary arteries (3) and enhancement of agonist-dependent vasoconstriction through a RhoA/ Rho kinase (ROCK)-mediated myofilament Ca 2ϩ sensitization pathway (14,20,34,54), responses that may contribute to the pathogenesis of pulmonary hypertension in this setting.RhoA is a small GTP-binding protein that is activated in response to stimulation of many G protein-coupled receptors (45). However, RhoA-mediated vascular smooth muscle (VSM) Ca 2ϩ sensitization can also be elicited by depolarizing stimuli (33,41,50,55). Membrane potential (E m ) depolarization-induced VSM contraction appears to be mediated, in part, via Ca 2ϩ -dependent stimulation of RhoA/ROCK (33, 41, 50, 55), thus establishing a dual role for Ca 2ϩ in regulation of VSM contraction: 1) activation of myosin light chain (MLC) kinase and 2) RhoA-media...

Mechanosensitive ion channels are thought to initiate pressure-induced vasoconstriction, however, the molecular identity of these channels is unknown. Recent work from our laboratory suggests that members of the Degenerin/Epithelial Na+ Channel (DEG/ENaC) family may be components of the mechanosensitive ion channel complex in vascular smooth muscle (VSM); however, the specific DEG/ENaC proteins mediating myogenic constriction are unknown. The goal of this study is to determine if specific knockdown of beta or gammaENaC, using dominant-negative (DN) or small-interference RNA (siRNA) molecules, inhibits pressure-induced vasoconstriction in mouse renal interlobar arteries. To address this goal, isolated arteries were transiently transfected with beta or gammaENaC DN-cDNA or siRNA molecules. After 24 h, vessels were either 1) cannulated and pressurized for pressure-diameter response curves or 2) dissociated and immunolabeled to determine VSM cell endogenous ENaC protein expression. We found that transfection of betaENaC DN-cDNA or siRNA suppresses beta-, but not gammaENaC protein expression. Similarly, gammaENaC DN-cDNA or siRNA suppresses gamma-, but not betaENaC protein expression. In addition, transfection of beta- or gammaENaC DN-cDNA or siRNA molecules inhibits pressure-induced vasoconstriction, but does not block agonist-induced vasoconstriction. Our results provide the first direct evidence that beta and gammaENaC proteins are essential in mediating myogenic vasoconstriction in mouse renal interlobar arteries.