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Pulmonary arterial hypertension (PAH) is a disease characterized by increased vasoconstriction and vascular remodeling. Pulmonary artery smooth muscle cells (PASMCs) highly express the transcription factor hypoxia-inducible factor-1α (HIF-1α), yet the role of PASMC HIF-1α in the development of PAH remains controversial. To study the role of SMC HIF-1α in the pulmonary vascular response to acute and chronic hypoxia, we used a gain-of-function strategy to stabilize HIF-1α in PASMC by generating mice lacking prolyl hydroxylase domain (PHD) 1 and 2 in SM22α-expressing cells. This strategy increased HIF-1α expression and transcriptional activity under conditions of normoxia and hypoxia. Acute hypoxia increased right ventricular systolic pressure (RVSP) in control, but not in SM22α-PHD1/2-/- mice. Chronic hypoxia increased RVSP and vascular remodeling more in control SM22α-PHD1/2+/+ than in SM22α-PHD1/2-/- mice. In vitro studies demonstrated increased contractility and myosin light chain phosphorylation in isolated PHD1/2+/+ compared with PHD1/2-/- PASMC under both normoxic and hypoxic conditions. After chronic hypoxia, there was more p27 and less vascular remodeling in SM22α-PHD1/2-/- compared with SM22α-PHD1/2+/+ mice. Hypoxia increased p27 in PASMC isolated from control patients, but not in cells from patients with idiopathic pulmonary arterial hypertension (IPAH). These findings highlight an SM22α-expressing cell-specific role for HIF-1α in the inhibition of pulmonary vasoconstriction and vascular remodeling. Modulating HIF-1α expression in PASMC may represent a promising preventative and therapeutic strategy for patients with PAH. NEW & NOTEWORTHY In a mouse model wherein hypoxia-inducible factor 1 alpha (HIF-1α) is stabilized in vascular smooth muscle cells, we found that HIF-1α regulates vasoconstriction by limiting phosphorylation of myosin light chain and regulates vascular remodeling through p27 induction. These findings highlight a cell-specific role for HIF-1α in the inhibition of pulmonary vasoconstriction and vascular remodeling. [ABSTRACT FROM AUTHOR]

Though survival rates for preterm infants are improving, the incidence of chronic lung disease of infancy, or bronchopulmonary dysplasia (BPD), remains high. Histologically, BPD is characterized by larger and fewer alveoli. Hypoxia-inducible factors (HIFs) may be protective in the context of hyperoxia-induced lung injury, but the cell-specific effects of HIF expression in neonatal lung injury remain unknown. Thus, we sought to determine whether HIF stabilization in SM22a-expressing cells can limit hyperoxiainduced neonatal lung injury. We generated SM22a-specific HIF-1a-stabilized mice (SM22a-PHD1/2/mice) by cross-breeding SM22a-promotor-driven Cre recombinase mice with prolyl hydroxylase PHD1flox/flox and PHD2flox/flox mice. Neonatal mice were randomized to 21% O2 (normoxia) or 80% O2 (hyperoxia) exposure for 14 days. For the hyperoxia recovery studies, neonatal mice were recovered from normoxia for an additional 10 wk. SM22a-specific HIF-1a stabilization mitigated hyperoxia-induced lung injury and preserved microvessel density compared with control mice for both neonates and adults. In SM22a-PHD1/2/mice, pulmonary artery endothelial cells (PAECs) were more proliferative and pulmonary arteries expressed more collagen IV compared with control mice, even under hyperoxic conditions. Angiopoietin-2 (Ang2) mRNA expression in pulmonary artery smooth muscle cells (PASMC) was greater in SM22a-PHD1/2/compared with control mice in both normoxia and hyperoxia. Pulmonary endothelial cells (PECs) cocultured with PASMC isolated from SM22a-PHD1/2/mice formed more tubes and branches with greater tube length compared with PEC cocultured with PASMC isolated from SM22a-PHD1/2þ/þ mice. Addition of Ang2 recombinant protein further augmented tube formation for both PHD1/2þ/þ and PHD1/2/PASMC. Cell-specific deletion of PHD1 and 2 selectively increases HIF-1a expression in SM22a-expressing cells and protects neonatal lung development despite prolonged hyperoxia exposure. HIF stabilization in SM22a-expressing cells preserved endothelial cell proliferation, microvascular density, increased angiopoietin-2 expression, and lung structure, suggesting a role for cell-specific HIF-1a stabilization to prevent neonatal lung injury. [ABSTRACT FROM AUTHOR]

Pulmonary artery smooth muscle cells (PASMCs) express endothelin (ET-1), which modulates the pulmonary vascular response to hypoxia. Although cross-talk between hypoxia-inducible factor-1α (HIF-1α), an O2-sensitive transcription factor, and ET-1 is established, the cell-specific relationship between HIF-1α and ET-1 expression remains incompletely understood. We tested the hypotheses that in PASMCs 1) HIF-1α expression constrains ET-1 expression, and 2) a specific microRNA (miRNA) links HIF-1α and ET-1 expression. In human (h)PASMCs, depletion of HIF-1α with siRNA increased ET-1 expression at both the mRNA and protein levels (P < 0.01). In HIF-1α−/− murine PASMCs, ET-1 gene and protein expression was increased (P < 0.0001) compared with HIF-1α+/+ cells. miRNA profiles were screened in hPASMCs transfected with siRNA-HIF-1α, and RNA hybridization was performed on the Agilent (Santa Clara, CA) human miRNA microarray. With HIF-1α depletion, miRNA-543 increased 2.4-fold (P < 0.01). In hPASMCs, miRNA-543 overexpression increased ET-1 gene (P < 0.01) and protein (P < 0.01) expression, decreased TWIST gene expression (P < 0.05), and increased ET-1 gene and protein expression, compared with nontargeting controls (P < 0.01). Moreover, we evaluated low passage hPASMCs from control and patients with idiopathic pulmonary arterial hypertension (IPAH). Compared with controls, protein expression of HIF-1α and Twist-related protein-1 (TWIST1) was decreased (P < 0.05), and miRNA-543 and ET-1 expression increased (P < 0.001) in hPASMCs from patients with IPAH. Thus, in PASMCs, loss of HIF-1α increases miRNA-543, which decreases Twist expression, leading to an increase in PASMC ET-1 expression. This previously undescribed link between HIF-1α and ET-1 via miRNA-543 mediated Twist suppression represents another layer of molecular regulation that might determine pulmonary vascular tone. [ABSTRACT FROM AUTHOR]

Accessory subunits associated with the calcium-sensitive potassium channel (BKCa), a major determinant of vascular tone, confer functional and anatomical diversity. The β1 subunit increases Ca2+ and voltagesensitivity of the BKCa channel and is expressed exclusively in smooth muscle cells. Evidence supporting the physiological significance of the β1 subunit includes the observations that murine models with deletion of the β1 subunit are hypertensive and that humans with a gain-of-function β1 mutation are at a decreased risk of diastolic hypertension. However, whether the β1 subunit of the BKCa channel contributes to the low tone that characterizes the normal pulmonary circulation or modulates the pulmonary vascular response to hypoxia remains unknown. To determine the role of the BKCa channel β1 subunit in the regulation of pulmonary vascular tone and the response to acute and chronic hypoxia, mice with deletion of the Kcnmb1 gene that encodes for the β1 subunit (Kcnmb1−/−) were placed in chronic hypoxia (10% O2) for 21–24 days. In normoxia, right ventricular systolic pressure (RVSP) did not differ between Kcnmb1+/+ (controls) and Kcnmb1−/− mice. After exposure to either acute or chronic hypoxia, RVSP was higher in Kcnmb1−/− mice compared with Kcnmb1+/+ mice, without increased vascular remodeling. β1 subunit expression was predominantly confined to pulmonary artery smooth muscle cells (PASMCs) from vessels ≤ 150 µm. Peripheral PASMCs contracted collagen gels irrespective of β1 expression. Focal adhesion expression and Rho kinase activity were greater in Kcnmb1−/− compared with Kcnmb1+/+ PASMCs. Compromised PASMC β1 function may contribute to the heightened microvascular vasoconstriction that characterizes pulmonary hypertension. [ABSTRACT FROM AUTHOR]

Compromised pulmonary endothelial cell (PEC) barrier function characterizes acute respiratory distress syndrome (ARDS), a cause of substantial morbidity and mortality. Survival from ARDS is greater in children compared with adults. Whether developmental differences intrinsic to PEC barrier function contribute to this survival advantage remains unknown. To test the hypothesis that PEC barrier function is more well-preserved in neonatal lungs compared with adult lungs in response to inflammation, we induced lung injury in neonatal and adult mice with systemic lipopolysaccharide (LPS). We assessed PEC barrier function in vivo and in vitro, evaluated changes in the expression of focal adhesion kinase 1 (FAK1) and phosphorylation in response to LPS, and determined the effect of FAK silencing and overexpression on PEC barrier function. We found that LPS induced a greater increase in lung permeability and PEC barrier disruption in the adult mice, despite similar degrees of inflammation and apoptosis. Although baseline expression was similar, LPS increased FAK1 expression in neonatal PEC but increased FAK1 phosphorylation and decreased FAK1 expression in adult PEC. Pharmacologic inhibition of FAK1 accentuated LPS-induced barrier disruption most in adult PEC. Finally, in response to LPS, FAK silencing markedly impaired neonatal PEC barrier function, whereas FAK overexpression preserved adult PEC barrier function. Thus, developmental differences in FAK expression during inflammatory injury serve to preserve neonatal pulmonary endothelial barrier function compared with that of adults and suggest that intrinsic differences in the immature versus pulmonary endothelium, especially relative to FAK1 phosphorylation, may contribute to the improved outcomes of children with ARDS. [ABSTRACT FROM AUTHOR]

Endothelin-1 (ET-1) increases pulmonary vascular tone through direct effects on pulmonary artery smooth muscle cells (PASMC) via membrane-bound ET-1 receptors. Circulating ET-1 contributes to vascular remodeling by promoting SMC proliferation and migration and inhibiting SMC apoptosis. Although endothelial cells (EC) are the primary source of ET-1, whether ET-1 produced by SMC modulates pulmonary vascular tone is unknown. Using transgenic mice created by crossbreeding SM22α-Cre mice with ET-1flox/flox mice to selectively delete ET-1 in SMC, we tested the hypothesis that PASMC ET-1 gene expression modulates the pulmonary vascular response to hypoxia. ET-1 gene deletion and selective activity of SM22α promoter-driven Cre recombinase were confirmed. Functional assays were performed under normoxic (21% O2) or hypoxic (5% O2) conditions using murine PASMC obtained from ET-1+/+ and ET-1-/- mic and in human PASMC (hPASMC) after silencing of ET-1 using siRNA. Under baseline conditions, there was no difference in right ventricular systolic pressure (RVSP) between SM22α-ET-1-/- and SM22α-ET-1+/+ (control) littermates. After exposure to hypoxia (10% O2, 21-24 days), RVSP was and vascular remodeling were less in SM22α-ET-1-/- mice compared with control littermates (P < 0.01). Loss of ET-1 decreased PASMC proliferation and migration and increased apoptosis under normoxic and hypoxic conditions. Exposure to selective ET-1 receptor antagonists had no effect on either the hypoxia-induced hPASMC proliferative or migratory response. SMC-specific ET-1 deletion attenuates hypoxia-induced increases in pulmonary vascular tone and structural remodeling. The observation that loss of ET-1 inhibited SMC proliferation, survival, and migration represents evidence that ET-1 derived from SMC plays a previously undescribed role in modulating the response of the pulmonary circulation to hypoxia. Thus PASMC ET-1 may modulate vascular tone independently of ET-1 produced by EC. [ABSTRACT FROM AUTHOR]

Bronchopulmonary dysplasia (BPD), a chronic lung disease of infancy, is characterized by arrested alveolar development. Pulmonary angiogenesis, mediated by the vascular endothelial growth factor (VEGF) pathway, is essential for alveolarization. However, the transcriptional regulators mediating pulmonary angiogenesis remain unknown. We previously demonstrated that NF-κB, a transcription factor traditionally associated with inflammation, plays a unique protective role in the neonatal lung. Therefore, we hypothesized that constitutive NF-κB activity is essential for postnatal lung development. Blocking NF-κB activity in 6-day-old neonatal mice induced the alveolar simplification similar to that observed in BPD and significantly reduced pulmonary capillary density. Studies to determine the mechanism responsible for this effect identified greater constitutive NF-κB in neonatal lung and in primary pulmonary endothelial cells (PEC) compared with adult. Moreover, inhibiting constitutive NF-κB activity in the neonatal PEC with either pharmacological inhibitors or RNA interference blocked PEC survival, decreased proliferation, and impaired in vitro angiogenesis. Finally, by chromatin immunoprecipitation, NF-κB was found to be a direct regulator of the angiogenic mediator, VEGFreceptor- 2, in the neonatal pulmonary vasculature. Taken together, our data identify an entirely novel role for NF-κB in promoting physiological angiogenesis and alveolarization in the developing lung. Our data suggest that disruption of NF-κB signaling may contribute to the pathogenesis of BPD and that enhancement of NF-κB may represent a viable therapeutic strategy to promote lung growth and regeneration in pulmonary diseases marked by impaired angiogenesis. [ABSTRACT FROM AUTHOR]

Previously, we observed that hypoxia increases the expression of the β1-subunit (KCNMB1) of the calcium-sensitive potassium channel (BKCa). Herein, we elucidate the mechanism whereby hypoxia increases KCNMB1 expression in human pulmonary artery smooth muscle cells (hPASMC). In response to hypoxia, the expression of both the transcription factor hypoxia-inducible factor 1-α (HIF-1α) and KCNMB1 are increased. Knockdown of HIF-1α using a shRNA plasmid blocked the hypoxic induction of KCNMB1 expression. Chromatin immunoprecipitation (ChIP) demonstrated HIF-1α binding to three discrete regions of the human KCNMB1 promoter known to contain hypoxia response elements (HREs). A KCNMB1 promoter reporter assay combined with site-directed mutagenesis identified two adjacent HREs located between -3,540 bp and -3,311 bp that are essential for the hypoxic induction of KCNMB1 promoter activity. Furthermore, additional ChIP assays demonstrated recruitment of the HIF-1α transcriptional coactivator, p300, to this same promoter region. Treatment of hPASMC with the histone deacetylase inhibitor, trichostatin, prolonged the increase in KCNMB1 observed with hypoxia, suggesting that alterations in chromatin remodeling function to limit the hypoxic induction of KCNMB1. Finally, KCNMB1 knockdown potentiated the hypoxia-induced increase in cytosolic calcium in hPASMC, highlighting the contribution of the β1-subunit in modulating vascular SMC tone in response to acute hypoxia. In conclusion, HIF-1α increases KCNMB1 expression in response to hypoxia in hPASMC by binding to two HREs located at -3,540 to -3,311 of the KCNMB1 promoter. We speculate that selective modulation of KCNMB1 expression may serve as a novel therapeutic approach to address diseases characterized by an increase in vascular tone. [ABSTRACT FROM AUTHOR]

At birth, pulmonary vasodilation occurs concomitant with the onset of air-breathing life. Whether and how Rho kinase (ROCK) modulates the perinatal pulmonary vascular tone remains incompletely understood. To more fully characterize the separate and interactive effects of ROCK signaling, we hypothesized that ROCK has discrete effects on both pulmonary artery (PA): 1) endothelial cell (PAEC) nitric oxide (NO) production and contractile state; and 2) smooth muscle cell tone independent of endothelial NO synthase (eNOS) activity. To test these hypotheses, NO production and endothelial barrier function were determined in fetal PAEC under baseline hypoxia and following exposure to normoxia with and without treatment with Y-27632, a specific pharmacological inhibitor of ROCK. In acutely instrumented, late-gestation ovine fetuses, eNOS was inhibited by nitro-l-arginine infusion into the left PA (LPA). Subsequently, fetal lambs were mechanically ventilated (MV) with 100% oxygen in the absence (control period) and presence of Y-27632. In PAEC, treatment with Y-27632 had no effect on cytosolic calcium but did increase normoxia-induced NO production. Moreover, acute normoxia increased PAEC barrier function, an effect that was potentiated by Y-27632. In fetal lambs, MV during the control period had no effect on LPA flow. In contrast, MV after Y-27632 increased LPA flow and fetal arterial Po2 (PaO2) and decreased PA pressure. In conclusion, ROCK activity modulates vascular tone in the perinatal pulmonary circulation via combined effects on PAEC NO production, barrier function, and smooth muscle tone. ROCK inhibition may represent a novel treatment strategy for neonatal pulmonary vascular disease. [ABSTRACT FROM AUTHOR]

Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O2) tension with a decrease in cytosolic calcium ([Ca2+]i), an acute increase in O2 tension has no net effect on [Ca2+]i in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O2 tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (≥4th generation) and maintained under hypoxic conditions (~25 Torr) in primary culture. After fura 2 loading, apparent [Ca2+]i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca2+]i more in PI-ITN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intra- cellular store depletion with CPA, followed by superfusion with Ca2+-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE. [ABSTRACT FROM AUTHOR]

At birth, the lung environment changes from low to relatively high O2 tension. Pulmonary blood flow increases and pulmonary artery pressure decreases. Recent data suggest that pulmonary vascular calcium-sensitive K+ channel (BKCa) activation mediates perinatal pulmonary vasodilation. Although BKCa channel expression is developmentally regulated, the molecular mechanisms responsible for BKCa expression remain unknown. We tested the hypothesis that the low-O2 tension environment of the normal fetus modulates BKCa channel expression. We analyzed BKCa expression under conditions of hypoxia and normoxia both in vitro and in vivo. BKCa α-subunit mRNA expression increased twofold in ovine pulmonary artery smooth muscle cell (PASMC) primary cultures maintained in hypoxia. In vivo, BKCa expression was similarly affected by hypoxia. When adult Sprague-Dawley rats were placed in hypobaric hypoxic chambers for 3 wk, hypoxic animals showed an increase of threefold in the expression of BKCa α- and more than twofold in the expression of BKCa β1-subunit mRNA. Immunochemical staining was consistent with the genetic data. To assess transcriptional activation of the β-subunit of the BKCa, both BKCa β1,- and β2-subunit luciferase (KCa β:luc+) reporter genes were constructed. Hypoxia increased PASMC KCa β1:luc+ reporter expression by threefold and KCa β2:luc+ expression by 35%. Fetal PASMC treated with the hypoxia-inducible factor-1 mimetic deferoxamine showed a 63 and 41% increase in BKCa channel α- and β1-subunit expression, respectively. Together, these results suggest that oxygen tension modulates BKCa channel subunit mRNA expression, and the regulation is, at least in part, at the transcriptional level. [ABSTRACT FROM AUTHOR]

In utero, blood shunts away from the lungs via the ductus arteriosus (DA) and the foramen ovale. After birth, the DA closes concomitant with increased oxygen tension. The present experimental series tests the hypothesis that oxygen directly increases DA smooth muscle cell (SMC) cytosolic calcium ([Ca2+]i) through inactivation of a K+ channel, membrane depolarization, and entry of extracellular calcium. To test the hypothesis, DA SMC were isolated from late-gestation fetal lambs and grown to subconfluence in primary culture in low oxygen tension (25 Torr). DA SMC were loaded with the calcium-sensitive fluorophore fura-2 under low oxygen tension conditions and studied using microfluorimetry while oxygen tension was acutely increased (120 Torr). An acute increase in oxygen tension progressively increased DA SMC [Ca2+]i by 11.7 ± 1.4% over 40 mm. The effect of acute normoxia on DA SMC [Ca2+]i was mimicked by pharmacological blockade of the voltage-sensitive K+ channel. Neither removal of extracellular calcium nor voltage-operated calcium channel blockade prevented the initial increase in DA SMC [Ca2+]i. Manganese quenching experiments demonstrated that acute normoxia initially decreases the rate of extracellular calcium entry. Pharmacological blockade of inositol triphosphate-sensitive, but not ryanodine-sensitive, intracellular calcium stores prevented the oxygen-induced in- crease in [Ca2+]i. Endothelin increased [Ca2+]i in acutely normoxic, but not hypoxic, DA SMC. Thus acute normoxia 1) increases DA SMC [Ca2+]i via release of calcium from intracellular calcium stores, and subsequent entry of extracellular calcium, and 2) potentiates the effect of contractile agonists. Prolonged patency of the DA may result from disordered intracellular calcium homeostasis. [ABSTRACT FROM AUTHOR]

Myeloperoxidase (MPO)-derived oxidants participate in the respiratory antimicrobial defense system but are also implicated in oxidant-mediated acute lung injury. We hypothesized that MPO contributes to lung injury commonly observed after bone marrow transplantation (BMT). MPO-sufficient (MPO+/+) and -deficient (MPO-/-) mice were given cyclophosphamide and lethally irradiated followed by infusion of inflammation-inducing donor spleen, T cells at time of BMT. Despite suppressed generation of nitrative stress, MPO-/- recipient mice unexpectedly exhibited accelerated weight loss and increased markers of lung dysfunction compared, with MPO+/+ mice. The increased lung injury, during MPO deficiency was a result of donor T cell-dependent inflammatory responses because, bronchoalveolar lavage fluids (BALF) from MPO-/- mice contained increased numbers of inflammatory cells and higher levels of the proinflammatory cytokine TNF-α and the monocyte chemoattractant protefrt-1 compared with wild-type mice. Enhancecr inflammation in MPO-/- mice was associated with suppressed apoptosis of BALF inflammatory cells. The inflammatory process in MPO-/- recipients was also associated with enhanced necrosis of freshly isolated alveolar type II cells, critical for preventing capillary leak. We conclude that suppressed MPO-derived oxidative/nitrative stress is associated with enhanced lung inflammation and persistent alveolar epithelial injury. [ABSTRACT FROM AUTHOR]

Chronic intrauterine pulmonary hypertension compromises fetal pulmonary artery smooth muscle cell O[sub 2] sensing. Am J Physiol Lung Cell Mol Physiol 285: L1354-L1361, 2003. First published July 25, 2003; 10.1152/ajplung.00091.2003.—To test the hypothesis that chronic intrauterine pulmonary hypertension (PHTN) compromises pulmonary artery (PA) smooth muscle cell (SMC) O[sub 2] sensing, fluorescence microscopy was used to study the effect of an acute increase in Po[sub 2] on the cytosolic Ca[sup 2+] concentration ([Ca[sup 2+]][sub i]) of chronically hypoxic subconfluent monolayers of PA SMC in primary culture. PA SMCs were derived from fetal lambs with PHTN due to intrauterine ligation of the ductus arteriosus. Acute normoxia decreased [Ca[sup 2+]][sub i] in control but not PHTN PA SMC. In control PA SMC, [Ca[sup 2+]][sub i] increased after Ca[sup 2+]-sensitive (K[sub Ca]) and voltage-sensitive (K[sub v]) K[sup +] channel blockade and decreased after diltiazem treatment. In PHTN PA SMC, K[sub Ca] blockade had no effect, whereas Ky blockade and diltiazem increased [Ca[sup 2+]][sub i]. Inhibition of sarcoplasmic reticulum Ca[sup 2+] ATPase activity caused a greater increase in [Ca[sup 2+]][sub i] in controls compared with PHTN PA SMC. Conversely, ryanodine caused a greater increase of [Ca[sup 2+]][sub i] in PHTN compared with control PA SMC. K[sub Ca] channel mRNA is decreased and Ky channel mRNA is unchanged in PHTN PA SMC compared with controls. We conclude that PHTN compromises PA SMC O[sub 2] sensing, alters intracellular Ca[sup 2+] homeostasis, and changes the predominant ion channel that determines basal [Ca[sup 2+]][sub i] from K[sub Ca] to K[sub v]. [ABSTRACT FROM AUTHOR]

K[sup +] channels play an important role in mediating pulmonary vasodilation caused by increased oxygen tension, nitric oxide, alkalosis, and shear stress. To test the hypothesis that lung K[sup +] channel gene expression may be altered by chronic increases in pulmonary blood flow, we measured gene and protein expression of calcium-sensitive (K[sub Ca]) and voltage-gated (Kv2.1) K[sup +] channels, and a pH-sensitive K[sup +] channel (TASK), in distal lung from fetal lambs in which an aortopulmonary shunt was placed at 139 days gestation. Under baseline conditions, animals with an aortopulmonary shunt showed elevated pulmonary artery pressure and pulmonary blood flow compared with twin controls. Hypoxia caused a greater increase in pulmonary vascular tone in shunt animals compared with controls. Alkalosis caused pulmonary vasodilation in control but not shunt animals. To determine lung K[sup +] channel mRNA levels, we performed quantitative RT-PCR. In comparison with control animals, lung K[sub Ca] channel mRNA content was increased in shunt animals, whereas TASK mRNA levels were decreased. There was no difference in Kv2.1 mRNA levels. Channel protein expression was consistent with these findings. We conclude that, in the presence of elevated pulmonary blood flow, K[sub Ca] channel expression is increased and TASK is decreased. [ABSTRACT FROM AUTHOR]

Ca[sup 2+]-sensitive K[sup +] (K[sub Ca]) channels play an important role in mediating perinatal pulmonary vasodilation. We hypothesized that lung K[sub Ca] channel function may be decreased in persistent pulmonary hypertension of the newborn (PPHN). To test this hypothesis, pulmonary artery smooth muscle cells (PASMC) were isolated from fetal lambs with severe pulmonary hypertension induced by ligation of the ductus arteriosus in fetal lambs at 125-128 days gestation. Fetal lambs were killed after pulmonary hypertension had been maintained for at least 7 days. Age-matched, sham-operated animals were used as controls. PASMC K[sup +] currents and membrane potentials were recorded using amphotericin B-perforated patch-clamp techniques. The increase in whole cell current normally seen in response to normoxia was decreased (333.9 ± 63.6% in control vs. 133.1 ± 16.0% in hypertensive fetuses). The contribution of the K[sub Ca] channel to the whole cell current was diminished in hypertensive, compared with control, fetal PASMC. In PASMC from hypertensive fetuses, a change from hypoxia to normoxia caused no change in membrane potential compared with a -14.6 ± 2.8 mV decrease in membrane potential in PASMC from control animals. In PASMC from animals with pulmonary hypertension, 4-aminopyridine (4-AP) caused a larger depolarization than iberiotoxin, whereas in PASMC from control animals, iberiotexin caused a larger depolarization than 4-AP. These data confirm the hypothesis that the contribution of the K[sub Ca] channel to membrane potential and O[sub 2] sensitivity is decreased in an ovine model of PPHN, and this may contribute to the abnormal porinatal pulmonary vasoreactivity associated with PPHN. [ABSTRACT FROM AUTHOR]

Examines the effect of oxygen-induced fetal pulmonary vasodilation on intracellular calcium activation of K[sub Ca] channels. Importance of oxygen sensing in fetal pulmonary artery smooth muscle; Role of caffeine and diltiazem; Decrease in intracellular calcium concentration.

Examines the roles of nitric oxide (NO) and superoxide (O[sub2]) imbalance in post-bone marrow transplantation (BMT) survival and inflammation. Method used to measure nitrite in bronchoalveolar lavage fluid (BALF); Suppressed production of proinflammatory cytokines in BALF of inducible nitric oxide synthase (iNOS) knockout mice after allogeneic transplantation; Comparison of iNOS-deficient and iNOS-sufficient mice to show the contribution of NO to post-BMT mortality.

Presents information on a study examined the effects of potassium channel antagonists on fetal and adult pulmonary artery smooth muscle cells (PASMC). Materials and methods; Results and discussion.

Focuses on a study which discussed the role of chronic intrauterine pulmonary hypertension in calcium-sensitive potassium channel messenger RNA expression. Role of calcium-sensitive channels in perinatal pulmonary vasodilation; Materials and methods used; Results; Conclusions.

Provides information on a study which examined the mechanisms underlying developmental changes in pulmonary vascular tone. Methodology; Effect of hypoxia on cytosolic concentration in adult and fetal pulmonary artery smooth muscle cells; Discussion.