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To test the hypothesis that an acute increase in O(2) tension increases cytosolic calcium ([Ca(2+)](i)) in fetal pulmonary artery endothelial cells (PAECs) via entry of extracellular calcium and subsequent calcium-induced calcium release (CICR) and nitric oxide release, low-passage PAECs (

At birth, the lung environment changes from low to relatively high O2tension. 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 BKCachannel expression is developmentally regulated, the molecular mechanisms responsible for BKCaexpression remain unknown. We tested the hypothesis that the low-O2tension environment of the normal fetus modulates BKCachannel expression. We analyzed BKCaexpression under conditions of hypoxia and normoxia both in vitro and in vivo. BKCaα-subunit mRNA expression increased twofold in ovine pulmonary artery smooth muscle cell (PASMC) primary cultures maintained in hypoxia. In vivo, BKCaexpression 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 BKCaα- and more than twofold in the expression of BKCaβ1-subunit mRNA. Immunochemical staining was consistent with the genetic data. To assess transcriptional activation of the β-subunit of the BKCa, both BKCaβ1- and β2-subunit luciferase (KCaβ:luc+) reporter genes were constructed. Hypoxia increased PASMC KCaβ1:luc+reporter expression by threefold and KCaβ2:luc+expression by 35%. Fetal PASMC treated with the hypoxia-inducible factor-1 mimetic deferoxamine showed a 63 and 41% increase in BKCachannel α- and β1-subunit expression, respectively. Together, these results suggest that oxygen tension modulates BKCachannel subunit mRNA expression, and the regulation is, at least in part, at the transcriptional level.

K+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+ channel gene expression may be altered by chronic increases in pulmonary blood flow, we measured gene and protein expression of calcium-sensitive (KCa) and voltage-gated (Kv2.1) K+ channels, and a pH-sensitive K+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+ channel mRNA levels, we performed quantitative RT-PCR. In comparison with control animals, lung KCa 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, KCachannel expression is increased and TASK is decreased.

Calcium-sensitive potassium (KCa) channels play a critical role in mediating perinatal pulmonary vasodilation. Because infants with persistent pulmonary hypertension of the newborn (PPHN) have blunted vasodilator responses to birth-related stimuli, we hypothesized that lung KCachannel gene expression is decreased in PPHN. To test this hypothesis, we measured KCa channel gene expression in distal lung homogenates from both fetal lambs with severe pulmonary hypertension caused by prolonged compression of the ductus arteriosus and age-matched, sham-operated animals (controls). After at least 9 days of compression of the ductus arteriosus, fetal lambs were killed. To determine lung KCa channel mRNA levels, primers were designed against the known sequence of the KCa channel and used in semiquantitative RT-PCR, with lung 18S rRNA content as an internal control. Compared to that in control lambs, lung KCa channel mRNA content in the PPHN group was reduced by 26 ± 6% ( P < 0.02), whereas lung voltage-gated K+ 2.1 mRNA content was unchanged. We conclude that lung KCa channel mRNA expression is decreased in an ovine model of PPHN. Decreased KCa channel gene expression may contribute to the abnormal pulmonary vascular reactivity associated with PPHN.

In cystic fibrosis the bronchiectatic conducting airways have large numbers of neutrophils in their walls and in their luminal contents. The neutrophil's primary granule enzyme activities of elastase and peroxidase are increased in the sputum of these patients. It has been postulated that these enzymes--together or individually--act to damage the airway epithelium. However, only peroxidase activity has consistently correlated with the degree of structural and functional airway disease in these patients with leakage of plasma protein into the airway lumen (Regelmann et al., Pediatr Pulmonol, 1995; 19:1-9). The present study was designed to test whether human neutrophil-derived myeloperoxidase can independently produce bronchial epithelial damage without the presence of proteases, as measured by increased permeability of the airway epithelium. Human peripheral blood neutrophils were purified, their primary granules isolated, and their peroxidase purified using affinity and ion exchange column chromatography. Activity of the proteinase-free peroxidase was measured using a chromogenic substrate. The effect of this peroxidase on the permeability of excised rat tracheas was measured using radioactive and fluorescent-labeled non-ionic molecules of varying molecular weight. Rat tracheas exposed to 15 minute treatments with either 130 U of peroxidase or hydrogen peroxide (10(-5) M) did not show a significant increase in the permeability of the epithelium to [3H]inulin, [14C]sucrose, and fluorescein isothiocyanate dextran 20 compared with control tracheas. However, those tracheas exposed to 130 U peroxidase followed by 10(-5) M hydrogen peroxide showed an increased permeability to each of the three test solutes. We conclude that proteinase-free myeloperoxidase, in the presence of non-toxic concentrations of its substrates, hydrogen peroxide and halide, produced increases in permeability to non-ionic molecules in the rat trachea within 15 minutes.

Patients with cystic fibrosis (CF) of the same age differ significantly in their degree of pulmonary disease. Based on preliminary observations, we postulated that the activity of my-eloperoxidase would be significantly increased in patients with greater structural lung damage than in those with less lung damage. Acid extracts of weighed sputum samples were assayed for lactoferrin concentrations by ELISA. Activities of peroxidase, cathepsin G, and elastase (with and without proteinase 3) were determined by kinetic analysis using chromogenic substrates. The patients were divided into quartiles based on their Brasfield chest-radiograph score. Patients in the first quartile (least amount of structural lung abnormality) were compared to those in the fourth quartile. The concentration of lactoferrin, a specific (secondary) granule protein of neutrophils, did not differ between the two patient groups. However, the activities of the neutrophil primary granule proteins, peroxidase, elastase, and elastase plus proteinase 3, were significantly elevated in the group with the most structural lung abnormality. Sputum albumin concentration was used to estimate leakages of plasma proteins into the airways. Peroxidase activity, but not the activity of cathepsin G, of elastase, or of elastase plus proteinase 3, correlated significantly with albuminig sputum in both quartile groups. To confirm the association of sputum peroxidase activity with differences in lung structure and to test its correlation with lung function, spirometry was performed in a second group of patients during the week prior to the time of sputum sampling. In this second group, increased sputum peroxidase activity was associated with worse Brasfield scores and with decreased percent-predicted forced expiratory volume in 1 sec. Lactoferrin concentration again did not differ between the groups. Bacterial factors also did not differ. The results indicate that the degree of lung damage in patients with CF was more closely related to sputum peroxidase activity than to age, gender, CFTR genotype, or sputum bacterial parameters. Further study is required to determine whether or not peroxidase activity in the airway causes this lung damage. © 1995 Wiley-Liss, Inc.

Freshly isolated monocytes from cystic fibrosis (CF) heterozygotes and homozygotes had significantly increased oxygen uptake and superoxide formation after surface glycoprotein stimulation than did monocytes from age- and sex-matched controls. Lack of differences among the genotypes in inhibition by simple sugars of the concanavalin A-stimulated superoxide production and lack of differences in concanavalin A-binding surface proteins suggested that different regulation of the oxidase pathway produced the increased oxygen uptake and superoxide formation in CF patients and carriers. This regulatory role is consistent with the predicted structure of the CF gene product. The results support the hypothesis that the mononuclear phagocytes of CF heterozygotes have a significantly increased ability to kill intracellular microbes and may confer a selective advantage to the host.

The transcriptional machinery involved in the transition of an infant from intrauterine to air-breathing life is developmentally regulated, as the fetus and adult manifest differential genetic expression. The low oxygen (O 2 ) environment of the mammalian fetus and the increase in O 2 tension that occurs at birth may account for the developmentally regulated alterations in gene expression. We tested the hypothesis that hypoxia-inducible factor 1 (HIF-1) expression, an O 2 -sensitive transcription factor, is developmentally regulated. We found that in fetal pulmonary artery (PA) smooth muscle cells (SMC), fetal HIF-1 protein levels were O 2 -insensitive, whereas in adult PA SMC, hypoxia increased HIF-1 protein expression. Surprisingly, hypoxia increased HIF-1 mRNA expression in fetal, but not in adult, PA SMC. HIF-1 degradation and transcriptional activity is contingent on prolyl- and asparagyl-hydroxylases. To determine whether developmental differences in O 2 sensitivity or expression of these enzymes accounts for the divergence of HIF-1 sensitivity between fetus and adult, we studied the expression of the three most well characterized prolyl-hydroxylases, PHD1, PHD2, and PHD3, and the expression of regulators of HIF-1 transcriptional activity, asparagyl-hydroxylase, factor inhibiting HIF, and the oncogenic factor, CITED2 (CREB-binding protein/p300 interacting transactivator with ED-rich tail). We found that, as in the case of HIF-1, these genes are differentially regulated in the fetus, enabling the mammalian fetus to thrive in the low O 2 tension intrauterine environment even while rendering a newborn infant uniquely well adapted to respond to the acute increase in O 2 tension that occurs at birth.

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 PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intracellular 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.

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+]iby 11.7 ± 1.4% over 40 min. The effect of acute normoxia on DA SMC [Ca2+]iwas 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 increase in [Ca2+]i. Endothelin increased [Ca2+]iin acutely normoxic, but not hypoxic, DA SMC. Thus acute normoxia 1) increases DA SMC [Ca2+]ivia 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.

To test the hypothesis that chronic intrauterine pulmonary hypertension (PHTN) compromises pulmonary artery (PA) smooth muscle cell (SMC) O2sensing, fluorescence microscopy was used to study the effect of an acute increase in Po2on the cytosolic Ca2+concentration ([Ca2+]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 [Ca2+]iin control but not PHTN PA SMC. In control PA SMC, [Ca2+]iincreased after Ca2+-sensitive (KCa) and voltage-sensitive (Kv) K+channel blockade and decreased after diltiazem treatment. In PHTN PA SMC, KCablockade had no effect, whereas Kvblockade and diltiazem increased [Ca2+]i. Inhibition of sarcoplasmic reticulum Ca2+ATPase activity caused a greater increase in [Ca2+]iin controls compared with PHTN PA SMC. Conversely, ryanodine caused a greater increase of [Ca2+]iin PHTN compared with control PA SMC. KCachannel mRNA is decreased and Kvchannel mRNA is unchanged in PHTN PA SMC compared with controls. We conclude that PHTN compromises PA SMC O2sensing, alters intracellular Ca2+homeostasis, and changes the predominant ion channel that determines basal [Ca2+]ifrom KCato Kv.

To address developmental regulation of pulmonary vascular O2 sensing, we tested the hypotheses that 1) fetal but not adult pulmonary artery smooth muscle cells (PASMCs) can directly sense an acute increase in O2, 2) Ca2+-sensitive K+(KCa) channel activity decreases with maturation, and 3) PASMC KCa channel expression decreases with maturation. We used fluorescence microscopy to confirm that fetal but not adult PASMCs are able to sense an acute increase in O2tension. Acute normoxia induced a 22 ± 2% decrease in cytosolic Ca2+ concentration ([Ca2+]i) in fetal PASMCs and no change in [Ca2+]i in adult PASMCs ( P < 0.01). The effects of K+channel antagonists were studied on fetal and adult PASMC [Ca2+]i. Iberiotoxin (10−9 M) caused PASMC [Ca2+]i to increase by 694 ± 22% in the fetus and caused no change in adult PASMCs. KCa channel expression and mRNA levels in distal pulmonary arteries from fetal and adult sheep were examined. Both KCachannel protein and mRNA expression in the distal pulmonary vasculature decreased with maturation. We conclude that maturation-dependent changes in PASMC O2 sensing render the fetal PASMCs uniquely sensitive to an acute increase in O2 tension at a biologically critical time point.

To examine mechanisms underlying developmental changes in pulmonary vascular tone, we tested the hypotheses that 1) maturation-related changes in the ability of the pulmonary vasculature to respond to hypoxia are intrinsic to the pulmonary artery (PA) smooth muscle cells (SMCs); 2) voltage-gated K+(Kv)-channel activity increases with maturation; and 3) O2-sensitive Kv2.1 channel expression and message increase with maturation. To confirm that maturational differences are intrinsic to PASMCs, we used fluorescence microscopy to study the effect of acute hypoxia on cytosolic Ca2+concentration ([Ca2+]i) in SMCs isolated from adult and fetal PAs. Although PASMCs from both fetal and adult circulations were able to sense an acute decrease in O2tension, acute hypoxia induced a more rapid and greater change in [Ca2+]iin magnitude in PASMCs from adult compared with fetal PAs. To determine developmental changes in Kv-channel activity, the effects of the K+-channel antagonist 4-aminopyridine (4-AP) were studied on fetal and adult PASMC [Ca2+]i. 4-AP (1 mM) caused PASMC [Ca2+]ito increase by 94 ± 22% in the fetus and 303 ± 46% in the adult. Kv-channel expression and mRNA levels in distal pulmonary arteries from fetal, neonatal, and adult sheep were determined through the use of immunoblotting and semiquantitative RT-PCR. Both Kv2.1-channel protein and mRNA expression in distal pulmonary vasculature increased with maturation. We conclude that there are maturation-dependent changes in PASMC O2sensing that may render the adult PASMCs more responsive to acute hypoxia.

Introduction: At birth, pulmonary artery (PA) blood flow increases 8–10 fold and PA pressure falls by 50% within 24 hours. The mechanisms responsible for postnatal adaptation of the pulmonary circulation remain incompletely understood. High serum endothelin-1 (ET-1) levels are found in infants with persistent pulmonary hypertension of the newborn. We hypothesized that chronic intrauterine pulmonary hypertension (PHT) potentiates agonist induced increase of cytosolic calcium concentration ([Ca2+]i) in fetal PA smooth muscle cells(SMC). Methods: Chronic intrauterine pulmonary hypertension was induced by intrauterine ligation of the ductus arteriosus (DA) in late-gestation fetal lambs. 7–10 days following DA ligation, animals were euthanized. Distal PAs (>4th generation) were removed from the lung and PA SMC were isolated and maintained in normoxic (95%O2) primary culture. Age-matched non-PHT controls were sacrificed at ∼132 days gestation and cells were cultured in the same manner. Fluorescence microscopy using fura-2 was used to determine the effect of ET-1 (10−6 -10−8 M) on PA SMC [Ca2+]i from PHT and control animals. To determine the effect of PHT on endothelin A and B (ETA and ETB) receptor mRNA expression, quantitative, internally controlled RT-PCR was performed on distal PAs and SMC in culture. Results: ET-1 (10−8 M) caused a monophasic increase in [Ca2+]i (206 +/- 47%, p < 0.01 vs baseline) above baseline in controls (n = 25), while PHT SMC [Ca2+]i did not increase significantly. When PHT SMC were treated with 10−6M ET-1, in those cells that responded, [Ca2+]i increased 174 +/- 81% above baseline(p < 0.01 vs baseline). Only 3/33 (9%) of PHT SMC responded to ET-1 compared to 90% of controls. Additionally, the duration of the increase in [Ca2+]i was longer in the PHT SMC: PHT SMC [Ca2+]i fell to baseline after 328 +/- 16 sec, where control [Ca2+]i fell to baseline after 117 sec. ETA and ETB receptor mRNA expression are both increased in PHT. Conclusions: PHT decreases PA SMC sensitivity to ET-1 by ∼100 fold, however, PHT increases the duration of the ET-1 induced increase in [Ca2+]i. We conclude that PHT prolongs the response of PA SMC to ET-1. This prolongation may lead to significant increases in PA SMC [Ca2+]i that results in attenuated postnatal adaptation of the pulmonary circulation.

Pulmonary Vascular Voltage-Sensitive K+ Channel Expression is Developmentally Regulated † 1630

The role of anti-neutrophil antibodies was investigated as a cause of functional abnormalities of circulating neutrophils in a child with persistent neutropenia and recurrent mouth sores. The patient's neutrophils were isolated by isopycnic centrifugation and compared with normal adult neutrophils. Functional abnormalities in the patient's PMNS included (1) decreased chemotaxis studied by under agarose method and by skin window, (2) decreased oxidative burst measured by luminol amplified chemi luminescence, and (3) decreased superoxide production measured by cytochrome C reduction assay. The ability of the patient's serum to bind normal neutrophils was determined by immunofluorescence. While the patient's serum bound normal PMNs, this binding did not interfere with their oxidative burst. In contrast, the patient's serum did not bind to his own PMNs in vitro even though his neutrophil specific antibody was anti-NAI and his PMNs were NAI positive. Therefore, the functional abnormalities of this patient's PMNs were not due to neutrophil antibody binding alone, but due to a subpopulation of dysfunctional neutrophils in circulation possibly selected out by their low affinity to the circulating autoantibodies.