Determination of free desmosine and isodesmosine as urinary biomarkers of lung disorder using ultra performance liquid chromatography-ion mobility-mass spectrometry

The elastin degradation products, desmosine (DES) and isodesmosine (IDES) are highly stable, cross-linking amino-acids that are unique to mature elastin. The excretion of DES/IDES in urine, in the free form and with associated peptide fragments, provides an indicator of lung damage in chronic obstructive pulmonary disease (COPD). A quantitative ion mobility-mass spectrometry (IM-MS) method has been developed for the analysis of free DES/IDES in urine with deuterated IDES as an internal standard. Resolution of DES/IDES isomers was achieved in less than five minutes using ultra performance liquid chromatography (UPLC) combined with ion pairing. The optimized UPLC-IM-MS method provided a linear dynamic range of 10-300 ng/mL and a limit of quantitation of 0.028 ng/mL for IDES and 0.03 ng/mL for DES (0.55 ng and 0.61 ng on column respectively). The method reproducibility (%RSD) was <4% for DES and IDES. The UPLC-IM-MS method was applied to the analysis of urine samples obtained from healthy volunteers and COPD patients. The DES/IDES concentrations in healthy and COPD urine showed an increase in DES (79%) and IDES (74%) in the COPD samples, relative to healthy controls. The incorporation of an IM separation prior to m/z measurement by MS was shown to reduce non-target ion responses from the bio-fluid matrix.     

Micellar electrokinetic chromatography for the determination of urinary desmosine and isodesmosine in patients affected by chronic obstructive pulmonary disease

The presence in urine of desmosine (DES) and isodesmosine (IDES), two crosslinked amino acids unique to the elastic fiber network, can be used as a specific indicator of degradation of mature elastin. Compared to methodologies so far available, the capillary electrophoretic technique reported here seems to be suitable and convenient for determining desmosines in urine of patients affected by chronic obstructive pulmonary disease (COPD). By using 35 mM sodium tetraborate pH 9.3 containing 65 mM SDS as the background electrolyte, the peaks of DES and IDES could be detected in hydrolyzed urine samples from controls and patients. Owing to the simultaneous determination of endogenous urinary creatinine used as appropriate internal standard, the amount of these amino acids could be accurately quantified. The results obtained were of the same order of magnitude as the data already reported in the literature for COPD patients. Thus micellar electrokinetic chromatography (MEKC) may be considered as a reliable technique for studying the turnover of the elastic fiber in clinical conditions.     

Elastin synthesis during perinatal lung development in the rat

The rate of soluble elastin synthesis was estimated in lung explants from rats of differing ages to better define periods in lung development important to the deposition of lung elastin. Lungs from rat pups at days 1, 3, 7, 9, 12, 15, and 21 post-parturition and from adult rats were incubated in a defined medium containing L-[3H]valine. Following incubation, labelled soluble elastin (tropoelastin) was separated from other soluble proteins by coacervation and electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The tropoelastin synthetic rate was then estimated after correcting for differences in recovery of radioactivity as tropoelastin and lung tissue L-[3H]valine specific activity. Maximal rates of elastin synthesis were observed in lung explants from 7-12-day-old rats. The rate of elastin synthesis during this period was 5-8-times the rate observed in adult rat lung (expressed per g of fresh lung) and represented approx. 2% of the total protein synthesis. Moreover, the values derived from lung explant culture for elastin synthesis were consistent with values for lung elastin deposition in the perinatal rat (5-10 micrograms elastin/h per g lung).     

Pulmonary mechanics during the ventilatory response to hypoxemia in the newborn monkey

Dynamic lung compliance (CL), inspiratory pulmonary resistance (RL), and functional residual capacity (FRC) were measured in 10 unanesthetized 48 h-old newborn monkeys and seven 21-day-old infant monkeys during acute exposures to an equivalent level of hypoxemia. End-expiratory airway occlusions were performed and the pressure developed by 200 ms (P0.2) was utilized as an index of central respiratory drive. P0.2 demonstrated a sustained increase throughout the period of hypoxemia on day 2 despite the fact that minute ventilation (VI) initially increased but then fell back to base-line levels. Dynamic lung compliance fell and FRC increased by 5 min of hypoxemia in the newborns. The 21-day-old monkeys exhibited a sustained increase in both VI and P0.2 throughout the hypoxic period with no change in CL and FRC. RL did not change at either postnatal age during hypoxemia. These data indicate that the neonatal monkey is subject to changes in pulmonary mechanics (decreased CL and increased FRC) during hypoxemia and that these changes are eliminated with maturation.     

Quantification of elastin cross-linking amino acids,desmosine and isodesmosine,in hydrolysates of rat lung by ion-pair liquid chromatography-mass spectrometry

The elastin cross-linking amino acids, desmosine (DES) and isodesmosine (IDE), in hydrolysates of rat lungs were quantified by ion-pair liquid chromatography-electrospray mass spectrometry. The column was a 2.0 mm i.d. x 150 mm Develosil UG3 (ODS) with a mobile phase A of 7 mM pentafluoropropionic anhydride (PFPA) using ultrapure water as the ion-pair reagent, and a mobile phase B of 7 mM PFPA in 80% methanol. The retention times of IDE and DES were 25.5 and 26.6 min, respectively. The mean concentrations of IDE and DES in the lung were 191.6+/-54.5 nmol/g lung (dry tissue) (+/-SD) and 184.0+/-39.3 nmol/g lung, respectively, and the IDE/DES ratio was 1.04, in Wistar Kyoto rats. Our results indicate that ion-pair liquid chromatography-mass spectrometry is a useful procedure for quantitation of DES and IDE in hydrolysates of rat lung.     

Hyperoxic inhibition of newborn rat lung development: protection by deferoxamine.

Prolonged exposure to hyperoxia markedly inhibits normal lung development (alveolarization and respiratory surface area expansion) in immature animals. Since (a) hyperoxia results in excess hydroxyl radical (OH.) formation, (b) (OH.) is implicated in O2-induced lipid peroxidation and DNA alterations, and (c) both OH. formation and its interaction with DNA are Fe++ dependent; chelation of Fe++ should act to protect against pulmonary O2 toxicity and hyperoxic inhibition of lung development. We therefore treated litters of newborn rats with the iron chelator Deferoxamine mesylate (DES) (150 mg/kg/day) during a 10-day exposure to greater than 95% O2. Morphometric analysis demonstrated that compared to the mean airspace size in air control rat pups (Lm = 44.5 microns), hyperoxic exposure resulted in a 34% larger mean air space diameter in O2-saline rat lungs (59.5 microns) versus only an 11% enlargement in O2-DES lungs (51.1 microns*). Lung internal surface area (cm2) per 100-g body weight were air control = 4480, O2-saline = 3570 (decreases 20.3%), and O2-DES = 4125* (decreases 7.9%) (*p less than 0.05 versus O2-saline group). DES-treated animals also had significantly decreased lung conjugated diene levels during hyperoxic exposure and increased lung elastin content (reflective of preserved lung alveolar formation) compared to O2-saline rats. These results indicate that DES treatment substantially ameliorated the inhibitory effects of neonatal hyperoxic exposure on normal lung development.     

Plasma and urinary endothelin-1 concentrations in asphyxiated newborns [corrected

The aim of this study was to determine if there is any correlation between the hypoxia induced deterioration of renal functions and urinary excretions of endothelin (ET). Therefore using a sensitive and specific radioimmunoassay, we have investigated plasma ET-1 concentrations and urine ET-1 excretions in healthy and asphyxiated newborns. Sixteen newborns (10 boys, 6 girls) with perinatal asphyxia or hypoxia of variable seriousness which were followed at Newborn Intensive Care Unit in Eskisehir Osmangazi University Faculty of Medicine were enrolled. Simultaneously, gestation and weight matched 10 newborns (6 boys, 4 girls) with no asphyxia (first minute Apgar score >7) were enrolled as controls. Plasma ET-1 concentrations of the asphyxiated infants (61.8+/-79.3 pg/ml, between 23.4-125.2 pg/ml) were higher than in the control group (29.3+/-22.1 pg/ml, between 12.3 and 50.8 pg/ml, p<0.05). However creatinine clearance values were not different between the two groups (p>0.05), mean fractional excretion of sodium levels (FeNa%) were higher in the study group than the controls (p<0.01). Urinary ET-1 concentrations in the asphyxiated infants were 144.6+/-63.4 pg/ml versus 70.1+/-27.7 pg/ml in the control group (p<0.001). The ET clearance were more elevated in the asphyxiated newborns than in the healthy infants (p<0.05). Urinary ET-1/Cr ratio in the hypoxic infants were significantly elevated in the first day of life when compared with those of healthy infants (p<0.05). Total ET excretion was negatively correlated with FeNa (%) (r=-0.603, p<0.05). Plasma ET-1 concentrations of the asphyxiated infants reduced at 48 hours of age (p<0.001). Fifth minute Apgar score was negatively correlated with urinary ET-1 levels (r=-0.615, p<0.01), urinary Na excretion (r=-0.583, p<0.01), FeNa (%) (r=-0.597, p<0.01) and total ET excretion (r=-0.560, p<0.01) and positively correlated with ET clearance (r=0.559, p<0.05). Urinary ET-1 levels were negatively correlated with umbilical artery BE levels (r=-0.612, p<0.05). To our study, elevated urinary ET-1 levels were observed during perinatal asphyxia and urinary ET-1 levels were negatively correlated with 5th minute Apgar score and cord blood base excess levels. For this reason urinary ET-1 levels could be a marker of perinatal asphyxia as cord blood ET-1 levels. With investigations showing renal production is independent from plasma and increased urinary ET-1/Cr levels in newborn with perinatal asphyxia and also negative correlation between the total ET excretion and FeNa, urinary ET-1 levels could be served as a useful marker to detecting also impaired renal functions in infants with perinatal asphyxia.     

Role of eicosanoids in relative oxygen tolerance of newborn rabbits

Prolonged exposure to hyperoxia can result in significant lung injury, although newborn animals are more oxygen-tolerant than adults. Mechanisms affording tolerance to the newborn are incompletely understood. This study examined the hypothesis that eicosanoids play a significant role in newborn oxygen tolerance. One litter of term newborn albino rabbits and 15 adult rabbits were exposed to 65 hours of greater than 95% O2. An additional litter of newborns served as a normoxic control. Normoxic newborn rabbits had very high quantities of 6-keto-PGF1a and low TXB2 in bronchoalveolar lavage (BAL) fluid. Sixty-five hours of oxygen exposure in newborn rabbits produced no evidence of lung injury on light microscopy, 97% of BAL white cells were alveolar macrophages and BAL protein was low. An equal period of oxygen exposure produced significant lung injury in adult rabbits. BAL fluid from oxygen-injured adults contained a 17-fold greater percentage of PMN and 16-fold higher protein than oxygen-exposed newborns. Hyperoxic adults had significantly lower 6-keto-PGF1a, and significantly higher LTB4 and LTC4 in BAL compared to hyperoxic newborns. This study confirms the hypothesis of relative oxygen tolerance in newborn rabbits compared to adults, and suggests that this tolerance may have been afforded by higher pulmonary levels of the protective prostacyclin metabolite.     

Milk ceruloplasmin is a valuable source of nutrient copper ions for mammalian newborns

This research focuses on the role of milk ceruloplasmin (Cp), the main extracellular copper-containing protein of vertebrates, as a source of copper for newborns. In the first part of the study, Cp concentration and Cp-associated copper were measured in human skimmed milk at the 1st and the 5th days postpartum. It was shown that most of the copper was associated with Cp and that the decrease in copper concentration during lactation was related to the drop of Cp levels. The following in vivo experiments demonstrated that milk [(125)I]Cp per os administered to 6-day-old rats (embryonic-type copper metabolism) was transported into their bloodstream. The electrophoretic mobility and relative molecular weight of [(125)I]Cp transferred through the cellular barrier remained unaltered. However, 22-day-old rats (adult-type copper metabolism) digested the administered milk [(125)I]Cp completely. In the final part of the study, newborn rats were fed with baby formula for 8d. It was found that these rats switched their copper metabolism from embryonic type to adult type earlier than their littermates fed by dams. Activation of Cp gene expression in the liver, increased Cp and copper concentrations in the blood, and reduced copper content of the liver were observed in the rats fed with baby formula. In the brain, no copper concentration change was observed, but Cp and copper concentrations were dramatically increased in the cerebrospinal fluid. The role of milk Cp as a source of copper adapted to embryonic-type copper metabolism is discussed.     

Alloparental Responsiveness to Newborns by Nonreproductive,Adult Male,Common Marmosets (Callithrix jacchus)

Parental care in mammals is influenced by sensory stimuli from infants, and by changes in the hormone levels of caretakers. To determine the responsiveness to infant cues in nonreproductive adult male common marmosets (Callithrix jacchus) with and without previous experience in caretaking, we exposed 12 males to newborn marmosets and assessed their cortisol plasma levels and behavioral response. Newborn marmosets housed in transparent enclosures were placed inside the cages of the adult male subjects. Males were exposed four times to two different experimental conditions: (a) newborn enclosures remained closed during the observation period and (b) newborn enclosures were opened during the observation period to allow direct social interaction by the adult males. Blood samples from adult males were collected after each behavioral observation trial to measure the levels of cortisol. The behavioral responses of adult males exposed to the closed and open newborn enclosures showed a significant difference only with respect to the frequency of displacements, where males moved among the quadrants of their own cages with greater frequency when the newborn enclosure was sealed. Experienced males approached newborn enclosures more frequently, spent more time in close proximity, and carried and recovered newborns more quickly than inexperienced males. The successive exposure to newborns increased the responsiveness in inexperienced males. The highest levels of plasma cortisol in adult males were recorded following periods of exposure to the sealed newborn enclosures. This suggests that successive exposure to newborns and previous alloparental caregiving experience while living in family groups influences the responsiveness of male marmosets to the sensory cues of newborns. Am. J. Primatol. 75:145‐152, 2013. © 2012 Wiley Periodicals, Inc.     

新生儿缺氧缺血性脑病血清S-100B蛋白与NBNA评分动态监测的临床研究

目的：观察新生儿缺氧缺血性脑病（hypoxic-ischemicencephalopathy,HIE）血清S-100B蛋白的动态变化规律,探讨其在HIE早期诊断中的价值,以及其浓度变化与病情严重程度及预后的关系。同时研究围产期高危因素以及NBNA评分在HIE发生发展与预后中的作用。方法：30例住院正常新生儿作为对照组,于出生后采血,55例HIE患儿（HIE组）分别于出生后1天、2天、7天采血,采用酶联免疫吸附试验、双抗体夹心法检测。收集并分析两组围产期相关资料。HIE组并于采血同时进行NBNA评分。结果：（1）HIE患儿生后第一天与第二天血清S-100B蛋白浓度明显高于对照组（P〈0.05）,生后第七天轻度HIE与对照组比较没有统计意义,中、重度HIE与对照组比较有统计学意义。（2）生后第一天与第二天不同病情组HIE患儿NBNA评分相互比较差异具有统计学意义（P〈0.05）,第七天轻、中和重度患儿NBNA评分〈35分的患儿分别占33.3%,47.1%,100%。结论：动态监测HIE患儿血清S-100B蛋白浓度和NBNA评分的变化,对HIE的早期诊断,严重程度的判断以及预后的估计有重要意义。     

Iron release, superoxide production and binding of autologous IgG to band 3 dimers in newborn and adult erythrocytes exposed to hypoxia and hypoxia-reoxygenation

Iron is released in a desferrioxamine (DFO)-chelatable form when erythrocytes are challenged by an oxidative stress. The release is increased when an accelerated removal of erythrocytes occurs such as in perinatal period, in which iron release is greater in hypoxic than in non-hypoxic newborns. This suggests that an hypoxic environment at birth promotes iron release. To test this possibility, iron release in a model of hypoxia, hypoxia-reoxygenation and normoxia was studied in newborn and adult erythrocytes. In newborn erythrocytes, hypoxia induced a much greater iron release compared to an equal period of normoxia. In adult erythrocytes, hypoxia also induced a greater iron release as compared to normoxia, but it was much lower than that seen with newborn erythrocytes. Methemoglobin (MetHb) formation roughly paralleled iron release. The phenylhydrazine-promoted superoxide anion (O(2)?(-)) production was greater with normoxic but lower with hypoxic erythrocytes from newborns as compared to that from adults. This discrepancy between iron release and O(2)?(-) production may be explained by the shift towards MetHb in hemoglobin autoxidation. Iron diffusion out of the erythrocytes was much higher with hypoxic erythrocytes from newborns as compared to that from adults. Also the binding of autologous IgG to band 3 dimers (AIgGB) is much greater with hypoxic erythrocytes from newborns as compared to that from adults, suggesting that the level of iron release is related to the extent of band 3 clustering and that hypoxia accelerates removal of erythrocytes from bloodstream in in vivo condition.     

Liberation of desmosine and isodesmosine as amino acids from insoluble elastin by elastolytic proteases

The development of atherosclerotic lesions and abdominal aortic aneurysms involves degradation and loss of extracellular matrix components, such as collagen and elastin. Releases of the elastin cross-links desmosine (DES) and isodesmosine (IDE) may reflect elastin degradation in cardiovascular diseases. This study investigated the production of soluble elastin cross-linking structures by proteinases implicated in arterial diseases. Recombinant MMP-12 and neutrophil elastase liberated DES and IDE as amino acids from insoluble elastin. DES and IDE were also released from insoluble elastin exposed to monocyte/macrophage cell lines or human primary macrophages derived from peripheral blood monocytes. Elastin oxidized by reactive oxygen species (ROS) liberated more unconjugated DES and IDE than did non-oxidized elastin when incubated with MMP-12 or neutrophil elastase. These results support the exploration of free DES and IDE as biomarkers of elastin degradation.     

Two new elastin cross-links having pyridine skeleton. Implication of ammonia in elastin cross-linking in vivo

Isolation and structure analysis of two amino acids from bovine ligamentum nuchae elastin hydrolysates revealed the presence of pyridine cross-links in elastin. The structures of these amino acids were determined to have 3,4,5- and 2,3,5-trisubstituted pyridine skeletons both with three carboxylic acids and a mass of 396 (C(18)H(28)N(4)0(6)) identified as 4-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)-pyridine and 2-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)-pyridine. We have named these pyridine cross-links desmopyridine (DESP) and isodesmopyridine (IDP), respectively. Structure analysis of these pyridine cross-links implied that the formation of these cross-links involved the condensation reaction between ammonia and allysine. The elastin incubated with ammonium chloride showed that DESP and IDP levels increased as the allysine content decreased. DESP and IDP were measured by high pressure liquid chromatography (HPLC) with UV detection and were found in a variety of bovine tissues. The DESP/desmosine (DES) and IDP/isodesmosine (IDE) ratios in aorta elastin were higher than in other tissues. DESP and IDP contents in human aorta elastin were found to be gradually increased with age. The concentration of IDP was significantly elevated in aorta elastin of rat with chronic liver cirrhosis induced by carbon tetrachloride (mean +/- S.D.; 11.1 +/- 0.9 nmol/mg elastin) when compared with normal rats (5.9 +/- 1.5 nmol/mg elastin). Although DESP and IDP are present at only trace concentrations in the tissue elastin, these pyridine cross-links may be useful biomarkers for the aortic elastin damaged by ammonia.     

Retinoic acid attenuates O2-induced inhibition of lung septation

Exposure of the newborn lung to hyperoxia is associated with impaired alveolar development. In newborn rats exposed to hyperoxia and studied at day 14 of life, retinoic acid (RA) treatment improved survival and increased lung collagen but did not improve alveolar development. To determine whether RA treatment during exposure to hyperoxia results in late improvement in alveolarization, we treated newborn rats with RA and hyperoxia from day 3 to day 14 and then weaned O2 to room air by day 20, and studied the animals on day 42. O2-exposed animals had larger mean lung volumes, larger alveoli, and decreased gas-exchange tissue relative to air-exposed animals, whereas RA-treated O2-exposed animals were not statistically different from air-exposed controls. Relative to control animals, elastin staining at day 14 was decreased in hyperoxia-exposed lung independent of RA treatment, and, at day 42, elastin staining was similar in all treatment groups. At day 14, elastin gene expression was similar in all treatment groups, whereas at day 42 lung previously exposed to hyperoxia showed increased elastin signal independent of RA treatment. These results indicate that RA treatment during hyperoxia exposure promotes septal formation without evidence of effects on elastin gene expression after 4 wk of recovery.     

子宫颈机能不全孕妇血清松弛素、弹性蛋白表达意义及对妊娠结局的评估价值研究

摘要 目的：分析子宫颈机能不全孕妇血清松弛素、弹性蛋白表达意义及对妊娠结局的评估价值。方法：选择我院于2020年5月至2022年5月接诊的120例子宫颈机能不全孕妇作为观察组，另选同期的120例正常妊娠孕妇作为对照组。分析血清松弛素、弹性蛋白表达水平，及其与子宫颈机能不全孕妇宫口扩张宽度、宫颈长度的关系，观察血清松弛素、弹性蛋白在早产者与非早产者间的差异性，使用受试者工作特征曲线(ROC)下面积(AUC)评价血清松弛素联合弹性蛋白对早产及围产儿死亡的预测效能。结果：观察组血清松弛素水平较对照组高，弹性蛋白水平较对照组低（P＜0.05）；经Pearson相关性分析，子宫颈机能不全孕妇宫口扩张宽度与血清松弛素水平呈正相关，与弹性蛋白水平呈负相关（P＜0.05）；宫颈长度与血清松弛素水平呈负相关，与弹性蛋白水平呈正相关（P＜0.05）；在120例子宫颈机能不全孕妇中，早产42例、非早产78例；早产组血清松弛素水平高于非早产组，弹性蛋白水平低于非早产组（P＜0.05）；经ROC曲线分析，血清松弛素联合弹性蛋白预测子宫颈机能不全孕妇发生早产的AUC为0.910，预测围产儿死亡的AUC为0.943。结论：血清松弛素水平升高和弹性蛋白水平降低均与子宫颈机能不全有关，两者联合预测早产及围产儿死亡的效能较好，值得进一步研究应用。     

Cellular immunity in newborn infants with hyperbilirubinemia]

In order to identify the possibility of prenatal or perinatal bacterial contact with immunization of the cellular immunity system as underlying cause of the "idiopathic" newborn icterus (without blood group incompatibility) the lymphocyte transformation test with addition of streptolysin O or E. coli antigen was carried out in 68 newborns with a birth weight ranging between 1260 and 4200 g. The sensitization rate identified among the newborns with hyperbilirubinaemia did not differ significantly from those of the control group. Thus an ensured connection between a prenatal streptococcus or E. coli contact and the appearance of an idiopathic newborn hyperbilirubinaemia could not be established.     

Elastin mRNA levels during foetal development of sheep nuchal ligament and lung. Hybridization to complementary and cloned DNA

Elastin mRNA levels were quantified in sheep nuchal ligament and lung during the latter half of foetal development with elastin-specific cDNA (complementary DNA) probes using both hybridization in solution (saturation analysis) and hybridization on a fixed support (Northern analysis). For the solution-hybridization studies, cDNA prepared from nuchal-ligament mRNA was enriched to 65% for elastin sequences by hybridizing it to its template at a R0t (mol X s X litre-1) value that included only the abundant class of mRNA sequences. Hybridization of this probe to RNA extracted from nuchal ligament between 70 and 138 days after conception demonstrated elastin sequences increased about 10-fold (from 0.047 to 0.438% of total RNA). In contrast, lung elastin mRNA levels increased only 3-fold (from 0.009 to 0.022% of total RNA) during the same period. Over this development period these values correspond to increases in the average number of elastin mRNA molecules from 950 to 20 000 molecules/ligament cell and from 130 to 330 molecules/lung cell. For Northern analysis, elastin mRNA was purified from near-term-sheep nuchal ligament on sucrose density gradients. Analysis of the translation products of this elastin mRNA showed that relative elastin precursor synthesis was at least 80% of total [3H]valine incorporation. The Mr of this elastin mRNA, determined by methylmercury-agarose-gel electrophoresis, was approx. 1.25 X 10(6). Northern hybridization of nuchal ligament and lung RNA to a [32P]cDNA probe, transcribed from this sucrose-gradient-purified elastin mRNA, confirmed the developmental changes in elastin mRNA levels detected by solution-hybridization techniques. The specificity of this method was confirmed by using a cloned elastin gene fragment. These studies demonstrate that elastin mRNA levels in organs such as nuchal ligament and lung increase with foetal development, but that there are significant differences in the average cellular elastin mRNA content of these two organs.     

Chronic intrauterine pulmonary hypertension decreases calcium-sensitive potassium channel mRNA expression

Calcium-sensitive potassium (K(Ca)) 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 K(Ca) channel gene expression is decreased in PPHN. To test this hypothesis, we measured K(Ca) 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 K(Ca) channel mRNA levels, primers were designed against the known sequence of the K(Ca) channel and used in semiquantitative RT-PCR, with lung 18S rRNA content as an internal control. Compared to that in control lambs, lung K(Ca) 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 K(Ca) channel mRNA expression is decreased in an ovine model of PPHN. Decreased K(Ca) channel gene expression may contribute to the abnormal pulmonary vascular reactivity associated with PPHN.