Basil O. Ibe

Effects of endothelin, platelet activating factor and thromboxane A2 in ferret lungs

We have compared the effects of three vasoactive agents, endothelin, platelet activating factor and thromboxane A2 analogue, U 46,619, in the pulmonary circulation of ferrets. Lungs of nine adult ferrets, body weight 1.06 +/- 0.27 kg, were isolated and perfused with sheep red blood cells suspended in Krebs solution with 2 g% Dextran 70 (hematocrit 33 +/- 6%), under conditions of constant flow in zone 3. Endothelin-1 (ET: 0.1-1.35 micrograms/kg), platelet activating factor (PAF: 0.5-5.5 micrograms/kg) or thromboxane A2 analogue (U 46,619: 1 microgram/kg), was infused into the pulmonary artery and the pressure response determined. To locate the site of action of the agents, the pulmonary circulation was partitioned into arteries, microvessels and veins by measuring pressures in 20-50 microns diameter subpleural arterioles and venules by micropuncture, both during baseline and after the peak response to each vasoactive drug. We found that the ferret pulmonary circulation constricted in response to ET, PAF and U 46,619, but the magnitude of constriction varied. Ferret lungs were most sensitive to U 46,619 and least sensitive to PAF. The major site of action also differed among the agents; U 46,619 and ET predominantly constricted veins whereas PAF predominantly constricted arteries. We conclude that the ferret pulmonary circulation demonstrates differential responsiveness to vasoactive agents, that venous constriction is a common feature and that the predominant site of action varies with the specific agent.

Antenatally administered PPAR-γ agonist rosiglitazone prevents hyperoxia-induced neonatal rat lung injury

The physiological development and homeostasis of the lung alveolus is determined by the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) by the interstitial lipofibroblast. We have recently shown (Dasgupta C et al., Am J Physiol Lung Cell Mol Physiol 296: L1031-L1041, 2009.) that PPAR-γ agonists administered postnatally accelerate lung maturation and prevent hyperoxia-induced lung injury. However, whether the same occurs antenatally is not known. The objective of this study was to test the hypothesis that the potent PPAR-γ agonist rosiglitazone (RGZ), administered antenatally, enhances fetal lung maturation and protects against hyperoxia-induced neonatal lung injury. Sprague-Dawley rat dams were administered either diluent or RGZ (3 mg/kg), at late gestation, to determine its effect on lung maturation and on hyperoxia (95% O(2) exposure for 24 h)-induced neonatal lung injury. The lungs were examined for the expression of specific markers of alveolar development (surfactant proteins A and B, cholinephosphate cytidylyltransferase-α, leptin receptor, triglyceride uptake, and [(3)H]choline incorporation into saturated phosphatidylcholine) and injury/repair, in particular, the markers of transforming growth factor-β signaling (activin receptor-like kinase-5, SMAD3, lymphoid enhancer factor-1, fibronectin, and calponin). Overall, antenatal RGZ accelerated lung maturation and blocked the inhibition of alveolar sacculation and septal wall thinning by hyperoxia. RGZ specifically stimulated the development of the alveolar epithelial type II cell, the lipofibroblast, and the vasculature. The increased expression of the transforming growth factor-β intermediates, such as SMAD3 and lymphoid enhancer factor-1, implicated in hyperoxic lung injury, was also blocked by antenatal RGZ treatment. In conclusion, PPAR-γ agonists can enhance fetal lung maturation and can effectively prevent hyperoxia-induced neonatal lung injury.

Levalbuterol inhibits human airway smooth muscle cell proliferation: therapeutic implications in the management of asthma.

Background: Racemic albuterol is a mixture of (R)- and (S)-enantiomers of albuterol. Its pharmacological activity and clinical efficacy reside in the (R)-enantiomer (levalbuterol), but the (S)-enantiomer exacerbates airway reactivity in nonclinical models. The role of albuterols in airway smooth muscle cell (SMC) proliferation is not well understood. Methods: The effect of levalbuterol on human bronchial SMC growth was compared with the effects of racemic albuterol and (S)-albuterol. Cells were fed albuterols and 3H-thymidine in 5% FBS and incubated for 24 h. The effect of (S)-albuterol on levalbuterol actions was also studied and so were the effects of cAMP/PKA, PI-3 kinase, NK-ĸB, and retinoblastoma (Rb) proteins on albuterols and human bronchial SMC proliferation. Results: Levalbuterol inhibited cell proliferation at low concentrations. The growth-inhibitory effect of levalbuterol occurs via activation of the cAMP/PKA pathway. Addition of (S)-albuterol to levalbuterol decreased the growth-inhibitory effect of levalbuterol, and (S)-albuterol attenuated levalbuterol-induced cAMP release by 65%. Levalbuterol inhibited NF-ĸB and Rb protein expressions. ICI-118551 abrogated the inhibitory properties of levalbuterol. The PAF receptor antagonist CV-3988 inhibited (S)-albuterol-induced cell growth, with no effect on levalbuterol. Conclusions: Levalbuterol inhibits cell growth by activating the cAMP/PKA pathway and inhibiting PI-3 kinase, NF-ĸB and Rb protein expression, and (S)-albuterol induces cell growth by activating PAF-receptor-mediated cell signaling.

Nebulized PPARγ agonists: a novel approach to augment neonatal lung maturation and injury repair in rats

Background:By stimulating lipofibroblast maturation, parenterally administered peroxisome proliferator-activated receptor γ (PPARγ) agonists promote lung homeostasis and injury repair in the neonatal lung. In this study, we determined whether PPARγ agonists could be delivered effectively via nebulization to neonates, and whether this approach would also protect against hyperoxia-induced lung injury.Methods:One-day old Sprague-Dawley rat pups were administered PPARγ agonists rosiglitazone (RGZ, 3 mg/kg), pioglitazone (PGZ, 3 mg/kg), or the diluent, via nebulization every 24 h; animals were exposed to 21% or 95% O2 for up to 72 h. Twenty-four and 72 h following initial nebulization, the pups were sacrificed for lung tissue and blood collection to determine markers of lung maturation, injury repair, and RGZ and PGZ plasma levels.Results:Nebulized RGZ and PGZ enhanced lung maturation in both males and females, as evidenced by the increased expression of markers of alveolar epithelial and mesenchymal maturation. This approach also protected against hyperoxia-induced lung injury, since hyperoxia-induced changes in bronchoalveolar lavage cell and protein contents and lung injury markers were all blocked by nebulized PGZ.Conclusion:Nebulized PPARγ agonist administration promotes lung maturation and prevents neonatal hyperoxia-induced lung injury in both males and females.

Leukotriene metabolism by intrapulmonary vessels of newborn lambs : Effect of platelet-activating factor

Leukotrienes (LTs) are potent vasoconstrictors in the pulmonary circulation. We investigated LTB4 and LTE4 metabolism by intrapulmonary arteries and veins of 2 to 9 days old lambs (n=6). Paired vessels were incubated under baseline, and stimulated conditions. LTB4 and LTE4Leukotrienes (LTs) are potent vasoconstrictors in the pulmonary circulation. We investigated LTB4 and LTE4 metabolism by intrapulmonary arteries and veins of 2 to 9 days old lambs (n = 6). Paired vessels were incubated under baseline, and stimulated conditions. LTB4 and LTE4 were extracted from media, quantfied by enzyme-linked immunosorbent assay (ELISA), normalized to tissue weight and presented as ng/mg tissue (means +/- SEMs). In arteries, baseline synthesis of LTB4 was 0.15+/-0.20 and increased to 0.96+/-0.04 on stimulation with 1.0 micromol/L A2318, and 1.74+/-0.25 with 0.1 mmol/L arachidonic acid (AA). In veins the corresponding values were 0.28+/-0.10, 2.50+/-0.51, and 5.36+/-0.70. Baseline production of LTB4 was higher in veins. LTE4 synthesis in arteries was 0.25+/-0.02, which increased to 0.42+/-0.05 with A23187, and further to 0.69+/-0.06 with AA. The corresponding values in veins were 0.23+/-0.05, 0.74+/-0.09, and 1.56+/-0.28. Baseline metabolism of LTE4 by the vessels was not different. Furthermore, stimulation of vessels with 50 nmol/L PAF led to over 3-fold increase in LTB4 and LTE4 metabolism by the vessels. Smooth muscle cells stimulated with A23187 metabolized LTB4 and LTC4, which was sequentially catabolized to LTD4 and LTE4. Generally, stimulated veins, whether vessels or smooth muscle cells, metabolized more leukotrienes. The selective 5-lipoxygenase inhibitor, AA-861, significantly attenuated synthesis of both leukotrienes. Western analysis of membrane protein showed gReater expression of 5-lipoxygenase in stimulated veins. Our data show that veins produce more leukotrienes due to greater expression of 5-lipoxygenase in the vessels, and suggest that veins of newborn lamb lungs may be more susceptible to LT-induced vascular reactivity in the pulmonary circulation.

Role of Epidermal Growth Factor Receptor in Ovine Fetal Pulmonary Vascular Remodeling Following Exposure to High Altitude Long-Term Hypoxia

The purpose of this study was to evaluate color vision during high altitude mountain climbing by applying the Mollon-Reffin Minimalist test to 14 climbers, all of whom were participating in the expedition to Ama Dablam (6,812 m) in Nepal. Before leaving for Nepal (at 300 m), all 28 eyes showed normal color vision in all 3 axes. At 1,300 m, 100% of eyes showed normal color vision in the protan and deutan axes, while 25% showed minimally reduced color discrimination in the tritan axis. At 4,000 m, 100% showed normal deutan axis, 4% minimally reduced protan axis, and 72% minimally reduced tritan axis discrimination. At 5,400 m 100% of eyes tested showed normal protan and deutan axis discrimination, while 75% showed minimally and 25% moderately reduced tritan axis discrimination. Back home at 300 m 3 days after return, 100% showed normal deutan, 4% minimally reduced protan, and 38% minimally reduced tritan axis discrimination. One year later, all eyes showed normal color vision in all three axes. Changes in tritan axis discrimination correlated well with increased heart rate (r = 0.69; p = 0.0001) and decreased oxygen saturation (r = 0.71; p = 0.001) at high altitude. This study shows that the tritan color vision axis is predominantly affected at high altitude, but that this reduced color discrimination is transient.

Platelet-Activating Factor Modulates Activity of Cyclic Nucleotides in Fetal Ovine Pulmonary Vascular Smooth Muscle

At birth, release of endogenous vasodilators such as nitric oxide and prostacyclin facilitate pulmonary vasodilation via the cyclic nucleotides, cGMP and cAMP. Interaction of cyclic nucleotides and platelet-activating factor (PAF)-mediated responses in pulmonary vascular smooth muscle is not known. We studied the effects of cGMP and cAMP on PAF-mediated responses in ovine fetal intrapulmonary venous smooth muscle cells. Studies were done in hypoxia or normoxia with buffer with 8-Br-cGMP (BGMP) and 8-Br-cAMP (BAMP), as well as cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA) inhibitors. All groups were treated with 1 nM PAF and incubated for 30 min for the binding assay or 20 min for measurement of inositol 1,4,5-phosphate (IP3) production. BGMP and BAMP decreased PAF binding in normoxia by 63 and 14%, respectively. Incubations with the PKG inhibitor Rp-8-(4-chlorophenylthio)-guanosine-3′,5′-cyclic monophosphorothioate sodium and the PKA inhibitor Rp-adenosine-3′,5′-cyclic monophosphorothioate abrogated the inhibitory effects of BGMP and BAMP. PAF-stimulated IP3 production was 8565 ± 314 dpm/106 cells in hypoxia and 5418 ± 118 dpm/106 cells in normoxia, a 40% decrease. BGMP attenuated PAF-stimulated IP3 production by 67 and 37% in hypoxia and normoxia, respectively; the value for BAMP was 44% under both conditions. Pretreatment with PKG or PKA inhibitor abrogated BGMP and BAMP inhibition of IP3 release. PAF receptor (PAFr) protein expression decreased in normoxia, but pretreatment with 10 nM PAF up-regulated PAFr expression. Pretreatment with PAF decreased expression and activities of PKG or PKA proteins in normoxia and hypoxia. Our data demonstrate the existence of cGMP/cAMP-PAF cross-talk in pulmonary vascular smooth muscle cells, which may be one mechanism by which PAFr-mediated vasoconstriction is down-regulated at birth.

Metabolism of Endogenous Arachidonic Acid by Isolated Lungs of Neonatal and Adult Rabbits Stimulated with Calcium Ionophore: Effect of Hypoxia

We have determined the effect of hypoxia on arachidonic acid metabolism by rabbit lungs stimulated with calcium ionophore A23187. Isolated lungs of neonatal and adult rabbits were perfused during normoxia (pO2 greater than 100 torr) or hypoxia (pO2 less than 40 torr) and arachidonic acid metabolism stimulated by the addition of A23187 (5 microM) to the perfusate. Cyclooxygenase metabolites PGE2, TxA2, and PGI2 were measured by radioimmunoassay and lipoxygenase metabolites LTB4, LTC4, LTD4, and LTE4 by HPLC. During normoxia, neonatal lungs synthesized the three cyclooxygenase metabolites that we measured in similar amounts. LTC4 constituted 56% of the leukotrienes produced. Hypoxia caused a 100% increase in the amount of PGI2 synthesized by neonatal lungs; however, total TxB2 and PGE2 production was not altered significantly. LTC4 production decreased significantly during hypoxia, whereas LTE4 production increased. Adult lungs synthesized significantly lower amounts of both cyclooxygenase and 5-lipoxygenase products than neonatal lungs. During normoxia, PGE2 was measured in highest amount in adult lungs. Similar to the neonatal lungs, LTC4 was the predominant leukotriene (56%) measured. During hypoxia, there was a 100% increase in PGI2 production by adult lungs, as was observed in neonatal lungs. There was also a small but significant increase in PGE2 production, with no change in TxA2 production. LTC4 production also decreased, but there was a marked increase in synthesis of LTB4 by adult lungs. Our data demonstrate that hypoxia alters the profile of arachidonic acid metabolites produced by rabbit lungs stimulated with A23187. Also, the age of the rabbit significantly affects both the amount and profile of metabolites synthesized by stimulated lungs during normoxia and hypoxia.

Mechanisms by Which S-Albuterol Induces Human Bronchial Smooth Muscle Cell Proliferation

Background: Racemic albuterol is a 50:50 mixture of the R-isomer, levalbuterol, and the S-isomer, S-albuterol. S-Albuterol increases airway hyperresponsiveness to spasmogens, exacerbates asthmatic conditions and stimulates cell growth, whereas levalbuterol attenuates cell growth in culture. The mechanisms of S-albuterol-induced cell proliferation are not well understood. We studied the role of albuterol isomers and intracellular cell cycle regulators on proliferation of human bronchial smooth muscle cells. Methods: Serum-starved cells (72 h) were fed test agents for 24 h and cell proliferation was measured. The expression of nuclear factor-ĸB inhibitory protein IĸBα, nuclear factor-ĸB, cyclin-dependent kinases 2 and 4, interleukin (IL)-6, and retinoblastoma and platelet-activating factor (PAF) receptor protein were measured by Western blotting. Results:S-Albuterol, PAF and platelet-derived growth factor stimulated cell proliferation, but levalbuterol and the racemic mixture inhibited cell proliferation compared with the effect of 5% fetal bovine serum alone. The proliferative effect of platelet-derived growth factor on S-albuterol was not additive, suggesting that the 2 mediators act by different mechanisms. S-Albuterol induced greater expression of all the measured proteins than either levalbuterol, the racemic mixture or 5% fetal bovine serum. S-Albuterol stimulated IL-6 secretion and abolished the ability of levalbuterol to inhibit IL-6 secretion. Conclusion: Our data show that S-albuterol stimulates cell proliferation by activating expression and phosphorylation of several intracellular mitogenic proteins and may exacerbate asthma by stimulating the release of IL-6. Induction of PAF receptor protein expression by S-albuterol strongly suggests that S-albuterol may exert its adverse effects by binding to a G protein-coupled receptor such as the PAF receptor.

Prolonged hypoxia modulates platelet activating factor receptor-mediated responses by fetal ovine pulmonary vascular smooth muscle cells

Hypoxia augments PAF receptor (PAFr) binding and PAFr protein expression in venous SMC (SMC-PV). We compared effect of acute and prolonged hypoxia (pO(2)<40 torr) on PAFr-mediated responses in arterial SMC (SMC-PA) and SMC-PV. Cells were studied for 30 min (acute) or for 48 h (prolonged) hypoxia and compared to normoxic (pO(2) ~100 torr) conditions. PAF binding was quantified in fmol/10(6) cells (mean ± SEM). PAF binding in normoxia were SMC-PA, 5.2 ± 0.2 and in SMC-PV, 19.3 ± 1.1; values in acute hypoxia were SMC-PA, 7.7 ± 0.4 and in SMC-PV, 27.8 ± 1.7. Prolonged hypoxia produced 6-fold increase in binding in SMC-PA, but only 2-fold increase in SMC-PV, but binding in SMC-PV was still higher. Acute hypoxia augmented inositol phosphate release by 50% and 40% in SMC-PA and SMC-PV, respectively. During normoxia, PAFr mRNA expression by both cell types was similar, but expression in hypoxia by SMC-PA was greater. In SMC-PA, hypoxia and PAF augmented intracellular calcium flux. Re-exposure of cells to 30 min normoxia after 48 h hypoxia decreased binding by 45-60%, suggesting immediate down-regulation of hypoxia-induced PAFr-mediated effects. We speculate that re-oxygenation immediately reverses hypoxia effect probably due to oxygen tension-dependent reversibility of PAFr activation and suggest that exposure of the neonate to prolonged state of hypoxia will vilify oxygen exchange capacity of the neonatal lungs.