RhoA-Rho kinase and platelet-activating factor stimulation of ovine foetal pulmonary vascular smooth muscle cell proliferation.

OBJECTIVES: Platelet-activating factor (PAF) is produced by pulmonary vascular smooth muscle cells (PVSMC). We studied effects of Rho kinase on PAF stimulation of PVSMC proliferation in an attempt to understand the role of RhoA/Rho kinase on PAF-induced ovine foetal pulmonary vascular remodelling. Our hypothesis is that PAF acts through Rho kinase, as one of its downstream signals, to induce arterial (SMC-PA) and venous (SMC-PV) cell proliferation in the hypoxic lung environment of the foetus, in utero. MATERIALS AND METHODS: Rho kinase and MAPK effects on PAF receptor (PAFR)-mediated cell population expansion, and PAFR expression, were studied by DNA synthesis, western blot analysis and immunocytochemistry. Effects of constructs T19N and G14V on PAF-induced cell proliferation were also investigated. RESULTS: Hypoxia increased PVSMC proliferation and Rho kinase inhibitors, Y-27632 and Fasudil (HA-1077) as well as MAPK inhibitors PD 98059 and SB 203580 attenuated PAF stimulation of cell proliferation. RhoA T19N and G14V stimulated cell proliferation, but co-incubation with PAF did not affect proliferative effects of the constructs. PAFR protein expression was significantly downregulated in both cell types by both Y-27632 and HA-1077, with comparable profiles. Also, cells treated with Y-27632 had less PAF receptor fluorescence with significant disruption of cell morphology. CONCLUSIONS: Our results show that Rho kinase non-specifically modulated PAFR-mediated responses by a translational modification of PAFR protein, and suggest that, in vivo, activation of Rho kinase by PAF may be a further pathway to sustain PAFR-mediated PVSMC proliferation.

Platelet-activating factor synthesis and receptor-mediated signaling are downregulated in ovine newborn lungs: relevance in postnatal pulmonary adaptation and persistent pulmonary hypertension of the newborn.

Platelet-activating factor (PAF) is a phospholipid with a wide range of biological activities. We studied PAF metabolism and PAF receptor (PAFR) signaling in perinatal ovine lungs to understand PAF's role in transition of the perinatal pulmonary hemodynamics and pathophysiology of persistent pulmonary hypertension of the newborn. We hypothesized that downregulation of PAF synthesis with upregulation of PAF catabolism by acetylhydrolase (PAF-Ah) in the newborn lung is needed for fetus-to-newborn pulmonary adaptation. Studies were conducted on fetal and newborn lamb pulmonary arteries (PA), veins (PV) and smooth muscle cells (SMC). PAF metabolism, PAFR binding and cell proliferation were studied by cell culture; gene expression was studied by qPCR. Fetal lungs synthesized 60% more PAF than newborn lungs. Compared with the fetal PVs and SMCs, PAF-Ah activity in newborn was 40-60% greater. PAF-Ah mRNA expression in newborn vessels was different from the expression by fetal PA. PAF-Ah gene clone activity confirmed deletion of hypoxia-sensitive site. PAFR mRNA expression by the PVs and SMC-PV of the fetus and newborn was greater than by corresponding PAs and SMC-PA. Q-PCR study of PAFR expression by the SMC-PV of both groups was greater than SMC-PA. Fetal SMCs bound more PAF than the newborn SMCs. PAFR antagonist, CV-3988, inhibited PAFR binding and DNA synthesis by the fetal SMCs, but augmented binding and DNA synthesis by newborn cells. We show different PAF-PAFR mediated effects in perinatal lungs, suggesting both transcriptional and translational regulation of PAF-Ah and PAFR expression in the perinatal lamb lungs. These indicate that the downregulation of PAF-mediated effects postnatally protects against persistent pulmonary hypertension of the newborn.

Platelet-activating factor stimulates ovine foetal pulmonary vascular smooth muscle cell proliferation: role of nuclear factor-kappa B and cyclin-dependent kinases.

OBJECTIVE: Platelet-activating factor (PAF) is implicated in pathogenesis of persistent pulmonary hypertension of the neonate (PPHN); PAF is a mitogen for lung fibroblasts. PAF's role in pulmonary vascular smooth muscle cell (PVSMC) proliferation and in hypoxia-induced pulmonary vein (PV) remodelling has not been established and mechanisms for PAF's cell-proliferative effects are not well understood. We investigated involvement of PAF and PAF receptors in PVSMC proliferation. MATERIALS AND METHODS: Cells from pulmonary arteries (SMC-PA) and veins (SMC-PV) were serum starved for 72 h in 5% CO2 in air (normoxia). They were cultured for 24 h more in normoxia or 2% O(2) (hypoxia) in 0.1% or 10% foetal bovine serum with 5 microCi/well of [(3)H]-thymidine, with and without 10 nm PAF. Nuclear factor-kappa B (NF-kappaB), CDK2 and CDK4 protein expression, and their roles in cell proliferation control were studied. RESULTS: PAF and hypoxia increased SMC-PA and SMC-PV proliferation. WEB2170 inhibited PAF-induced cell proliferation while lyso-PAF had no effect. SMC-PV proliferated more than SMC-PA and PAF plus hypoxia augmented NF-kappaB protein expression. NF-kappaB inhibitory peptide attenuated PAF-induced cell proliferation by 50% and PAF increased CDK2 and CDK4 protein expression. The data show that hypoxia and PAF up-regulate PVSMC proliferation via PAF receptor-specific pathway involving NF-kappaB, CDK2 and CDK4 activations. CONCLUSION: They suggest that in vivo, in foetal lung low-oxygen environment, where PAF level is high, proliferation of PVSMC will occur readily to modulate PV development and that failure of down-regulation of PAF effects postnatally may result in PPHN.

Maturational changes in ovine pulmonary metabolism of platelet-activating factor: implications for postnatal adaptation.

We recently reported that PAF acetylhydrolase (PAF-Ah) mRNA level and PAF-Ah activity in lamb lungs are up-regulated in the immediate newborn period, thereby facilitating the fall in postnatal PAF levels as well as a fall in pulmonary vascular resistance (B. O. Ibe, F. C. Sardar, and J. U. Raj, Mol Genet Metab 69:46-55, 2000). We have studied hypoxia effects on PAF synthesis and PAF-Ah activity in fetal lamb pulmonary arterial smooth muscle cells (FPASMC) and endothelial cells (FPAEC). We also studied PAF synthesis by platelets, and PAF-Ah activity in plasma of perinatal lambs at different ages. PAF synthesis (means +/- SEM, pmol/10(6) cells) by SMC in baseline was 168 +/- 27 and increased 3-fold on stimulation with A23187. Hypoxia augmented A23187-stimulated PAF synthesis by 30%. In FPAEC, baseline synthesis was 0.54 +/- 0.062 and increased 3-fold to 1.72 +/-.34. Hypoxia had no effect on PAF synthesis by EC. FPASMC produced over 300-fold more PAF than FPAEC. PAF synthesis by platelets was 47.02 +/- 7.1, 63.4 +/- 6.6, 71.5 +/- 9.9, and 62.2 +/- 5.2 for fetal, and newborn lambs <2 h, <1 day, and 6-12 days, old, respectively. PAF synthesis by platelets of <1 day-old lambs was different from that of fetal lambs. PAF-Ah activity (nmol lyso-PAF/min/mg protein) by FPASMC in normoxia was 3.41 +/- 0.38 which was 50% higher than the rate in hypoxia. Activity in FPAEC was 1.75 +/- 0.37 which was not different from hypoxia. PAF-Ah activity in fetal lamb plasma was 47.83 +/- 6.87 which was different from 155.32 +/- 12.10, the activity in plasma of newborn <1 day old. Activity in the other perinatal lambs did not differ from fetal or newborn <1 d. Our data suggest that lower pulmonary vascular PAF synthesis in normoxia together with higher PAF-Ah activity during immediate postnatal period is necessary to ensure rapid catabolism of PAF in vivo so as to facilitate postnatal adaptation of the pulmonary and systemic circulations.

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 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.

Platelet-activating factor receptors in lamb lungs are downregulated immediately after birth.

Platelet-activating factor (PAF) is a phospholipid with diverse biological functions mediated by a G protein-coupled receptor. We determined PAF receptor binding in lung membranes of four groups of perinatal lambs. Membrane protein (100 microg/ml) was incubated for 60 min at 30 degrees C with 0.5-24 nM of acetyl-[(3)H]PAF in 30 mM Tris buffer, pH 7.2, containing 0.25% BSA, 10 mM MgCl(2), and 125 mM choline chloride. PAF bound to membrane was isolated and quantified by scintillation spectrometry, followed with Scatchard analysis for receptor density (B(max)). The B(max) (means +/- SE, fmol/mg protein) were 445.8 +/- 12.3, 244.2 +/- 3.3, 250.6 +/- 3.6, and 419. 9 +/- 8.6 for the fetal, 90-min-old, <1-day-old, and 6- to 12-day-old lambs, respectively. The B(max) for the 90-min-old and <1-day-old lambs were not different but were significantly lower than those of either the term fetal or 6- to 12-day-old lambs. These data show a significant decrease in PAF binding to its receptor and in PAF B(max) in lung membranes of immediate newborn lambs. The dissociation constants (K(D), nM) were 7.7 +/- 0.52, 11.5 +/- 0.34, 6.9 +/- 0.48, and 5.0 +/- 0.53 for fetal, 90-min-old, <1-day-old, and 6- to 12-day-old newborn lamb lungs, respectively. The K(D) of the 90-min-old lamb was the highest of all. PAF receptor gene measured by RT-PCR showed a significant downregulation of PAF receptor gene mRNA in lungs of lambs <1 day old, suggesting a transcriptional regulation of PAF receptor gene expression in the immediate newborn period. We speculate that decreased PAF receptor binding immediately after birth will facilitate the fall in pulmonary vascular resistance in the immediate newborn period.

Platelet activating factor acetylhydrolase activity in lamb lungs is up-regulated in the immediate newborn period.

We recently showed that platelet activating factor (PAF) is an important modulator of pulmonary vasomotor tone in the fetus, with a significant decrease in circulating PAF levels in the immediate newborn period. In this study, we have determined PAF catabolism by PAF acetylhydrolase (PAF-Ah) in lungs of near-term fetal and newborn 2- to 16-h (<1 day) and 6- to 12-day-old lambs. The rate of PAF catabolism by lung homogenate protein from the three groups of lamb lungs was studied at 37 degrees C in 30 mM Tris buffer, pH 7.5, containing 0.01% BSA. Each lung homogenate protein was incubated for 10 min with 50 microM [(3)H]acetyl-PAF at pO(2) <50 Torr (hypoxia) and approximately 100 Torr (normoxia). PAF-Ah activity was quantified as amount of lyso-PAF produced. PAF-Ah activity (means +/- SEM, nmol lyso-PAF/min/mg protein) in fetal lung homogenate was 1.19 +/- 0.14 and 2.46 +/- 0.05 during hypoxia and normoxia, respectively. The corresponding values for the newborns were newborn <1 day, 1.65 +/- 0.26 and 2.95 +/- 0.07 and newborn 6-12 days, 1.25 +/- 0.10 and 2.84 +/- 0.05. In all groups, PAF-Ah activity was higher in normoxia than in hypoxia. During normoxia, PAF-Ah activity in newborn <1 day was significantly higher than the activity in fetus, but similar to the activity in newborn 6- to 12-day-old lamb lungs. These data show a significant up-regulation of PAF-Ah activity in lungs in the immediate newborn period. PAF-Ah gene expression measured by RT-PCR showed a significant up-regulation of the PAF-Ah gene in lungs of lambs <1 day old, suggesting a transcriptional regulation of the PAF-Ah gene in the immediate newborn period. These results suggest that up-regulation of PAF-Ah activity after birth with oxygenation will result in a decrease in circulating PAF levels, thereby facilitating the fall in pulmonary vascular resistance in the immediate newborn period.

Phosphodiesterase activity in intrapulmonary arteries and veins of perinatal lambs.

The transition from fetal to newborn life is marked by a reduction in pulmonary vascular tone mediated by the intracellular second messengers, cGMP and cAMP. We have compared the rates of phosphodiesterase (PDE)-catalyzed hydrolysis of cGMP and cAMP in intrapulmonary vessels of fetal (146 +/- 2 days gestation) and newborn (3-7-day-old) lambs, each n = 6. Lung vessels of second to sixth generations were dissected and cytosol was prepared by differential centrifugation. PDE activity in cytosol was determined by radiometric assay of the hydrolysis of exogenous nucleotides at 30 degrees C for 10 min. Rates of hydrolysis (pmol/min/mg protein) of cGMP were 225 +/- 38 in fetal arteries and different from 151 +/- 7 in veins. In newborn vessels, the rates were 155 +/- 49 and 63 +/- 13 in arteries and veins, respectively. Rates of cAMP hydrolysis by the fetus were 80 +/- 11 in arteries and 45 +/- 16 veins. In newborn lambs the rates were 69 +/- 10 in arteries and different from 18 +/- 4 in veins. Inhibition of PDE activity by zaprinast, a cGMP-specific PDE inhibitor, and rolipram, a cAMP-specific PDE inhibitor, was more in veins of fetal and newborn lambs. Our data show that rates of hydrolysis of the cyclic nucleotides were faster in fetal vessels than in the newborn. We speculate that this would result in a greater accumulation of the cyclic nucleotides in newborn vessels, particularly the veins, and therefore endow the veins with less vascular tone.

Platelet-activating factor modulates pulmonary vasomotor tone in the perinatal lamb.

Eight near-term fetal lambs were studied acutely in utero to determine role of platelet-activating factor (PAF) in the regulation of vasomotor tone in systemic and pulmonary circulations in the immediate perinatal period. Four fetal lambs were studied predelivery and 2 h postdelivery to determine circulating PAF levels. Aortic and pulmonary arterial pressures and cardiac output were measured continuously, and systemic and pulmonary vascular resistances were calculated. Left pulmonary arterial blood flow was also measured in four fetal lambs. After delivery and oxygenation, circulating PAF levels fell significantly. When WEB-2170, a specific PAF-receptor antagonist, was infused to block effect of endogenous PAF in the eight near-term fetal lambs, systemic vascular resistance fell 30% but pulmonary vascular resistance fell dramatically by 68%. Specificity of WEB-2170 was tested in juvenile lambs and was found to be very specific in lowering vasomotor tone only when tone was elevated by action of PAF. Our data show that endogenous PAF levels in the fetus contribute to maintain a high basal systemic and pulmonary vasomotor tone and that a normal fall in circulating PAF levels after birth and oxygenation may facilitate fall in pulmonary vascular resistance at birth.

Platelet-activating factor in plasma of patients with sickle cell disease in steady state.

The role of platelet-activating factor (PAF) in the pathogenesis of microvascular vaso-occlusion in sickle cell disease (SCD) is not known. In order to assess a role for PAF in vaso-occlusion in patients with SCD in steady state conditions, we measured plasma PAF level and plasma PAF acetylhydrolase activity as indices of PAF metabolism in vivo. We also studied PAF synthesis, from (3H)-acetate, by purified platelets stimulated with A23187. PAF was extracted from plasma of ten patients with SCD in steady state and from age-matched controls. PAF, purified by thin-layer chromatography, was quantitated by radioimmunoassay. PAF level (mean +/- SEM, pg/ml) in plasma of controls was 393 +/- 65, which was significantly lower than the 797 +/- 62 measured in plasma of patients with SCD. There was no difference in acetylhydrolase activity between the two groups. PAF synthesis (mean +/- SEM, nmol/10(6) cells) by platelets of controls without exogenous lyso-PAF was 1.69 +/- 0.24, higher than the 0.59 +/- 0.038 synthesized by platelets of patients with SCD. Incubation of platelets with 1.0 micromol/L lyso-PAF increased PAF synthesis by controls to 8.93 +/- 1.76, still higher than the 4.59 +/- 0.98 synthesized by platelets of patients with SCD. Our data show that patients with SCD are susceptible to a higher circulating levels of PAF in vivo during steady-state conditions. We speculate that higher levels of PAF may be a contributing factor to the persistent stress and inflammatory state of the microcirculation of patients with SCD.

Leukotriene synthesis by isolated perinatal ovine intrapulmonary vessels correlates with age-related changes in 5-lipoxygenase protein.

Leukotrienes (LT) are potent vasoconstrictors in the pulmonary circulation. We have investigated the synthesis of LTC4 by intrapulmonary arteries and veins of perinatal lambs. Paired vessels of near-term fetal 146 +/- 2- and 2- to 7-day-old newborn lambs (each n = 7), were incubated for 10 min at 37 degrees C in baseline, with 1 mumol/L A32187 or 0.1 mmol/L arachidonic acid. Produced leukotriene C4 was assayed from media by ELISA. Baseline production of leukotriene (ng/mg tissue, means +/- SEM) by vessels for arteries was 0.006 +/- 0.001 and 0.059 +/- 0.009 for fetus and newborn, respectively. In veins, the values were 0.013 +/- 0.003 and 0.073 +/- 0.007 for fetus and newborn, respectively. On stimulation with the calcium ionophore A23187, production by arteries increased 25-fold in the fetus, but 4-fold in the newborn. The corresponding values for stimulated veins were 37-fold and 9-fold in fetus and newborn, respectively. Generally, production by veins was greater than production by the matching arteries. In all instances, the fetal vessels produced less leukotrienes than the newborn vessels. Western analysis of stimulated and unstimulated vessel membrane protein showed greater expression of 5-lipoxygenase in veins than in arteries (P < 0.05). Our data show that veins produce more LTC4 due to greater expression of 5-lipoxygenase in the vessels and thus suggest that veins of perinatal lamb lungs may be more susceptible to LT-induced vasoreactivity in the perinatal pulmonary circulation. We speculate that a higher production of LTC4 by fetal veins may be necessary to maintain a high venous tone in fetal lungs.

Sickle erythrocytes induce prostacyclin and thromboxane synthesis by isolated perfused rat lungs.

The role of eicosanoids in the pathogenesis of acute or chronic lung syndrome in sickle cell disease is unknown. We investigated the synthesis of prostacyclin (PGI2), thromboxane (Tx) A2, and prostaglandin (PG) E2 by three groups of isolated rat lungs perfused with buffer (GPBS), normal (HbAA), and sickle (HbSS) erythrocyte suspensions. Isolated lungs were perfused at a constant pressure and flow rate (Q) of 40 ml x kg(-1) x min(-1) with GPBS or 7% erythrocyte suspensions for 15 min. Autologous platelet-rich plasma (PRP) was added, and perfusion was continued for 15 min and then at two times Q for another 15 min. Perfusate samples were assayed for the specific eicosanoids. Perfusate level of PGI2 in GPBS lungs was the least among the three groups. However, the PGI2 level in HbSS lungs was 90% higher than from HbAA lungs after 15 min of perfusion and was 180% higher on perfusion with PRP. Additionally, coperfusion of erythrocytes and PRP augmented perfusate levels of TxA2 and PGE2 over 1,000% more in HbSS than HbAAlungs. These data show that HbSS erythrocytes increased perfusate levels of the eicosanoids, suggesting increased synthesis, perhaps due to aberrant erythrocyte-endothelium interactions.

Prostaglandins E2 and I2 cause greater relaxations in pulmonary veins than in arteries of newborn lambs.

Prostaglandins E2 (PGE2) and I2 (PGI2) are important vasoactive mediators in pulmonary vessels. The present study was designed to determine whether the responses of pulmonary arteries to these prostanoids are different from those of veins in newborn lambs. Fourth-generation pulmonary arterial and venous rings without endothelium were suspended in organ chambers filled with modified Krebs-Ringer bicarbonate solution (95% O2-5% CO2, 37 degrees C), and their isometric force was measured. During contraction with endothelin-1 or U-46619 (indomethacin was present to eliminate the possible involvement of endogenous cyclooxygenase products), PGE2, PGI2, and carbacyclin (a stable analogue of PGI2) induced greater relaxations in veins than in arteries. In both vessel types, relaxations induced by PGE2 were greater than those induced by PGI2 or carbacyclin. Forskolin, an activator of adenylate cyclase, also induced greater relaxation of veins than of arteries. Relaxation induced by 8-bromoadenosine 3',5' -cyclic monophosphate, an analogue of adenosine 3',5' -cyclic monophosphate (cAMP), was comparable in both vessel types. Radioimmunoassay revealed that the basal and calcium ionophore A-23187-induced releases of PGE2 or 6-ketoprostaglandin F1 alpha (the stable breakdown product of PGI2) were similar between arteries and veins. Measurement of cAMP (in the presence of isobutylmethylxanthine) showed that PGE2 and forskolin induced greater increase in cAMP in veins than in arteries. Our results demonstrate that PGE2 and PGI2 are more potent vasodilators in pulmonary veins than in arteries in newborn lambs. A difference in the activity of adenylate cyclase may contribute to the differential responses to PGE2 and PGI2 between pulmonary arteries and veins. Furthermore, PGE2 appears play an more important role than does PGI2 in modulating pulmonary vascular tone of newborn lambs.

Heterogeneity in prostacyclin and thromboxane synthesis in ovine pulmonary vascular tree: effect of age and oxygen tension.

Intrapulmonary arteries and veins of 8 near-term fetal lambs (141-145 days gestation) and 8 ewes were isolated into segments of > 3mm, 1-3 mm, and < mm in diameter. Vessels were incubated in Krebs' buffer at 37 degrees C at PO2 approximately 100 torr (normoxia) and PO2 < 50 torr (hypoxia) to study local vascular production of prostanoids. Prostacyclin and thromboxane (Tx) A2 produced were measured by radioimmunoassay and expressed in ng/mg dry wt, means +/- SEM. During normoxia, fetal arteries > 3 mm synthesized more prostacyclin than adult arteries of the same size (1.71 +/- 0.3 vs 0.45 +/- 0.04). However, fetal arteries < 1 mm synthesized less prostacyclin than adult arteries < 1 mm (0.47 +/- 0.1 vs 1.75 +/- 0.16). Prostacyclin production by veins > 3 mm was similar in the fetus and adult (0.49 +/- 0.06 vs 0.67 +/- 0.08), but in veins < 1 mm was greater in adult than in fetal vessels (1.73 +/- 0.17 0.54 +/- 0.06). Hypoxia-attenuated prostacyclin production by fetal arteries and veins of all sizes, but only in 1 to 3-mm-size adult arteries. In general, production of TxA2 by segments of fetal and adult vessels was less than 50% of that of prostacyclin. Protein and DNA concentrations in similar sized fetal and adult vessels were similar. The data show that there is heterogeneity in the production of prostacyclin and TxA2 along the ovine pulmonary vascular tree. Prostanoid synthesis of fetal vessels is markedly influenced by hypoxia, with a greater suppression of prostacyclin synthesis. Similar protein and DNA concentrations in fetal and adult vessels suggest that differences in prostanoid production by vessel segments may be due to differences in enzyme activity rather than cell number or tissue mass.

Hypoxia attenuates metabolism of platelet activating factor by fetal and newborn lamb lungs.

The metabolism of platelet activating factor (PAF) by lungs of near-term fetal and 5- to 9-day-old lambs was studied during normoxia and hypoxia at 37 degrees C in 30 mM Tris buffer. PAF synthesis was studied in lung cytosol and membrane using 250 microM [3H]acetyl CoA, 40 microM lyso-PAF, and 50 micrograms protein. PAF catabolism was studied in lung homogenate (LH) using 50 microM [3H]alkyl-PAF. PAF was extracted and assayed by thin-layer chromatography (TLC) and liquid scintillation spectrometry. Levels of PAF synthesized (nmol/min per mg protein) by fetal lung membrane versus cytosol were 1.35 +/- 0.07 versus 0.61 +/- 0.08, which were greater than those by newborn which were 0.33 +/- 0.07 versus 0.17 +/- 0.03. Hypoxia did not alter PAF synthesis by the lungs. PAF catabolism (nmol lyso-PAF/min per mg protein) by fetal LH was 0.07 +/- 0.01, which increased to 0.24 +/- 0.02 during normoxia. In newborn LH, the rate was 0.24 +/- 0.04 and increased to 0.33 +/- 0.01 during normoxia. PAF catabolism was higher in newborn than in fetal LH. An increase in pO2 augmented PAF catabolism, more in fetal than in newborn LH. Thus rate of PAF synthesis decreases from fetus to newborn, but PAF catabolism increases from fetus to newborn. The higher rate of PAF synthesis coupled with a low rate of PAF catabolism in the hypoxic environment of fetal lungs may predispose the fetus to a high PAF level, which may contribute to the high basal vasomotor tone in fetal lungs. A fall in PAF level with oxygenation, due to increased PAF catabolism, may facilitate the normal fall in pulmonary vascular resistance at birth.

Antenatal glucocorticoid treatment attenuates immediate postnatal prostacyclin and thromboxane levels in plasma of very preterm lambs.

The effect of antenatal glucocorticoid treatment on the production of cyclooxygenase metabolites was studied in very preterm lambs. Seven fetal lambs, 121 days of gestation, received a single dose of betamethasone, 0.5 mg/kg i.m., 48 h prior to delivery. Five age-matched controls received saline intramuscularly. Each fetus was delivered and ventilated for 3 h and sacrificed. Plasma was prepared from blood drawn from the umbilical cord of each fetus, and 60, 120 and 180 min after delivery. Mesenteric (MESA) and femoral (FEMA) arteries were isolated and incubated in Krebs' buffer for 10 min at 37 degrees C. Samples were extracted for prostacyclin (PGI2), and thromboxane (Tx)A2, purified by HPLC and measured by specific radioimmunoassay. Amounts of metabolites measured postnatally from betamethasone-treated preterm lambs were significantly lower (p < 0.05) than the amounts from saline lambs. Prostacyclin production by MESA and FEMA of betamethasone-treated lambs was lower than by vessels of saline-treated lambs. There was no difference in TxA2 production by vessels from the two groups of preterm lambs. Our data show that antenatal betamethasone treatment decreased systemic prostanoid production suggesting a decreased reactivity of the vascular membrane.

Developmental differences in production of lipoxygenase metabolites by ovine lungs.

This study determined age-related differences in production of lipoxygenase metabolites by ovine lungs. Lungs of near-term fetal (146 +/- 2 days gestation), neonatal (8 +/- 2 days), and adult sheep were homogenized and 9000g fraction (9000g) cytosol, and microsomes were prepared by differential centrifugation. These subcellular fractions were incubated for 10 min at 37 degrees C, either alone, with [14C]arachidonic acid ([14C]AA), or with A23187. Hydroxyeicosatetraenoic acids (HETEs) and leukotrienes (LTs) were extracted from incubation media and quantitated by HPLC. All three lung subcellular fractions metabolized [14C]AA into 5-, 12-, and 15-HETEs. Production of each HETE was greater in neonatal and adult 9000g and microsomes than in fetal microsomes. Leukotriene production from [14C]AA by 9000g, cytosol, and microsomes was greater, the older the animal. Production of LTs by microsomes was greater than that by cytosol at all ages. LT production from endogenous AA increased significantly on stimulation with 5 microM A23187, and was inhibited by 50 microM NDGA. In each case, LTC4 was metabolized to LTD4 and LTE4. The data show that ovine lungs metabolize exogenous and endogenous AA into 5-, 12-, and 15-HETEs and LTs and that production of lipoxygenase metabolites by the lung increases with age. Also, in the ovine lung, 5-lipoxygenase is more membrane bound than cytosolic.

Elevated urinary levels of thromboxane and prostacyclin metabolities in sickle cell disease reflects activated platelets in the circulation.

There is evidence for increased factor VII turnover and the associated increased thrombin generation and fibrinolytic activities in sickle cell disease (SCD) that may affect in vivo platelet and endothelial cell reactivity. We studied the release of specific eicosanoids that are indicative of in vivo platelet activation and endothelial cell injury. The circulating and urinary levels of 2,3-dinor thromboxane B2(2,3-dinor-TxB2),TxB2,PGI2 [as 6-keto-PGF1 alpha], and PGE2 were measured in 15HbSS patients, eight HbAA non-haemolytic anaemic individuals and 12 healthy HbAA controls using specific RIAs. The mean urinary 2,3-dinor-TxB2 in the HbSS patients was significantly higher than in both the healthy HbAA and the anaemic controls. 6-keto-PGF1 alpha was undetected in the urines of the healthy HbAA controls, but was measured insignificant amounts in the HbSS and the HbAA anaemic patients. The urinary concentrations of PGE2 and TxB2 in HbSS patients' samples were also significantly higher than those of both control groups (P < 0.05). PGE2 and TxB2 levels were below the detection limit in the plasmas of the HbAA subjects, but were measurable in the HbSS and HbAA anaemic plasmas. The plasma level of 6-keto-PGF1 alpha in the HbSS patients was also significantly higher than in the control groups. The data indicates a persistent inflammatory process in the HbSS patients, and is consistent with the hypothesis that there is platelet and endothelial cell activation in SCD.

Metabolism of N-methylcarbazole by rat lung microsomes.

N-methylcarbazole (NMC) is a procarcinogenic component of tobacco smoke particulate matter. It is metabolized by liver microsomes into some hydroxylated metabolites such as the potent mutagen N-hydroxymethylcarbazole (NHMC). Lung metabolism and toxicity of NMC is not known. Since the lung is the primary organ of inhalation of tobacco smoke, NMC metabolism by lung microsomes was studied in comparison with the metabolism by liver microsomes. Liver or lung microsomes (1 mg/mL) were incubated with 0.5 mM NMC for 30 min at 37 degrees C. NMC metabolites were extracted with ethyl acetate and analyzed by reversed-phase high-performance liquid chromatography. Rat lung microsomes metabolized NMC with a similar profile to liver microsomes, although lung microsomes produced greater number of metabolites. The potent mutagen NHMC was also the major NMC metabolite produced by lung microsomes, as confirmed by particle beam mass spectrometry. However, lung microsomes produced only 10% of NHMC produced by liver microsomes. Metabolism of NMC by lung microsomes also led to depletion of the endogenous antioxidant glutathione by 34% compared to controls, indicating a significant generation of some reactive intermediates during NMC metabolism by lung microsomes. The data show that the lung participates directly in producing the potent mutagen NHMC from NMC present in tobacco smoke.

Mechanisms by which Candida albicans induces endothelial cell prostaglandin synthesis.

One strategy for improving resistance to opportunistic pathogens is to determine host cellular responses during the invasion process and upregulate those responses that are relevant to host defense mechanisms. Within this context, we have shown previously that invasion of endothelial cells by Candida albicans in vitro causes increased production of prostaglandins. As a prerequisite for modulating endothelial cell prostaglandin production, we now characterize the mechanisms through which this process occurs. Endothelial cell invasion by C. albicans appeared to stimulate the conversion of arachidonic acid into prostaglandins by upregulating the synthesis of endothelial cell cyclooxygenase and increasing the activity of the endothelial cell phospholipase. The enhanced activities of these two enzymes were independent of calphostin C-sensitive protein kinase C and resulted in the increased production and extracellular secretion of prostaglandin I2 (PGI2), PGF2 alpha, and PGE2. The secretion of these prostaglandins had no effect on the amount of endothelial cell injury induced by C. albicans. The role of the increased prostaglandin secretion by endothelial cells is likely related to modulation of the leukocyte response at the candida-leukocyte-endothelial cell interface.