The role of the blood in metabolism of prostaglandin E1 in the cat lung

We found that when 15-keto-PGE1 was added to cat blood, it was converted to 13,14-dihydro-15-keto-PGE1 (dihydro-keto-PGE1) by a NADH-dependent enzyme associated with some formed element(s) in the blood. When PGE1 was injected into the pulmonary artery of blood-perfused lungs, the only metabolite detectable in the pulmonary venous blood was the dihydro-keto-PGE1. However, when the lungs were perfused with an artificial perfusate containing no blood cells, a small amount of 15-keto-PGE1 was detected in the venous effluent. Therefore it would appear that a blood-borne Δ13 reductase was partially responsible for the conversion of PGE1 to dihydro-keto-PGE1 on passage through blood-perfused cat lungs.     

Hypotensive response to prostacyclin and 6-keto-PGE1 following hepatectomy in the rat

Prostacyclin (PGI2) is metabolized to 6-keto-prostaglandin E1 (6-keto-PGE1) which is more stable yet equipotent to PGI2 in lowering systemic arterial blood pressure in the dog. In this study, partial hepatectomy was performed to determine the role of the liver in the vasodepressor response to both intravenously administered PGI2 and 6-keto-PGE1. The magnitude and the duration of systemic hypotensive responses were measured in hepatectomized and sham-operated male Wistar rats following less than maximal, equidepressor doses of PGI2 (0.3 microgram/kg), 6-keto-PGE1 (1.0 microgram/kg), and also PGE1 (3.0 micrograms/kg) and PGE2 (3.0 micrograms/kg). Hepatectomy did not significantly alter the magnitude of the systemic hypotensive response to any of the prostaglandins tested. This indicates that the liver and hepatic circulation do not contribute significantly to the hypotensive effect of these prostaglandins by alterations of systemic vascular resistance, venous pooling of blood, or the generation of additional vasoactive metabolites as may be expected following administration of these prostaglandins. However, hepatectomy did significantly increase the duration of the hypotensive response to PGI2 and 6-keto-PGE1 but not PGE1 or PGE2. We conclude that in vivo, the liver has a more significant role in PGI2 and 6-keto-PGE1 inactivation than in the inactivation of PGE1 and PGE2 when administered intravenously. These results also support the relatively greater significance of the lung in the inactivation of PGE1 and PGE2 in vivo.     

6-Keto-prostaglandin E1 and the decidual cell reaction in rats

Although there is considerable evidence that prostaglandins (PGs) are involved in the decidual cell reaction in rats, which PGs are involved is uncertain. In the present study, we investigated the possibility that 6-keto-PGE1 is involved. To determine its ability to induce decidualization, 6-keto-PGE1 was infused unilaterally from Alzet osmotic minipumps into the uterine lumen of ovariectomized rats treated with estrogen and progesterone to sensitize their uteri for the decidual cell reaction. To reduce endogenous PG production, indomethacin was injected 2-3 h prior to pump insertion and was included in the vehicle for PG infusion. As determined by uterine weights 5 days after pump insertion, 6-keto-PGE1 and PGE2 produced decidualization which was equivalent. As indicated by a dose-response study, 6-keto-PGE1 and PGE2 did not differ in their ability to bring about decidualization. To determine if a deciduogenic stimulus resulted in increased uterine production of 6-keto-PGE1, as assessed by uterine concentrations, 6-keto-PGE1 and PGE concentrations in the uterus were determined after the unilateral intrauterine injection of 100 microliters sesame oil. There were no significant differences between stimulated and non-stimulated horns in 6-keto-PGE1 concentrations, whereas the concentrations of PGE2 were elevated in the stimulated horns. These data indicate that while both exogenous 6-keto-PGE1 and PGE2 induce decidualization, only uterine PGE concentrations are elevated by deciduogenic stimuli. Thus it is unlikely that 6-keto-PGE1 plays a role in decidualization.     

Pulmonary microvascular responses to arachidonic acid in isolated perfused guinea pig lung

We examined the effects of arachidonic acid (AA) on pulmonary hemodynamics and fluid balance in Ringer- and blood-perfused guinea pig lungs during constant-flow conditions. Mean pulmonary arterial (Ppa), venous (Pv), and capillary pressures (Pcap, estimated by the double-occlusion method) were measured, and arterial (Ra) and venous resistances (Rv) were calculated. Bolus AA injection (500 micrograms) caused transient increases (peak response 1 min post-AA) in Ppa, Pcap, and Rv without affecting Ra in both Ringer- and blood-perfused lungs. The response was sustained in blood-perfused lungs. AA had no effect on the capillary filtration coefficient in either Ringer- or blood-perfused lungs. AA stimulated the release of thromboxane B2 and 6-ketoprostaglandin F1 alpha in both Ringer- and blood-perfused lungs, but the responses were sustained only in the blood-perfused lungs. Meclofenamate (1.5 X 10(-4) M), a cyclooxygenase inhibitor, abolished the AA-induced pulmonary hemodynamic responses in both Ringer- and blood-perfused lungs, whereas U-60257 (10 microM), a lipoxygenase inhibitor, attenuated the response only in the blood-perfused lungs. In conclusion, AA does not alter pulmonary vascular permeability to water in either Ringer- or blood-perfused lungs. AA mediates pulmonary venoconstriction and thus contributes to the rise in Pcap. The venoconstriction results from the generation of cyclooxygenase-derived metabolites from lung parenchymal cells and blood-formed elements. Lipoxygenase metabolites may also contribute to the vasoconstriction in the blood-perfused lungs.     

Omega-hydroxylation of prostaglandin E1 in the isolated perfused lungs of pregnant rabbits

Cytochrome P450PG omega is induced in the rabbit lung in a gestational age-dependent manner and hydroxylates certain eicosanoids at their terminal, or omega (omega), carbon. This enzyme has been isolated from microsomal fractions and its activity has been characterized (Williams, D.E., et al., J. Biol. Chem. 259; 14600-14608, 1984). The experiments presented here examine the omega-hydroxylation activity of the intact lung during presentation of an eicosanoid substrate, prostaglandin E1 (PGE1), to the lung vasculature. Isolated, perfused lungs from three pregnant and four nonpregnant rabbits were injected with [3H]-PGE1. One-second fractions were collected from the perfusion effluent and were analyzed for metabolism of PGE1. Lungs isolated from pregnant rabbits metabolized PGE1 mainly to two polar derivatives, 20-hydroxy-PGE1 and 13,14-dihydro-15-keto-20-hydroxy-PGE1, whereas lungs from nonpregnant rabbits yielded mainly a relatively nonpolar metabolite, 13,14-dihydro-15-keto-PGE1. These metabolites were identified by coelution with standards that were generated enzymatically in vitro and whose structures were confirmed by gas chromatography/mass spectrometry (GC/MS).     

Pulmonary and systemic vascular responses to 6-keto-PGE1 in the conscious lamb

6-Keto-PGE1 is a potent direct dilator of the pulmonary and systemic circulations of the newborn lamb under both normoxic and hypoxic conditions. Its threshold dose is similar to that of PGI2 and PGE1. Under hypoxia, 6-keto-PGE1 appears equally effective on the pulmonary and systemic circulations, while under normoxia it predominantly affects the systemic circulation.     

Mechanism of peptidoleukotriene-induced increases in pulmonary transvascular fluid filtration

We examined the effects of leukotrienes C4 (LTC4) and D4 (LTD4) (1 microgram) on the pulmonary vascular filtration coefficient, a measure of vessel wall conductivity to water, and the alterations in pulmonary vascular resistance (PVR) in isolated-perfused guinea pig lungs. We also assessed whether LTC4 and LTD4 increased the permeability to albumin in cultured monolayers of pulmonary artery endothelial cells. In Ringer-perfused and blood-perfused lungs, LTC4 resulted in increases in pulmonary arterial pressure (Ppa) and the pulmonary capillary pressure (Pcap) measured as the equilibration pressure after simultaneous pulmonary arterial and venous occlusions. Pulmonary venous resistance (Rv) increased to a greater extent than arterial resistance (Ra) in both Ringer-perfused and blood-perused lungs challenged with LTC4. The greater increase in PVR in blood-perfused lungs corresponded with a greater elevation of lung effluent thromboxane B2 (TxB2) concentration. The LTC4-stimulated increase in PVR was prevented by pretreatment with meclofenamate (10(-4) M). LTD4 also induced rapid increases in Ppa and Pcap in both Ringer-perfused and blood-perfused lungs; however, Ppa decreased before stabilizing at a pressure higher than base line. The increases in Rv with LTD4 were greater than Ra. The LTD4-stimulated increases in Ra and Rv also paralleled the elevation in TxB2 concentration. As with LTC4, the increases in Ppa, Pcap, PVR, and TxB2 concentration were greater in blood-perfused than in Ringer-perfused lungs. Pretreatment with meclofenamate reduced the magnitude of the initial increase in Ppa, but did not prevent the response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improvement of radioimmunoassays for prostaglandins in bovine blood plasma and their application to monitor reproductive functions

Efficient RIA procedures are required for determination of prostaglandins (PGF(2alpha), PGE(2), PGI(2) and their metabolites) in bovine blood plasma to elucidate their significance in reproductive endocrinology. A new rapid efficient prepurification was developed using commercial octadecyl silicagel cartridges. Prepurification is especially necessary for the determination of 13,14-dihydro-15-keto-PGE(2) (PGEM). After prepurification, PGEM was first converted into the more stable 13,14-dihydro-15-keto-PGA(2) (PGAM) and measured in a RIA-system for PGAM. For PGF(2alpha), 13,14-dihydro-15-keto-PGF(2alpha) (PGFM), PGE(2) and 6-keto-PGF(1alpha) direct tests using 50 mul plasma per tube were elaborated. The validity of the tests was monitored by high performance liquid chromatography radioimmunoassay (HPLC RIA ). Infusion studies using PGF(2alpha) and PGE(2) showed that about 10% of these hormones remained unmetabolized after the first passage through the lungs. The biological half life of the metabolites PGFM and PGEM in bovines was estimated to be 4 min. Thus, PGFM and PGEM measurements in the peripheral circulation reflect even short-term secretory changes of PGF(2alpha) and PGE(2). During the infusion of PGF(2alpha) the levels of progesterone decreased but were not affected by PGE(2). Both prostaglandins caused increased oxytocin secretion. In the cow peripartum first PGEM elevations were measured 5 to 8 d ante partum, whereas PGFM increased 1 to 2 d ante partum. Then both prostaglandins increased simultaneously until parturition. In the postpartal phase PGFM was higher than PGEM, and both prostaglandins remained elevated for several days. Prostacyclin levels remained unchanged during the peripartal period.     

Metabolism of N-acetyl PGE2 carboxamide in the rat

After intratracheal administration to rats, the bronchodilator N-acetyl PGE2 carboxamide was converted rapidly to PGE2 and 13,14-dihydro-15-keto-PGE2, the major plasma metabolite. Oxidation of the N-acetyl carboxamide by prostaglandin dehydrogenase and hydrolysis of the imide bond were demonstrated in vitro.     

Chinese hamster monomeric carbonyl reductases of the short-chain dehydrogenase/reductase superfamily

Chinese hamster monomeric carbonyl reductases (CHCRs) belong to the short-chain dehydrogenase/reductase (SDR) superfamily, which is a family of enzymes that metabolize many endogenous and xenobiotic compounds. We previously cloned three carbonyl reductase cDNAs-Chcr1, Chcr2, and Chcr3. By performing spectrophotometric analyses, we indicated that the enzymes CHCR1, CHCR2, and CHCR3 had similar specificities toward steroids; only CHCR3 did not show any reactivity with prostaglandins (PGs). In the present study, we investigated the characteristics of CHCRs in detail, that is, the differences in their expression patterns, physicochemical properties, and enzymatic activities. CHCR1 exhibited sex-dependent expression patterns. CHCRs showed multiple surface potentials in the zeta potential analysis and CHCR3 exhibited an isatin reductase activity with a high K(m) value. By the present HPLC-analysis, all the three enzymes exhibited PGF(2alpha) dehydrogenase activity and could oxidize PGF(2alpha) to PGE(2) and 15-keto-PGF(2alpha), i.e., the three enzymes exhibited 9- and 15-hydroxy PG dehydrogenase activities. Moreover, 15-keto-PGE(2) was detected in a comparatively higher amount in the dehydrogenase reaction products of CHCR2 than in those of CHCR1 and CHCR3, suggesting that CHCR2 can oxidize PGE(2) and/or 15-keto-PGF(2alpha) to 15-keto-PGE(2); however, these two PGs did not seem to be efficient substrates of CHCR1. Despite the differences in the dehydrogenase activities between CHCR1 and CHCR2, PGE(2) reductase activities of the two enzymes were similar, and PGF(2alpha) was predominantly produced from PGE(2) as a result of the PG 9-keto reductase activity. On the other hand, CHCR3 exhibited a reduced PGE(2) reductase activity. In conclusion, although the CHCRs share a high degree of sequence identity (>70%), they clearly differed in their enzymatic characteristics.     

Pulmonary microvascular response to LTB4: effects of perfusate composition

We examined the effects of leukotriene B4 (LTB4) on pulmonary hemodynamics and vascular permeability using isolated perfused guinea pig lungs and cultured monolayers of pulmonary arterial endothelial cells. In lungs perfused with Ringer solution, containing 0.5 g/100 ml albumin (R-alb), LTB4 (4 micrograms) transiently increased pulmonary arterial pressure (Ppa) and capillary pressure (Pcap). Pulmonary edema developed within 70 min after LTB4 injection despite a normal Pcap. The LTB4 metabolite, 20-COOH-LTB4 (4 micrograms), did not induce hemodynamic and lung weight changes. In lungs perfused with autologous blood hematocrit = 12 +/- 1%; protein concentration = 1.5 +/- 0.2 g/100 ml), the increases in Ppa and Pcap were greater, and both pressures remained elevated. The lung weight did not increase in blood-perfused lungs. In lungs perfused with R-alb (1.5 g/100 ml albumin) to match the blood perfusate protein concentration, LTB4 induced similar hemodynamic changes as R-alb (0.5 g/100 ml) perfusate, but the additional albumin prevented the pulmonary edema. LTB4 (10(-11)-10(-6) M) with or without the addition of neutrophils to the monolayer did not increase endothelial 125I-albumin permeability. Therefore LTB4 induces pulmonary edema when the perfusate contains a low albumin concentration, but increasing the albumin concentration or adding blood cells prevents the edema. The edema is not due to increased endothelial permeability to protein and is independent of hemodynamic alterations. Protection at higher protein-concentration may be the result of LTB4 binding to albumin.     

Formation of 13,14-dihydro-prostaglandin E1 during intravenous infusions of prostaglandin E1 in patients with peripheral arterial occlusive disease.

Formation of 13,14-dihydro-prostaglandin (PG) E1 during intravenous infusions of PGE1 in patients with peripheral arterial occlusive disease was investigated. Using both high performance liquid chromatography (h.p.l.c.) combined with radioimmunoassay and gas chromatography/triple stage quadrupole mass spectrometry (GC/MS/MS) basal levels of 13,14-dihydro-PGE1 were found to be close to or below the detection limits of the assay methods. Levels of the PGE1 metabolite increased significantly during the infusion periods and decreased after their end. Since 13,14-dihydro-PGE1, in contrast to its precursors 15-keto-PGE1 and 15-keto-13,14-dihydro-PGE1, is biologically active, its formation could contribute to the beneficial effects of PGE1 administered intravenously in patients with peripheral arterial occlusive disease.     

Acetylcholine's effect on vascular resistance and compliance in the pulmonary circulation

Acetylcholine's effect on the distribution of vascular resistance and compliance in the canine pulmonary circulation was determined under control and elevated vascular tone by the arterial, venous, and double occlusion techniques in isolated blood-perfused dog lungs at both constant flow and constant pressure. Large and small blood vessel resistances and compliances were studied in lungs given concentrations of acetylcholine ranging from 2.0 ng/ml to 200 micrograms/ml. The results of this study indicate that acetylcholine dilates large arteries at low concentrations (less than or equal to 20 ng/ml) and constricts small and large veins at concentrations of at least 2 micrograms/ml. Characterization of acetylcholine's effects at constant pulmonary blood flow indicates that 1) large artery vasodilation may be endothelial-derived relaxing factor-mediated because the dilation is blocked with methylene blue; 2) a vasodilator of the arachidonic acid cascade (blocked by ibuprofen), probably prostacyclin, lessens acetylcholine's pressor effects; 3) when vascular tone was increased, acetylcholine's hemodynamic effects were attenuated; and 4) acetylcholine decreased middle compartment and large vessle compliance under control but not elevated vascular tone. Under constant pressure at control vascular tone acetylcholine increases resistance in all segments except the large artery, and at elevated vascular tone the pressor effects were enhanced, and large artery resistance was increased.     

Predominance of locally-produced prostaglandin E2 over prostacyclin in skin incisions in man

Blood was collected from skin incisions made by the 'Simplate' technique in 8 healthy men. Prostaglandin (PG) E2, but not 6-oxo-PGF1 alpha, the stable hydrolysis product of prostacyclin (PGI2), was tentatively identified using capillary column gas chromatography/electron capture mass spectrometry. It was not possible to quantify PGE2 because of the small volumes of blood generated by this method. Larger blood samples were then collected from 22 skin incisions in 13 patients undergoing cardiothoracic surgery. Concentrations of PGE2 were substantially greater than 6-oxo-PGF1 alpha in every sample. 13,14-Dihydro-15-oxo-PGF2 alpha, a pulmonary metabolite of PGE2, was not elevated, indicating that the PGE2 was synthesised locally at the site of incision. In 6 further patients undergoing cardiac surgery, blood sampled from an antecubital vein before and during operation contained little or no PGE2. We conclude that a substantial proportion of the PGE2 in blood emerging from skin incisions may be formed locally by the traumatised microvessels, consistent with the hypothesis that PGE2 is the principle prostaglandin synthesised by human cutaneous microvessels in vivo.     

Demonstration of prostaglandin activity in lungs of the rainbow lizard (Agama agama) by bioassay

Extracts and perfusate effluents of lungs of the rainbow lizard (Agama agama) were assayed for prostaglandin-like activity. Results of differential bioassay and thin-layer chromatography suggested that the prostanoid was predominantly PGE2-like. The mean PGE2-like content of 10 lizard lung extracts was 2.9 micrograms g-1 wet weight compared with 146 ng g-1 in rat lungs. Mechanical pressure applied to the lung during perfusion through the pulmonary vasculature provoked the release of large quantities of PGE2-like material. This release was blocked by fatty acid cyclooxygenase inhibitors. Compared with guinea-pig and rat lungs, lizard lungs exhibited a markedly low capacity for inactivating PGE2. In view of an apparently high prostaglandin-forming and a low inactivating capacity, we speculate that under certain circumstances, lizard lungs may release vasoactive substances into the circulation.     

Phorbol myristate acetate-stimulated release of cyclooxygenase products in rat pleural cells: derivatization of prostaglandins with 9-anthryldiazomethane for fluorometric determination by high performance liquid chromatography

White cells were collected from the wash of rat pleural cavity after exsanguination. The incubation mixture of the pleural cells with 1 microM phorbol myristate acetate (PMA) was extracted with acidified ethanol and purified with a Sep-pak C18. The resultant fraction containing prostaglandins (PG) and thromboxane (TX) was allowed to react with 9-anthryldiazomethane (ADAM). After removing contaminants and degraded reagent by silica gel Sep-pak, samples were applied to reversed phase high performance liquid chromatography of octadecylsilyl silica gel and monitored by a fluorescent detector. ADAM derivatives of the authentic PGD2, PGE2, PGF2 alpha, 6-keto-PGF1 alpha, 6-keto-PGE1, TXB2, 15-keto-PGE2, 13,14-dihydro-15-keto-PGF2 alpha and 13,14-dihydro-15-keto-PGE2 showed linear regression lines of peak heights within a range of 0.5-25 ng. By using this method PGD2, 6-keto-PGF1 alpha and TXB2 were detected in the incubation mixture of the rat pleural cells with PMA. The result clarified the origin of these PGs and TX found in the exudate of rat pleurisy induced by PMA. ADAM method for HPLC with a help of clean-up by Sep-pak could be a useful tool for detection of a series of arachidonate metabolites in biological materials.     

Impact of microvascular circulation on peripheral lung stability

The involvement of pulmonary circulation in the mechanical properties was studied in isolated rat lungs. Pulmonary input impedance (ZL) was measured at a mean transpulmonary pressure (Ptpmean) of 2 cmH2O before and after physiological perfusion with either blood or albumin. In these lungs and in a group of unperfused lungs, ZL was also measured at Ptpmean values between 1 and 8 cmH2O. Airway resistance (Raw) and parenchymal damping (G) and elastance (H) were estimated from ZL. End-expiratory lung volume (EELV) was measured by immersion before and after blood perfusion. The orientation of the elastin fibers relative to the basal membrane was assessed in additional unperfused and blood-perfused lungs. Pressurization of the pulmonary capillaries significantly decreased H by 31.5 +/- 3.7% and 18.7 +/- 2.7% for blood and albumin, respectively. Perfusion had no effect on Raw but markedly altered the Ptpmean dependences of G and H < 4 cmH2O, with significantly lower values than in the unperfused lungs. At a Ptpmean of 2 cmH2O, EELV increased by 31 +/- 11% (P = 0.01) following pressurization of the capillaries, and the elastin fibers became more parallel to the basal membrane. Because the organization of elastin fibers results in smaller H values of the individual alveolus, the higher H in the unperfused lungs is probably due to a partial alveolar collapse leading to a loss in lung volume. We conclude that the physiological pressure in the pulmonary capillaries is an important mechanical factor in the maintenance of the stability of the alveolar architecture.     

Prostaglandin release by slow reacting substance from guinea pig and human lung tissue.

Slow reacting substance (SRS) injected into the pulmonary artery released prostaglandins E (PGE) and F2alpha (PGF2alpha) and the 15-keto-13, 14-dihydro PG metabolites from non-sensitized and ovalbumin sensitized, isolated, perfused guinea pig lungs. PGs were also released from lungs incubated with SRS. Sensitized lungs released more PGs in both types of preparations. Indomethacin inhibited the effect of SRS. Passively sensitized human lung fragments, in parallel to guinea pig lung, released PGE, PGF2alpha and the metabolites when incubated with SRS or antigen. In in vivo experiments, SRS and arachidonic acid given intravenously increased the airway insufflation pressure in anesthetized quinea pigs. These effects, but not the action of injected PGF2alpha and histamine, were abolished by indomethacin. The results indicate that one of the modes of SRS action is by release of PGs, and are consistent with the hypothesis that PGs are predominantly "secondary" mediators (in the temporal sense) of the antigen-antibody reaction.     

Effect of mercaptoethanol in the radioactive thin layer chromatography assay of NAD+-15-hydroxyprostaglandin in dehydrogenase.

The use of mercaptoethanol in the assay of rat kidney 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to have minimal effect on activity assayed with the spectrophotometric and substrate loss assays. However, mercaptoethanol appeared to inhibit PGDH when assayed by thin-layer chromatography, based upon conversion of 3H-PGE1 to 15-keto-3H-PGE1. Mercaptoethanol reacted with 15-keto-PGE1 to alter its chromatographic mobility and to suppress the U.V. absorption spectrum of 15-keto-PGE1. The implication of the use of ME in radiometric assays is discussed.