Effect of an upstream grid on the fluidization of pharmaceutical carrier powders.

The influence of grid generated mixing on the fluidization of pharmaceutical carrier powders is studied in a channel-flow experiment using direct high-speed imaging and particle image velocimetry (PIV). Four different lactose powders with mass median diameters that range between 61 µm and 121 µm are used. The degree of powder mixing in the flow as a function of grid position relative to the powder bed and grid area blockage ratios (ranging from ~25% to ~40%) is studied for a range of flow-rates. The study presents comprehensive mappings of how pharmaceutical powders are fluidised under the influence of mixing, by examining powder bed morphology, powder emptying rate, and the local flow-field surrounding the pocket. The use of a grid results in higher evacuation percentages (void fraction) and a faster evacuation rate but is associated with randomized evacuation behaviour as observed from the powder bed morphology. Use of a grid can enable evacuation of powder at lower overall flow-rates, which may have important implications on respiratory drug delivery. PIV results show the trend of mean velocities with the mass median powder diameter and demonstrates how a grid with lower blockage ratio can increase the degree of mixing of the evacuating powder and make the evacuation process more rapid. This study contributes towards a better understanding of fluidization processes as relevant to dry powder inhaler devices and sheds light on how simple design alterations, such as adding an upstream grid, can be incorporated to optimise device effectiveness.

Effect of Pseudomonas infection on weight loss, lung mechanics, and cytokines in mice.

Poor growth, Pseudomonas aeruginosa endobronchitis, pulmonary inflammation, and decline of lung function are hallmarks of cystic fibrosis (CF), yet the relationship between these features is poorly understood. Because animal models of chronic bronchopulmonary infection with P. aeruginosa used to study pulmonary inflammation in CF have also been associated with weight loss, we sought to determine whether this weight loss was due to the inflammatory process and/or to changes in lung function. P. aeruginosa-laden agarose beads were instilled into the lungs of mice. Weight loss was greatest 3 d after Pseudomonas infection. Infected mice had a rapid though transient rise in absolute neutrophil counts, mTNF-alpha, mIL-1beta, mIL-6, mip-2, and KC in bronchoalveolar lavage fluid. There was no difference in lung resistance or lung compliance measured by body plethysmography between infected and control mice. Weight loss did correlate with the concentration of proinflammatory cytokine levels 3 d after inoculation of mice with Pseudomonas, and body composition analysis revealed loss of skeletal muscle mass. These results suggest that weight loss in P. aeruginosa-infected mice was associated with the inflammatory process and not with altered pulmonary responsiveness. These findings may provide insights into the cause of cachexia and weight loss seen in patients with CF.

15-HETE-substituted diglycerides selectively regulate PKC isotypes in human tracheal epithelial cells.

Human tracheal epithelial (TE) cells selectively incorporate their major lipoxygenase product, 15-hydroxyeicosatetraenoic acid (15-HETE), into the sn-2 position of phosphatidylinositol (PI) (S. E. Alpert and R. W. Walenga. Am. J. Respir. Cell Mol. Biol. 8: 273-281, 1993). Here we investigated whether 15-HETE-PI is a substrate for receptor-mediated generation of 15-HETE-substituted diglycerides (DGs) and whether these 15-HETE-DGs directly activate and/or alter conventional diacylglycerol-induced activation of protein kinase C (PKC) isotypes in these cells. Primary human TE monolayers incubated with 0.5 microM 15-[3H]-HETE or 15-[14C]HETE for 1-2 h were stimulated with 1 nM to 1 microM platelet-activating factor (PAF) for 30 s to 6 min, and the radiolabel in the medium, cellular phospholipids, and neutral lipids was assessed by high-performance liquid and thin-layer chromatography. PAF mobilized radiolabel from PI in a dose-dependent manner (22 +/- 5% decrease after 1 microM PAF) without a concomitant release of free intra- or extracellular 15-HETE. 14C-labeled DGs were present in unstimulated TE monolayers incubated with 15-[14C]HETE, and the major 14C band, identified as sn-1,2-15-[14C]HETE-DG, increased transiently in response to PAF. Western blots of freshly isolated and cultured human TE cells revealed PKC isotypes alpha, betaI, betaII, delta, epsilon, and zeta. In vitro, cell-generated sn-1, 2-15-[14C]HETE-DG selectively activated immunoprecipitated PKC-alpha and inhibited diacylglycerol-induced activation of PKC-alpha, -delta, -betaI, and -betaII. Our observations indicate that 15-HETE-DGs can modulate the activity of PKC isotypes in human TE cells and suggest an intracellular autocrine role for 15-HETE in human airway epithelia.

Ozone inactivates cyclooxygenase in human tracheal epithelial cells without altering PGHS-2 mRNA or protein.

Exposure of human tracheal epithelial (TE) cells to ozone (0.1-0.5 ppm) leads to a transient increase followed by decreased production of prostaglandin (PG) E2 concomitant with dose-dependent loss and delayed recovery of cyclooxygenase (CO) activity [S.E. Alpert and R.W. Walenga. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L734-L743, 1995]. Formation of reactive oxygen species (ROS) in cultured tracheobronchial epithelial cells during ozone exposure was recently demonstrated (L.C. Chen and Q.Qu. Toxicol. Appl. Pharmacol. 143: 96-101, 1997). In the present study, we investigated if ROS generated by ozone-exposed human TE cells contribute to PGE2 production and/or CO inactivation and whether the delay in recovery of CO activity after ozone reflects impaired gene transcription and/or protein synthesis. Rapid, dose-dependent ROS generation, assessed by fluorescence of dihydrorhodamine 123, was detected in human TE monolayers exposed to 0.21-0.63 ppm ozone. In a different system, TE cells were exposed to air or 0.5 ppm ozone for 1 h by serial renewal/collection of an adherent film of media. Ozone-induced ROS formation, the transient increase and decline in PGE2, and CO inactivation were attenuated by an intracellular hydroxyl radical scavenger, 1,3-dimethyl-2-thiourea. Ibuprofen, a reversible CO inhibitor, prevented PGE2 release during ozone exposure (and hence autocatalytic CO inactivation) but not loss of CO activity. Although CO activity remained depressed for hours after ozone exposure, compared with air-exposed cultures, no differences were detected in mRNA and protein levels of prostaglandin endoperoxide G/H synthase 2 (PGHS-2), the only CO isoform present in human TE cells, or in the rate of de novo PGHS-2 synthesis. Our findings suggest that ozone-induced PGE2 production and CO inactivation are primarily the result of formation of intracellular oxidant molecules and that delayed recovery of CO activity in human TE cells after short-term ozone exposure is due to persistent inactivation of PGHS-2, rather than to interference with its synthesis.

Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa.

In cystic fibrosis (CF), defective function of the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells and submucosal glands results in chronic pulmonary infection with Pseudomonas aeruginosa. The pulmonary infection incites an intense host inflammatory response, causing progressive suppurative pulmonary disease. Mouse models of CF, however, fail to develop pulmonary disease spontaneously. We examined the effects of bronchopulmonary infection on mice homozygous for the S489X mutation of the CFTR gene using an animal model of chronic Pseudomonas endobronchial infection. Slurries of sterile agarose beads or beads containing a clinical isolate of mucoid P. aeruginosa were instilled in the right lung of normal or CF mice. The mortality of CF mice inoculated with Pseudomonas-laden beads was significantly higher than that of normal animals: 82% of infected CF mice, but only 23% of normal mice, died within 10 d of infection (P = 0.023). The concentration of inflammatory mediators, including TNF-alpha, murine macrophage inflammatory protein-2, and KC/N51, in bronchoalveolar lavage fluid in CF mice 3 d after infection and before any mortality, was markedly elevated compared with normal mice. This inflammatory response also correlated with weight loss observed in both CF and normal littermates after inoculation. Thus, this model may permit examination of the relationship of bacterial infections, inflammation, and the cellular and genetic defects in CF.

Constitutive expression of prostaglandin endoperoxide G/H synthetase (PGHS)-2 but not PGHS-1 in hum an tracheal epithelial cells in vitro.

Primary cultures of human tracheal epithelial (HTE) cells cultured in vitro, in defined serum-free media, express prostaglandin endoperoxide G/H synthase (PGHS) activity and produce prostaglandin E2 (PGE2). In contrast to every other cell type studied to date, HTE cells appear to constitutively express PGHS-2, the 'inducible' form of the enzyme, while expressing little or no PGHS-1, the 'housekeeping' isoenzyme in vitro. Prostaglandin synthesis in HTE cells was reduced by a selective PGHS-2 inhibitor, N-[2-cyclohexyloyl-4-nitrophenyl] methane-sulfonamide (NS398), with an IC50 of approximately 1 microM. Immunoblotting and immunoprecipitation of enzymatic activity with isozyme-specific antisera revealed only the PGHS-2 isoform. Full length human cDNA probes detected only PGHS-2 message in Northern blots. Neither PGHS-2 activity nor mRNA levels were dependent on, nor stimulated by peptide growth factors present in the defined serum-free growth medium, or by serum. Prolonged maintenance in the absence of retinoic acid, however, lead to a decline in PGHS activity. Phorbol-myristate acetate (PMA) induced PGHS-2 activity and mRNA and neither PMA-induced, nor constitutive PGHS-2 expression was suppressed by corticosteroids. Actinomycin D-treatment for six hours reduced the PGHS-2 activity and mRNA to only 50% that of untreated cells, suggesting that PGHS-2 mRNA is extremely stable in these cells. HTE cells, at least in vitro, appear unique among prostaglandin-producing cells in that they express PGHS-2, constitutively, independent of regulation by growth factors, serum, or corticosteroids and fail to express PGHS-1 under any culture condition studied.

Ozone exposure of human tracheal epithelial cells inactivates cyclooxygenase and increases 15-HETE production.

We assessed the immediate and prolonged effects of ozone on arachidonic acid (AA) metabolism by primary cultured human tracheal epithelial (TE) cells. TE monolayers were exposed at a gas-fluid interface to air or 0.1, 0.25, or 0.5 ppm ozone (15 min air, then 45 min air/ozone), and serially collected effluents were analyzed by thin-layer chromatography (TLC) and/or high-performance liquid chromatography. Release of prostaglandin E2 (PGE2) and AA, but not 15-hydroxyeicosatetraenoic acid (15-HETE) or its metabolites, was detected from cultures prelabeled with [14C]AA. PGE2 production, measured by immunoassay, was nearly constant during air exposure. In contrast, PGE2 increased two- to threefold during the first 15-min exposure to all concentrations of ozone, but then progressively declined to 78 +/- 17, 57 +/- 12 (P < or = 0.05), and 45 +/- 15% (P < or = 0.05) of air controls after exposure to 0.1, 0.25, and 0.5 ppm ozone. Ozone did not induce a new spectrum of AA metabolites; only PGE2, lesser amounts of PGF2 alpha, and 15-HETE were present in media and cell extracts of air- or ozone-exposed cultures provided with 30 microM exogenous AA. However, cyclooxygenase (CO) activity (PGE2 produced from 30 microM AA) decreased to 82 +/- 9, 53 +/- 8 (P < or = 0.05), and 28 +/- 6% (P < or = 0.05) vs. controls after 0.1, 0.25, and 0.5 ppm ozone, whereas 15-HETE production was unimpaired. When cells exposed to 0.5 ppm ozone were maintained for up to 6 h in 5% CO2-air, spontaneous PGE2 production remained decreased and recovery of CO activity was extremely slow. TLC analysis of lipid extracts from [14C]AA-labeled cells revealed a nearly twofold increase in free intracellular 15-HETE, and hydrolysis of phospholipids demonstrated increased esterified 15-HETE. Exposure of human TE cells to ozone leads to a transient increase followed by prolonged decrease in PGE2 production and increased intracellular retention of 15-HETE. Loss of the bronchodilator and anti-inflammatory properties of epithelial PGE2, with or without increased 15-HETE, might contribute to ozone-induced airway dysfunction.

Leukotriene B4 markedly elevated in the epithelial lining fluid of patients with cystic fibrosis.

Persistent neutrophil infiltration into the airways of patients with cystic fibrosis (CF) results in lung destruction. Eicosanoid lipid mediators, particularly leukotriene B4 (LTB4), may play a role in neutrophil influx and activation. We compared the eicosanoid content of epithelial lining fluid (ELF) obtained by bronchoalveolar lavage (BAL) from 17 patients with CF and 10 healthy subjects. LTB4 was the predominant eicosanoid in the CF airway (16.7 +/- 9.1 ng/ml ELF in CF versus 0.5 +/- 0.1 ng/ml ELF in healthy subjects). Prostaglandins (PG) and thromboxane (TX) were also elevated in CF (PGE2, 8.5 +/- 2.2; PGF2 alpha, 6.0 +/- 2.0; and TXB2, 14.0 +/- 3.0 ng/ml ELF) compared with healthy subjects (PGE2, 0.4 +/- 0.2; PGF2 alpha, 0.5 +/- 0.2; and TXB2, 1.2 +/- 0.4 ng/ml ELF). We also developed a protocol for the storage and subsequent analysis of BAL fluid that assures accurate and reproducible measurements of these eicosanoids. BAL samples stored for up to 8 months retain greater than 80% of their original eicosanoid content if the BAL fluid is immediately treated with methanol, concentrated, and stored at -70 degrees C without further purification. These data suggest that CF airways contain sufficient amounts of LTB4 both to recruit additional neutrophils into the airways and to stimulate neutrophils to release their injurious products. Therapies aimed at interfering with the production or action of LTB4 may be beneficial in CF and other lung diseases with a significant neutrophil response.

Human tracheal epithelial cells selectively incorporate 15-hydroxyeicosatetraenoic acid into phosphatidylinositol.

15-hydroxyeicosatetraenoic acid (15-HETE) is the major lipoxygenase metabolite of arachidonic acid produced by human airway epithelial cells. Because HETEs have been shown to be rapidly metabolized and/or incorporated into cellular lipids in other cell types, we investigated the uptake, metabolism, and intracellular distribution of exogenous 15-HETE by primary monolayer cultures of human tracheal epithelial (HTE) cells. At concentrations of 0.1 microM, [3H]15-HETE was rapidly incorporated by HTE cells and also metabolized primarily by beta-oxidation to several more polar products that were released extracellularly. The majority of cell-associated [3H]15-HETE radiolabel was distributed into phospholipids, with phosphatidylinositol (PI) accounting for approximately 75% of phospholipid radiolabel. Exogenous 5- and 12-HETE were also metabolized by HTE cells but were less extensively incorporated into phospholipids and were distributed primarily into phosphatidylcholine and phosphatidylethanolamine. Phospholipase A2 hydrolysis indicated selective esterification of unmodified 15-HETE to the sn-2 position of phospholipids. 15-HETE incorporation into total phospholipids and into PI was saturable (half maximal incorporation at 0.82 and 0.68 microM, respectively), while incorporation into neutral lipids continued to increase at concentrations of 15-HETE up to 5 microM. The incorporation of 15-HETE into PI was metabolically stable, with an intracellular half-life of 12 h, and was not subject to mobilization in response to 5 microM calcium ionophore A23187. HTE cells can incorporate and metabolize HETEs that the cells themselves produce as well as those that might be released by inflammatory cells recruited into the airway.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin E2 attenuates hyperoxia-induced injury in cultured rabbit tracheal epithelial cells.

We assessed the kinetics of hyperoxia-induced prostaglandin E2 (PGE2) production by cultured rabbit tracheal epithelial (TE) cells with different inherent capacities to generate PGE2 and the role of endogenous PGE2 production in protecting these cells from hyperoxic injury. Rabbit TE cells grown to confluence with or without lipid supplements [0.1% Excyte III (Miles-Pentex) and 1 microM arachidonic acid] were exposed for 2 h to control (5% CO2/air) or hyperoxic (5% CO2/90% O2) atmospheres at a gas-fluid interface. Serial cell culture effluents collected during exposure were analyzed for PGE2 by enzyme-linked immunoassay. Basal PGE2 production by lipid-supplemented cells was approximately 3-fold greater than that by unsupplemented cultures (p less than 0.01). In lipid-supplemented cells, PGE2 production doubled after 15 min of hyperoxic exposure (p less than 0.05) and then declined to approximately 50% of initial levels, whereas exposure to 5% CO2/air did not significantly change PGE2 production. In unsupplemented cells, neither control nor hyperoxic exposure altered PGE2 production. Hyperoxia-exposed TE cells had decreased ability to convert 10 microM exogenous arachidonic acid to PGE2, suggesting hyperoxia-induced inhibition of the enzymes involved in PGE2 synthesis. Lipid-supplemented cells were less susceptible to hyperoxic injury than unsupplemented monolayers, as evidenced by increased viability (trypan blue exclusion) and decreased generation of lipid peroxides (thiobarbituric acid reactive substances). Addition of exogenous PGE2 to unsupplemented cultures at concentrations that were produced by lipid-supplemented cells (2 ng/mL every 15 min) during hyperoxic exposure eliminated these differences in hyperoxia-induced lipid peroxidation and cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Tracheal epithelial cell fatty acid composition modulates prostaglandin E2 and cAMP production.

Tracheal epithelial (TE) cells from both rabbits and humans, when cultured in defined serum-free media without lipid supplements, develop fatty acid profiles significantly different from freshly isolated epithelium, including a markedly decreased cellular content of arachidonic acid (AA). In rabbit TE cells, supplementation of media with a phospholipid-rich lipoprotein extract (Excyte III) plus 1 microM bovine serum albumin-complexed AA (Excyte/AA) restored the fatty acid composition of the cultured cells more similar to that of native airway epithelium than did supplementation of media with 5% fetal bovine serum (FBS). In human TE cells, Excyte/AA or 5% FBS increased AA content, but neither lipid supplement completely "normalized" the fatty acid profiles. Compared with lipid-unsupplemented cultures, basal production of prostaglandin E2 (PGE2) was increased by approximately four- to eightfold in rabbit and human TE cells supplemented with 5% FBS or Excyte/AA. In Excyte/AA-supplemented human TE cells, PGE2 production induced by 5 microM calcium ionophore A23187 was more than threefold greater than that of companion ionophore-stimulated unsupplemented monolayers, but PGE2 production was similar in both culture conditions in response to 10 microM exogenous AA. Thus increased cellular content and availability of AA, rather than changes in cyclooxygenase activity, appear to be responsible for the elevated PGE2 production in Excyte/AA-supplemented human TE cells. Secondary effects of lipid supplementation were also observed; Excyte/AA-supplemented human TE cells produced significantly less adenosine 3',5'-cyclic monophosphate (cAMP) in response to exogenous PGE2 and isoproterenol than did lipid-unsupplemented cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of prostaglandins and hydroxyeicosatetraenoic acids in kitten retina: comparison with other species.

Conversion of arachidonic acid to eicosanoids by kitten retinae was investigated to evaluate whether the pattern of kitten retinal eicosanoids simulates that found in the human and other animal species. Freshly isolated kitten retinae were incubated with 20 microM radiolabeled arachidonic acid, and the metabolites were analysed by reverse phase-high pressure liquid chromatography, thin-layer chromatography and gas chromatography-mass spectroscopy. Kitten retinal tissues converted arachidonic acid into prostaglandins (PGs), thromboxane (Tx) and hydroxyeicosatetraenoic acids (HETEs). The major eicosanoid identified was 6kPGF1 alpha--the stable non-enzymatic hydrolysis product of prostacyclin. Other eicosanoids identified included TxB2, PGE2, PGF2 alpha, 12-hydroxy-heptadecatrienoic acid, 12-HETE, and 15-HETE. The spectrum of kitten retinal cyclooxygenase metabolites is similar to those obtained from bovine retina and human retinal vascular endothelium with prostacyclin being the major cyclooxygenase metabolite produced.

A prostacyclin analogue crosses the in vitro perfused human placenta and improves transfer in some pathologic states.

Placental blood flow is reduced in pregnancies complicated by hypertension, intrauterine growth retardation, maternal smoking, or diabetes. Umbilical-placental production of the potent vasodilator prostacyclin is also reduced in these pathologic states and this deficiency may contribute to an associated increase in the incidence of low infant birthweight by affecting a reduction in placental nutrient transfer. We have studied the effects of the prostacyclin analogue carbacyclin on diffusional transfer in the human placenta perfused in vitro. We have found that carbacyclin crosses the human placenta and can significantly increase diffusional transfer in placenta from pregnancies complicated by hypertension or maternal smoking and in the normal term placenta in which prostacyclin production has first been reduced through the administration of ibuprofen. Carbacyclin had no effect, however, in untreated placenta from normal pregnancies or in placenta from diabetic pregnancies. These results suggest that the prostacyclin-deficient perfused placenta may serve as a model for several placental insufficiency syndromes and that the possibility that prostacyclin analogues may improve deficient nutrient transfer in some pathologic pregnancies warrants further investigation.

Functional consequences of abnormal fatty acid profiles in cultured airway epithelial cells.

Maintenance of serum-free conditions for the culture of TE or other airway epithelial cells provides a defined environment in which to explore the regulation of cellular functions. Yet TE cells appear to be dependent on the medium for essential, if not all, polyunsaturated fatty acids. At present, some laboratories routinely use serum to support the growth of airway epithelial cells, presumably in part through recognition that cells of mammalian origin require an exogenous source of lipids. While 5% FBS can increase the linoleic and arachidonic acid content of cultured rabbit and human TE cells, it does not fully restore the fatty acid composition of cultured TE cells to that of freshly isolated cells, particularly in the case of human TE cells. Equally good, if not better, repair of membrane fatty acid composition can be achieved by addition of a defined, commercial non-serum source of lipids (Excyte III) plus exogenous arachidonic acid. Cultured TE cells maintained in serum-free medium have been shown to be deficient in prostaglandin and HETE production, both at baseline and in response to physiological stimuli compared to TE cells with greater endogenous content of arachidonic acid. Differences between lipid supplemented and unsupplemented cultured TE cells in cAMP response to PGE2 and in susceptibility to hyperoxic injury have been observed. Other cellular functions regulated by the fatty acid composition of membrane lipids may also be impaired in lipid unsupplemented cells. It is evident that the maintenance of as normal as possible membrane fatty acid content is essential to the use of cultured TE cells as experimental models of airway epithelium.

15-Hydroxyeicosatetraenoic acid stimulates migration of human retinal microvessel endothelium in vitro and neovascularization in vivo.

We evaluated 15-hydroxyeicosatetraenoic acid (15-HETE), a major arachidonic acid product of vascular endothelium and leukocytes, for its effect on neovascularization. In a modified Boyden chamber assay, 15-HETE (10-7 M) stimulated human retinal microvessel endothelial cell migration by 42 +/- 10% (mean +/- S.E.M., p less than 0.01). 12-HETE, a major arachidonic acid metabolite of platelets, had no such effect. Further studies in the rabbit corneal pocket assay revealed that 15-HETE stimulated neovascularization in vivo. Concentrations at which the in vivo effects were observed are within the range generated by several cell types and are achievable in human serum. 15-HETE stimulation of human endothelial cell migration in vitro and neovascularization in vivo suggests that it may play a role in vasoproliferative disorders.

Effects of changes in oxygen tension on lipoxygenase metabolites. Serum 15-HETE is increased in kittens exposed to hyperoxia.

The chronic phase of O2-induced retinopathy is characterized by retinal neovascularization. We have previously demonstrated that 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), a product of white cells, is proangiogenic. In this study, kittens exposed to in vivo hyperoxia produced increased amounts of 15-HETE. Nine litters of 30 kittens (aged 6-8 days) were used. Control kittens were left in room air; hyperoxic kittens were placed in 80% oxygen for 48 h; recovery kittens were returned to room air for 24 h following hyperoxic exposure. Following treatments, the animals were sacrified, and blood was evaluated for 15-HETE. Stimulated serum 15-HETE levels were assayed by high-performance liquid chromatography and GC-selected ion monitoring. While controls produced 0.48 +/- 0.16 (SE) nmol/ml of 15-HETE, values in the hyperoxic and recovery animals were increased at 0.7 +/0 0.2 and 0.68 +/- 0.15 nmol/ml (p less than 0.05 and p = 0.05, respectively). Increased production of this proangiogenic metabolite by WBCs (which can migrate out of blood vessels to set up extravascular angiogenic foci) may play a role in the genesis of the neovascularization process that occurs in response to oxygen-induced injury.

Trans-placental transport and metabolism of carbacyclin by perfused human placental in vitro.

When carbacyclin (5E-6a-carba-prostaglandin I2) was added to the maternal afferent circulation of in vitro perfused placentae from normal term pregnancies, relatively little carbacyclin was found in either the maternal or fetal efferent circulations. When carbacyclin was added to the perfusate at 1.0 microM, the peak level in the maternal effluent was only 0.06 microM and in the fetal effluent, 0.026 microM. When infused at 10 microM, 0.77 microM carbacyclin was measured in the maternal effluent and 0.13 in the fetal effluent. These findings demonstrate that carbacyclin is transferred across the placenta from the maternal side to the fetal, but that the net transfer is small. The assay procedure employed HPLC resolution, followed by capillary gas chromatography and selected ion monitoring using PGB as an internal standard. The low levels of carbacyclin detected in the effluents did not result from poor recovery in the analyses. When carbacyclin was added to maternal or fetal effluents at 1 microM, the recovery averaged 85.4 +/- 14.1% (SD); at 10 microM recovery averaged 97.3 +/- 4.2%. Much of the loss of carbacyclin on passage through placental circulation resulted from metabolism. Extracts of both fetal and maternal effluents from placenta perfused with carbacyclin contained a component which on reverse phase HPLC appeared less polar than carbacyclin. When analyzed by GC/MS as the methyl ester-trimethylsilyl ether, this component had a mass spectrum expected for 15-dehydro-carbacyclin. When the presumed metabolite was further converted to the methoxime, the mass spectrum was identical to published spectra for that derivative of 15-dehydro-carbacyclin. When extracts of fetal effluents were analyzed for 15-dehydro-carbacyclin metabolite as well as carbacyclin, it appeared that the metabolite accounted for the majority of the carbacyclin recovered. Most of the metabolite was apparently not formed in the fetal circulation, since when carbacyclin was added to the fetal afferent circulation, little 15-dehydro-carbacyclin was observed in either efferent fluid, and most of the perfused carbacyclin was recovered unaltered in the fetal effluent.

Formation of hydroxyeicosatetraenoic acids (HETE) in blood from adults versus neonates: reduced production of 12-HETE in cord blood.

Hydroxyeicosatetraenoic acids (HETE) are major arachidonic acid metabolites of a number of cells found in blood and blood vessels. These products have been implicated in physiologic responses as diverse as platelet aggregation, cell migration, and cell proliferation. Using a sensitive and specific assay, GC/selected ion monitoring after high-performance liquid chromatography separation, we have measured the levels of three HETE isomers of biologic significance 12-HETE, 15-HETE, and 5-HETE in plasma, serum and stimulated serum (formed in the presence of arachidonic acid and calcium ionophore), obtained from normal adults and cord blood from normal neonates. Whereas there were no significant differences between the two groups for 5- or 15-HETE in any of the samples, stimulated serum from adults produced 12 times as much 12-HETE when compared to cord blood. When platelets were isolated from adult and cord blood, 12-HETE production by neonatal platelets, stimulated with 10 microM arachidonic acid, was less than one-fourth that of adults. Although no role for 12-HETE in normal platelet responses has yet been established, it has been reported that those individuals with myeloproliferative syndromes who demonstrate a concomitant decrease in platelet 12-HETE synthetic ability have an increased bleeding tendency. It needs to be further evaluated if this already depressed level of 12-lipoxygenase in neonatal platelets may contribute to pathologic bleeding in those infants subjected to additional stress (such as prematurity or birth asphyxia).

Nerve growth factor rapidly stimulates arachidonate metabolism in PC12 cells: potential involvement in nerve fiber growth.

Homogenates prepared from pheochromocytoma (PC12) cells that are extending nerve fibers in response to nerve growth factor (NGF) have an increased capacity to metabolize exogenous arachidonate compared with homogenates prepared from cells untreated with NGF. These changes are not a consequence of cell attachment, since they are also seen in NGF-treated PC12 cells grown in suspension and are not found in attached cells grown in the absence of NGF. This NGF-stimulated increase in arachidonate metabolic capacity occurs rapidly and before the extension of nerve fibers. In contrast to NGF, epidermal growth factor does not alter the metabolism of exogenous arachidonate by PC12 cells. Radioimmunoassay of medium from PC12 cultures indicates that intact cells produce and release increased amounts of prostaglandin (PGE) in response to NGF. Drugs that inhibit arachidonate liberation from membrane phospholipids (mepacrine or 4-bromphenacyl bromide) block NGF-stimulated nerve fiber growth by PC12 cells. Selective inhibitors of cyclooxygenase metabolism of arachidonate (indomethacin and aspirin) fail to block growth, but inhibitors of lipoxygenase metabolism (baicalein, BW755, and eicosatetraynoic acid) are potent blockers. In cultures of dorsal root ganglion neurons, inhibitors of arachidonate release (mepacrine, 4-bromphenacyl bromide) or its subsequent metabolism by lipoxygenases (nordihydroquaiaretic acid, eicosatetraynoic acid) also prevent the early morphological events of nerve fiber growth. Our data suggest that NGF rapidly and specifically increases the capacity of PC12 cells to synthesize arachidonate metabolites, and that arachidonate metabolism may be important in nerve fiber growth by both PC12 cells and dorsal root ganglion neurons.