Fetal lung disaturated phosphatidylcholine. Ostensible increase following exposure to dexamethasone

Fetal rat lung removed at 15 days gestation and placed in organ culture incorporates choline into phosphatidylcholine. Addition of 10(-9) M dexamethasone resulted in increased rates of choline incorporation per micrograms protein after both 6 and 12 days culture. This concentration of dexamethasone did not increase tissue phosphatidylcholine or disaturated phosphatidylcholine. Thus, at a culture time when dexamethasone had a significant effect on choline incorporation, there was no change in either the total phospholipid or disaturated phosphatidylcholine content of the lung tissue. The transplacental administration of dexamethasone decreased fetal lung DNA and phospholipid content. At the mid-range dosage tested (400 micrograms), dexamethasone depressed DNA (51%) appreciably more than total phosphatidylcholine (28%) and disaturated phosphatidylcholine (33%). These results show that the hormone does not increase the total amount of surfactant per lung. The increased disaturated phosphatidylcholine per mg DNA results in an ostensible beneficial effect of dexamethasone on surfactant and may reflect an increased proportion of Type II cells in fetal lung both in vitro and in vivo following hormone exposure. Disaturated phosphatidylcholine per Type II alveolar cell is no doubt increased but the trade-off is fewer total cells in the lung.     

Regulation of phospholipid synthesis by intracellular phospholipases in fetal rabbit type II pneumocytes

Exposure of fetal type II pneumocytes to phospholipase A2 inhibitors led to significantly reduced choline uptake and decreased synthesis of total and disaturated phosphatidylcholines from both [methyl-14C]choline and [9,10(n)-3H]palmitate precursors. The percentage of the total synthesized phosphatidylcholine recovered as disaturated phosphatidylcholine was increased when compared to that in control cultures, suggesting that unsaturated phosphatidylcholine synthesis was reduced to a greater extent than that of the disaturated species. Synthesis of sphingomyelin and phosphatidylethanolamine from labeled palmitate was also reduced, whereas that of phosphatidylinositol and phosphatidylglycerol was significantly increased. Addition of phospholipase C resulted in increased synthesis of phosphatidylcholine from both labeled precursors; no significant changes were found in synthesis of most of the other 3H-labeled lipids. Added phospholipase A2 did not lead to any changes in either choline or palmitate incorporation. However, when melittin (a phospholipase A2 activator) was added to the cultures, greater incorporation of both palmitate and choline was observed, along with a significant increase in the percentage of total cellular radioactivity in 14C-labeled lipids, indicating also stimulation of phosphatidylcholine synthesis. A marked increase in CTP: phosphorylcholine cytidylyltransferase activity was found after treatment of the cultures with phospholipase C. Exposure to quinacrine also increased the activity of this enzyme. Addition of phospholipase C and melittin to prelabeled pneumocyte cultures accelerated degradation of cell phospholipids and the release of free fatty acids as the main degradation products. These findings suggest that intracellular phospholipases are regulators of synthesis of surfactant phospholipids in fetal type II pneumocytes, and that activation or inhibition of these phospholipases could represent a mechanism through which hormones and pharmacological agents modify surfactant and other phospholipid synthesis.     

A study of the molecular species of diacylglycerol, phosphatidylcholine and phosphatidylethanolamine and of cholinephosphotransferase and ethanolaminephosphotransferase activities in the type II pneumocyte

The percent distributions of the molecular species of diacylglycerol, phosphatidylcholine and phosphatidylethanolamine in rat whole lung and type II pneumocytes were found to differ significantly. Diacylglycerol from the type II pneumocyte is enriched in the disaturated species and diminished in the polyenoic species compared to whole lung. Type II pneumocyte phosphatidylcholine is enriched in the disaturated species and diminished in all other species compared to whole lung. Relative to whole lung, type II pneumocyte phosphatidylethanolamine is greatly enriched in monoenoic and depleted in polyenoic fatty acid species. Analysis of the fatty acid composition of the molecular species in general indicated differences in relative amounts of fatty acids which were most pronounced in palmitic, palmitoleic, stearic and oleic acids, both within and between type II pneumocyte and whole lung glycerolipids. Significant differences between molecular species also existed within type II pneumocyte glycerolipids. In this cell type, phosphatidylcholine is enriched in disaturated and diminished in monoenoic species compared to diacylglycerol. Phosphatidylethanolamine is enriched in monoenoic and polyenoic species relative to diacylglycerol. In order to determine whether differences observed in type II pneumocyte glycerolipid molecular species were attributable to differences in the specificities of cholinephosphotransferase and ethanolaminephosphotransferase, the selectivity of these enzymes was examined. While cholinephosphotransferase showed diminished activity towards 1-stearoyl-2-linoleoyl-sn-glycerol, neither enzyme showed selectivity towards other tested diacylglycerols under a variety of conditions. Therefore, while in the type II pneumocyte significant amounts of phosphatidylcholine (particularly the disaturated species) and phosphatidylethanolamine may be synthesized de novo, enzymes responsible for remodeling (phospholipase A2 and acyltransferases) may play an important role in establishing the final molecular species composition of both phosphatidylcholine and phosphatidylethanolamine.     

Alveolar pre-type II cells from the fetal rabbit lung: effect of confluence on the production of disaturated phosphatidylcholine

Pre-type II alveolar cells isolated from the fetal rabbit lung on the 24th gestational day have been maintained in vitro for 14 days in a chemically defined medium supplemented with hormone-stripped serum. These cells replicate in culture. Measurement of the incorporation of [14C]choline into cellular disaturated phospholipid indicated that those cells grown in vitro under standard conditions for 8 days (pre-confluent) incorporate the radioactive precursor at a similar rate to cells maintained for 14 days (post-confluent). Both dexamethasone and serum-free medium conditioned by monolayer cultures of fetal rabbit lung fibroblasts stimulated [14C]choline incorporation into disaturated phosphatidylcholine (PC) by the pre- and post-confluent cultures after 24 or 48 h of exposure: the conditioned medium was more effective than the steroid. These treatments had little effect on choline incorporation into disaturated phosphatidylcholine of preconfluent cells during the first 12 h. A marked response occurred by 24 h after which the labelling of disaturated phosphatidylcholine plateaued. In contrast, with post-confluent cells labelling of disaturated PC increased in a more linear fashion and only plateaued after 72 h. Determination of the ratio of incorporation of [14C]choline into disaturated versus unsaturated phospholipid indicated that serum-free medium conditioned by monolayer cultures of fetal lung fibroblasts specifically increased the level of radioactive precursor in the disaturated phospholipid in both the pre- and post-confluent cell monolayers.     

Human fetal and adult spleen phospholipids and their fatty acid composition

Total phospholipid contents and the individual phospholipid components of human adult and fetal spleens from 17--18 and 23--24 week's pregnancies composition of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, diphosphatidylglycerol and phosphatidic acid studied in human adult, 17--18 and 23--24 week fetal spleens.     

Role of α-glucosidase in fetal lung maturation

The role of lysosomal enzyme acid α-glucosidase in fetal lung development was investigated with the aid of a specific inhibitor, the pseudosaccharide acarbose. The drug was added to a Waymouth culture medium of fetal rat lung expiants cultivated for 48 h from gestational stage 19.5 days, an in vitro system previously shown to allow morphological and biochemical maturation of alveolar epithelium. Glycogenolysis was reduced by 40% as compared with tissue cultivated on control medium, which means that α-glucosidase could account for as much as 40% of fetal lung glycogenolysis, the remaining 60% being presumably achieved by cytosolic phosphorylase and by a microsomal neutral α-glucosidase. By the same time, the increase of phospholipids of surfactant fraction extracted from cultivated expiants was partially inhibited: total and saturated phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were about 30–40% lower than in lungs cultivated on control medium. It should be emphasized that DNA concentration and increases in non-surfactant phospholipids were unchanged by the drug. α-Glucosidase activity was evidenced in the lysosomal fraction, in the microsomal fraction and, although in lower amounts, in the surfactant fraction extracted from term fetal lung. The results suggest that lysosomal α-glucosidase plays a major role in lung maturation and could facilitate glycogenolysis for the specific use of glycogen stores in providing substrates for surfactant phospholipid biosynthesis.     

Surfactant peptides stimulate uptake of phosphatidylcholine by isolated cells

To determine whether small hydrophobic surfactant peptides (SP-B and SP-C) participate in recycling of pulmonary surfactant phospholipid, we determined the effect of these peptides on transfer of 3H- or 14C-labelled phosphatidylcholine from liposomes to isolated rat alveolar Type II cells and Chinese hamster lung fibroblasts. Both natural and synthetic SP-B and SP-C markedly stimulated phosphatidylcholine transfer to alveolar Type II cells and Chinese hamster lung fibroblasts in a dose- and time-dependent fashion. Effects of the peptides on phospholipid uptake were dose-dependent, but not saturable and occurred at both 4 and 37 degrees C. Uptake of labelled phospholipid into a lamellar body fraction prepared from Type II cells was augmented in the presence of SP-B. Neither SP-B nor SP-C augmented exchange of labelled plasma membrane phosphatidylcholine from isolated Type II cells or enhanced the release of surfactant phospholipid when compared to liposomes without SP-B or SP-C. Addition of native bovine SP-B and SP-C to the phospholipid vesicles perturbed the size and structure of the vesicles as determined by electron microscopy. To determine the structural elements responsible for the effect of the peptides on phospholipid uptake, fragments of SP-B were synthesized by solid-phase protein synthesis and their effects on phospholipid uptake assessed in Type II epithelial cells. SP-B (1-60) stimulated phospholipid uptake 7-fold. A smaller fragment of SP-B (15-60) was less active and the SP-B peptide (40-60) failed to augment phospholipid uptake significantly. Like SP-B and SP-C, surfactant-associated protein (SP-A) enhanced phospholipid uptake by Type II cells. However, SP-A failed to significantly stimulate phosphatidylcholine uptake by Chinese hamster lung fibroblasts. These studies demonstrate the independent activity of surfactant proteins SP-B and SP-C on the uptake of phospholipid by Type II epithelial cells and Chinese hamster lung fibroblasts in vitro.     

Epidermal growth factor in the rat lung

Epidermal Growth Factor (EGF) in pharmacological doses is able to induce precoccious lung maturation in rabbits and sheeps. As EGF is probably acting in a para- or autocrine way, we have searched for EGF in the lungs. We report EGF immunoreactivity to be present in the type II pneumocytes of the rat from a couple of days prior to birth and throughout life. Further, we report EGF immunoreactivity to be present in cells in the bronchi and the bronchioles from day 20-21 of gestation and throughout life. G-200 gelchromatography of lung extracts indicates that the EGF-reactive material is a high molecular weight form of EGF. Since previous studies have shown that EGF in pharmacological doses is able to promote lung maturation, our results may imply a physiological role for EGF in the lungs.     

Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture

Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.     

Purification, characterization and substrate specificity of rabbit lung phospholipid transfer proteins.

Three phospholipid transfer proteins, namely proteins I, II and III, were purified from the rabbit lung cytosolic fraction. The molecular masses of phospholipid transfer proteins I, II and III are 32 kilodaltons (kDa), 22 kDa and 32 kDa, respectively; their isoelectric point values are 6.5, 7.0 and 6.8, respectively. Phospholipid transfer proteins I and III transferred phosphatidylcholine (PC) and phosphatidylinositol (PI) from donor unilamellar liposomes to acceptor multilamellar liposomes; protein II transferred PC but not PI. All the three phospholipid transfer proteins transferred phosphatidylethanolamine poorly and showed no tendency to transfer triolein. The transfer of [14C]PC from unilamellar liposomes to multilamellar liposomes facilitated by each protein was affected differently by the presence of acidic phospholipids in the PC unilamellar liposomes. In an equal molar ratio of acidic phospholipid and PC, phosphatidylglycerol (PG) reduced the activities of proteins I and III by 70% (P = 0.0004 and 0.0032, respectively) whereas PI and phosphatidylserine (PS) had an insignificant effect. In contrast, the protein II activity was stimulated 2-3-times more by either PG (P = 0.0024), PI (P = 0.0006) or PS (P = 0.0038). In addition, protein II transferred dioleoylPC (DOPC) about 2-times more effectively than dipalmitoylPC (DPPC) (P = 0.0002), whereas proteins I and III transferred DPPC 20-40% more effectively than DOPC but this was statistically insignificant. The markedly different substrate specificities of the three lung phospholipid transfer proteins suggest that these proteins may play an important role in sorting intracellular membrane phospholipids, possibly including lung surfactant phospholipids.     

Epidermal growth factor in the rat lung

Summary Epidermal Growth Factor (EGF) in pharmacological doses is able to induce precoccious lung maturation in rabbits and sheeps. As EGF is probably acting in a para- or autocrine way, we have searched for EGF in the lungs.We report EGF immunoreactivity to be present in the type II pneumocytes of the rat from a couple of days prior to birth and throughout life. Further, we report EGF immunoreactivity to be present in cells in the bronchi and the bronchioles from day 20–21 of gestation and throughout life. G-200 gelchromatography of lung extracts indicates that the EGF-reactive material is a high molecular weight form of EGF.Since previous studies have shown that EGF in pharmacological doses is able to promote lung maturation, our results may imply a physiological role for EGF in the lungs.     

Glycerol metabolism in type II pneumocytes isolated from streptozotocin-diabetic rats

Glycerol utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozocinin-diabetic rats. With glucose in the incubation medium, incorporation of exogenous [1,3-14C]glycerol into disaturated phosphatidylcholine, total phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) was increased 4-fold in cells from diabetic rats. In the absence of glucose, glycerol incorporation was 5-fold greater than in its presence in cells from normal animals, but was further increased 2.2-fold in cells from diabetic rats. Insulin treatment of diabetic rats returned all incorporation rates to control values. The increased glycerol incorporation rates were not due to differences in either phospholipid turnover or the size of the glycerol 3-phosphate precursor pool. Kinetic analysis of glycerol entry into the acid-soluble cell fraction indicated that glycerol transport occurred largely by simple diffusion, and was not rate limiting for its entry into lipids. Glycerol entry into the total lipid fraction was saturable, reaching a Vmax of 48 pmol/micrograms DNA per h in normal cells and 120 pmol/micrograms DNA per h in cells from diabetic rats, with no change in the Km (0.31 mM). While glycerol oxidation was reduced 23% in cells from diabetic rats in the presence of glucose and by 44% in the absence of glucose, glycerol kinase activity in sonicates of cells from diabetic animals was increased 210% and was reversed by in vivo insulin treatment. These results suggest that glycerol utilization in type II pneumocytes is a hormonally regulated function of both glycerol oxidation and glycerol phosphorylation.     

Sex-specific differences in rabbit fetal lung maturation in response to epidermal growth factor.

Males and females exhibit different stages of lung development at the same gestation with males lagging behind. We hypothesized that one of the mechanisms responsible for the sex-specific difference in fetal lung maturation is a delay in the onset of epidermal growth factor (EGF) activity in the male fetal lung. EGF influences growth and differentiation during development. We studied the effects of EGF on the incorporation of glycerol into lamellar body disaturated phosphatidylcholine (DSPC) in sex-specific fetal rabbit lung explants prepared at 21 and 24 days gestation (term 31 days). The explants were maintained in Waymouth's media + 10% stripped fetal calf serum with or without EGF (10 ng/ml). The incorporation of [1,3-14C]glycerol into lamellar body DSPC was assessed after 3, 5, or 7 days of culture. Female lung explants prepared at 21 days of gestation had increased incorporation of glycerol into DSPC over time in response to EGF treatment. Male lung explants prepared at 21 days did not respond to EGF treatment. In explants prepared at 24 days gestation, baseline glycerol incorporation into DSPC was higher in female as compared to male fetal lung explants. EGF-responsiveness was also sex-specific in these more mature explants, with the male explants now responding to EGF with a consistent increase in the incorporation of glycerol into lamellar body DSPC. We conclude that one of the mechanisms responsible for the lag in male fetal lung development is a delay in the onset of EGF activity.     

Isolation of lamellar bodies from rat granular pneumocytes in primary culture

A lamellar body fraction was isolated from rat alveolar granular pneumocytes in primary culture by upward flotation on a discontinuous sucrose gradient and compared with a similar fraction isolated from lung homogenates. Lamellar bodies from granular pneumocytes were free of detectable contamination with either succinate dehydrogenase or NADPH-cytochrome c reductase. There was an enrichment of acid phosphatase activity, which, based on distribution of enzyme activity on the gradient, did not appear to be a contamination from other fractions. The lamellar body fraction of granular pneumocytes yielded approx. 1 microgram protein/10(6) cells with a phospholipid-to-protein ratio (mg/mg) of 9.6 +/- 0.4 (n = 7). Composition with respect to total phospholipids was 71.0% phosphatidylcholine (disaturated phosphatidylcholine, 45.2%), 8.4% phosphatidylglycerol and 12.8% phosphatidylethanolamine. Palmitic acid comprised 66% of the fatty acids in phosphatidylcholine and 34% of those in phosphatidylglycerol. The lamellar body fraction from granular pneumocytes was similar to that from whole lung with respect to phospholipid-to-protein ratio and phospholipid composition and showed only minor differences in fatty acid composition. Ultrastructurally, lamellar bodies showed generally intact limiting membranes and lamellated structure. Lamellar bodies from granular pneumocytes showed occasional multinucleated whorls which were not seen in those isolated from lung homogenates. This study describes a method for preparing a homogeneous fraction of intact lamellar bodies from small amounts of material (6 X 10(7) granular pneumocytes). The yield on a per cell basis was higher when compared with a similar preparation from whole lung, although overall yield is small, due to loss of cells during the cell isolation procedure. This preparation may be useful to evaluate the role of lamellar bodies in the synthesis and secretion of lung surfactant by isolated granular pneumocytes.     

Stimulation of lung maturity: investigation of ambroxol in various animal models

The surfactant or phospholipid lining of the alveolar surface is essential for lung function and airway stability. In premature neonates, the idiopathic respiratory distress syndrome (IRDS) is usually due to a prenatal deficiency in pulmonary surfactant. Experimental and clinical investigations with corticosteroids and other drugs were conducted to study various aspects of lung maturation. The present study of Ambroxol, a new compound with surfactant stimulating properties, was carried out on adult and foetal animals. In adult experimental animals an increase in the activity of Type II pneumocytes was shown by measuring various biochemical parameters. In models of premature animals comparable to clinical IRDS conditions, the antenatal treatment of foetal lambs and rabbits with Ambroxol enhanced lung maturation.     

Depletion of alveolar glycogen corresponds with immunohistochemical development of CD208 antigen expression in perinatal lamb lung.

CD208 DC lysosomal-associated protein is a marker of activated human dendritic cells; however, recently it was described as a marker of adult type II pneumocytes in many species including humans and sheep. Our hypothesis was that CD208 is developmentally regulated in lung pneumocytes. Lamb lungs at varying stages of development were stained immunohistochemically for CD208 and with Nile red (a fluorescent stain for lamellar bodies of type II cells) along with pulmonary markers of maturation (glycogen stores and surfactant protein A [SP-A] expression) or proliferation (Ki-67). CD208 staining and Nile red were localized to rare pneumocytes in young fetal lambs (day 115), increasing in frequency and stain intensity with age. Periodic acid-Schiff staining of glycogen granules was most prominent in the young lambs (day 115) with reduced staining through advancing lung development. SP-A was detected in pulmonary epithelia and staining in alveoli increased through gestation with decreased staining at 2 weeks of age. Intranuclear Ki-67 staining decreased through late gestation but was increased in 2-week-old lambs. Ontogeny of CD208 staining and depletion of glycogen were correlated (p<0.0001) and consistent with the premise that CD208 is localized to developing lamellar bodies. The findings suggest that CD208 antigen expression may serve as a marker for pneumocyte maturation in the developing fetal lung.     

Lack of change in the composition of fetal lamb lung surfactant during gestation

Fetal surfactant from lamb lung fluids collected daily from day 114 to day 146 of gestation, was isolated by centrifugation (pellet material) and further purified by sucrose density gradient centrifugation. The concentration of the pellet material from lung fluid (crude surfactant) increased from day 125 till day 135 and fluctuated strongly from that period onwards, whereas lung fluid secretion increased linearly until a few days before parturition. The pellet phospholipid composition changed with gestational age, suggesting biochemical maturation of the surfactant-producing system. The purified surfactant fraction, of which approximately 85% was phosphatidylcholine, did not change however from day 122 onwards except for a small increase in the percentage of phosphatidylglycerol. Alveolar wash surfactant or the lamellar body material, isolated from fetal lungs at different gestational ages had the same composition as surfactant from lung fluids. Only the composition of lamellar bodies of '125 day' lungs differed slightly from that of the lung fluid surfactant. The similar characteristics of all purified surfactant fractions throughout gestation indicate that, in the fetal lamb, lung maturation is associated with an increase in surfactant production no significant changes in phospholipid composition.     

The effect of betamethasone and fetal sex on the synthesis and maturation of lung surfactant phospholipids in rabbits

In the present study we investigated the maturation of the surfactant phospholipids and the role of fetal sex on the effect of betamethasone in male and female rabbit fetuses. Betamethasone was administered to the doe (0.2 mg/kg intramuscularly) 42 and 18 h prior to killing. The fetuses were studied at 27 and 28 days from conception. Results from the alveolar lavage show that male fetuses tended to have a lower disaturated phosphatidylcholine/sphingomyelin ratio and lower levels of phosphatidylinositol. Phosphatidylglycerol was detected in trace amounts. This was apparently due to the high extracellular levels of myo-inositol inhibiting the synthesis of surfactant phosphatidylglycerol while increasing the synthesis of surfactant phosphatidylinositol. Betamethasone increased the recovery of disaturated phosphatidylcholine and phosphatidylinositol from the lung lavage in both sexes. As studied in lung slices in vitro, the betamethasone treatment decreased the incorporation of glucose into phospholipids, including into the fatty acid moiety of disaturated phosphatidylcholine, although it had no significant effect on the incorporation of glucose into the glycerol moiety of disaturated phosphatidylcholine. However, the addition of palmitate increased the incorporation of glucose into the glycerol moiety of disaturated phosphatidylcholine. The betamethasone treatment did not increase the incorporation of [1-14C]pyruvate into disaturated phosphatidylcholine. Following betamethasone administration, the availability of fatty acids may become rate-limiting for the synthesis of surfactant phospholipids. Betamethasone increased the activities of phosphatidic acid phosphohydrolase and phosphatidate cytidyltransferase in a fraction of microsomal membranes. The present evidence suggests that the glucocorticoid-induced lung maturation and the maturation of the normal lung are associated with an increase in the activity of the enzymes which are involved in metabolizing phosphatidic acid to neutral and acidic surfactant secretion of the male fetus was not explained by possible sex-related differences in the biosynthesis of the phospholipids.     

Effect of incubation of guinea-pig taenia coli in potassium-free media on arachidonate release and lipid metabolism

We studied the effects of immersion of guinea-pig taenia coli strips in potassium-free media on arachidonate stores and other lipid fractions. Control studies obtained with the strips in Krebs solution showed that greater than 97% of arachidonate was found esterified in phospholipid with the following distribution: phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol. 30 min incubation of the strips with [3H]arachidonate complexed to albumin resulted in incorporation of this isotope into phospholipid and neutral lipid fractions, phosphatidylcholine greater than neutral lipid greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. 30 min incubations with 32PO4(2-)-resulted in an isotope incorporation into phospholipids, phosphatidylcholine greater than phosphatidylserine plus phosphatidylinositol greater than phosphatidylethanolamine. After 'loading' with [3H]arachidonate and 32P, placing the strips in potassium-free media caused the following: there was an increased release of [3H]arachidonate from the tissue into the bathing solution. [3H]Arachidonate and 32P radioactivity in phosphatidylinositol fell without a change in phosphatidylinositol content. [3H]Arachidonate and 32P radioactivity in other phospholipid fractions was unchanged. Arachidonate specific activity fell and arachidonate content increased in the phosphatidylserine plus phosphatidylinositol fraction. [3]Arachidonate in neutral lipid did not change significantly. We conclude that exposure of taenia coli to potassium-free media activates turnover of phosphatidylinositol, which results in release of arachidonate.     

Phospholipid molecular species in human umbilical artery and vein endothelial cells

Molecular species of several phospholipid classes and subclasses were quantitatively determined in human umbilical artery and vein endothelial cells. Both types of endothelial cells were similar in phospholipid class composition, whereas they were markedly different in phospholipid subclass and molecular species composition. The amounts of two ether subclasses in phosphatidylcholine and phosphatidylethanolamine were higher in artery endothelial cells than those in vein endothelial cells. The relative content of alkylacyl subclass in phosphatidylcholine, a precursor of platelet-activating factor, was about three times higher in artery endothelial cells than in vein endothelial cells. In artery endothelial cells, arachidonic acid was in highest amounts in alkenylacyl phosphatidylethanolamine, followed by diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, and phosphatidylinositol. In the vein endothelial cells, arachidonic acid was highest in phosphatidylinositol, followed by diacyl phosphatidylethanolamine, diacyl phosphatidylcholine, and alkenylacyl phosphatidylethanolamine. Artery endothelial cells had higher amounts of molecular species containing arachidonic acid than vein endothelial cells in all phospholipid classes and subclasses. These differences are thought to reflect the functional differences of artery and vein endothelial cells.