John M. Johnston

Initiation of human parturition

Arachidonic acid (the precursor of prostaglandins of the 2-series and related compounds) is released from phosphatidylinositol in a reaction sequence catalyzed by three enzymes, i.e., phospholipase C, diacylglycerol lipase, and monoacylglycerol lipase. Diacylglycerol, an intermediate in this pathway, was found in human amnion tissue. The fatty acid composition of the diacylglycerol fraction of amnion tissue was very similar to that of the phosphatidylinositol fraction. The diacylglycerol content of human amnion tissue obtained during early labor was greater than that of amnion tissue obtained before the onset of labor. These findings are supportive of the proposition that arachidonic acid is released from the phosphatidylinositol of amnion tissue during human parturition.

Identification of phospholipid platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in human amniotic fluid and urine

Human amniotic fluid and fetal urine were examined for the presence of phospholipid platelet-activating factor (PAF). PAF was detected in lipid extracts of some samples of amniotic fluid obtained from women in labor but it was undetectable in samples of amniotic fluid obtained before the onset of labor. PAF was identified by chromatographic mobility, platelet aggregation and chemical modifications. LysoPAF was also present in amniotic fluid at higher concentrations than those of PAF. Both PAF and lysoPAF were identified also in newborn and adult urine.

Prevention of glomerulonephritis and prolonged survival in New Zealand Black/New Zealand White F1 hybrid mice fed an essential fatty acid-deficient diet.

Female B/W mice spontaneously develop an autoimmune disease that is similar to systemic lupus erythematosus. Antibodies to doublestranded DNA (dsDNA) and antinuclear antibodies develop in aging animals; death from immune complex-mediated glomerulonephritis occurs from 8 to 12 mo of age. It has been reported that prostaglandin (PG)E(1) treatment of such mice prolongs survival. In the present study, four groups of female B/W mice were studied beginning at 6-11 wk of age on the following regimens: (a) a synthetic diet that contained 20% safflower oil, (b) a standard laboratory chow diet, (c) a standard diet together with injections of PGE(1), and (d) an essential fatty acid-deficient synthetic diet that contained 20% coconut oil. All animals were tested monthly for antinuclear antibodies and anti-dsDNA. Kidney tissue was obtained for light and immunofluorescence microscopy when animals were dying. All disease manifestations were altered strikingly in the essential fatty acid (EFA)-deficient animals. Intermediate benefit was seen in PGE(1)-treated animals. 7% of the control animals and 18% of safflower oil-fed animals survived to 10 mo. In contrast, the PGE(1)-treated and EFA-deficient mice had a similar survival rate (78-88%). At age 16 mo, 78% of EFA-deficient mice and 45% of PGE(1)-treated mice were alive. 25% of the PGE(1)-treated and 55% of the EFA-deficient animals survived to 20 mo. Serum anti-dsDNA appeared at age 5 mo in safflower oil-fed and control animals, but not until 9 and 12 mo for PGE(1)-treated and EFA-deficient animals, respectively. All kidneys from 7- to 9-mo-old safflower oil-fed and control animals and the majority of kidneys from PGE(1)-treated animals were abnormal by light and immunofluorescence microscopy. Kidneys from EFA-deficient animals were essentially normal at 10 mo. At 13 mo, all PGE(1)-treated animals examined had significant kidney involvement, whereas none of the EFA-deficient animals had glomerulonephritis. These findings demonstrate that an EFA-deficient diet has a beneficial effect on murine lupus erythematosus.

Differentiation of type II cells of human fetal lung in vitro.

SummaryLung tissue expiants from mid-trimester human abortuses were maintained for 8 days in organ culture in medium with or without serum. Before the start of culture the cells lining the pre-alveolar ducts were undifferentiated and contained no lamellar bodies, the intracellular organelle that contains surfactant. After 4 days in organ culture, the epithelium lining the pre-alveolar ducts was composed of differentiated type II cells containing numerous lamellar bodies. During the 8-day culture period there was increased incorporation of [3H]choline into phosphatidylcholine and disaturated phosphatidylcholine. In addition, the specific activity of phosphatidate phosphohydrolase, a regulatory enzyme in lung phospholipid synthesis, increased 4-fold during the culture period. Lamellar bodies isolated by differential centrifugation from expiants maintained in culture for 7 days had the characteristic ultrastructure described for this organelle. Lamellar bodies were isolated from expiants which had been incubated with [14C]glycerol. When the glycerophospholipid composition of lamellar bodies was analyzed it was found that the majority of the radiolabeled glycerol (74%) was incorporated into phosphatidylcholine and into the anionic phospholipids, phosphatidylglycerol (5%) and phosphatidylinositol (6%). Thus, human fetal lung expiants maintained in organ culture contain differentiated type II cells which synthesize surfactant characteristic of human fetal lung at 36 to 38 weeks of gestation.

Platelet-Activating Factor–Induced Ischemic Bowel Necrosis: The Effect of Platelet-Activating Factor Acetylhydrolase

ABSTRACT: In the present investigation, the rat model of necrotizing enterocolitis (NEC) was further developed by injection of platelet-activating factor (PAF) in the descending aorta. The role of the plasma PAF-acetylhydrolase (PAF-AH) was examined in the prevention of this disease. PAF (0.35 μg) caused ischemic intestinal necrosis when administered intraaortically. The effects of PAF injection on the small intestine were examined histologically in samples of the duodenum, jejunum, and ileum. The administration of PAF resulted in extensive hemorrhagic damage in all regions of the small bowel and a marked hemoconcentration. Pretreatment of the rats with dexamethasone or medroxyprogesterone significantly increased plasma PAF-AH activity. Dexamethasone and medroxyprogesterone prevented the gross and histologic features of NEC as well as the hemoconcentration. In contrast, lower amounts of PAF were sufficient to cause bowel necrosis and hemoconcentration when decreased activities of plasma PAF-AH were induced by 17α-ethynylestradiol or 4-aminopyr-azolopyrimidine administration. We have recently reported that PAF-AH is present in human milk. The beneficial effect of breast feeding in preventing the development of NEC in the newborn is discussed and a mechanism proposed to explain this finding. It is suggested that PAF may play an important role in the pathogenesis of NEC and that the increased plasma activity of PAF-AH caused by dexamethasone and the presence of this enzyme, of milk origin, in the lumen of the small bowel may prove to be beneficial in the prevention of this disease.

Effects of endotoxins and cytokines on the secretion of platelet-activating factor-acetylhydrolase by human decidual macrophages

Objective: The aim was to clarify the role of platelet-activating factor in parturition, preterm labor, and premature rupture of membranes. Study Design: Decidual macrophage populations were obtained by enzymic digestion, Ficoll-Paque centrifugation, or flow cytometric sorting. The effects of endotoxins and cytokines on platelet-activating factor-acetylhydrolase secretion by these cells were examined. Results: Lipopolysaccharide inhibited the platelet-activating factor-acetylhydrolase secretion by decidual macrophages. The inhibition was partially reversed by interleukin-1 receptor antagonist or by neutralizing antibodies against interleukin-1α, interleukin-1β, or tumor necrosis factor-α. Tumor necrosis factor-α, interleukin-1α, and interleukin-1β also decreased the enzyme secretion. The inhibitory actions of tumor necrosis factor-α and interleukin-1β were specifically neutralized by the corresponding antibodies. The effect of interleukin-1α or interleukin-1β on the secretion was abolished by interleukin-1 receptor antagonist. Conclusion: It is suggested that platelet-activating factor is involved in the pathogenesis of preterm labor or premature rupture of membranes caused by endotoxins and the subsequent activation of cytokine network.

The effect of cortisol on rabbit fetal lung maturation in vitro.

Abstract Explants of lung tissue from 19-day gestational age fetal rabbits were maintained in organ culture in medium with or without fetal calf serum for 1 to 11 days. Based on the results of biochemical and morphological studies it was apparent that the type II pneumonocyte differentiated in vitro at a time similar to that which occurs with maturation in vivo . The epithelial cells of the presumptive alveoli were undifferentiated at the start of incubation, but within 9 days developed increased amounts of Golgi apparatus and rough endoplasmic reticulum, many microvilli on the luminal surface and numerous lamellar bodies. Secreted lamellar bodies and tubular myelin figures were observed in the lumina of cultured explants. The incorporation of [ 3 H]choline into phosphatidylcholine by lung tissue explants maintained in medium containing 10% fetal calf serum remained relatively constant for 7 days of incubation but thereafter increased two-fold. When explants were maintained in fetal calf serum-containing medium and cortisol (10 −7 M ) or betamethasone (10 −7 M ), the incorporation of choline into phosphatidylcholine was two to three times greater than that of explants maintained in serum-containing medium without cortisol. When explants of fetal lung tissue were incubated in the presence of cortisol without fetal calf serum there was no stimulatory effect of cortisol on phosphatidylcholine biosynthesis. Therefore, serum cofactors are necessary for the stimulatory effects of cortisol on fetal lung development. The specific activity of phosphatidate phosphohydrolase (PAPase) increased to very high levels during the culture period. In the presence of serum, cortisol or betamethasone had no effect on the specific activity of phosphatidate phosphohydrolase.

Phosphatidylinositol-specific Phospholipase C in Fetal Membranes and Uterine Decidua

An assay procedure was developed in which phosphatidyl[2-(3)H]inositol was employed as substrate for the measurement of phosphatidylinositol-specific phospholipase C activity. Employing this assay, phosphatidylinositol-specific phospholipase C activity in human fetal membranes and uterine decidua was identified and characterized. The specific activity of this enzyme in amnion (4.4 mumol x mg(-1) protein x h(-1)) was three times that in uterine decidua and more than five times that in chorion laeve. No difference was found between the specific activity of phosphatidylinositol-specific phospholipase C in placental amnion and that in reflected amnion. The products of phosphatidylinositol hydrolysis in short-term incubations were stoichiometric amounts of diacylglycerol and inositol-1,2-cyclic-phosphate plus inositol-1-phosphate. After longer periods of incubation, monoacylglycerol also was detected. Diacylglycerol lipase activity also was demonstrated in these tissues. More than 90% of phosphatidylinositol-specific phospholipase C activity of amnion tissue was recovered in the 105,000-g supernatant fraction, and optimal enzymatic activity in vitro was observed at pH 6.5-7.5 in the presence of Ca(2+) (8 mM) and mercaptoethanol (4 mM). Phosphatidylinositol-specific phospholipase C activity was stimulated by fatty acids in low concentrations, but was inhibited by lysophosphatidylcholine and a variety of detergents. No effect of labor on the specific activity of phosphatidylinositol-specific phospholipase C in either fetal membranes or uterine decidua could be detected. The finding of an active phosphatidylinositol-specific phospholipase C activity in human fetal membranes and uterine decidua is complementary to our previous finding of a selective loss of arachidonic acid from phosphatidylinositol of human fetal membranes during labor. The action of phosphatidylinositol-specific phospholipase C, coupled to diacylglycerol lipase action, could provide a mechanism for the release of arachidonic acid for prostaglandin biosynthesis during parturition.

Phospholipid biosynthesis in lung lamellar bodies.

Summary Lung Type II epithelial cells contain lamellar bodies, the storage form of surfactant, which is predominately dipalmitoyl-phosphatidylcholine. Isolated lamellar bodies, free of microsomal and mitochondrial contamination have significant phosphatidic acid phosphohydrolase activity. Calculations based on enzyme distribution indicate that as much as 40% of the phosphatidic acid phosphohydrolase activity within the Type II cell is associated with the lamellar bodies. This high concentration of phosphatidic acid phosphohydrolase, a key enzyme in phospholipid biosynthesis, suggests that the phosphatidylcholine stored within the lamellar bodies may be synthesized at the perilamellar surface.

Mesenchyme-epithelial interactions in human endometrium. Prostaglandin synthesis in separated cell types.

Glandular epithelium and stromal cells of human endometrium were separated and maintained in monolayer culture. At the time the cells became confluent, cell suspensions were prepared and incubated with [14C]arachidonic acid. Radiolabeled prostaglandin E2 and, to a lesser extent, prostaglandin F2 alpha and metabolites of these prostaglandins, were formed principally in stromal cells. There was considerably less prostaglandin formation in endometrial glands either after maintenance in monolayer culture or in freshly separated glands. In stromal cells of endometrium prostaglandin formation was linear with time of incubation for 2.5 min and with [14C]arachidonic acid concentrations up to 8 microM. When stromal cells and epithelial cells were combined, all prostaglandin formation could be accounted for by that produced in stromal cells. Little or no prostaglandin formation was detected in stromal cells from human adipose tissue or in fibroblasts from human genital or abdominal skin or human fallopian tube.