Regulation by prostaglandin E2 of interleukin release by T lymphocytes in mucosa

Regulation of immune cell activation in lymphocyte-bearing human tissues is a pivotal host function, and metabolites of arachidonic acid (prostaglandin E₂ in particular) have been reported to serve this function at non-mucosal sites. However, it is unknown whether prostaglandin E₂ is immunoregulatory for the large lymphocyte population in the lamina propria of intestine; whether low (nM) concentrations of prostaglandin E₂ modulate immune responses occurring there; and whether adjacent inflammation per se abrogates prostaglandin E₂'s regulatory effects. To address these issues, intestine-derived lymphocytes and T hybridoma cells were assessed, T cell activation was monitored by release of independently quantitated lymphokines, and dose-response studies were performed over an 8-log prostaglandin E₂concentration range. IL-3 release by normal intestinal lamina propria mononuclear cells was reduced (up to 78%) in a dose-dependent manner by prostaglandin E₂, when present in as low a concentration as 10⁻¹⁰M. PGE₂ also inhibited(by ≥ 60%) mucosal T lymphocytes' ability to destabilize the barrier function of human epithelial monolayers. Further, with an intestine-derived T lymphocyte hybridoma cell line, a prostaglandin E₂ dose-dependent reduction in IL-3 and IL-2 (90 and 95%, respectively) was found; this was true for both mitogen- and antigen-driven T cell lymphokine release. Concomitant [³H] thymidine uptake studies suggested this was not due to a prostaglandin E²-induced reduction in T cell proliferation or viability. In contrast, cells from chronically inflamed intestinal mucosa were substantially less sensitive to prostaglandin E², e.g., high concentrations (10⁻⁶ M) of prostaglandin E² inhibited IL-3 release by only 41%. We conclude that prostaglandin E² in nM concentrations is an important modulator of cytokine release from T lymphocytes derived from the gastrointestinal tract, and it may play a central role in regulation of lamina propria immunocyte populations residing there. © 1996 Wiley-Liss, Inc.     

Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Saline washed red blood cells of the toadfish convert [1-¹⁴C] arachidonic acid to products that cochromatograph with prostaglandin E₂ and prostaglandin F_2α. This synthesis is inhibited by indomethacin (10 μg/ml). Conversion of arachidonic acid to prostaglandin E₂ was confirmed by mass spectrometry. When saline washed toadfish red blood cells were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15,-³H]-arachidonic acid, comparison of the isotope ratios of the radioactive products indicated that prostaglandin F_2α was produced by reduction of prostaglandin E₂. The capacity of toadfish red blood cells to reduce prostaglandin E₂ to prostaglandin F_2α was confirmed by incubation of the cells with [1-¹⁴C] prostaglandin E₂.     

Effects of prostaglandin E1 and isoproterenol on cyclic AMP content of human fibroblasts modified by time and cell density in subculture

In confluent cultures of “young” (< 30 generations) human fibroblasts, maximally effective concentrations of prostaglandin E₁ (5.6 μM) and isoproterenol (2 μM) increased cyclic AMP content several hundred-fold and approximately 30-fold, respectively. On the first day after initiation of cultures at either low (approx. 3 · 10⁵ cells) or high (approx. s · 10⁶ cells) cell density the magnitude of the isoproterenol effect was similar to that in confluent cultures. It increased during the next few days, reaching a maximum around day 2–3, and then declined. On any day during the period of subculture, the magnitude of the isoproterenol effect was inversely related to cell density. Alterations in response to prostaglandin E₁ as a function of time in subculture or cell density were less dramatic. The effects of prostaglandin E₁ were, however, smaller at some point during the first few days of subculture than after day 7, and when effects of prostaglandin E₁ were minimal, those of isoproterenol were maximal and approached those of prostaglandin E₁. On any day of subculture, cells in cultures of higher density tended to accumulate more cyclic AMP in response to prostaglandin E₁ than did those in low density cultures. The effects of prostaglandin E₁ and isoproterenol on cyclic AMP content were qualitatively similar in “young” and in “old” (> 60 generations in culture) human fibroblasts although the changes associated with duration of subculture and cell density tended to be less marked with “old” cells. In the “young” fibroblasts responsiveness to isoproterenol and prostaglandin E₁ appears to correlate with cell morphology and with the fractional rate of growth in subcultures. It is suggested that the capacities of the fibroblasts to respond to these two agents may be altered independently during growth of human fibroblasts.     

Specific prostaglandine E1 and A1 binding sites in rat a dipocyte plasma membranes

Rat adipocyte plasma membranes sacs have been shown to be a sensitive and specific system for studying prostaglandin binding. The binding of prostaglandin E₁ and prostaglandin A₁ increases linearly with increasing protein concentration, and is a temperature-sensitive process. Prostaglandin E₁ binding is not ion dependent, but is enhanced by GTP. Prostaglandin A₁ binding is stimulated by ions, but is not affected by GTP.Discrete binding sites for prostaglandin E₁ and A₁ were found. Scatchard plot analysis showed that the binding of both prostaglandins was biphasic, indicating two types of binding sites. Prostaglandin E₁ had association constants of 4.9 · 10⁹ 1/mole and 4 · 10⁸ 1/mole, while the prostaglandin A₁ association constants and binding capacities varied according to the ionic composition of the buffer. In Tris-HCl buffer, the prostaglandin A₁ association constants were 8.3 · 10⁸ 1/mole and 5.7 · 10⁷ 1/mole, while in the Krebs—Ringer Tris buffer, the results were 1.2 · 10⁹ 1/mole and 8.6 · 10⁶ 1/mole.Some cross-reactivity between prostaglandin E₁ and A₁ was found for their respective binding sites. Using Scatchard plot analysis, it was found that a 10-fold excess of prostaglandin E₁ inhibited prostaglandin A₁ binding by 1–20% depending upon the concentration of prostaglandin A₁ used. Prostaglandin E₁ competes primarily for the A prostaglandin high-affinity binding site. Similar Scatchard analysis using a 20-fold excess of prostaglandin A₁ inhibited prostaglandin E₁ binding by 10–40%. Prostaglandin A₁ was found to compete primarily for the E prostaglandin low-affinity receptor.All of the bound [³H]prostaglandin E₁, but only 64% of the bound [³H]-prostaglandin A₁ can be recovered unmetabolized from the fat cell membrane. There is no non-specific binding of prostaglandin E₁, but 10–15% of prostaglandin A₁ binding to adipocyte membranes is non-specific. Using a parallel line assay to measure relative affinities for the E binding site, prostaglandin E₁ > prostaglandin A₂ > prostaglandin F_2α. Prostaglandin E₂ and 16,16-dimethyl prostaglandin E₂ were equipotent with prostaglandin E₁, while other prostaglandins had lower relative affinities. 7-Oxa-13-prostynoic acid does not appear to antagonize prostaglandin activity in adipocytes at the level of the receptor.     

Prostaglandin E1 binding and adenylate cyclase activation in normal and transformed fibroblasts

The binding of [³H]prostaglandin E₁ to membranes of clones of normal rat kidney fibroblasts (NRK cells) has been measured. Cell lines that responded to prostaglandin E₁, such as NRK and NRK transformed with Schmitt-Ruppin strain of Rous sarcoma virus (SR-NRK cells), have a high affinity prostaglandin E₁ binding site. Murine-sarcoma-virus-transformed lines of NRK cells are unresponsive to prostaglandin E₁ and have reduced prostaglandin E₁ binding. Exposure of cells to prostaglandin E₁ results both in decreases prostaglandin E₁ responsiveness and reduced prostaglandin E₁ binding.Activation of adenylate cyclase is correlated to binding of prostaglandin E₁ to receptors in both NRK and SR-NRK cell membranes. Mathematical models suggest that GTP decreases the affinity of hormone for its receptor while increasing the catalytic efficiency of adenylate cyclase, and that aggregates of occupied receptors may play an important role in the activation of adenylate cyclase.     

Hormone-specific responses and biosynthesis of sulfolipids in cell lines derived from mammalian kidney

The established cell lines isolated from mammalian kidney were characterized by its receptor activities against hormones and the ability to synthesize sulfolipids localized in the renal tubule.The level of 3′: 5′-cyclic AMP in JTC-12.P3 (monkey kidney) cells increased in 2 min as much as 2.5–5-fold on activation with 1.0 unit/ml of bovine parathyroid hormone or 1.9 units/ml of synthetic parathyroid hormone (1–34) resulting in intracellular cyclic AMP concentration of more than 40 pmol/mg protein. Prostaglandin E₁ (14 μM) and isopropylnorepinephrine (10 μM) were also found to increase the concentration of cyclic AMP by more than 30- and 9-fold, respectively. Addition in medium of calcitonin, arginine vasopressin, adrenocorticotropic hormone and glucagon caused no significant changes of cyclic AMP level in the cell.In contrast, MDCK, a cell line isolated from canine kidney, reacted to arginine vasopressin, isopropylnorepinephrine and prostaglandin E₁ and only slightly to parathyroid hormone. MDBK cell line derived from bovine kidney or fibroblast cell lines from rat lung and guinea pig kidney did not react to any of the hormones specific to kidney, i.e. arginine vasopressin, calcitonin or parathyroid hormone in the presence of theophylline. However, in the presence of 2 mM isobutylmethylxanthine, small but significant elevation of cellular cyclic AMP levels in response to calcitonin, arginine vasopressin, isopropylnorepinephrine and prostaglandin E₁ was observed.The cell lines JTC-12, MDCK and MDBK, when incubated with H₂³⁵SO₄, incorporated the isotope into sulfolipids assigned as sulfatides and ceramide dihexoside sulfate or in MDCK also into cholesterol sulfate.The results suggested that JTC-12, MDCK and MDBK cell lines are epithelial origin and also JTC-12 and MDCK originated most probably from renal tubular cells of cortex and medulla, respectively.     

Beta-adrenergic stimulation of prostaglandin production by human amnion tissue

Arachidonic acid is released from specific glycerophospholipids in human amnion and is used to synthesize prostaglandins that are involved in parturition. In an investigation of the regulation of prostaglandin production in amnion, the effects of isoproterenol on discs of amnion tissue maintained were examined. Isoproterenol caused a large but transitory increase in the amount of cyclic AMP in amnion discs and this was accompanied by a sustained stimulation of the release of arachidonic acid (but not palmitic acid or stearic acid) and prostaglandin E₂. The dependencies of cyclic AMP accumulation, arachidonic acid mobilization and prostaglandin E₂ release on the concentration of isoproterenol were similar, each response was maximal at 10⁻⁶ M isoproterenol and was inhibited by propranolol. Dibutyryl cyclic AMP stimulated the release of prostaglandin E₂ from amnion discs. Although prostaglandin E₂, when added to amnion discs caused an accumulation of cyclic AMP, it did not appear to mediate isoproterenol-induced accumulation of cyclic AMP since the latter effect was insensitive to indomethacin in concentrations at which prostaglandin production was inhibited greatly. These data support the proposition that catecholamines, found in increasing amounts in amniotic fluid during late gestation, my be regulators of prostaglandin production by the amnion.     

Prostaglandin Production by Experimental Tumours and Effects of Anti-inflammatory Compounds

THE presence of small quantities of prostaglandin-like material has been demonstrated in medullary carcinoma of the thyroid¹ and in Kaposi's sarcoma²; the diarrhoea often associated with these tumours has been attributed to prostaglandins. Diarrhoea is also often present in mice bearing the BP8/P₁ tumour. Preliminary investigations of the ascitic fluid from mice inoculated with this tumour showed the presence of pharmacologically active substances and prostaglandin E₂-like activity was found in small amounts³. Subsequent examination of the BP8/P₁ cells has revealed concentrations of prostaglandin E₂ in excess of 100 times that of the fluid. Significant amounts have also been found in another tumour, sarcoma 180 (S180).     

Bradykinin-stimulated release of [3H]arachidonic acid from phospholipids of HSDM1C1 cells: Comparison of diacyl phospholipids and plasmalogens as sources of prostaglandin precursors

Ethanolamine plasmalogens (1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamines) of many tissues contain high levels of arachidonate at their 2-position, and in certain tissues have been implicated as possible donors of arachidonate required in the synthesis of prostaglandins and thromboxanes. In the present study, [³H]arachidonate-labeled phospholipids of HSDM₁C₁ cells, a cell line derived from a mouse fibrosarcoma, were examined to determine the donor of the arachidonic acid released upon bradykinin stimulation of the synthesis of PGE₂. HSDM₁C₁ cells labeled with [³H]arachidonic acid for 24 hr in serum-free medium were used in most of the experiments and had the following distribution of label among the cellular lipids; phosphatidylcholine (33%), phosphatidylinositol (20%), diacyl-sn-glycero-3-phosphoethanolamine (15%), ethanolamine plasmalogen (15%), and less polar lipids (16%). Bradykinin treatment stimulated a rapid hydrolysis of [³H]arachidonate from the cellular lipids and conversion of the released acid to PGE₂, which was secreted into the medium. The label was released predominantly from phosphatidylinositol and possibly from phosphatidylcholine with no detectable change in the labeling of diacyl- or 1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The ethanolamine plasmalogens, therefore, do not appear to be involved in the stimulated release of arachidonate in the HSDM₁C₁ cells. Indomethacin blocked the bradykinin-stimulated synthesis of PGE₂ and to a lesser degree inhibited the release of [³H]-arachidonate from the cellular lipids into the medium.     

Activation of protein kinase C is coupled to prostaglandin E2 synthesis in swine granulosa cells

We have examined the role of phospholipid-sensitive calcium-dependent protein kinase (protein kinase C) in prostaglandin E₂ synthesis by monolayer cultures of swine granulosa cells. Specific phorbol ester derivatives known to activate protein kinase C significantly augmented the production of prostaglandin E₂. These stimulatory actions were dose and time-dependent, and could be abolished by the cyclooxygenase inhibitor, indomethacin, or the protein synthesis inhibitor, cycloheximide. Moreover, the rank order of potency of phorbol esters in enhancing prostaglandin E₂ production was concordant with that demonstrated for activation of protein kinase C. Phorbol ester in conjunction with the divalent cation ionophore, A23187, increased prostaglandin E₂ production synergistically. In addition, a non-phorbol stimulator of protein kinase C, 1-octanoyl-2-acetylglycerol, also significantly enhanced prostaglandin E₂ biosynthesis. The stimulated synthesis of prostaglandin E₂ was confirmed by high-pressure liquid chromatographic purification of this radiolabeled metabolite of ³H-arachidonic acid, and by capillary gas chromatography high-resolution mass spectrometry. Thus, the present studies indicate that the protein kinase C effector pathway is functionally coupled to prostaglandin E₂ production in the swine granulosa cell.     

Bradykinin-stimulated release of [H]arachidonic acid from phospholipids of HSDM1C1 cells: Comparison of diacyl phospholipids and plasmalogens as sources of prostaglandin precursors

Ethanolamine plasmalogens (1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamines) of many tissues contain high levels of arachidonate at their 2-position, and in certain tissues have been implicated as possible donors of arachidonate required in the synthesis of prostaglandins and thromboxanes. In the present study, [³H]arachidonate-labeled phospholipids of HSDM₁C₁ cells, a cell line derived from a mouse fibrosarcoma, were examined to determine the donor of the arachidonic acid released upon bradykinin stimulation of the synthesis of PGE₂. HSDM₁C₁ cells labeled with [³H]arachidonic acid for 24 hr in serum-free medium were used in most of the experiments and had the following distribution of label among the cellular lipids; phosphatidylcholine (33%), phosphatidylinositol (20%), diacyl-sn-glycero-3-phosphoethanolamine (15%), ethanolamine plasmalogen (15%), and less polar lipids (16%). Bradykinin treatment stimulated a rapid hydrolysis of [³H]arachidonate from the cellular lipids and conversion of the released acid to PGE₂, which was secreted into the medium. The label was released predominantly from phosphatidylinositol and possibly from phosphatidylcholine with no detectable change in the labeling of diacyl- or 1-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The ethanolamine plasmalogens, therefore, do not appear to be involved in the stimulated release of arachidonate in the HSDM₁C₁ cells. Indomethacin blocked the bradykinin-stimulated synthesis of PGE₂ and to a lesser degree inhibited the release of [³H]-arachidonate from the cellular lipids into the medium.     

An in vitro system to screen for diarrheagenic chemicals

We examined an in vitro system to screen for diarrheagenic chemicals using an established intestinal cell line (T₈₄ human colonic carcinoma). The cells were grown on Millicell-PCF (polycarbonate membrane) wells. The cells were seeded at approximately 5 × 10₆ cells/30mm well and incubated for 9–11 days in a 5% C0₂ incubator saturated with water at 37°C. The culture medium was a 1:1 mixture of Ham's F12 and Dulbecco's MEM with 5% fetal bovine serum and 25 pglml gentamicin sulfate. The well containing cells was removed from the incubator and mounted in a modified Ussing chamber for measurement of shortcircuit current (I_sc). Chemical-induced increases in Psc are usually indicative of electrogenic epithelial Cl^– secretion, which is associated with diarrheagenic effects in animals and humans. T₈₄ cells grown on Millicell-PCF membrane responded with an increase in Isc after basolateral addition of the cholinergic (muscarznic) agonist carbachol, prostaglandin E₂, 16,16-dimethylprostaglandin E₂, and forskolin, while non-diarrheagenic prostaglandin D₂ did not affect I_sc. Based on our results, this in vitro system has the potential to be adapted as a rapid screen for detecting diarrheagenic chemicals.Abbreviations dmPGE₂ 16,16-dimethylPGE₂ - EC₅₀ 50% of maximum effective concentration - EDTA ethylenediaminetetraacetate - I_SC short-circuit current - PGD₂ prostaglandin D₂ - PGE₂ prostaglandin E₂ - PD potential difference - R_T transepithelial resistance     

Changes in prostaglandin E1 stimulation of glucose oxidation in rat adipocytes during maturation and aging

Prostaglandin E₁ stimulates glucose oxidation in isolated rat adipocytes in a time and concentration dependent manner. Maximal stimulation requires 2 hours exposure to prostaglandin, although effects can be detected by 0.5 hours or earlier. In contrast to prostaglandin E₁, prostaglandin F₂α has essentially no effect on glucose oxidation. Maximal stimulation by prostaglandin E₁ at all ages tested occurs at concentrations of 10^?5 ? 10^?4M. Stimulation is greatest in cells of mature (10–12 month old) animals at 81 ± 9% above basal levels of glucose oxidation. This is to reduced to 48 ± 8% in cells of senescent (23–26 month old) animals, and at 23 ± 18% in cells of young (2–3 month old) rats is not significantly different from basal oxidation in most animals. These results are consistent with data for adipocytes and other cell types indicating that responsiveness to certain hormones is altered during maturation and aging.     

Bradykinin-stimulated release of arachidonate from phosphatidyl inositol in mouse fibrosarcoma cells

We recently proposed a new pathway by which arachidonate is released from platelet phosphatidyl inositol after stimulation by either thrombin or calcium ionophore A23187. The initial step in arachidonate liberation involves hydrolysis of phosphatidyl inositol to form 1,2-diacylglycerol which is subsequently hydrolyzed by a diacylglycerol lipase to liberate arachidonate for the prostaglandin and lipoxygenase pathways. Whether this pathway is unique to platelets or accounts for arachidonate release from other tissues has not been previously studied. Thus we have now investigated arachidonate metabolism in mouse fibrosarcoma cells (HSDM₁C₁) grown in culture. These cells contain approximately 7.6% of their total phospholipid as phosphatidyl inositol in the resting state (range 6.5–8.3%). When bradykinin (12 μM) is added to the fibrosarcoma cells, there is a rapid depletion of membrane phosphatidyl inositol reaching 62 ± 8% S.D. of baseline values by 15 seconds, falling to 36 ± 6% by 15 minutes. The drop in membrane phosphatidyl inositol is accompanied by release of arachidonate and PGE₂ into the culture medium. The time course of phosphatidyl inositol breakdown and PGE₂ formation supports the idea that phosphatidyl inositol breakdown provides the arachidonate for prostaglandin synthesis in mouse fibrosarcoma cells. Crude extracts of HSDM₁C₁ cells contained sufficient phosphatidyl inositol-specific phospholipase C activity and diacylglycerol lipase activity to account for arachidonate release in these cells.     

In vitro synthesis of prostaglandins and related lipids by populations of human peripheral blood mononuclear cells

This report focuses on the identification of the human peripheral blood mononuclear cells that do or do not produce prostaglandins (PGs) and related arachidonic acid metabolites. Our results, using two different assay systems, indicate that the monocyte/macrophage (MØ) is the major and possibly sole source of thromboxane (TXB₂) and prostaglandin E₂ (PGE₂) among peripheral blood mononuclear cells. Adherent peripheral blood monocytes (> 95% esterase positive) produced substantial amounts of these compounds. Quantitation of products which had incorporated exogenous ¹⁴C-arachidonic acid and radioimmunoassay of adherent cell culture fluids demonstrated that the amount of TXB₂ produced by these cells was appreciably greater than the amount of PGE₂ produced. Additional confirmation of TXB₂ synthesis was shown by abolishing the TXB₂ peak on TLC and TXB₂ activity detected by RIA by treating cells with a specific inhibitor of thromboxane synthetase. In contrast, non-adherent T cells failed to synthesize either PGE₂ or TXB₂. Non-adherent B cells (95% Ig positive) incubated with ¹⁴C-arachidonic acid produced a small peak of radioactivity co-chromatographing with TXB₂, and no PGE₂. All three cell populations incorporated similar amounts of ¹⁴C-arachidonic acid into hydroxy-fatty acids. We were unable to detect 6-keto-F_1α, the hydrolysis product of prostacyclin (PGI₂) in any of the cell types tested. The absence of PG synthesis among normal peripheral blood T and B cells was also noted among established human lymphoid cell lines. Neither a human T (CCRF), nor a human B-cell line (GM-130), produced PGE₂ or TXB₂. Three murine macrophage cell lines, P388D₁, J774.2, and WHI-3 produced PGE₂ and the latter TXB₂ as well.     

Conformational changes in erythrocyte membranes by prostaglandins as measured by circular dichroism

Membranes were prepared from fresh, washed human erythrocytes by hemolysis and washing with 5 mm sodium phosphate buffer (pH 7.4). The mean residue ellipticity, [θ], of erythrocyte membrane circular dichroism was altered by prostaglandin E₁ or prostaglandin F_2α at 37 °C when observed from 250 nm to 190 nm. The decrease in negativity of [θ] with 10^?6m prostaglandin E₁ was 12.7% at 222 nm and 17.7% at 208 nm, and with 10^?6m prostaglandin F_2α 22.5% and 34.2%, respectively (P < 0.01). Similar changes in [θ] were observed at lower concentrations of prostaglandins. No strict relationship between amount of change of [θ] and prostaglandin concentrations of 3 × 10^?5m to 3 × 10^?12m was evident. A persistent alteration of [θ] with prostaglandin was observed at 37 °C. Transient change of [θ] occurred at 25 °C with prostaglandin. No change of [θ] was observed at 15 or 20 °C. Buffer or palmitic acid were without effect on membrane [θ]. Phosphatidyl inositol or methyl arachidonate caused an increase in negativity of membrane spectra. The observed alterations of membrane [θ] did not arise from changes in light scattering as the OD₇₀₀–OD₂₀₀ of membranes was not changed by prostaglandin. Effects of prostaglandin were not dependent on light path length. The prostaglandin E₁ antagonist, 7-oxa-13-prostynoic acid, at 10^?7m produced no change of [θ] of membrane spectra and prevented the otherwise demonstrable effects of 10^?10m prostaglandin E₁ on [θ]. The decrease in negativity of [θ] at 222 nm is indicative of a decrease in ellipticity of membrane protein. These studies suggest that prostaglandins may act by inducing a conformational change in membrane protein.     

Comparison of the effects of prostaglandin I2 and prostaglandin E2 stimulation of the rat kidney adenylate cyclase-cyclic AMP systems

Prostacyclin (Prostaglandin I₂) effects on the rat kidney adenylate cyclase-cyclic AMP system were examined. Prostaglandin I₂ and prostaglandin E₂, from 8 · 10^?4 to 8 · ^?7 M stimulated adenylate cyclase to a similar extent in cortex and outer medulla. In inner medulla, prostaglandin I₂ was more effective than prostaglandin E₂ at all concentrations tested. Both prostaglandin I₂ and prostaglandin E₂ were additive with antidiuretic hormone in outer and inner medulla. Prostaglandin I₂ and prostaglandin E₂ were not additive in any area of the kidney, indicating both were working by similar mechanisms. Prostaglandin I₂ stimulation of adenylate cyclase correlated with its ability to increase renal slice cyclic AMP content. Prostaglandin I₂ and prostaglandin E₂ (1.5 · 10^?4 M) elevated cyclic AMP content in cortex and outer medulla slices. In inner medulla, with Santoquin® (0.1 mM) present to suppress endogenous prostaglandin synthesis, prostaglandin I₂ and prostaglandin E₂ increased cyclic AMP content. 6-Ketoprostaglandin F_1α, the stable metabolite of prostaglandin I₂, did not increase adenylate cyclase activity or tissue cyclic AMP content. Thus, prostaglandin I₂ activates renal adenylate cyclase. This suggests that the physiological actions of prostaglandin I₂ may be mediated through the adenylate cyclase-cyclic AMP system.     

The mechanisms of preterm labour; the interaction between amnion cells and leukocytes in the metabolism of arachidonic acid

Chorioamnionitis is frequently associated with preterm labour. We have used a cell culture model system to examine the effects of leukocytes upon the metabolism of endogenous arachidonic acid from within amnion cells. We have demonstrated that activated leukocytes release substances which increase the overall release and metabolism of endogenous arachidonic acid within amnion cells causing an increase in prostaglandin E₂ production as well as a smaller increase in non-cyclooxygenase metabolism. When amnion cells and leukocytes are cultured together, in addition to prostaglandin E₂ production by amnion cells, arachidonic acid released by the amnion cells appears to be metabolised by leucocytes to prostaglandin F_2α, prostacyclin and thromboxane A₂. Prostaglandins E₂ and F_2α are the principal cyclo-oxygenase products of this interaction.We postulate that chorioamnionitis stimulates preterm labour not only by causing an increase in prostaglandin E2 synthesis by amnion cells but by metabolism of amnion derived arachidonic acid to the powerfully oxytocic prostaglandin F_2α by leukocytes.     

Augmentation of prostaglandin and thromboxane production in vitro by monocytes exposed to histamine-induced suppressor factor (HSF)

A possible mechanism to explain the suppression of mitogen-induced lymphocyte proliferation in vitro by histamine-stimulated mononuclear cells was investigated. In initial experiments, the inhibitory action of histamine-induced suppressor factor (HSF) on lymphocyte proliferation was documented to be reduced by the addition of indomethacin (1 μg/ml). Moreover, the addition of exogeneous PGE₂ (10^?7-10^?8 M) to mononuclear cell cultures reconstituted HSF activity in the presence of indomethacin. In order to ascertain the nature of the target cell responding to HSF, control and suppressor supernatants were incubated with human lymphocytes or monocytes (5 × 10⁶ cells/ml) for 24 hr. Following incubation, the supernatants were assayed for their content of prostaglandin E₂, F_2α, and thromboxane B₂. Monocytes (but not lymphocytes) incubated with supernatants containing HSF increased their production of prostaglandin E₂, F_2α, and thromboxane B₂ by 169, 53, and 49%, respectively. Suppressor supernatants were generated with histamine or an H-2 agonist (dimaprit) and chromatographed by gel filtration on Sephadex G-100. The elution profiles for the factor(s) inducing suppression of lymphocyte proliferation (25–40,000 daltons) and augmenting PGE₂ production (25,000 daltons) overlapped but were not identical. Collectively, these data suggest that HSF-mediated inhibition of lymphocyte proliferation may occur in part through the augmented production of prostaglandins and/or thromboxane B₂ by human monocytes.