Modulation of fibroblast proliferation by sulfidopeptide leukotrienes: effect of indomethacin

Because infiltration of mononuclear cells and fibroblast proliferation are associated in chronic inflammatory lesions, we tested the hypothesis that leukotrienes (LT), a product of activated mononuclear cells, may modulate fibroblast growth. Proliferation of cultured human skin fibroblasts was estimated by [3H]thymidine incorporation and cell count at increasing concentrations (0.1 nM to 0.1 microM) of LTC4 or LTD4. LTC4 and LTD4 stimulated cell growth in a dose-dependent manner only in the presence of 50 microM indomethacin. Under similar conditions, LTE4 but not LTB4 (0.1 microM) was active. Both asynchronous, growing cells and synchronous, quiescent cells were sensitive to LT when prostaglandin (PG) synthesis was suppressed by indomethacin. Other blockers of cyclooxygenase such as ibuprofen and aspirin exhibited identical permissive activity, and the effect of indomethacin was totally abolished by addition of PGE2. LTC4 modified neither [3H]arachidonic acid release from prelabeled fibroblasts nor PGE2 production by fibroblasts. These results demonstrate that the sulfidopeptide LT stimulate fibroblast proliferation only when the endogenous synthesis of PG is blocked, but they do not enhance the synthesis of PG in their target cells showing no evidence for a negative feed-back loop. Nevertheless, it seems likely that the initiation and development of the fibrotic process in the different tissues depends in part on the local balance between PG and LT productions.     

Prostaglandins mediate suppression of lymphocyte proliferation and cytokine synthesis in acute Trypanosoma cruzi infection

Suppression of host lymphoproliferative responses to mitogens and Ag is characteristically seen during acute infection with the protozoan parasite Trypanosoma cruzi. We investigated the reciprocal regulation of prostaglandins (PG), TNF-alpha, and nitric oxide (NO) production and their effects on cytokine production and lymphoproliferative responses to parasite Ag and to Con A by spleen cells (SC) from T.-cruzi-infected mice. Large amounts of PGE2, TNF-alpha, and NO were produced during infection. TNF-alpha stimulated PG and NO synthesis, while both mediators inhibited TNF-alpha synthesis. Blocking PG also reduced NO synthesis indicating that PG stimulate NO production. Treatment with indomethacin or NMLA stimulated lymphoproliferation on days 6 and 22 of infection; on day 14, when suppression of proliferation and NO production was maximal, combined inhibition of NO and PG production restored parasite Ag specific and Con A proliferative responses. Blocking PG or NO production increased IL-2, IFN-gamma, and TNF-alpha, but not IL-12 production by SC; IL-10 levels were not reduced. Indomethacin-treated infected mice had higher mortality compared to untreated infected animals. The data indicate that PG, together with NO and TNF-alpha, participate in a complex circuit that controls lymphoproliferative and cytokine responses in T. cruzi infection.     

Splenic macrophage function in early syphilitic infection is complex. Stimulation versus down-regulation

Macrophages are important regulatory cells that can both stimulate and down-regulate various immune functions. During syphilitic infection, these cells phagocytize, kill, and lyse Treponema pallidum. They also modulate early T cell activation by decreasing IL-2 production through secretion of PG. This report focuses on additional complexities of macrophage regulation. Non-adherent splenic cells were stimulated with Con A to induce IFN-gamma synthesis. High levels were detected in preparations from normal rabbits and much lower levels in preparations from infected rabbits. The organisms also readily stimulated IL-1 synthesis by adherent spleen preparations from normal but not from infected rabbits. When indomethacin was added to these latter preparations, this IL-1 defect was reversed, implicating PG in this down-regulation. Spleen cells were obtained from normal rabbits and from rabbits infected testicularly for 9 to 12 days. Infection elevated basal levels of class II Ia Ag on adherent cells. In addition, macrophage Ia expression was increased during 4 days of in vitro incubation with treponemes. Non-adherent spleen cells from infected animals inhibited two different macrophage functions. First, culture filtrates obtained after 48 h of incubation contained a soluble factor that subsequently decreased LPS-induced IL-1 synthesis. Second, when macrophages were co-incubated with non-adherent cells, treponemal stimulation of macrophage Ia expression was inhibited; this inhibition was reversed by indomethacin implicating prostaglandins in this down-regulation. In further experiments an exogenous source of IFN-gamma was incubated with adherent cells from infected rabbits. This stimulated macrophage function as shown by increased IL-1 synthesis and Ia expression and decreased PGE2 secretion. Results are discussed in terms of the complexities of immunoregulation by macrophages during syphilitic infection.     

Decreased sensitivity of atopic mononuclear cells to prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2)

The effect of PGE2 and PGD2 on several lymphocyte functions in vitro was evaluated in nonatopic and atopic subjects. Both PGE2 and PGD2 inhibited phytohemagglutinin-induced protein synthesis ([3H] leucine uptake) by nonatopic mononuclear cells and T cells in a dose-dependent manner (10(-6) to 10(-12) M). Protein synthesis by atopic mononuclear cells was not significantly suppressed by the above concentration of PGE2. Although PGD2 effectively suppressed protein synthesis by atopic mononuclear cells and T cells at 10(-6) M, lower concentrations were ineffective. Kinetic studies revealed significant differences in the suppressive effects of PGE2 and PGD2 on atopic and nonatopic mononuclear cells at 24 and 48 h, but not at 72 or 96 hr. Protein synthesis by T helper-enriched populations (suppressor cell depletion by anti-Leu-2b + complement) obtained from nonatopics was significantly reduced by PGE2 and PGD2, suggesting that these mediators may be directly inhibiting the responding population. By contrast, protein synthesis by T suppressor-enriched populations (helper cell depletion by OKT4 + complement) obtained from nonatopics was enhanced by PGE2 and PGD2, suggesting that the PG were activating these cells. Atopic T helper and T suppressor cells exhibited decreased responsiveness to PGE2 and PGD2 compared with nonatopic cells. PGE2 and PGD2 inhibited the phytohemagglutinin-stimulated proliferative response ([3H]thymidine uptake) by both atopic and nonatopic mononuclear cells in a dose-dependent manner and to the same extent. However, although PGE2 and PGD2 generated functional suppressor activity (when using a coculture technique) in nonatopic mononuclear cells, these mediators failed to activate atopic suppressor cells. These results suggest that reduced responses by atopic T cells to signals provided by PGE2 and PGD2 are not solely restricted to suppressor cell function, and could indicate an impaired ability to regulate immune and/or inflammatory reactions.     

Methyl xanthine phosphodiesterase inhibitors behave as prostaglandin antagonists in a perfused rat mesenteric artery preparation.

The methyl xanthines, theophylline, caffeine and 3-isobutyl-1 methyl xanthine (MIX) inhibited the pressure responses to noradrnealine, angiotensin II and potassium ions in the isolated perfused mesenteric vascular bed of the male rat. The ID50s for inhibition of responses to noradrenaline were 1.85 mug/ml (0.83 x 10(-5) M) for MIX, 18 mug/ml (1 x 10(-4)M) for theophylline and 133 mug/ml (6.8 x 10(-4) M) for caffeine. Similar ID50 concentrations were found for responses to angiotensin II and potassium. We have previously found that substances which inhibit the three pressor agents equally may be prostaglandin (PG) synthesis inhibitors or PG antagonists. Xanthine itself, cyclic AMP and dibutyrl cyclic AMP had no inhibitory effects on the preparation up to concentrations of 10-2 M. Partial inhibition of PG synthesis by indomethacin shifted the % inhibition/log concentration curve to the left, while addition of exogenous PGE2 shifted it to the right. In preparations completely inhibited by sufficient indomethacin added to the perfusate to block PG synthesis, and then restored by adding 1 or 5 ng/ml PGE2 in addition to the indomethacin, the methyl xanthines again inhibited responses suggesting that they were PG antagonists rather than inhibitors of synthesis or release. In preliminary experiments MIX also inhibited effects of PGF2alpha on rat uterus and PGE1 on guinea pig ileum. Effective concentrations of theophylline were similar to the therapeutic levels in human plasma. PG antagonists may be a major action of methyl xanthines requiring reinterpretation of many experiments which have attributed their effects to PDE inhibition. PGs may also be involved in regulating PDE action.     

A prostaglandin-mediated suppressive activity of cord as compared to maternal or other adult adherent cells in OKT3 antibody-induced proliferation

In a previous study we reported that cord blood lymphocytes show lower OKT3 responses as compared to their mothers and to other, unrelated adults. In the study reported here, we investigated the interactions between lymphocytes and adherent accessory cells in OKT3-stimulated cultures of newborn (cord), maternal, and other adult peripheral blood mononuclear leukocytes (PBML) and determined the following. (1) Removal of adherent cells (AC), by two cycles of plastic adherence or by nylon wool columns, impaired the OKT3-induced proliferation of maternal/adult cells, but significantly enhanced the OKT3 responsiveness of cord cells. (2) Addition of indomethacin, and other prostaglandin (PG) synthesis inhibitors, caused a more than twofold augmentation of cord PBML OKT3 responses, but had only a small, if any, enhancing effect on maternal/adult PBML. Cord PBML cultures deprived of AC were no longer enhanced by indomethacin. (3) Exogenous PGE2 (1.4 X 10(-6) through 1.4 X 10(-9) M) strongly inhibited OKT3-induced proliferation of maternal, cord, and adult PBML, at a wide range of antibody concentrations (5-100 ng/ml). However, an obvious difference in the extent of PG-mediated inhibition was observed among these three populations, and the order of PG sensitivity, from most to least sensitive, was cord greater than maternal greater than adult. (4) Purified interleukin-1 (IL-1) could not replace the accessory function of AC in the OKT3-induced proliferation of maternal/adult lymphocytes. In contrast, IL-1 increased by greater than 50% the OKT3 responsiveness of cord PBML in the absence, but not in the presence, of cord monocytes. Our observations strongly argue for a distinct, predominantly suppressive function of cord monocytes as compared to maternal/adult monocytes in OKT3-induced mitogenesis, and indicate prostaglandins as major mediators of this suppression.     

Rat mammary preadipocytes in culture produce a trophic agent for mammary epithelia-prostaglandin E2

Cell strains and cell lines rat mammary (Rama) 350-353 have been isolated from the slowly adherent stromal fraction of enzymatically digested rat mammary glands. Primary cultures of this fraction yield fat cels on extended culture. Their proportion can be increased with horse serum or growth hormone in the medium, and this increase is associated with a 100-fold or more increase in the release of radioimmunoassayable prostaglandins of the E type (PGE). The stromal cell strains and lines that are capable of yielding fat cells also secrete elevated levels (greater than 100 ng/mg/24 hr) of PGE; the fast-sticking epithelial fraction in primary cultures and the epithelial cell lines derived from it secrete 10-100 times less. Chromatography and radioisotopic labeling of the culture media from Rama 352 cells identify the PG as PGE2. PGE2 with insulin and hydrocortisone maximally stimulates [3H]DNA synthesis of epithelial cell lines and primary cultures from normal and tumorous glands by 2-4-fold at concentrations (10-20 ng/ml) well below those released by the preadipocytic stromal cells (20-100 ng/ml). Medium exposed to most cultured cells stimulates [3H]DNA synthesis of one epithelial cell line, Rama 25, by 2-4-fold. Prevention of the synthesis of PGE2 in Rama 352 cultures with indomethacin or flurbiprofen abolishes the mitogenic activity present in the culture medium, and the PG receptor antagonist polyphloretin phosphate inhibits completely the mitogenic activity for Rama 25 cells. Myoepithelial-like cell lines normally secrete moderate levels of PGE (10-100 ng/mg/24 hr) but the mitogenic activity for Rama 25 cells released from one such line, Rama 29, is not abolished by preventing the synthesis of PG's nor by PG-receptor antagonists.     

Methyl xanthine phosphodiesterase inhibitors behave as prostaglandin antagonists in a perfused rat mesenteric artery preparation

The methyl xanthines, theophylline, caffeine and 3-isobutyl-1 methyl xanthine (MIX) inhibited the pressure responses to noradrenaline, angiotensin II and potassium ions in the isolated perfused mesenteric vascular bed of the male rat. The ID50s for inhibition of responses to noradrenaline were 1.85 μg/ml (0.83 × 10⁻⁵M) for MIX, 18 μg/ml (1 × 10⁻⁴M) for theophylline and 133 μg/ml (6.8 × 10⁻⁴M) for caffeine. Similar ID50 concentrations were found for responses to angiotensin II and potassium. We have previously found that substances which inhibit the three pressor agents equally may be prostaglandin (PG) synthesis inhibitors or PG antagonists. Xanthine itself, cyclic AMP and dibutyryl cyclic AMP had no inhibitory effects on the preparation up to concentrations of 10⁻²M. Partial inhibition of PG synthesis by indomethacin shifted the % inhibition/log concentration curve to the left, while addition of exogeneous PGE2 shifted it to the right. In preparations completely inhibited by sufficient indomethacin added to the perfusate to block PG synthesis, and then restored by adding 1 or 5 ng/ml PGE2 in addition to the indomethacin, the methyl xanthines again inhibited responses suggesting that they were PG antagonists rather than inhibitors of synthesis or release. In preliminary experiments MIX also inhibited effects of PGF2α on rat uterus and PGE1 on guinea pig ileum. Effective concentrations of theophylline were similar to the therapeutic levels in human plasma. PG antagonism may be a major action of methyl xanthines requiring reinterpretation of many experiments which have attributed their effects to PDE inhibition. PGs may also be involved in regulating PDE action.     

Regulation of prostaglandin production by osteoblast-rich calvarial cells

The effect of various factors upon prostaglandin (PG) production by the osteoblast was examined using osteoblast-rich populations of cells prepared from newborn rat calvaria. Bradykinin and serum, and to a lesser extent, thrombin, were all shown to stimulate PGE2 and 6-keto-PGF1 alpha (the hydration product of PGI2) secretion by the osteoblastic cells. Several inhibitors of prostanoid synthesis, dexamethasone, indomethacin, dazoxiben and nafazatrom, were tested for their effects on the calvarial cells. All inhibited PGE2 and PGI2 (the major arachidonic acid metabolites of these cells) production with half-maximal inhibition by all four substances occurring at approximately 10(-7) M. For dazoxiben and nafazatrom, this was in contrast to published results from experiments in vivo which have indicated that the compounds stimulated PGI2 production. Finally, since the osteoblast is responsive to bone-resorbing hormones, these were tested. Only epidermal growth factor (EGF) was shown to modify PG production. At early times EGF stimulated PGE2 release, however, the predominant effect of the growth factor was an inhibition of both PGE2 and PGI2 production by the osteoblastic cells. The present results suggest that the bone-resorbing hormones do not act to cause an increase in PG by the osteoblast and that any increase in PG production by these cells may be in response to vascular agents.     

Regulation of human natural killing. III. Mechanism for interferon induction of loss of susceptibility to suppression by cyclic AMP elevating agents

In this study we demonstrated that human NK cells activated by IFN or poly I:C were partially resistant to suppression by PGE2, PGD2, PGA2, PGI2, dibutyryl cAMP, isoproterenol, and theophylline. This partial loss of inhibition was not due to endogenous PG production because the addition of indomethacin to cultures stimulated with IFN or poly I:C did not prevent the partial loss of sensitivity to PGE2. NK cells incubated in the presence of PGE2 overnight, however, were not sensitive to inhibition. IFN or poly I:C did not stimulate PG synthesis nor elevate intracellular cAMP levels of NK cells. On the other hand, IFN or poly I:C diminished the accumulation of intracellular cAMP levels in NK cells in response to PGE2 stimulation. Dibutyryl cAMP and theophylline suppressed the cytolytic activity of the unstimulated cells more than that of the activated cells. A possible mechanism for the IFN-induced unresponsiveness to PGE2 may be a compartmentalized loss of cAMP responsiveness. Cycloheximide, puromycin, emetine, and actinomycin D blocked NK activation by IFN and poly I:C as well as the acquisition of resistance to PGE2-mediated suppression.     

Cellular immunity in Q fever: modulation of responsiveness by a suppressor T cell-monocyte circuit

Human infection with the rickettsia Coxiella burnetii presents as an acute flulike primary Q fever, as a subacute granulomatous hepatitis, or, rarely, as chronic endocarditis. We have previously described lymphocyte unresponsiveness to Coxiella antigen in patients with Q fever endocarditis. This unresponsiveness was antigen specific and was mediated in part by adherent suppressor cells. In this report we show that the adherent suppressor cells work via prostaglandin E2 (PGE2)4 production. Addition of the cyclooxygenase inhibitor indomethacin to cultures of PBMC from patients with endocarditis or chronic laboratory exposure resulted in consistent increases in Coxiella-specific lymphocyte proliferation. The degree of increase in proliferation induced by indomethacin correlated strongly with the amount of PGE2 produced in a 4-hr culture stimulated by Coxiella antigen, but it also correlated with the sensitivity to inhibition of mitogenesis by PGE2. The suppressor mechanism was antigen nonspecific, because induction of suppression in vitro by Coxiella antigen also suppressed Candida-induced proliferation when both antigens were present in the same culture. Addition of indomethacin to these antigen cocultures totally reversed the Coxiella-induced suppression, confirming the evidence above that the nonspecific effector mechanism of suppression was prostaglandin (PG)-mediated. Elicitation of suppression, however, was antigen specific and involved a T cell-monocyte suppressor circuit. Supernatants from Coxiella-stimulated immune T cells and from the suppressor subset (OKT8+-enriched) of those T cells, but not unstimulated immune cells, induced augmented PGE2 production by unrelated nonimmune PBMC. We conclude that the lymphocyte unresponsiveness characterizing patients with Q fever endocarditis is modulated in part by an antigen-specific T suppressor cell which secretes a lymphokine to stimulate PGE2 production by adherent cells.     

Effect of physical stress on sensitivity of lymphocytes to inhibition by prostaglandin E2

Physical stress is associated with depressed cellular immune function. We have found that lymphocytes from subjects undergoing either of 2 stressful events, cardiac surgery or childbirth, are more sensitive to inhibition by PGE2. For example, the concentration of PGE2 required for 50% inhibition of 3H-thymidine incorporation (ID50) into phytohemagglutinin-stimulated lymphocytes from patients undergoing cardiac surgery went from 1.5 X 10(-8) M on the day before surgery to 3 X 10(-9) M on the day after surgery. This increase in sensitivity to PGE2 was accompanied by a significantly decreased lymphocyte proliferative response (27 to 68% of control, depending on mitogen dose) and a 50% increase in the percentage of E rosette-positive cells with receptors for the Fc portion of IgG. The increased sensitivity to PGE and the depressed mitogen responses returned to preoperative values by day 10. The depressed mitogen responses of the postoperative patients were completely restored to normal by removal of glass-adherent cells before culture. In addition, the responses of the postoperative patients and the women in labor were partially restored by the addition of indomethacin, a prostaglandin synthetase inhibitor, to the cultures. Thus it would appear that physical stress causes lymphocytes to become more sensitive to prostaglandin E2, and the increased sensitivity to inhibition by this immunomodulator is responsible in part for the depressed cellular immune function after physical stress.     

Two signals are required for accessory cells to induce B cell unresponsiveness. Tolerogenic Ig and prostaglandin

We previously demonstrated that a lymphoid dendritic cell-like tumor line (P388AD.2) presented a normally tolerogenic signal, fluoresceinated sheep gamma-globulin (FL-SGG), as an immunogenic one. In contrast, macrophages derived from the peritoneal cavity potentiated the ability of FL-SGG to induce B cell unresponsiveness. In this paper we examined whether two different Ia+ splenic accessory cells differentially presented tolerogen to spleen cells or fluorescein (FL)-binding B cells. Interestingly, lymphoid dendritic cells presented FL-SGG to spleen cells and elicited augmented anti-FL antibody responses, whereas splenic macrophages presented this same moiety and elicited hapten-specific B cell unresponsiveness. The mechanism of splenic macrophage-elicited B cell negative signaling was investigated, and it was found that B cell unresponsiveness was abrogated in the presence of the cyclooxygenase inhibitor indomethacin. This observation suggested a crucial role for PG in B cell negative signaling. The addition of 10 nM PGE2 restored unresponsiveness in cultures treated with indomethacin and tolerogen-pulsed macrophages, even though this dose of PG had no effect on the ability of B cells to be triggered by an immunogenic signal. A role for T cells was excluded, inasmuch as purified hapten-specific B cells were specifically tolerized by FL-SGG-pulsed macrophages. Lymphoid dendritic cells pulsed with FL-SGG did not deliver a tolerogenic or immunogenic signal to FL-specific B cells. However, when PGE2 was supplied, B cell unresponsiveness was induced. Finally, we tested whether "non-tolerogenic" doses of FL-SGG could render hapten-specific B cells unresponsive in the presence of PGE2, but in the absence of accessory cells. Interestingly, the combination of non-tolerogenic amounts (10 to 1000 pg/ml) of FL-SGG in conjunction with PGE2 induced unresponsiveness, whereas neither moiety alone was effective. These results suggest that splenic macrophages and lymphoid dendritic cells exert opposing effects on the immune system as evidenced by the induction of negative or positive B cell signaling. Our observations suggest that one of the key factors in controlling whether an accessory cell delivers a tolerogenic signal is the ability to secrete PG.     

Stimulation of adenosine 3':5'-monophosphate formation by prostaglandins in human astrocytoma cells. Inhibition by nonsteroidal anti-inflammatory agents.

Prostaglandins (PG) of the E series and catecholamines stimulate adenosine 3':5'-monophosphate (cAMP) formation in human astrocytoma cells (1321N1). These two classes of effectors activated adenylate cyclase upon interaction with different receptor systems. No evidence for a mediatory role for PG in the action of catecholamines was found. PG interacted with 1321N1 cells with an order of potency of PGE1 = PGE2 greater than PGA1 greater than PGF2 alpha. The effect of combinations of the various PG indicated that all efficacious PG interacted with a common receptor. 7-Oxa-13-prostynoic acid and indomethacin were shown to be competitive inhibitors of the effect of PGE1 with Ki values of 4 and 150 micron, respectively. These two compounds did not inhibit the effect of isoproterenol. Polyphloretin phosphate caused a complex pattern of inhibition of the effects of PGE1 and at higher concentrations also inhibited the effects of isoproterenol. The mefenamate class of nonsteroidal anti-inflammatory agents was found to inhibit the effects of PGE1 with a potency order of meclofenamic acid greater than flufenamic acid = mefenamic acid. The inhibitory action of meclofenamic acid was complex involving specific, but partial, insurmountable antagonism of PGE1 as well as competitive inhibition of PGE1 effects. At higher concentrations of meclofenamic acid a nonspecific inhibition of the effects of both PGE1 and isoproterenol was observed. These studies suggest that the inhibition by nonsteroidal anti-inflammatory agents of the physiological effects of PGE1 in animals may occur, at least in part, at the level of adenylate cyclase. The possibility that multiple classes of adenylate cyclase-linked PGE receptors might exist in nature is discussed.     

Prostaglandin production in myotube cultures. Influence on protein turnover.

The production of prostaglandins (PG) E2 and F2 alpha and their possible role in regulation of protein turnover in cultured skeletal-muscle cells were examined. Primary chick myoblasts and myotubes, and L8 myotubes, produced PGE2 and PGF2 alpha from endogenous arachidonic acid. PG production by all three cell types was increased manyfold by the addition of exogenous arachidonic acid. Arachidonate-stimulated PG production was inhibited by the addition of indomethacin (0.1 mM). When L8 and chick myotubes were treated with PGE2, PGF2 alpha, arachidonic acid (0.01 mM) or indomethacin (0.1 mM), no significant alterations in rates of protein synthesis or degradation were observed. Rates of protein synthesis and degradation in these cells were responsive to the addition of 10% fetal-bovine serum under identical experimental conditions. Thus, in contrast with incubated adult skeletal muscle, it appears that the production of prostaglandin metabolites from arachidonic acid is unrelated to regulation of protein turnover in cultured muscle cells.     

Inhibition of 15-hydroxy prostaglandin dehydrogenase and increase of prostaglandin E2: effect of sofalcone on rat gastric mucosa

The effect of sofalcone, an anti-ulcer agent, on gastric mucosal prostaglandin (PG) metabolism was studied. Gastric mucosal PGE2 was determined in rats in which PGE2 synthesis was inhibited by preadministration of indomethacin. Oral administration of sofalcone at doses of 200 and 400 mg/kg significantly inhibited the PG metabolizing enzyme, 15-hydroxy-PG-dehydrogenase (15-OH-PG-DH) activity and increased PGE2 contents in the rat gastric mucosa. The inhibition of 15-OH-PG-DH activity was accompanied by an increase of PGE2 contents up to 6 hours after the administration of sofalcone. These changes, however, were not observed 12 hours after its administration. Intraperitoneally administered sofalcone also inhibited 15-OH-PG-DH activity and increased PGE2 content. The inhibition of 15-OH-PG-DH activity by sofalcone was noncompetitive and uncompetitive against substrates NAD and PGE1, respectively. These results suggest that the increase of the gastric PGE2 level is mainly due to the inhibition of 15-OH-PG-DH activity, and this increase in PGE2 may be involved in the anti-ulcer effect of sofalcone.     

Role of cyclooxygenase-2 in Helicobacter pylori- induced gastritis in Mongolian gerbils

Cyclooxygenase (COX)-2 expression is induced in the gastric mucosa of Helicobacter pylori-infected patients, but its role remains unclear. We examined the effects of NS-398 and indomethacin on gastric pathology in H. pylori-infected Mongolian gerbils. COX-1 was detected in both normal and H. pylori-infected mucosa, whereas COX-2 was expressed only in the infected mucosa. PGE(2) production was elevated by H. pylori infection, and the increased production was reduced by NS-398, which did not affect PGE(2) production in normal mucosa. Indomethacin inhibited PGE(2) production in both normal and infected mucosa. Hemorrhagic erosions, neutrophil infiltration, lymphoid follicles, and epithelium damage were induced by H. pylori infection. NS-398 and indomethacin aggravated these pathological changes but did not increase viable H. pylori number. H. pylori-increased production of neutrophil chemokine and interferon-gamma was potentiated by NS-398 and indomethacin. Neither NS-398 nor indomethacin caused any pathological changes or cytokine production in normal animals. These results indicate that COX-2 as well as COX-1 might play anti-inflammatory roles in H. pylori-induced gastritis.     

The role of endogenous prostaglandins in the regulation of gastric secretion in rhesus monkeys

Prostaglandins (PG) are known to alter a variety of gastrointestinal functions, but the physiological role of endogenous PG remains unclear. This experiment was designed to evaluate changes in gastric secretion following both acute and chronic inhibition of PG synthesis with indomethacin (5 mg/kg s.c.). Gastric juice was collected by continuous aspiration in 8 conscious chair-adapted male rhesus monkeys following treatment with saline or indomethacin for one or four days. The gastric juice was analyzed for H+, Na+, K+, and Cl- concentrations. The amount of soluble mucus in the gastric juice was estimated using Alcian Blue dye binding of acidic glycoproteins and Periodic Acid Schiff reaction with neutral glycoproteins. PG levels were measured in the plasma and in biopsy samples of fundus, antrum and duodenum. Both one and four days of indomethacin significantly (p less than 0.05) decreased tissue PG levels in the fundus, antrum and duodenum. Plasma levels of PGF2 alpha were significantly (p less than 0.05) decreased after both one and four days of indomethacin, while PGE2 and 6-keto PGF1 alpha were significantly inhibited only after four days of indomethacin. Both acute and chronic inhibition of PG synthesis was accompanied by a decrease in the concentration of sodium and mucus in the gastric juice but by an increase in the output and concentration of hydrogen ion. These changes suggest a possible mechanism by which endogenous PG play a role in the regulation of gastric secretion and in the protection against gastrointestinal damage.     

An Ex Vivo Method for Evaluating Prostaglandin Synthetase Activity in Cortical Slices of Mouse Brain

The release of prostaglandin E2 (PGE2) from cortical slices of mice into incubation medium is followed for 3 h and compared to PGE2 levels in the corresponding slice. Immediately after decapitation, the rate of PGE2 released into the incubation medium is elevated and a steady low rate of spontaneous release is gained within 1-2 h of incubation. PGE2 synthesis and release is blocked in a dose-dependent manner by either indomethacin (3 X 10(-6) -3 X 10(-4) M) or flufenamic acid (2.6 X 10(-6) M) either when added in vitro or administered in vivo. Full recovery of PGE2 synthesis is reached after 3 h incubation of slices following in vivo administration of indomethacin. In vivo administration of flufenamic acid results in prolonged inhibition of PGE2 released in vitro. The inhibition of PGE2 released by indomethacin is also correlated with the slice PGE2 content. Administration of lipopolysaccharide (LPS), a known activator of phospholipase A2, results in a fivefold increase in PGE2 and a twofold increase in 6-keto-PGF1 alpha released into the medium. The release of thromboxane B2 is not affected by LPS.     

Influence of sulfasalazine, 5-aminosalicylic acid and sulfapyridine on prostanoid synthesis and metabolism in rabbit colonic mucosa

The effects of sulfasalazine (SASP) and its cleavage products 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP) on prostanoid (PG) synthesis and degradation were determined in rabbit colonic mucosa fractions in vitro. When the microsomal fraction was incubated with (14C)arachidonic acid, 10(-3) M SASP and SP did not markedly change the formation of labeled PGE2, PGF2 alpha, TxB2 and 6-keto-PGF1 alpha X 10(-4) M 5-ASA increased synthesis about 2.7-fold; the pattern of PG identified was unaltered. In the presence of the 10-fold higher concentration of 5-ASA, PG synthesis remained elevated at a similar level. When the cytosolic fraction was incubated with (3H)PGE2, 10(-3) M 5-ASA was without influence and 10(-3) M SP decreased slightly PGE2 breakdown. However, SASP showed a pronounced inhibitory effect at 10(-5) M and inhibition of PGE2 degradation was complete at 10(-3) M SASP. The results are compatible with the assumption that stimulation of PG synthesis by 5-ASA is related to therapeutic benefit in the treatment of ulcerative colitis.