Effects of 16,16-dimethyl prostaglandin E2 and indomethacin on leukotriene B4 and inflammation in rabbit colitis

The role of increased prostaglandin production and the effects of exogenous prostaglandins on inflammation of colitis are not established. We administered intramuscular 16,16-dimethyl prostaglandin E2 (DiM-PGE2) and indomethacin to rabbits with formalin immune-complex colitis and measured leukotriene B₄ (LTB4), prostaglandin E2 (PGE2) and severity of inflammation. DiM-PGE2 (100 ug/kg/BID) reduced LTB4 production (from 401±108 to 216±58 pg/ml) and infiltration of neutrophils, mucosal necrosis, inflammatory exudate and edema (all P<0.05). Other studies determined that parenteral DiM-PGE2 did not reduce the initial chemical damage induced by formalin, suggesting that cytoprotection of chemical insult was not the mechanism of suppressed inflamation in the immune colitis model. Indomethacin (10 mg/kg/d) reduced endogenous PGE2 by 80%, but did not reduce leukotriene production or inflammation. Exogenous prostaglandins cause a dose-dependent suppression of inflammation in experimental colitis, by a mechanism other than cytoprotection of chemical-induced mucosal injury.     

Indomethacin worsens and a leukotriene biosynthesis inhibitor accelerates mucosal healing in rat colitis.

The implication of leukotrienes as mediators of inflammation and recent evidence that prostaglandin analogues provide a beneficial effect during experimental colitis led to the speculation that (i) leukotrienes may be injurious and (ii) prostaglandins may be protective to colonic mucosa. Using a 2% acetic acid induced rat colitis model, we administered specific cyclooxygenase (indomethacin) and leukotriene biosynthesis inhibitors (MK-886) to examine the effect of endogenous prostaglandins and leukotrienes on colonic macroscopic injury, mucosal inflammation as measured by myeloperoxidase activity, net in vivo intestinal fluid absorption, and colonic PGE2 and LTB4 levels as measured by in vivo rectal dialysis. Indomethacin treatment prior to induction of colitis reduced endogenous mucosal PGE2 levels and exacerbated macroscopic ulceration and net fluid absorption. Addition of the exogenous PGE1 analogue misoprostol to the indomethacin-exacerbated colitis completely healed colonic macroscopic ulceration and inflammation but only partially improved fluid absorptive injury. The specific leukotriene biosynthesis inhibitor MK-886 administered prior to induction of colitis healed macroscopic ulceration and inflammation but not fluid absorptive injury. This mucosal reparative effect of MK-886 occurred at a dose that reduced colonic LTB4 synthesis while concomitantly enhancing PGE2 levels. Combining MK-886 with misoprostol treatment improved not only macroscopic ulceration and inflammation but also provided a synergistic effect that maintained net colonic fluid absorption at noncolitic control levels. These studies suggest that, during the induction of experimental colitis, endogenous prostaglandins play a pivotal role in providing a mucosal healing effect, and that leukotriene biosynthesis inhibitor may manifest part of its beneficial effect by shifting arachidonic acid metabolism towards production of prostaglandins.     

Inhibition by 16,16-dimethyl PGE2 of ethanol-induced gastric mucosal damage and leukotriene B4 and C4 formation

The effects of PGE2 and its stable analogue, 16,16 dimethyl PGE2 (dmPGE2) were investigated on ethanol-induced gastric mucosal haemorrhagic lesions and leukotriene formation in the rat. Exposure of the rat gastric mucosa to ethanol in-vivo, produced a concentration-related increase in the mucosal formation of leukotriene B4 (LTB4) which was correlated with macroscopically-apparent haemorrhagic damage to the mucosa. Challenge with absolute ethanol likewise enhanced the mucosal formation of LTC4 whereas the mucosal formation of 6-keto-PGF1 alpha was unaffected. Challenge of the rat gastric mucosa in vitro with ethanol induced a concentration-dependent increase in the formation of LTB4 and LTC4, but not 6-keto PGF1 alpha. Pretreatment with PGE2 (200-500 micrograms/kg p.o.) prevented the haemorrhagic mucosal damage induced by oral administration of absolute ethanol but not the increased formation of leukotrienes by the mucosa. In contrast, pretreatment with a high dose of dmPGE2 (20 micrograms/kg p.o.) prevented both the gastric mucosal lesions and the increase mucosal leukotriene formation. The differences in the effects of these prostaglandins may be related to the nature or degree of protection of the gastric mucosa. Thus, high doses of dmPGE2 but not PGE2 may protect the cells close to the luminal surface of the mucosa and hence reduce the stimulation of leukotriene synthesis by these cells.     

Dendritic cells produce eicosanoids,which modulate generation and functions of antigen-presenting cells

Eicosanoids have been shown to be potent immunoregulatory arachidonic acid (AA) metabolites. AA is the precursor of prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) which are able to modulate both inflammation and the immune response. Dendritic cells process and present antigens to T lymphocytes. They are highly specialized antigen-presenting cells (APC) and usually considered as 'professional APC'. In the present paper, we report some data on the biosynthetic capacity of murine APC from the bone marrow (BM-DCs) to produce AA metabolites. Using an ELISA we have observed that BM-DCs spontaneously produce both PGE(2) and LTB(4) whose production increased in response to bacterial lipopolysaccharides (LPS). In addition we found that LTB(4) production was twice as high when both COX pathways were blocked with selective COX-inhibitors. We have also investigated the effect of PGE(2) and LTB(4) on the in vitro generation of the so-called BM-DCs. Exogenous PGE(2) and LTB(4) added to bone marrow cultures inhibit and promote, respectively, BM-DC generation. PGE(2) added to the maturing BM-DCs reduces their MHC class-II expression.     

Effect of nimesulide on acetic acid- and leukotriene-induced inflammatory bowel disease in rats

Inflammatory bowel disease (IBD) is a relapsing inflammation of intestine, which is mediated by release of inflammatory mediators. Both cyclo-oxygenase product prostaglandin (PGE2) and lipo-oxygenase product leukotriene (LTB4), may contribute to the pathogenesis of the inflammatory response. Nimesulide, a preferential COX-2 inhibitor was evaluated for its efficacy against experimental colitis in two different models (acetic acid- and LTB4-induced IBD) in rats. Inflammatory response was induced by intrarectal single administration of acetic acid or LTB4. Nimesulide (9 and 18 mg/kg, p.o.) significantly prevented development of inflammatory changes, decreased myeloperoxidase (MPO) activity, and also restored the altered contractility response of the isolated colon segment to KCl. The results suggested the involvement of both cyclo-oxygenase (COX) and lipo-oxygenase-mediated proinflammatory agents in colonic inflammatory process associated with IBD. Further, this study suggests that such therapeutic interventions may be of value in the treatment of IBD.     

The effect of essential fatty acid deficiency on eicosanoid production in the inflamed rat colonic mucosa

Eicosanoids are potent mediators of inflammation and are synthesized in increased quantity in active ulcerative colitis. To elucidate the role of prostaglandin E2, thromboxane A2, prostaglandin I2, and leukotriene B2 in acute chemical colitis induced by 4% acetic acid, we utilized an animal model which has a deficiency of arachidonic acid, the precursor of eicosanoids due to an essential fatty acid deficient diet. Forty-eight hours after colitis was induced, mucosal synthesis of the cyclooxygenase products, prostaglandin E2, thromboxane A2, and prostaglandin I2, was significantly decreased in essential fatty acid deficient rats compared to normal controls. However, the 5-lipoxygenase product, leukotriene B4, was not different between groups. The decrease in cyclooxygenase products did not correlate with any change in the severity of colonic inflammation as assessed by gross morphology, histology, or myleoperoxidase activity. Thus inhibition of formation of the cyclooxygenase products of arachidonate metabolism does not appear to improve the degree of inflammation under the experimental conditions employed in this study.     

Oxidative stress and lipid mediators induced in alveolar macrophages by ultrafine particles

In ambient aerosols, ultrafine particles (UFP) and their agglomerates are considered to be major factors contributing to adverse health effects. Reactivity of agglomerated UFP of elemental carbon (EC), Printex 90, Printex G, and diesel exhaust particles (DEP) was evaluated by the capacity of particles to oxidize methionine in a cell-free in vitro system for determination of their innate oxidative potential and by alveolar macrophages (AMs) to determine production of arachidonic acid (AA), including formation of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), reactive oxygen species (ROS), and oxidative stress marker 8-isoprostane. EC exhibiting high oxidative potential induced generation of AA, PGE2, LTB4, and 8-isoprostane in canine and human AMs. Printex 90, Printex G, and DEP, showing low oxidative capacity, still induced formation of AA and PGE2, but not that of LTB4 or 8-isoprostane. Aging of EC lowered oxidative potential while still inducing production of AA and PGE2 but not that of LTB4 and 8-isoprostane. Cellular ROS production was stimulated by all particles independent of oxidative potential. Particle-induced formation of AA metabolites and ROS was dependent on mitogen-activated protein kinase kinase 1 activation of cytosolic phospholipase A2 (cPLA2) as shown by inhibitor studies. In conclusion, cPLA2, PGE2, and ROS formation was activated by all particle types, whereas LTB4 production and 8-isoprostane were strongly dependent on particles' oxidative potential. Physical and chemical parameters of particle surface correlated with oxidative potential and stimulation of AM PGE2 and 8-isoprostane production.     

Effects of n-3 polyunsaturated fatty acids on indomethacin-induced changes in eicosanoid production and blood flow in the gastric mucosa of rats

We investigated the effects of n-3 polyunsaturated fatty acids (PUFAs) on non-steroidal anti-inflammatory drug (NSAID)-induced changes in microcirculation and eicosanoid production in the gastrointestinal mucosa. We measured gastric mucosal blood flow using laser Doppler flowmetry, assessed the fatty acid composition in the mucosal phospholipids, and quantified the production of prostaglandin E2 (PGE2), leukotriene B4, and leukotriene C4 (LTB4 and C4) from the mucosa with the stimulation of calcium ionophore 20 min after an injection of indomethacin or vehicle in rats fed a diet containing different compositions of alpha-linolenic acid. Four weeks after the initiation of the test diet the arachidonic acid level in gastric mucosal phospholipids was significantly lower in the perilla group than in the other three groups. Conversely, alpha-linolenic acid and eicosapentaenoic acid (EPA) were significantly higher in the perilla group than in the other three groups. The percent of gastric mucosal blood flow in the three groups administered indomethacin were significantly lower than that in the control group injected with vehicle alone. The percent of gastric mucosal blood flow in the perilla group was significantly higher than that in the corn group. LTB4 and LTC4 production from the gastric mucosa in the soybean and corn groups were significantly higher than those in the control group, and the LTC4 production in the perilla group was significantly lower than that in the corn group. There were no significant differences in PGE2 production among the four groups. Our results suggest that alpha-linolenic acid affectively suppressed the indomethacin-induced decreases in gastric mucosal blood flow by increasing EPA and decreasing the levels of arachidonic acid and LTC4 in the gastric mucosa.     

Failure of prostaglandins E1 and E2 to alter canine gastric mucosal cyclic nucleotides.

The action of prostaglandins and indomethacin on gastric mucosal cyclic nucleotide concentrations was evaluated in 18 anesthetized mongrel dogs. Prostaglandins E1 (PGE1) and E2 (PGE2) (25 microgram/kg bolus, then 2 micrograms/kg/min) were administered both intravenously (4 experiments; femoral vein) and directly into the gastric mucosal circulation (10 experiments; superior mesenteric artery). The possible synergistic effect of pre-treatment and continuous arterial infusion of indomethacin (5 mg/kg bolus for 5 min, then 5 mg/min), a prostaglandin synthetase inhibitor, with PGE2 was studied in 4 experiments. Antral and fundic mucosa were biopsied and measured by radioimmunoassay for cyclic nucleotides. Doses of PGE1 and PGE2 which inhibited histamine-stimulated canine gastric acid secretion did not significantly alter antral or fundic mucosal cyclic nucleotide concentrations. Concomitant infusion of PGE2 with indomethacin did not potentiate the mucosal nucleotide response compared to PGE2 alone. These studies fail to implicate cyclic nucleotides as mediators of the inhibitory acid response response induced by PGE1 or PGE2 in intact dog stomach.     

Effect of Cleome arabica leaf extract, rutin and quercetin on soybean lipoxygenase activity and on generation of inflammatory eicosanoids by human neutrophils

The effects of Cleome arabica leaf extract, rutin and quercetin on soybean lipoxygenase (Lox) activity and on calcium ionophore (A23187)-stimulated generation of the leukotriene B4 and prostaglandin E2 by human neutrophils were examined. The extract (25 microg/ml), rutin (25 microM) and quercetin (25 microM) inhibited LTB4 synthesis at all concentrations of A23187 used. The extract at 1-100 microg/ml and rutin at 1-100 microM inhibited LTB4 generation by neutrophils stimulated with 1 microM A23187 by about 50%. PGE2 production in response to different concentrations of A23187 was affected in a biphasic manner by the extract and rutin. Quercetin at 1-100 microM caused concentration-dependent inhibition of LTB4 and PGE2 production. The extract, rutin and quercetin caused concentration-dependent inhibition of soybean Lox activity. These results indicate that rutin, quercetin and an extract of C. arabica containing these compounds inhibit Lox activity, consequently decreasing LTB4 production. Thus, these compounds or extracts containing them may be beneficial for the treatment of inflammatory conditions, particularly those characterised by excessive leukotriene generation.     

Effect of 5-lipoxygenase inhibitor against lipopolysaccharide-induced hypothermia in mice

Bacterial endotoxin produces sepsis associated with alterations in body temperature (fever or hypothermia). The intraperitoneal administration of bacterial endotoxin, lipopolysaccharide (LPS; 50 microg/mouse) led to a decrease in colonic temperature starting 1 hr after the injection. The hypothermic effect was accompanied by a significant increase in hypothalamic leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) levels. 5-lipoxygenase inhibitor, zileuton (200 and 400 mg/kg, po) administered 30 min before LPS challenge significantly prevented hypothermia. However, non-selective cyclooxygenase inhibitor, indomethacin (10, 20 mg/kg, po) did not reverse the hypothermic response. Further, pretreatment of mice with zileuton prevented LPS-stimulated increase in hypothalamic LTB4 levels and caused a relatively small increase in PGE2 levels. Indomethacin had no effect on LTB4 levels but it reduced PGE2 levels. These results suggest a possible involvement of leukotrienes in LPS-induced hypothermia and the potential protective role of 5-lipoxygenase inhibitors in endotoxemia.     

Regulation of interleukin 1 production by mouse peritoneal macrophages. Effects of arachidonic acid metabolites, cyclic nucleotides, and interferons

The effects of prostaglandin E2 (PGE2), cyclic nucleotides, leukotriene B4 (LTB4), and interferons on interleukin 1 (IL 1) production by lipopolysaccharide (LPS)-stimulated C3H/HeNCrl mouse peritoneal macrophages were studied. IL 1 production was inhibited by PGE2, the adenosine 3':5'-monophosphate analog dibutyryl cAMP, the cAMP agonist isoproterenol, and the phosphodiesterase inhibitor isobutylmethylxanthine. These agents were more inhibitory when added early in the latent phase of IL 1 synthesis following stimulation with LPS rather than just prior to release of IL 1 into the medium. Production of both the intracellular and extracellular forms of IL 1 was blocked by PGE2 and cAMP. Suppression of LPS-induced IL 1 production by PGE2 was prevented by leukocyte alpha-interferon. Moreover, alpha-interferon augmented LPS-induced IL 1 production but did not stimulate IL 1 production in the absence of LPS. Immune gamma-interferon markedly inhibited LPS-stimulated IL 1 production. The lipoxygenase inhibitor eicosa-5,8,11,14-tetraynoic acid suppressed, whereas 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline augmented, LPS-induced IL 1 production. The opposing effects of these agents suggested that lipoxygenase metabolites do not act as inducers of IL 1 production. Purified LTB4 did not stimulate base-line or augment LPS-induced IL 1 production (both intracellular and extracellular forms). Moreover, calcium ionophore A23187 (a lipoxygenase activator) did not stimulate IL 1 production, alone or in combination with LTB4. Thus, net IL 1 production by macrophages may be regulated by a balance between the effects of PGE2, cAMP, alpha-interferon, and gamma-interferon, but not LTB4.     

Pharmacological characterisation of arthritis induced by Bothrops jararaca venom in rabbits: a positive cross talk between bradykinin, nitric oxide and prostaglandin E2

BACKGROUND: Our previous results showed that nitric oxide (NO) and bradykinin (BK) mediate the arthritis induced by Bothrops jararaca venom (BjV) in rabbits. In this study, we investigated the contribution of each receptor of BK as well as the inter-relationship between NO and eicosanoids in BjV-induced arthritis. METHODS: The arthritis was induced in rabbits with 16 microg of BjV injected intra-articularly. Prostaglandin E2 (PGE2), thromboxane B2 (TxB2), leukotriene B4 (LTB4) (radioimmunoassay) and nitrite/nitrate concentrations (NO2/NO3) (Griess reaction) were evaluated in the synovial fluid 4 h later. The animals were prior treated with NO synthase inhibitor (L-NAME; 20 mg/kg/day for 14 days), the B2 antagonist of BK (HOE-140) and the B1 antagonist of BK (des-Arg9[Leu8]-bradykinin), both at a dose of 0.3mg/kg, 30 min prior to the venom injection. RESULTS: Data show that L-NAME and HOE-140 treatment were equally able to reduce PGE2 and NO2/NO3 levels without interfering with TxB2 and LTB4 production. On the contrary, the B1 antagonist of BK inhibited TxB2 and LTB4 production, and did not alter PGE2 and NO metabolites levels in the inflamed joint. DISCUSSIONS: The results presented clarify the contribution of the kinin system, mainly through the B2 receptor, to the local inflammatory response induced by BjV, as well as its positive interaction with PGE2 and NO production.     

Release of leukotrienes C4 and B4 and prostaglandin E2 from human monocytes stimulated with aggregated IgG, IgA, and IgE

Purified human peripheral blood monocytes were stimulated with aggregated human myeloma proteins of different classes or the calcium ionophore A23187 and the release of leukotrienes C4 and B4 (LTC4, LTB4), and prostaglandin E2 (PGE2) into the supernatant was determined. The ionophore induced release of 10 +/- 5 ng LTC4/10(6) cells and 25 +/- 8 ng LTB4/10(6) cells. Aggregated IgG, IgA, and IgE, but not IgM or monomeric immunoglobulins (Ig), induced release of LTC4 and LTB4 that was approximately 10 to 20% of that induced by ionophore. In addition, IgG, IgA, and IgE, but not IgM, induced release of PGE2 (range 0.015 to 0.22 ng/10(6) cells). Aggregated Ig induced LTC4, LTB4, and PGE2 release in a dose-dependent manner; maximal leukotriene (LT) release was observed by 30 min, in contrast to PG release, which continued to increase up to 2.5 hr. Both ionophore- and Ig-induced LTC4 and LTB4 release were completely inhibited by removal of calcium from the media and by preincubation of cells with nordihydroguaiaretic acid. Indomethacin inhibited Ig-induced PGE2 release by 80%. Phagocytosis of the Ig aggregates was not required for LT or PGE2 release, since release was not inhibited by cytochalasin B. Release of LTC4, LTB4, and PGE2 induced by IgG, IgA, and IgE, but not IgM, correlated with the presence or absence of monocyte Fc receptors (FcR) as determined by rosette assays. The data suggest that IgG, IgA, and IgE immune complexes mostly likely induce monocyte arachidonic acid metabolism via cross-linking of FcR. The ability of monocytes to release eicosanoids in the absence of phagocytosis suggests that interaction of monocytes with immobilized immune complexes, such as those deposited in blood vessel walls or glomerular basement membranes, could initiate metabolism of arachidonic acid by monocytes. Such a mechanism could contribute to inflammatory reactions characterized by mononuclear cell infiltrates.     

The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein

Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E2 (PGE2) or leukotriene B4 (LTB4) by subchondral osteoblasts, PGE2 levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible for the shunt from prostaglandins to leukotrienes. FLAP mRNA levels varied in low and high OA groups compared with normal, whereas mRNA levels of 5-LO were similar in all osteoblasts. Selective inhibition of cyclooxygenase-2 (COX-2) with NS-398-stimulated FLAP expression in the high OA osteoblasts subgroup, whereas it was without effect in the low OA osteoblasts subgroup. The addition of PGE2 to the low OA osteoblasts subgroup decreased FLAP expression but failed to affect it in the high OA osteoblasts subgroup. LTB4 levels in OA osteoblasts were stimulated about twofold by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plus transforming growth factor-beta (TGF-beta), a situation corresponding to their effect on FLAP mRNA levels. Treatments with 1,25(OH)2D3 and TGF-beta also modulated PGE2 production. TGF-beta stimulated PGE2 production in both OA osteoblast groups, whereas 1,25(OH)2D3 alone had a limited effect but decreased the effect of TGF-beta in the low OA osteoblasts subgroup. This modulation of PGE2 production was mirrored by the synthesis of COX-2. IL-18 levels were only slightly increased in a subgroup of OA osteoblasts compared with normal; however, no relationship was observed overall between IL-18 and PGE2 levels in normal and OA osteoblasts. These results suggest that the shunt from the production of PGE2 to LTB4 is through regulation of the expression of FLAP, not 5-LO, in OA osteoblasts. The expression of FLAP in OA osteoblasts is also modulated differently by 1,25(OH)2D3 and TGF-beta depending on their endogenous low and high PGE2 levels.     

The effect of cocaine on gastric mucosal PGE(2), LTC(4) and ulcerations

The association between cocaine use and acute gastroduodenal perforation is known. The effect of cocaine and stress on gastric mucosal ulceration and the levels of prostaglandin E(2) (PGE(2)) and leukotriene C(4) (LTC(4)) was studied in 40 Sprague-Dawley rats. Controls received intraperitoneal (i.p.) saline, ten received i.p. cocaine (35 mg/kg), ten were stressed by the cold restraint method, and ten had i.p. cocaine and stress. Cocaine alone did not induce ulceration, but decreased PGE(2) levels. Stress alone caused ulceration, but was not associated with a change in either PGE(2) or LTC(4) levels. When combined with stress, however, cocaine caused a three-fold increase in ulceration and a significant increase in PGE(2) and LTC(4) levels. Stress may predispose the cocaine addict to loss of gastroduodenal mucosal integrity, which is related to an imbalance of PGE(2) and LTC(4) synthesis.     

Interaction between prostaglandin E2 and leukotriene D4 on the excretion of electrolytes by the dog kidney in vivo

Recent experiments indicate that prostaglandin E2 potentiates the vasodilatory properties of leukotrienes in the skin microcirculation. The present experiments were undertaken to study the effect of leukotriene D4 and prostaglandin E2 on renal hemodynamics and urinary electrolytes in the dog. Experiments were performed in three groups of anesthetized Mongrel dogs: the first group was studied under hydropenia, whereas the two remaining groups were studied during water diuresis with (Group 3) or without indomethacin (Group 2). LTD4 (100 ng/min) and PGE2 (3 ug/min) were infused in the left renal artery to minimize systemic effects of these compounds. LTD4 alone failed to influence urinary sodium excretion in all 3 groups. In Group 1, urinary sodium increased from 77 +/- 6 to 393 +/- 74 uEq/min during PGE2, and further increased to 511 +/- 52 uEq/min during LTD4 + PGE2. No change occurred in the contralateral right kidney. In this group, glomerular filtration as well as renal plasma flow were not statistically influenced. In Group 2, the same phenomenon was observed for urinary sodium. The combined infusion of LTD4 + PGE2 increased urinary sodium without significant changes in glomerular filtration and renal plasma flow. Finally, in Group 3, indomethacin was shown to reduce the natriuretic effects of LTD4 and PGE2: during PGE2 alone, urinary sodium increased from 90 +/- 14 to 260 +/- 66 uEq/min, and only rose from 80 +/- 10 to 175 +/- 19 uEq/min during the combined infusion of LTD4 and PGE2. In groups 2 and 3, free water clearance was utilized as an index of sodium chloride reabsorption in the thick ascending limb: this parameter increased from 2.35 +/- 0.25 to 4.70 +/- 0.30 ml/min, while urinary volume was increasing from 3.55 +/- 0.25 to 10.05 +/- 0.65 ml/min, during LTD4 + PGE2. Indomethacin, administered in Group 3, (3 mg/kg/hr) again abolished the effect of combined PGE2 + LTD4. These results indicate a potentiating effect of leukotriene D4 on the PGE2-induced natriuresis in the anesthetized dog. These phenomena occurred in the absence of significant changes in renal hemodynamics, therefore suggesting a direct tubular effect of these arachidonic acid metabolites. Finally, the water diuresis experiments suggest a proximal site of action of PGE2 and LTD4.     

Quantitation of arachidonic acid metabolites in small tissue biopsies by reversed-phase high-performance liquid chromatography

Arachidonic acid metabolites exert a variety of distinct biological effects on the initiation and resolution of inflammatory diseases and their measurements in tissue can be critical to evaluate their regulatory function during the course of inflammation and to supplement in vitro experiments. The aim of this study was the detection and quantitative analysis of four arachidonic acid metabolites in small-sized biopsies of human periodontal tissues. The biopsies were homogenized and injected directly into a single analytical column of a RP-HPLC system. Detection was performed by a photodiode array detector. Calibration was established by dilutions of authentic standards of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). A total of 38 specimens weighing between 19 and 191 mg (wet tissue) were analyzed (mean = 59.9 +/- 30.2 mg). The detection limits were 1 pg for LTB4 and 12-HETE, 0.5 pg for 15-HETE, and 10 ng for PGE2. The concentrations of PGE2 and LTB4 were significantly higher in inflamed than in healthy periodontal tissues (P = 0.0079; P = 0. 0114). 12-HETE was detected in one biopsy (30 pg/g); 15-HETE was not detected. This method of homogenization, extraction, and analysis of arachidonic acid metabolites by RP-HPLC appears to be well suited for studies of human oral biopsies. Only small tissue samples and minimal laboratory equipment were required for a sensitive analysis.     

In vitro locomotion of allosensitized T lymphocyte clones in response to metabolites of arachidonic acid is subset specific

The effects of the arachidonic acid metabolites prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) on the in vitro random migration of cloned murine T lymphocytes (derived from limiting dilution analysis of a C57BL/6 anti-DBA/2 mixed leukocyte culture) were examined. Experiments were also performed to study the effects of the cyclooxygenase inhibitor indomethacin on both random lymphocyte migration and lymphocyte migration in the presence of PGE2. The responses of cloned lymphocytes to PGE2 and LTB4 were compared with those of unsensitized lymph node lymphocytes. PGE2 at 100 ng/ml significantly inhibited (p less than 0.001) the in vitro migration of helper clones of T lymphocytes, but had no effect on random migration of cytotoxic T cells or helper independent cytotoxic (HIT) cloned cells. In contrast, LTB4 significantly (p less than 0.001) enhanced the random locomotion of helper, cytotoxic, and "HIT" cloned cells at 0.1 and 0.3 ng/ml. The effects of both PGE2 and LTB4 were found to be completely reversible by cell washing. Indomethacin (10(-7) M) did not alter random migration of any of the clones, and in particular, did not affect the inhibition of helper lymphocyte migration induced by PGE2. Unsensitized bulk lymph node lymphocyte migration was not affected by either PGE2 or LTB4. The results suggest that modulation of lymphocyte locomotor function by environmental stimuli may depend on cellular activation, and the locomotor responses of activated lymphocytes to arachidonic acid metabolites may be subset specific.     

Ciliary responsiveness in allergic and nonallergic airways

Allergic asthma is associated with airway (smooth muscle) hyperresponsiveness to several chemical mediators of anaphylaxis; however, it is not known whether this is accompanied by mucociliary hyperresponsiveness. The purpose of this study was therefore to determine if airway ciliary activity, a component function of mucociliary clearance, exhibits exaggerated responses to prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), and leukotriene D4 (LTD4) in allergic sheep when compared with nonallergic sheep, and the effects of LTD4 are direct or involve the generation of cyclooxygenase products of arachidonate metabolism. Ciliary beat frequency (CBF) was measured in a perfusion chamber with a microscopic technique using tracheal epithelial cells obtained from brushing of "allergic" (positive cutaneous reaction and previous bronchospastic response to inhaled specific antigen) and "nonallergic" (negative cutaneous reaction, no previous inhalation challenge with antigen) sheep. Mean base-line CBF was not different among the groups; PGE1, PGE2, and LTD4 induced dose-dependent increases in CBF, and these increases were not different in allergic and nonallergic sheep. At the highest agonist concentration the mean increase in CBF from base line varied between 13 and 16% (P less than 0.05). The ciliostimulatory effect of LTD4 was significantly blunted by both the sulfidopeptide leukotriene antagonist FPL-55712 and the cyclooxygenase inhibitor indomethacin. These results suggest that allergic sheep fail to exhibit ciliary hyperresponsiveness to selected chemical mediators of anaphylaxis and the ciliostimulatory effect of LTD4 depends on the activation of cyclooxygenase and possibly the generation of prostaglandins.