Influence of angiotensins (I, II, & III), bradykinin and arachidonic acid on renomedullary PGE production in vitro.

Renomedullary tissue from rabbit or rat was incubated with angiotensin I, II, III, arachidonic acid, bradykinin, indomethacin and meclofenamate to study their effect on PGE2 production. Arachidonic acid and bradykinin enhanced PGE2 production significantly. Indomethacin and meclofenamate inhibited PGE2 production by more than 70%. Angiotensin I, II and III did not influence PGE2 production. These results suggest that bradykinin and arachidonic acid stimulate PGE2 production by a direct cellular action whereas the angiotensins do not.     

Dissociation of aldosterone and prostaglandin biosynthesis in the rat adrenal glomerulosa

The relationship between aldosterone production and prosta-glandin E2 synthesis was evaluated using the responses of isolated rat adrenal glomerulosa cells to angiotensin II, ACTH and potassium. Simultaneous PGE2 and aldosterone measurements were made during timed incubations with these stimuli, and in incubations with arachidonic acid, meclofenamate, indomethacin, and aminoglutethamide. PGE2 and aldosterone production were assessed by radioimmunoassay. We were not able to demonstrate stimulation of PGE2 by angiotensin II, ACTH, or potassium despite significant increments in aldosterone production with these stimuli. Arachidonic acid enhanced PGE2 synthesis, but had no effect on aldosterone realease. Indomethacin and meclofenamate inhibited aldosterone secretion. Aminoglutethimide depressed aldosterone production, but had little effect on PGE2 levels in the media. These studies demonstrate that dienoic prostaglandins play no direct role in aldosterone production stimulated by angiotensin II, ACTH, or potassium in rat adrenal glomerulosa cells. Since inhibitors of cyclo-oxygenase decreased aldosterone synthesis, it is possible that fatty acids other than arachidonic acid may be cyclo-oxygenated to products which regulate aldosterone production.     

Cholera toxin-induced PGE(2) activity is reduced by chemical reaction with L-histidine

Mediators of cholera toxin (CT)-induced fluid secretion include 3',5'-adenosine monophosphate (cAMP), prostaglandin E(2) (PGE(2)), and 5-hydroxytryptamine (5-HT). Administration of L-histidine significantly reduced the net secretory response of the small intestine of mice challenged with CT and reduced the capacity of PGE(2) to stimulate Na+ transport in Ussing chambers. We demonstrated that L-histidine chemically modified the structure of PGE(2) but had no direct effect on cAMP or 5-HT. L-Histidine and imidazole reacted with PGE(2) in vitro in cell-free mixtures incubated at 37 degrees C and pH 7.0 under an atmosphere of N(2) with the formation of PGE(2)-imidazole and PGE(2)-histidine covalent adducts. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) analysis of the purified adduct showed that imidazole catalyzed the dehydration of PGE(2). A Michael adduct then was formed between C11 of 11-deoxy-Delta(10) PGE(2) (PGA(2)) and the tau nitrogen in the imidazole ring of L-histidine. Importantly, the isolated PGE(2)-imidazole and PGE(2)-histidine adducts inhibited CT-induced fluid loss and cAMP accumulation in mouse intestinal loops. The protection provided by PGE(2)-imidazole, PGE(2)-histidine, and L-histidine against intestinal fluid loss could provide a basis for future therapy against cholera.     

Arachidonic acid evokes epithelium-dependent relaxations in canine airways

Responses to arachidonate were examined in rings with and without epithelium of lobar, segmental, and subsegmental canine bronchi. Arachidonate evoked epithelium-dependent relaxations, which were less pronounced in subsegmental bronchi and abolished by indomethacin and meclofenamate. Nordihydroguairetic acid (NDGA) and nafazatrom reduced epithelium-dependent relaxations only in lobar but unmasked epithelium-independent relaxations to arachidonate in all bronchi. Prostaglandin E2 and prostacyclin relaxed all tissues similarly. In lobar bronchi without epithelium, basal release of prostaglandin E2 was reduced by indomethacin but unaffected by NDGA. Arachidonate augmented prostaglandin E2 release more in subsegmental than in lobar bronchi with epithelium; in bronchi without epithelium the rise was absent (lobar) or attenuated (subsegmental). Arachidonate augmented the release of 6-ketoprostaglandin F1 alpha more in lobar bronchi with than without epithelium; this was inhibited by indomethacin, but not NDGA. Thus arachidonate releases prostaglandin E2 (possibly produced by cyclooxygenase inaccessible to inhibitors and activated by lipoxygenase products) but not prostacyclin from the epithelium. Heterogeneity in response to arachidonate is not due to different sensitivity to, or production of, prostaglandins.     

Effects of cyclooxygenase and lipoxygenase inhibition on basal- and serotonin-induced ion transport in rat colon

The purpose of this study was to determine the effect of a selective cyclooxygenase (COX)-2 inhibitor as compared to non-selective COX and lipoxygenase (LOX) inhibitors in rat colon. Basal- and serotonin (5-hydroxytryptamine, 5-HT)-induced electrogenic ion transport (short circuit current, SCC), prostaglandin E2 (PGE2) release and histological characteristics were measured. Muscle-stripped mucosal sheets of the proximal and distal segment of rat colon were investigated by employing the Ussing chamber technique, radioimmunoassays for PGE2 and light microscopy examinations for control of tissue integrity. 5-HT and PGE2 both induced a concentration-dependent increase in SCC by activation of multiple receptors. The response to 5-HT was bumetanide-sensitive. Neither the non-selective COX inhibitor piroxicam, nor the selective COX-2 inhibitor SC-'236, altered basal- SCC or 5-HT-induced SCC. Indomethacin reduced both basal- and 5-HT-induced SCC in both segments. Nordihydroguaiaretic acid reduced the 5-HT-induced increase in SCC, but did not change basal SCC. 5-HT-induced a concentration-dependent release of PGE2. Only high concentrations of piroxicam and indomethacin reduced basal PGE2 release and 5-HT-induced PGE2 release. Histological examination of the specimens demonstrated only minor changes following mounting in chambers. There were no apparent differences in the morphology following treatment with COX or LOX inhibitors. These results suggest that in rat colon only the COX-1 enzyme is expressed under basal conditions. Furthermore, data suggest neither the COX-1 nor the COX-2 enzyme to be of major importance for 5-HT-induced ion transport in rat colon in vitro. In conclusion, this study supports 5-HT as a mediator of chloride secretion by activating several receptor subtypes and the LOX enzyme, releasing mediators such as leucotrienes.     

Prostaglandin E2 modulates a non-inactivating potassium current in rat neurohypophyseal nerve terminals.

A non-inactivating voltage dependent K+ channel current was observed in neuro-hypophyseal nerve terminals. This current was sensitive to inhibition by 4-aminopyridine and tetraethyl ammonium chloride, but was not sensitive to inhibition by alpha- or beta-dendrotoxin. Prostaglandin E2 (PGE2) modulated the voltage-dependent K+ channel, through a receptor-mediated process, as indicated by meclofenamate sensitivity, and this involved the activation of G protein(s), as indicated by sensitivity to guanosine-5'-O-(2-thiodiphosphate) (GDPfS). After short periods of incubation (e.g. 5 min), PGE2 increased the non-inactivating current. Following longer incubation periods with PGE2 (e.g. 20 min), the non-inactivating current declined. Forskolin and the cyclic adenosine monophosphate (AMP) analogs 8-bromo- and dibutyryl cyclic AMP, and Sp-cyclic AMPs inhibited the current, but did not mimic the increase in current caused by PGE2. Also, the cyclic AMP antagonist Rp-cyclic AMPs did not block the increase in current induced by PGE2. These results indicate that activation of cyclic AMP-dependent protein kinase (PKA) is not involved in mediating the stimulatory actions of PGE2. These observations provide evidence that PGE2 may contribute to the regulation of hormone release from the posterior pituitary by modulating K+ channels. However, the post-receptor mechanisms of subcellular signal transduction underlying this effect remain unknown.     

Urinary prostaglandin E excretion: Effect of chronic alterations in sodium intake and inhibition of prostaglandin synthesis in the rabbit

On the basis of acute experiments in animals, a role for prostaglandin E (PGE) in the regulation of urinary sodium excretion has been suggested. Limited information is available, however, concerning the possible role of PGE in chronic adjustments to sodium intake. These studies were designed to determine whether chronic changes in sodium balance would modify renal PGE excretion and whether partial inhibition of prostaglandin synthesis would after the ability of the kidney to adjust to an alteration in sodium intake. Thus, we measured sodium and PGE excretion in rabbits on chronic high and low salt diets before and after inhibition of prostaglandin synthesis with indomethacin or meclofenamate. Although the alterations in salt intake resulted in large changes in sodium excretion there was no significant change in urinary PGE excretion. After administration of either indomethacin or meclofenamate for several days there was a significant fall in PGE excretion, but no significant change in sodium excretion. These results suggest that in the rabbit 1) chronic changes in sodium excretion can occur without modifying PGE excretion (and presumably renal PGE synthesis) and 2) inhibition of PGE synthesis does not impair the kidney's ability to adjust to a chronic high or low sodium intake.     

Modulation by opiates of small intestinal prostaglandin E2 and 3',5'cyclic adenosine monophosphate levels and of indomethacin-induced ulceration in the rat.

We studied the effect of parenteral morphine and naloxone administration on intestinal mucosal Prostaglandin E2 (PGE2) and 3',5' cyclic adenosine monophosphate (cAMP) levels and on indomethacin-induced intestinal ulceration in the rat. Compared to the control group, morphine significantly decreased whereas naloxone markedly increased both PGE2 and cAMP mucosal levels, respectively. Morphine or naloxone alone did not cause mucosal injury. However, when given with indomethacin, morphine significantly potentiated the ulcerogenic effect of indomethacin while naloxone exerted a protective effect. These results suggest that opioid peptides may play a role in modulation of intestinal mucosal PGE2 and cAMP levels. In addition, enhancement of indomethacin-induced ulcer formation by morphine and amelioration by naloxone might be in part mediated through their effect on mucosal PGE2 and cAMP levels.     

Na+ influx and cell growth in cultured human fibroblasts. Effect of indomethacin

The effect of indomethacin on Na+ influx and cell growth in human diploid fibroblasts (HSWP) has been investigated. It was found that both indomethacin and aspirin block serum-stimulated Na+ influx in a dose-dependent manner (Ki = 0.34 +/- 0.04 mM and 11 +/- 1 mM respectively) while having no effect on influx of Na+ in the absence of serum. The Ki for inhibition of [3H]thymidine incorporation into HSWP cells (0.28 +/- 0.02 mM) closely correlated with the Ki for inhibition of Na+ influx. The onset of action of indomethacin is rapid (within 2 min) and inhibition of Na+ flux is readily reversed (within 5 min). Other workers have reported that indomethacin is cytostatic for human fibroblasts presumably via a slowly developing inhibition of "A" system amino acid transport [6]; however, present results indicate that inhibition of Na+ influx in HSWP cells occurs much more rapidly than the inhibition of amino acid transport observed in other human foreskin fibroblasts and therefore may be more closely related to the primary cellular locus of indomethacin action.     

炎症介质对离体大鼠肠系膜动脉降钙素基因相关肽释放的影响

本工作目的是在离体大鼠肠系膜动脉床灌流模型上,观察几种常见炎症介质：前列腺素Ｅ２（ＰＧＥ２）、缓激肽（ＢＫ）、组胺（ＨＩＳ）、血小板活化因子（ＰＡＦ）及５－羟色胺（５－ＨＴ）对血管周围感觉神经介质ＣＧＲＰ释放的直接影响。结果显示：ＰＧＥ２（１－１００μｍｏｌ／Ｌ）和ＢＫ（５－１０μｍｏｌ／Ｌ）能引起大鼠肠系膜动脉床时间和浓度依赖性地释放ＣＧＲＰ。ＨＩＳ,ＰＡＦ和５－ＨＴ则未见明显作用。结果提示,ＰＧＥ２与ＢＫ可能是引起血管周围感觉神经兴奋和ＣＧＲＰ释放的主要炎症介质。     

Mechanism of steroid action in inflammation: Inhibition of prostaglandin synthesis and release

Acute arthritis was induced by injection of cell-free extract of group A Streptococci into the knee joints of mature male rats. Slices of control and inflamed synovia were incubated for 30 to 240 minutes and the rate of prostaglandin E (PGE) released into the medium was measured by radioimmunoassay. PGE release from inflamed synovia was 5–8 fold higher than that in normal tissue. Incubation of inflamed synovia with corticosterone acetate, dexamethasone or prednisone (100 μg/ml) for one or four hours reduced PGE release by 33% and 55% respectively. Lower concentrations of corticosterone (10 – 30 μg/ml) were ineffective. Aldosterone and progesterone (100 μg/ml) had no effect on PGE release throughout the incubation period. Chloroquine (10 μg/ml) inhibited PGE release from inflamed synovia by 50%. Indomethacin (1 μg/ml) abolished PGE release by 90%. Corticosterone, dexamethasone and prednisone reduced PGE content of inflamed synovia by approximately 45% during a 4-h incubation period. Aldosterone and progesterone were ineffective, while indomethacin reduced PGE content by 70%. The suppressive action of corticosterone on PGE release was prevented by addition to the medium of arachidonic acid (2 μg/ml). By contrast, the inhibitory action of indomethacin was not affected by provision of exogenous substrate. We suggest that glucocorticosteroids reduce PGE release by limiting the availability of the substrate for prostaglandin biosynthesis, and this may well explain some of their anti-inflammatory properties.     

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.     

Characterization of prostaglandin production in tissue culture of rat renal medullary cells

Renal medullary cells from the rat were used to establish a cell culture line. The morphologic characteristics of these cells were similar to renal medullary interstitial cells. They produced substantial amounts of PGE when provided with arachidonic acid or fetal calf serum. PGE production was inhibited 80–90% by indomethacin or meclofenamate. PGE release by the cells was sensitive to and stimulated by changing the culture media. Stable levels of PGE in the media could be achieved if media changes were avoided during the experimental period.     

Cytostatic and cytolytic activities of macrophages regulation by prostaglandins

Murine peritoneal macrophages (M phi), activated in vivo or in vitro, remarkably inhibited the uptake of thymidine by a lens epithelial cell line, while resident M phi, or M phi induced by thioglycollate, exhibited much lower or no cytostatic capacity. The target cells were partially protected from the cytostatic activity by the anti-inflammatory agents indomethacin, aspirin, and dexamethasone, but not by lipoxygenase inhibitors. The protective activity of indomethacin and aspirin, but not of dexamethasone, was completely counteracted by prostaglandin E2 (PGE2). Yet, PGE2 alone has no effect on the uptake of [3H]thymidine by lens epithelial cells. PGE1 resembled PGE2 in its effect on this system, whereas PGA2, PGB2, or PGF2 alpha had no detectable activity. The counteracting effect of PGE2 was mimicked by dibutyryl cAMP or by cholera toxin, an agent which increases cAMP levels. These findings suggest that PGEs are not direct cytostatic agents, but rather, are essential mediators for the development of the cytostasis. Activated M phi did not lyse cells of the original lens epithelial cell line, but caused substantial cytolysis of cells of a subline derived from it. In contrast to its aforementioned effect on the cytostasis, PGE2 inhibited the cytolytic activity of M phi. Thus, this study provides a first demonstration in a single system of the opposite effects of PGEs on M phi activity on target cells, i.e., mediating the cytostasis and inhibiting the cytolysis.     

TNF and PGE(2) in human monocyte-derived macrophages infected with Chlamydia trachomatis

In this study levels of prostaglandin E(2) (PGE(2)), tumour necrosis factor (TNF) and interleukin-1 (IL-1) alpha in medium from monocyte derived macrophages (MdM) infected with Chlamydia trachomatis (L(2)/434/Bu or K biovars). TNF and PGE(2) were found in both cases while IL-1 alpha was not detected. Both TNF and PGE(2) levels were higher in the medium of the MdM infected with K biovars. TNF reached maximum levels 24 h postinfection, and then declined, while PGE(2) levels increased continuously during the infection time up to 96 h post-infection. Addition of dexamethasone inhibited production of TNF and PGE(2). Inhibition of PGE(2) production by indomethacin resulted in increased production of TNF, while addition of PGE(2) caused partial inhibition of TNF production from infected MdM.     

Endogenous prostaglandin formation in the field-stimulated guinea-pig vas deferens: comparison of the inhibitory effects of indomethacin and NS-398

Prostaglandin (PG) E2 inhibited both phases of contraction produced by electrical field stimulation of the guinea-pig vas deferens. PGF2alpha and PGD2 were without effect on this preparation. Carbacyclin (a PGI2) analogue inhibited the first phase of contraction at higher concentrations, whereas U46619 (a thromboxane mimetic) potentiated both phases of contraction. As exogenous arachidonic acid inhibits both phases of contraction of the electrically field-stimulated guinea-pig vas deferens, it is likely that the arachidonic acid is converted to PGE2 in the vas deferens. Indomethacin, a non-specific inhibitor of prostaglandin H synthase (PGHS), attenuated the inhibitory actions of exogenous arachidonic acid when examined on the first phase of contraction. NS-398, a relatively specific inhibitor of PGHS-2, also prevented the inhibitory action of exogenous arachidonic acid. However, NS-398 was much less effective than indomethacin in this respect even though NS-398 and indomethacin inhibit PGHS-2 with similar potencies. Consequently, the findings suggest that exogenous arachidonic acid is converted to PGE2 in the guinea-pig vas deferens by the actions of PGHS-1 and, to a lesser extent, by PGHS-2.     

Reduction of the hyperthermic effect of prostaglandin E2 by cholinomimetics, monoamines and calcium ions]

It was shown in experiments on unanesthetized rats with prostaglandin E2 (PGE2) hyperthermia, not preventable by aspirin that intraventricular (into the lateral ventricle) injections of arecoline, noradrenaline, 5-hydroxytryptamine (5-HT), histamine, and calcium ions and intraperitoneal eserine injection were capable of decreasing body temperature. PGE2 hyperthermia was not prevented by aspirin, but it was reduced by eserine. After the administration of arecoline and nicotine into the third ventricle of unanesthetized rabbits with PGE2 hyperthermia body temperature decreased as well. The effect of arecoline and 5-HT was reproducible in the same animals. The data are suggestive of the existence in the heat loss centre of mechanisms including cholinergic neurons whose activity was not completely suppressed by PGE2.     

Effect of insulin, prostaglandin E1 and uptake inhibitors on glucose transport in the perfused guinea-pig placenta

The effects of insulin, prostaglandin E1 (PGE1) and uptake inhibitors on unidirectional D-glucose influx at brush border (maternal) and basal (fetal) sides of the guinea-pig syncytotrophoblast were investigated in the intact, perfused guinea-pig placenta by rapid, paired-tracer dilution. Experiments were performed in either an in situ preparation artificially perfused through the umbilical vessels (intact maternal circulation) or in the fully isolated dually-perfused placenta in which both interfaces were studied simultaneously. Kinetic characterization of unidirectional D-glucose influx gave apparent Km values (mean +/- SEM) at maternal and fetal sides of 70 +/- 6 and 87 +/- 16 mM respectively; corresponding Vmax values were 53 +/- 3 and 82 +/- 6 mumol min-1g-1. At the fetal side (singly-perfused placenta) cytochalasin B (50 microM), ethylidene-D-glucose (100 mM) and PGE1 (1 microM) partially inhibited D-glucose uptake whereas cortisol (50 microM) and progesterone (100 microM) had no effect. Abolition of the sodium gradient across the fetal interface did not modulate the kinetics of influx. In the presence of 150 mu units ml-1 insulin (dually-perfused placenta), unidirectional uptake into the trophoblast and transplacental D-[3H]glucose transfer were unaltered. In contrast, prostaglandin E1 (1 microM) markedly reduced the Km and Vmax for D-glucose at both interfaces and the inhibitory effect was reflected in a reduction in specific transplacental D-glucose transfer. Further experiments showed that the isolated placenta releases prostaglandins (PGE; PGF2 alpha) into both circulations. Bilateral insulin perfusion did not affect either lactate release by the placenta or rapid metabolism of D-[14C]glucose to [3H]lactate (usually less than 10% effluent [14C]lactate in 5 min). An asymmetric degradation of exogenous insulin was observed in the dually-perfused placenta: uterine venous samples contained 24 +/- 7 microunits ml-1 immunoreactive insulin when compared to the arterial concentration (151 +/- 3 microU ml-1 perfusate) while no change was measureable in the fetal circulation within the same time period (152 +/- 5 microU ml-1). This asymmetry was confirmed in experiments employing [125I]insulin. These results demonstrate that glucose transport in the intact guinea-pig placenta occurs by a sodium-independent, cytochalasin B-inhibitable system which is insulin-insensitive. Prostaglandin E1 appeared to be a potent transport inhibitor which suggests that prostaglandins may be involved in the 'down' regulation of placental glucose transport in vivo.     

Indomethacin inhibits responses to all vasoconstrictors in the rat mesenteric vascular bed: Restoration of responses by prostaglandin E2

Indomethacin added to the perfusing buffer inhibited pressor responses to noradrenaline, angiotensin II, arginine vasopressin, histamine, serotonin, calcium ions and potassium ions in the male rat mesenteric vascular bed. For every pressor agent the indomethacin concentration which inhibited response amplitude by 50% was about 7 microg/ml (2.1 × 10^?5 M). With every pressor agent, prostaglandin (PG) E2 could restore normal responsiveness in indomethacin-blocked preparations even while the indomethacin was still present in the buffer. The concentration of PGE2 required was proportional to the concentration of indomethacin. Preparations completely inhibited by indomethacin needed about 5ng/ml PGE2 for complete restoration of normal responses. Aspirin and mefenamic acid could also inhibit responses to all pressor agents tested but with these drugs only a partial restoration could be achieved by PGE2.     

Prostaglandins may not mediate inhibition of gastric acid secretion by somatostatin in the rat

The role of prostaglandins as mediators of the inhibitory effect of somatostatin on gastric acid secretion has been evaluated in conscious and anesthetized rats. The effect of somatostatin on bethanechol-stimulated gastric acid secretion was determined with or without indomethacin pretreatment. Prostaglandin synthesis inhibition (less than 90%) by indomethacin was verified with PGE2-generation assay on gastric mucosal tissue. In both conscious and anesthetized rats somatostatin significantly inhibited the stimulated acid output in the control and indomethacin pretreated groups. The present findings do not support a role for prostaglandins in the inhibition of gastric acid secretion by somatostatin in the rat.