Endogenous Prostaglandin Biosynthesis Research Articles

OP0183 Rebamipide: A potential useful therapeutic addition in the treatment of recurrent oral ulcers in behÇet’s disease

BackgroundRecurrent oral ulceration is one of the hallmarks of Behçet’s disease (BD). To date, various clinical manifestations, including aphthous ulcers are treated with limited success. Rebamipide is an anti-ulcer, gastro-protective...

Suppression of Type I Collagen Gene Expression by Prostaglandins in Fibroblasts Is Mediated at the Transcriptional Level

Tissues undergoing a chronic inflammatory process, such as the synovium in rheumatoid arthritis, are characterized by the infiltration of lymphocytes of different subsets and activation of monocyte/macrophages. Interleukin-1 (IL-1), a monocyte/ macrophage product that stimulates synovial fibroblasts to produce matrix metalloproteinases (MMPs), prostaglandins, and other cytokines, also has profound effects on the synthesis of extracellular matrix components such as type I collagen. In previous studies, we have shown that synovial fibroblasts and chondrocytes isolated from human joint tissues are particularly sensitive to prostaglandins, which modulate the effects of IL-1 on collagen gene expression in an autocrine manner. BALBc/3T3 fibroblasts were treated with IL-1 and prostaglandins in the absence and presence of indomethacin to inhibit endogenous prostaglandin biosynthesis. Collagen synthesis was analyzed by SDS-PAGE as [3H]proline-labeled, secreted proteins, and prostaglandin production and cyclic adenosine 3',5'-cyclic monophosphate (camp) content were assayed. The expression of type I collagen gene (Col1a1) promoter-reporter gene constructs was examined in transient transfection experiments, and the binding of nuclear factors to the Col1a1 promoter region spanning -222 bp/+ 116 bp was analyzed by DNase I footprinting and electrophoretic mobility shift (EMSA) assays. IL-1 increased the synthesis of type I and type III collagens in BALBc/3T3 fibroblasts; greater increases were observed when IL-1-stimulated synthesis of PGE2 was blocked by indomethacin. Transient transfection experiments demonstrated dose-dependent inhibition of the-222 bp Col1a1 promoter by exogenously added prostaglandins with the order of potency of PGF2alpha > PGE2 > PGE1 DNase I footprinting showed increased protection, which extended from the region immediately upstream of the TATA box, owing to the binding of nuclear factors from PGE2- or PGE1-treated BALBc/3T3 cells. EMSA analysis showed zinc-dependent differences in the binding of nuclear factors from untreated and prostaglandin-treated cells to the -84 bp/-29 bp region of the Col1a1 promoter. These results show that the inhibition of Col1a1 expression by IL-1 in fibroblasts is mediated by prostaglandins at the transcriptional level and suggest that PGE-responsive factors may interact directly or indirectly with basal regulatory elements in the proximal promoter region of the Col1a1 gene.

Interleukin-1 beta induces interstitial collagenase gene expression and protein secretion in renal mesangial cells.

Degradation and tissue remodeling of the extracellular matrix in the normal glomerulus occur through the coordinate action of neutral metalloproteinases, which are in turn regulated by specific inhibitors. Many of these proteins can be secreted by mesangial cells. In the current study, gene regulation of a rat matrix metalloproteinase, interstitial collagenase and its tissue inhibitor of metalloproteinase-1 (TIMP-1), was investigated by Northern blot analysis. Stimulation of rat mesangial cell (RMC) collagenase by interleukin 1 beta (IL-1 beta) produced an increase (> 45-fold) in mRNA which peaked at 12 h. Lesser effects on the TIMP-1 mRNA expression were observed in response to IL-1 beta. Indomethacin did not influence the effect of IL-1 beta on collagenase, and exogenous prostaglandin E2 had no significant effect either on basal or IL-1 beta-stimulated mRNA levels. Collagenase was secreted into the media and showed minimal gelatinolytic activity at 36-h stimulation with IL-1 beta by zymography. By Western immunoblotting, we demonstrated with 24 h of stimulation the secretion of the active form of collagenase, which further increased after 36 h with IL-1 beta compared with the control. When RMC were retreated with genistein and herbimycin A, both inhibited collagenase mRNA induction by IL-1 beta. These data suggest that IL-1 beta stimulates interstitial collagenase synthesis and activation and that a tyrosine kinase pathway is involved in the signal transduction mechanisms and is not dependent on endogenous prostaglandin biosynthesis. Recently, a third interstitial collagenase (collagenase-3) has been identified from breast carcinoma. This cDNA is 84% identical to the rat interstitial collagenase cDNA probe we have utilized in this study and thus may represent the rat homologue of the human collagenase-3 now called matrix metalloproteinase (MMP)-13.

Antiulcer activity of eel-calcitonin (elcatonin) is associated with enhanced gastric mucosal PGE2 generation in the rat.

Elcatonin is a newly synthetized peptide that prevents experimental ulcer formation. In this study, we examined the influence of elcatonin on endogenous prostaglandin biosynthesis by the gastric mucosa with respect to its antiulcer activity. Control levels of PGE2 generation for the fundic and antral mucosa were 302 +/- 12 and 465 +/- 28 ng/g tissue/min, respectively. Pretreatment with elcatonin resulted in a dose-dependent increment in gastric mucosal PGE2 generation. We conclude that the antiulcer effect of elcatonin is probably due to its ability to enhance endogenous prostaglandin synthesis in the gastric mucosa.

Antiulcer Activity of Eel-Calcitonin (Elcatonin) is Associated with Enhanced Gastric Mucosal PGE2 Generation in the Rat

Elcatonin is a newly synthetized peptide that prevents experimental ulcer formation. In this study, we examined the influence of elcatonin on endogenous prostaglandin biosynthesis by the gastric mucosa with respect to its antiulcer activity. Control levels of PGE2 generation for the fundic and antral mucosa were 302±12 and 465±28 ng/g tissue/min, respectively. Pretreatment with elcatonin resulted in a dose-dependent increment in gastric mucosal PGE2 generation. We conclude that the antiulcer effect of elcatonin is probably due to its ability to enhance endogenous prostaglandin synthesis in the gastric mucosa.

15-Keto-13, 14-dihydroprostaglandin E 2-and F 2α-metabolite levels in blood from men and women given prostaglandin E 2 orally

Prostaglandin E 2 (PGE 2) was administered orally in a dose of 1 mg to healthy males (n = 20) and females (n = 10). Blood levels of 15-keto-13, 14-dihydroprostaglandin F 2α (PGF 2α-M) and 15-keto-13, 14- dihydroprostaglandin E 2 (PGE 2-M), determined as the rearrangement product 11-deoxy-15-keto-13,14-dihydro-11β,16-cycloprostaglandin E 2 (PGE 2-cyclo-M), were measured. The levels of the two PG metabolites increased already 10 minutes after ingestion of the tablet and the mean peak value for PGE 2-cyclo-M in the men was 4.64 nmol/l which was reached 50 minutes after PGE 2 administration. The mean peak value in women was 4.99 nmol/l which was obtained after 30 minutes. The increase in PGE 2-cyclo-M concentration was significantly faster (p < 0.05) in women than in the men. The mean plasma concentration of PGF 2α in males was 0.20 nmol/l prior to treatment and rose after PGE 2 ingestion to a mean peak level of 0.84 nmol/l after 70 minutes. The corresponding values for the females were 0.18 nmol/l and 0.88 nmol/l 50 minutes into treatment. When the data from both sexes were amalgamated PGE 2-cyclo-M peak levels were reached significantly (p = 0.004) sooner than the PGF 2α-M peak. The two PG metabolites returned to baseline levels in 70% of the individuals after 240 minutes. The increase in PGF 2α-M concentration following oral administration of PGE 2 indicates that part of the PGE 2 was reduced to PGF 2α. The fact that no difference was observed in the kinetics of the PGE 2-cyclo-M and PGF 2α-M in blood following PGE 2 administration before and after one week of naproxen treatment supports this assumption. An increase in PGE 2-cyclo-M blood levels following intravenous injection of PGF 2α in two women indicated that PGF 2α could be oxidized to PGE 2. However, it cannot be excluded that PGE 2 or PGF 2α administration also might affect endogenous prostaglandin biosynthesis.

Effect of linoleic acid infusion on blood pressure in normotensive and hypertensive rats.

High dietary intake of linoleic acid lowers arterial pressure, and, in vitro, linoleic acid inhibits the enzymatic activity of renin. The purpose of the present study was 1) to evaluate the effect of intravenous infusion of linoleic acid on blood pressure in normotensive and hypertensive Sprague-Dawley rats and 2) to determine whether the hypotensive response to linoleic acid infusion is caused by inhibition of circulating renin. Blood pressure was decreased (P less than 0.01) by linoleic acid infusion in normotensive sodium chloride-deprived animals and in animals with two-kidney, one-clip hypertension. In contrast, linoleic acid infusion did not affect blood pressure in normotensive rats on a "normal" or high sodium chloride intake, in rats with deoxycorticosterone acetate (DOCA)-salt hypertension, and in anephric rats. In sodium chloride-deprived rats, the reduction of blood pressure by linoleic acid infusion was associated with increased plasma renin activity (P less than 0.05); serum angiotensin-converting enzyme activity was unchanged. The in vitro enzymatic activity of exogenous renin in plasma of anephric rats was not affected by linoleic acid infusion. In two-kidney, one-clip hypertensive animals, pretreatment with indomethacin did not alter the hypotensive response to linoleic acid. Thus, although linoleic acid infusion lowered blood pressure in high renin but not in low renin states, the reduction of blood pressure was not related to inhibition of circulating renin or to alterations of endogenous prostaglandin biosynthesis.

Effect of sialoadenectomy on adaptive cytoprotection in the rat

In the present study we have investigated the effect of sialoadenectomy in the rat on adaptive cytoprotection induced by intragastric instillation of an irritant concentration of ethanol (10% wt/vol). Gastric mucosae were examined 1 h after oral administration of 100% ethanol. In animals with intact salivary glands, pretreatment with saline was followed 15 min later by 100% ethanol. This treatment was associated with a significant degree of mucosal damage. However, prior administration of 10% ethanol in place of saline reduced the area of ulceration. In sialoadenectomized rats, the irritant concentration of ethanol did not protect the gastric mucosa from the damaging actions of absolute ethanol. The effect was partially restored if sialoadenectomized rats were treated for 3 days with an aqueous extract of rat salivary gland tissue. Histologic examination revealed no differences in the extent of epithelial damage or adherent mucus between rats with intact salivary glands and sialoadenectomized rats following pretreatment with either saline or 10% ethanol. Although adaptive cytoprotection in animals with intact salivary glands was not associated with increases in mucosal capacities for prostaglandin E2 or 6-keto prostaglandin F1α biosynthesis, sialoadenectomy resulted in a reduction in the mucosal biosynthetic capacities for both prostanoids. Salivary gland extract administration to sialoadenectomized rats did not significantly alter mucosal prostaglandin biosynthesis levels. These data suggest that salivary gland factors affected adaptive cytoprotection induced by an irritant dose of ethanol. Although endogenous prostaglandin biosynthesis capabilities do not appear to be altered in adaptive cytoprotection, salivary factors appear to influence mucosal generation of prostaglandins.

Eicosanoids: role in experimental renal disease.

Because of their vasodilator and vasoconstrictor properties, vasoactive prostaglandins and thromboxane A2 have been proposed as modulators of the hemodynamic changes that occur in experimental models of renal disease. Increased synthesis of vasodilatory prostaglandins (PGE2) and perhaps prostaglandin I2 (PGI2) play a role in the maintenance of renal blood flow and GFR during states of impaired perfusion. In contrast, thromboxane A2 has been implicated as the vasoconstrictor responsible for the reduction of renal blood flow and GFR in certain animal models of experimental renal disease. These products and other metabolites of arachidonic acid may also participate in the immunological events underlying the onset and/or progression of experimental renal disease. It is evident that the pathophysiologic role of eicosanoids in experimental renal disease is not fully understood. Additional studies and further understanding of the many other potential roles of eicosanoids on immunological events, hemodynamic states, mesangial cell physiology, etc. are needed to comprehend more fully the extent of the participation of eicosanoids in the pathogenesis and pathophysiology of renal disease.

Pharmacological characterization of the histamine receptor in the isolated muscularis mucosae of the guinea-pig oesophagus.

To characterize the histamine receptors in the muscularis mucosae, the isotonic responsiveness of the isolated muscularis mucosae of the guinea-pig oesophagus to histamine receptor agonists and antagonists was examined in vitro. Histamine (0.1-100 microM) produced a concentration-dependent contraction of the muscularis mucosae (EC50 = 1.6 +/- 0.2 microM). The contractions were rapid in onset, sustained, reversible by washing and the preparation did not show tachyphylaxis. 2-Methylhistamine (2-MH), 2-pyridylethylamine (PEA) and 4-methylhistamine (4-MH) produced similar sustained contractions of the muscularis mucosae. The order of sensitivity was histamine greater than 2-MH greater than PEA greater than 4-MH. Impromidine (10-300 microM) and dimaprit (10-300 microM) caused no response in this tissue. The contractile responses to histamine, 2-MH, and PEA were competitively antagonized by diphenhydramine, and the pA2 values were almost the same (approximately 8.1). Cimetidine (100 microM) could not modify the contractile response to these agonists. The contractile response to histamine was slightly inhibited by tetrodotoxin (0.3 microM), atropine (1 microM), indomethacin (0.1-3 microM) or aspirin (30-300 microM), and the EC50 value was increased about 2-6 times by these drugs. When the preparation was incubated in Tyrode solution containing various calcium concentrations (0, 0.45, 0.9 and 1.8 mM), the concentration-response curve to histamine was shifted to the right and downward; the effect was inversely dependent on the calcium concentration, and in a calcium-free medium the response to histamine was abolished. Verapamil (1-10 microM) partially inhibited the contractile response to histamine. 7 The present results indicate that the contraction of the guinea-pig oesophageal muscularis mucosae to histamine is mediated mainly by a direct action on the smooth muscle and partly by indirect actions via the stimulation of either endogenous prostaglandin biosynthesis or intramural cholinergic nerves. The histamine receptors responsible for contractions of this tissue are probably mainly of the H,- subtype with H2-receptors having a negligible role.

Regulation of vasopressin action by prostaglandins. Evidence for prostaglandin synthesis in the rabbit cortical collecting tubule.

The present studies examined whether vasopressin increases prostaglandin biosynthesis in isolated rabbit cortical collecting tubules (CCT) and whether endogenous prostaglandin biosynthesis plays a role in modulating the response of this nephron segment to vasopressin. Three groups of studies were performed. In the first group, CCT and proximal straight tubules (PST) were incubated with [(3)H]arachidonic acid, and metabolites were separated and identified using silica gel thin-layer chromatography. CCT were capable of producing all of the major prostaglandins (PG) (PGE(2) > thromboxane B(2)[TxB(2)] > PGF(2alpha) > PGI(2)). PST produced significantly lesser quantities of these lipids. In the second group, radiolabeled arachidonic acid was incorporated into the phospholipid pool of both CCT and PST, vasopressin was added to the incubation medium, and metabolities were separated and identified as above. Vasopressin stimulated the release of all of the major prostaglandins in CCT but had no effect on PST. PGE release into the incubation medium, as assessed by a radioreceptor assay, increased 108%, and a vasopressin analogue, 1-desamino-8-d-arginine vasopressin, had a quantitatively similar effect. In the third group, a submaximal dose of vasopressin was administered to isolated, perfused CCT studied in the presence and absence of indomethacin to assess whether endogenous prostaglandins play a role in modulating the antidiuretic response to vasopressin. Studies were performed in rabbits on a normal diet and in desoxycorticosterone acetate (DOCA)- or KCl-loaded animals. In the state of mineralocorticoid excess, basal prostaglandin synthesis was 63% lower, and vasopressin-stimulated prostaglandin synthesis 76% lower, than the synthesis observed in rabbits on a normal diet. Cyclooxygenase inhibition exposed a significant hydroosmotic response to a submaximal dose of vasopressin in CCT from DOCA- or KCl-loaded animals. With arachidonic acid in the bath, the same dose of vasopressin failed to elicit a hydroosmotic response in CCT from rabbits on a normal diet even in the presence of a cyclooxygenase inhibitor. However, removal of exogenous arachidonic acid, with a consequently lower rate of prostaglandin synthesis, allowed the cyclooxygenase inhibitor to enhance the hydroosmotic response to vasopressin in these tubules.We conclude from these studies that the rabbit CCT has the capacity to synthesize all of the major prostaglandins and that the rate of synthesis of these lipids is enhanced by vasopessin. Prostaglandin synthesis by the CCT is postulated to modulate the antidiuretic action of vasopressin via a closed feedback loop. The effectiveness of this feedback regulation is dependent upon the mineralocorticoid status of the animal, which determines the level of basal and vasopressin-stimulated prostaglandin synthesis by the CCT.

Augmented acute hypotensive effect of dihydralazine and clonidine after linoleic acid rich diet in normotensive conscious rats

The influence of dietary linoleic acid (LA) content on the cardiovascular effects of dihydralazine and clonidine was investigated in conscious rats. Male normotensive rats were fed either e, diet rich in linoleic acid (LA) (13.3 cal-% LA) or a diet deficient in LA (0.5cal % LA) for a period of 5 months beginning in the pregnant mothers. Dihydralazine (1 mg/kg iv) or clonidine (10 μg/kg iv) were injected into conscious rats and blood pressure and heart rate were studied during a 20 min investigation period. The blood pressure lowering effects were higher in the rats fed the LA rich diet than in those fed the LA deficient diet 2 5, 10 and 20 min after dihydralazine and 5 min after clonidine injection. The increase in heart rate per 10 mmHg blood pressure reduction after dihydralazine injection was lower in the rats fed on the diet rich in LA, We assume that the change in the cardiovascular effects of dihydralazine and clonidine by dietary LA may be caused by alterations of endogenous prostaglandin biosynthesis and sympathetic activity.

A synthetic analog of prostaglandin E 2 is able to induce in vivo theta antigen on spleen cells of adult thymectomized mice

The effect of prostaglandins on the in vivo induction of theta antigen in splenic spontaneous rosette-forming cells derived from adult thymectomized mice was studied. A long-acting synthetic analog of prostaglandin E 2, di-M-PGE 2, mimicked the effects of thymic hormone and was active when mice were treated with as little as 0.1 μg ip. In addition, indomethacin, a potent inhibitor of prostaglandin biosynthesis, was able to reverse the inductive effects of exogenous thymic hormone and inhibit the expression of theta antigen in normal mice, presumably by interfering with the effect of endogenous thymic factors. Finally, indomethacin also partially suppressed the stimulatory effects of exogenously administered di-M-PGE 2, suggesting that this agent is effective, at least in part, because it stimulates endogenous prostaglandin biosynthesis. Possible mechanisms of action for the effects of prostaglandins are presented.

Induction of Labor by Oral PGE2 Administration—Evaluation of Different Dose Schedules

The efficacy and safety of different oral doses of PGE2 in tablet form were evaluated in 30 women admitted to the hospital for labor induction at or near term. The aim was to select a recommendable dose schedule based on recording of uterine contractility, clinical outcome and measurement of the resulting plasma levels of the two prostaglandin metabolites 15-keto-13, 14-dihydro-PGE2 and 15-keto-13, 14-dihydro-PGE2 alpha by gas-chromatography-mass spectrometry and by radio-immunoassay. Measurements of uterine contractility and of plasma levels of the PGE2 metabolite both showed that the preferable interval between oral doses of PGE2 tablets is one hour. Following 1.0 mg PGE2, the plasma concentration peaked after 45 to 60 minutes and had returned to approximately pretreatment levels after 120 minutes. With a two-hour interval between doses there was above all a decreased frequency of contractions in the last part of the interval. No such variation in the contractility pattern was seen when 1.0 mg PGE2 was administered every hour. When the individual dose was increased to 2.0 mg, signs of overstimulation of uterine contractility were observed. The plasma concentration of the E2 metabolite increased in accordance with the oral dose. A slow rise in the plasma concentrations of the E2 and F2 alpha metabolites was found some hours following initiation of treatment, possibly indicating an increased endogenous prostaglandin biosynthesis, probably secondary to the stimulated uterine contractility.

Regional and species differences in endogenous prostaglandin biosynthesis by brain homogenates

Endogenously formed prostaglandins (PGs) D 2, E 2 and F 2α were determined in homogenates of brain regions from rat, guinea-pig, rabbit and cat, using gas-chromatography-mass spectrometry. The main PGs formed in the brain regions of the rat were PGD 2, in the guinea-pig PGD 2 and PGF 2α, in the rabbit PGF 2α and in the cat PGE 2. Brain regions from the same animal species showed the same pattern of PG formation. They varied, however, in the amount of total PGs formed, the limbic system and the cerebral cortex being highest and cerebellum lowest.

Influence of Dietary Linoleic Acid Content on the Cytoplasmic Granularity of Renomedullary Interstitial Cells

A study of the influence of different diets (rich and deficient in polyunsaturated fatty acids) and of indomethacin on the mean count of renomedullary interstitial cell (RIC) lipid granules was undertaken. A correlation between dietary linoleic acid (LA) content (the main precursor of endogenous prostaglandin biosynthesis) and the number of granules in RIC was demonstrated: in comparison to the salt-loaded rats fed standard pellet diet, the number of RIC lipid granules in salt-loaded rats fed LA in excess increased, while in salt-loaded rats on LA-deficient diet the RIC granularity tended to decrease. Indomethacin treatment caused an increase in RIC granularity in salt-loaded rats fed a standard pellet diet and in those fed LA in excess, but had no effect in salt-loaded rats fed LA-deficient diet.

Effects of biogenic amines and psychotropic drugs on endogenous prostaglandin biosynthesis in the rat brain homogenates

Phenylalkylamine and indolalkylamine derivatives, as well as several drugs acting on the central nervous system, were tested for their effects on endogenous prostaglandin, (PG) biosynthesis in the rat brain homogenates. In the particulate suspension obtained by the removal of the soluble fraction from the rat brain homogenates PG-biosynthesis could be stimulated by noradrenaline. dopamine, adrenaline, serotonin, tryptamine and to a slight extent by tyramine. Isoprenaline, DOPA. α-methyl noradrenaline, α-methyl dopamine, α-methyl tryptamine and 5-hydroxy tryptophan were ineffective. PG-biosynthesis stimulated by catecholamines or indolalkylamines responsively could be inhibited by compounds with monoamine oxidase blocking properties. In the total rat brain homogenates another type of PG-biosynthesis could be demonstrated in the absence of catecholamine or indolalkylamine that could not, or but to a slight extent, be inhibited by monoamine oxidase blocking agents. Apomorphine, oxypertine, α-methyl noradrenaline, promethazine, DOPA, reserpine, chlorpromazine, desipramine, yohimbine and tetrabenazine inhibited this type of PG-biosynthesis, though they failed to influence PG-formation stimulated by catecholamine or indolalkylamine. A correlation could be established between the PG-formation inhibitory and lipid peroxidation antagonizing effects of these compounds. Non-steroidal anti-inflammatory agents, such as indomethacin, acetylsalicylic acid and dipyrone, inhibited both types of PG-biosynthesis. The results permit the conclusion that psychotropic drugs exert their effects on endogenous PG biosynthesis in the rat brain homogenates by inhibiting various activation processes.

PROLONGATION OF MURINE SKIN ALLOGRAFTS BY PROSTAGLANDIN E

The effects of 16,16-dimethyl prostaglandin E2 methyl ester (di-M-PGE2) and indomethacin (an inhibitor of endogenous prostaglandin biosynthesis) on mouse skin allograft survival were studies in B10.D2 female mice receiving skin allografts from (B10.BR X B10.D2)F1 mice. Control animals with and without i.p. diluent injections had a mean allograft survival of 13.8+/-0.6 and 13.5+/-0.5 days, respectively. Daily administration of di-M-PGE2 (200 microng/kg) prolonged mean allograft survival, both when administered alone, 16.7+/-0.6 days (P less than 0.001), or with indomethacin, 4 mg/kg thrice weekly, 16.0+/0.6 days (P less than 0.005). Increasing concentrations of indomethacin (4, 6, and 8 mg/kg thrice weekly) were inversely corrleated with allograft survival ((12.7+/-0.2, 11.8+/-0.2, and 10.9+/-0.4 days, respectively), coefficient of correlation=-0.6986; P less than 0.001. Mean plasma PGE levels at the time of total allograft rejection were 879+/-80 pg/ml in control, 717+/-59 pg/ml in 100 micron g of indomethacin-treated mice, and 654+/-59 pg/ml in 200 microng of indomethacin-treated mice (P less than 0.05). Exogenous di-M-PGE2 prolonged skin allograft survival in mice. Inhibition of endogenous prostaglandin biosynthesis by indomethacin chortened allograft survival, but this effect was completely abrogated by concurrent injection of di-M-PGE2.