Radiation protection of murine intestine by WR-2721, 16,16-dimethyl prostaglandin E2, and the combination of both agents

The survival of murine intestinal clonogenic cells (ICC) and the survival of mice after whole-body exposure to 137Cs irradiation were used to measure radiation protection by ethiophos (WR-2721), 16,16-dimethyl prostaglandin E2, and the combination of the two. Doses from 2 to 12.5 mg/mouse of WR-2721 increased cell survival linearly from 3.2 +/- 0.3 in controls given 15.0 Gy to 93.1 +/- 5.2 per jejunal circumference. In contrast, 16,16-dm PGE2 increased ICC survival at 15.0 Gy rapidly from 1 to 10 micrograms/mouse, followed by a plateau up to 100 micrograms/mouse. Animal survival at 6 days (LD50/6) increased from 16.3 +/- 0.4 Gy (95% confidence limits) in controls to 20.3 +/- 0.6 Gy in the PG-treated animals. WR-2721 increased the LD50/6 to 26.1 +/- 1.4 Gy. The dose modification factors were 1.25 and 1.60, respectively. The combination of agents increased ICC survival above that seen with each agent alone up to 8 mg WR-2721, above which no additional protection was seen. Animals given 10 micrograms PG plus 10 mg WR-2721 survived longer than with either agent given alone. The LD50/6 was 36.3 +/- 1.8 Gy for a dose modification factor (DMF) of 2.23. In addition, the slope of the probit curve was reduced from those of each agent alone. PG-induced changes in villus epithelial cell morphology and survival may account, in part, for these observations. The results suggest that either the mechanisms for these two types of radiation protectors are different or they act on separate subcellular targets which are critical to survival from radiation injury.     

Radiation-induced DNA single-strand breaks in the intestinal mucosal cells of mice treated with the radioprotectors WR-2721 or 16-16 dimethyl prostaglandin E2

Both S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721) and 16-16 dimethyl prostaglandin E2 (dm PGE2) protected the intestinal clonogenic cells to some degree from the effects of 137Cs gamma-irradiation. The D0 was increased from 1.1 +/- 0.12 Gy in controls to 1.55 +/- 0.48 Gy in 16-16 dm PGE2 treated and 2.12 +/- 0.20 Gy in WR-2721 treated mice. Both agents also increased the shoulder of the clonogenic-cell survival curve. Studies were done to measure the effects of these two different radioprotectors on radiation-induction of DNA single-strand breaks in cells comprising the murine intestinal mucosa. The number of DNA single-strand breaks increased with increasing doses of gamma-rays in animals killed immediately following exposure. WR-2721 reduced the number of initial radiation-induced DNA single-strand breaks when given one-half hour before exposure; the time of maximum protection. In contrast, 16-16 dm PGE2 given 1 hour before irradiation (the time required to afford maximum protection from radiation cytotoxicity) did not reduce the number of initial DNA breaks. Both agents impeded the rate of rejoining of DNA breaks with increasing time after irradiation. However, the relationship between these effects on the rate of strand rejoining and cell survival is unknown. These results suggest that either both agents are similarly distributed within the cells but the mechanisms of radioprotection are different, or the mechanisms by which these agents protect are similar, but the two agents affect different subcellular targets, the protection of which contributes to increased cell survival.     

Determination of the radioprotective effects of topical applications of MEA, WR-2721, and N-acetylcysteine on murine skin

Topical applications of MEA (beta-mercaptoethylamine or cysteamine), WR-2721 [S-2-(3-aminopropylamino)-ethylphosphorothioic acid], and N-acetylcysteine (NAC) were tested for their ability to protect the normal skin of the hind legs of mice against acute and late damage from single doses of 137Cs radiation. No significant protection was observed with either WR-2721 or NAC. MEA was shown to offer significant protection against acute skin damage in both buffered and unbuffered forms, but no significant protection against late contraction. The use of topical MEA on unanesthetized animals breathing carbogen (95% O2, 5% CO2) appears to give an enhanced level of radioprotection over that shown for anesthetized, air-breathing animals.     

Effect of misoprostol, a synthetic equivalent of prostaglandin E1, on bronchodilation in bronchial spasm]

A effect of a single dose 400 micrograms misoprostol (Cytotec-Searle) on a degree of the bronchospasm relief has been investigated in 8 patients, and the results have been compared with those produced by aminophylline i.v. infusion in the rate of 500 mg/hour. Prior to and 10, 20, 30, 60, 90, 120, 150 and 180 minutes after administration of both drugs, the following parameters of pulmonary ventilation have been determined: VC, VC%, FEV1, FEV1%, MVV, and MEFR. The results have shown that misoprostol given in a single oral dose of 400 micrograms to patients with bronchospasm has the bronchodilating activity which is statistically significant. However, this activity is rather weak and significantly lower than that of intravenous aminophylline infusion.     

Inhibition of anti-CD3 monoclonal antibody-induced T-cell proliferation by dexamethosone,isoproterenol, or prostaglandin E2 either alone or in combination

1. The purpose of these studies was to investigate the modulation of the proliferation of human T cells obtained from peripheral blood by dexamethasone (DEX), isoproterenol (ISO), and prostaglandin E2 (PGE2). The former two substances interact with T cells via the glucocorticoid and beta-adrenergic receptors respectively. When occupied by their natural ligands, glucocorticosteroids and catecholamines, these receptors have a role in modulating T-cell function during stress. During the inflammatory response increased levels of PGE2 bind to their receptors on T cells and thus alter responsiveness. Proliferation of T cells was induced by immobilized anti-CD3 monoclonal antibody (mAb) in the presence or absence of an additional costimulatory signal delivered by anti-CD28 mAb. 2. Various physiologic concentrations of DEX, ISO, or PGE2 were added at the time of initiation of the cultures and subsequent proliferation of the unstimulated T cells was determined. The results demonstrate that physiologic concentrations of all three of these agents inhibit the anti-CD3 mAb-induced proliferation of T cells. 3. Although DEX and PGE2 were equipotent in suppressing T-cell proliferation, ISO was much less effective. 4. Because concomitant elevations in the peripheral levels of these substances may occur, experiments were performed to determine the T-cell inhibitory effects of DEX together with either PGE2 or ISO. Synergistic suppression of T-cell proliferation was observed when various concentrations of DEX and PGE2, but not DEX and ISO, were added to cultures. This synergistic suppression could not be explained by an increase in cAMP accumulation in T cells stimulated with DEX and PGE2. 5. Finally, the addition of anti-CD28 mAb to anti-CD3 mAb-stimulated T cells overcame much of the suppression of proliferation induced by PGE2 or ISO but less so than that induced by DEX.     

In vivo and in vitro radioprotective effects of the prostaglandin E1 analogue misoprostol in DNA repair-proficient and -deficient rodent cell systems.

The radioprotective effect of a stable prostaglandin E(1) analogue, misoprostol, was studied in cells from mice with severe combined immunodeficiency (SCID) and in normal cells using X-ray-induced chromosomal aberrations and/or cell killing as the end points. The results clearly show misoprostol-induced radioprotective effects in spermatocytes of the first meiotic division when analyzed for X-ray-induced chromosomal aberrations. The protective effect was independent of Trp53 (formerly known as p53) status. Since spermatocytes are relatively easy to isolate, this appears to be a suitable in vivo model that will allow biochemical studies of the mechanisms involved in radioprotection mediated by misoprostol. Using transfected CHO-K1 cells that stably express a PGE(2) receptor (CPE cells), significant radioprotection mediated by misoprostol from both chromosome breakage and cell death could be demonstrated under in vitro conditions. In addition, evidence was obtained indicating that the degree of radioprotection was dependent on the cell cycle and that S-phase cells were less responsive to misoprostol-mediated radioprotection. These results suggest that CPE cells may be a suitable in vitro model for further studies on the cellular pathways involved in radioprotection by misoprostol in particular and prostaglandins in general.     

Sodium transport by isolated bullfrog small intestine. Effect of prostaglandin E1.

Addition of 446 micron prostaglandin E1 (PGE1) to the serosal medium of isolated short-circuited bullfrog small intestine elicited small increases transmural potential difference and short-circuit current while addition of PGE1 to the mucosal medium caused no change in the electrical parameters. Addition of 100 micron indomethacin to the mucosal medium inhibited both potential difference and short-circuit current with a resultant increase in steady-state tissue resistance. In the presence of mucosal 100 micron indomethacin, serosal 60 micron PGE1 markedly stimulated transmural potential difference and short-circuit current with a resultant decrease in steady-state tissue resistance. Serosal arachidonic acid (330 micron) stimulated transmural potential difference and short-circuit current and this effect was abolished by the addition of 100 micron indomethacin to the mucosal medium. Serosal 60 micron PGE1 only stimulated the M (mucosa) leads to S (serosa) unidirectional flux of sodium. These results strongly suggest that the PGE1 action is mediated either via a series of metabolic reactions which possibly increase the permeability of the mucosal membrane to sodium or via direct stimulation of rheogenic sodium pump activity.     

Inhibition of the graft-versus-host response by BCGcw-induced suppressor cells or prostaglandin E1

Immunization of C57BL/6 mice with BCGcw stimulated a population of "suppressor cells" which had a decreased capacity to induce the graft-versus-host response. The graft-versus-host response was quantitated using the Simonsen splenomegaly assay. F1 mice (C57BL/6 X CBA) were inoculated intraperitoneally with 1 X 10(8) parental (C57BL/6) or (CBA) spleen cells. The F1 mice were sacrificed 13 days later and the resulting splenomegaly was 3-4 times the normal amount. F1 mice which were injected with parental BCGcw-primed C57BL/6 spleen cells had a 50% inhibition of splenomegaly, whereas BCGcw-primed CBA spleen cells (a strain which does not develop suppressor cells) did not show this inhibition. In vitro results also confirmed that only C57BL/6 mice and not CBA mice developed suppressor cells after BCGcw immunization. A second study showed that X-irradiated (1000 R) BCGcw-primed "suppressor cells" could inhibit splenomegaly caused by the inoculation of normal parental C57BL/6 cells into F1 mice. The mechanism by which BCGcw-primed "suppressor cells" caused this inhibition of splenomegaly was delineated and found to be dependent upon the secretion of prostaglandin (PGE-1). Indomethacin and aspirin, potent inhibitors of prostaglandin synthesis, blocked the activity of C57BL/6 BCGcw "suppressor cells" and splenomegaly resulted. Systemic administration of the prostaglandin (15S)-15-methyl PGE-1 reduced splenomegaly approximately 50% in F1 mice which were injected with C57BL/6 or CBA cells. These results indicated that immunization with BCGcw stimulated a population of "suppressor cells" which could cause a decrease in graft-versus-host response and that the secretion of prostaglandin was responsible for this inhibition.     

Effect on airways resistance of prostaglandin E1 given by aerosol to healthy and asthmatic volunteers

The forced expiratory volume in one second (F.E.V.₁) was measured in healthy and asthmatic volunteers and the inhalation of prostaglandin E₁ (PGE₁) was compared with that of isoprenaline, using metered aerosols.In healthy volunteers PGE₁, either as the free acid or the neutral triethanolamine salt, did not affect the F.E.V.₁; the free acid was irritant to the upper respiratory tract. In five out of six asthmatic volunteers with reversible airways obstruction, inhalation of 55 μg of PGE₁ (triethanolamine salt) produced an increase in F.E.V.₁ comparable in both degree and duration to that produced by an inhalation of 550 μg. of isoprenaline sulphate.Though the triethanolamine salt was well tolerated in most of the asthmatic subjects studied, in one asthmatic subject this preparation caused coughing and there was a progressive reduction in the F.E.V.₁ associated with bronchospasm.     

Induction of luteolysis in the mare with a prostaglandin analogue

Five mares were administered 0.5 to 2.0 mg of a prostaglandin analogue, RS 9390 (Syntex), during nine estrous cycles in February and March. Luteolysis as measured by peripheral plasma progesterone occurred in four cycles, transitory luteolysis following 0.5 mg RS 9390 in two cycles, while functional corpora lutea were not present in three cycles. In 8 out of 9 of these cycles the mares returned to estrus 1.5 to 5 days following treatment. It appears that RS 9390 can be used as a regulator of cycle length in mares.     

Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis

Here we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E(2) synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE(2) biosynthetic pathway. GSH-dependent PGES activity in the cytosol of rat brains, but not of other tissues, increased 3-fold after lipopolysaccharide (LPS) challenge. Peptide microsequencing of purified enzyme revealed that it was identical to p23, which is reportedly the weakly bound component of the steroid hormone receptor/hsp90 complex. Recombinant p23 expressed in Escherichia coli and 293 cells exhibited all the features of PGES activity detected in rat brain cytosol. A tyrosine residue near the N terminus (Tyr(9)), which is known to be critical for the activity of cytosolic GSH S-transferases, was essential for PGES activity. The expression of cPGES/p23 was constitutive and was unaltered by proinflammatory stimuli in various cells and tissues, except that it was increased significantly in rat brain after LPS treatment. cPGES/p23 was functionally linked with COX-1 in marked preference to COX-2 to produce PGE(2) from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A(2) in the immediate response. Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE(2) that plays a role in maintenance of tissue homeostasis.     

Antiulcer effect of rioprostil, a prostaglandin E1 analog, in combination with antacid

Rioprostil, a primary alcohol prostaglandin E1 analog, is currently undergoing clinical evaluation for use in the treatment of peptic ulcer disease. Since antacids are often used in conjunction with other antiulcer agents, studies were conducted to determine if concomitantly administered antacid modifies the antiulcer activity of rioprostil. This investigation showed that concomitant administration of antacid (0.25-1.0 ml Maalox) does not inhibit the ability of rioprostil (0.125-4.0 micrograms/kg, p.o.) to prevent ethanol-induced gastric lesions in rats. The antiulcer effect of the drug combination was additive, suggesting that each compound acts independently to prevent gastric bleeding. These results in animals suggest that clinically the use of antacid will not compromise the efficacy of rioprostil and that the combination may be a useful mode of therapy for the treatment of peptic ulcer disease.     

Ultra sensitive determination of limaprost, a prostaglandin E1 analogue, in human plasma using on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometry

A highly sensitive and selective method has been developed and validated to determine limaprost, a prostaglandin (PG) E(1) analogue, in human plasma by on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometry (2D-LC/MS/MS) due to the lack of efficient methods to determine very low levels of limaprost in plasma. Limaprost and its deuterium derivatives, used as internal standard, were extracted by protein precipitation and following three-step solid phase extractions. After extraction procedure, samples were analyzed by on-line 2D-LC/MS/MS with electrospray ionization in negative mode. The 2D-LC system consists of Phenyl column at first dimension and ODS at second dimension with a trapping column placed between the separation columns. The linear dynamic range of this method was 0.1-10 pg/ml with 3 ml of plasma (r >0.9987). Acceptable precision and accuracy were obtained over the calibration curve ranges. The assay has been successfully used in analyses of human plasma samples to support clinical pharmacokinetics studies.     

The adenylate cyclase of rheumatoid synovial fluid macrophages is more sensitive for dl-5E, 19,14-di dehydro-carbo-prostacyclin, a stable prostacyclin analogue, than for prostaglandin E2

dl-5E, 19,14-di dehydro-carbo-prostacyclin (DDH-carbo PGI2), a stable prostacyclin (PGI2) derivative, but not prostaglandin (PG) E2, stimulated the adenylate cyclase of synovial fluid macrophages, isolated from rheumatoid patients with an active synovitis, in a dose dependent manner (10-1000 ng/ml). DDH-carbo PGI2 also stimulated synovial macrophage cAMP synthesis when injected into the knee joint. Exogenous arachidonic acid (AA) had little effect on cyclic-AMP (cAMP) formation or PGI2 release (assayed as 6ketoPGF1 alpha). It stimulated, however, the release of PGE2 and, to a lesser extent, thromboxane (Tx) A2 (measured as TxB2).     

Liver X receptor ligands inhibit the lipopolysaccharide-induced expression of microsomal prostaglandin E synthase-1 and diminish prostaglandin E2 production in murine peritoneal macrophages

Microsomal prostaglandin E synthase (mPGES)-1, which is dramatically induced in macrophages by inflammatory stimuli such as lipopolysaccharide (LPS), catalyzes the conversion of cyclooxygenase-2 (COX-2) reaction product prostaglandin H(2) (PGH(2)) into prostaglandin E(2) (PGE(2)). The mPGES-1-derived PGE(2) is thought to help regulate inflammatory responses. On the other hand, excess PGE(2) derived from mPGES-1 contributes to the development of inflammatory diseases such as arthritis and inflammatory pain. Here, we examined the effects of liver X receptor (LXR) ligands on LPS-induced mPGES-1 expression in murine peritoneal macrophages. The LXR ligands 22(R)-hydroxycholesterol (22R-HC) and T0901317 reduced LPS-induced expression of mPGES-1 mRNA and mPGES-1 protein as well as that of COX-2 protein. However, LXR ligands did not influence the expression of microsomal PGES-2 (mPGES-2) or cytosolic PGES (cPGES) protein. Consequently, LXR ligands suppressed the production of PGE(2) in macrophages. These results suggest that LXR ligands diminish PGE(2) production by inhibiting the LPS-induced gene expression of the COX-2-mPGES-1 axis in LPS-activated macrophages.     

Microsomal prostaglandin E synthase-1 is a major terminal synthase that is selectively up-regulated during cyclooxygenase-2-dependent prostaglandin E2 production in the rat adjuvant-induced arthritis model

To better define the role of the various prostanoid synthases in the adjuvant-induced arthritis (AIA) model, we have determined the temporal expression of the inducible PGE synthase (mPGES-1), mPGES-2, the cytosolic PGES (cPGES/p23), and prostacyclin synthase, and compared with that of cyclooxygenase-1 (COX-1) and COX-2. The profile of induction of mPGES-1 (50- to 80-fold) in the primary paw was similar to that of COX-2 by both RNA and protein analysis. Quantitative PCR analysis indicated that induction of mPGES-1 at day 15 was within 2-fold that of COX-2. Increased PGES activity was measurable in membrane preparations of inflamed paws, and the activity was inhibitable by MK-886 to >or=90% with a potency similar to that of recombinant rat mPGES-1 (IC(50) = 2.4 microM). The RNA of the newly described mPGES-2 decreased by 2- to 3-fold in primary paws between days 1 and 15 postadjuvant. The cPGES/p23 and COX-1 were induced during AIA, but at much lower levels (2- to 6-fold) than mPGES-1, with the peak of cPGES/p23 expression occurring later than that of COX-2 and PGE(2) production. Prostacyclin (measured as 6-keto-PGF(1alpha)) was transiently elevated on day 1, and prostacyclin synthase was down-regulated at the RNA level after day 3, suggesting a diminished role of prostacyclin during the maintenance of chronic inflammation in the rat AIA. These results show that mPGES-1 is up-regulated throughout the development of AIA and suggest that it plays a major role in the elevated production of PGE(2) in this model.     

Differential suppression of menstrual fluid prostaglandin F2a, prostaglandin E2, 6-keto prostaglandin F1a and thromboxane B2 by suprofen in women with primary dysmenorrhea

Eleven women with primary dysmenorrhea completed a randomized, double-blind, placebo-controlled, three-way cross-over study comparing 200 and 400mg suprofen. Menstrual fluid volume did not change. Mean+/-S.E.M. menstrual fluid PGF2a was significantly suppressed from 18.9+/-1.9 microg (placebo) to 10.9+/-1.7 and 9.3+/-2.1 microg with 200 and 400 mg suprofen, respectively (p=<0.005). PGE2 dropped from 7.8+/-0.9 to 4.6+/-0.8 and 4.6+/-1.1 microg (p=<0.05) and TxB2 from 17.5+/-4.3 to 7.5+/-2.9 and 3.6+/-1.3 microg (p=<0.01), respectively. 6-Keto PGF1a was significantly suppressed (2.7+/-0.4 to 1.9+/-0.5 microg, p=<0.025) with only 400 mg suprofen. Six subjects rated placebo poor and five fair to very good. In contrast, nine rated suprofen excellent to fair while two rated poor. Thus, suprofen was clinically effective but the differential suppression of prostanoids favors 200mg which spares 6-keto PGF1a.