Prostaglandins最新文献

A sensitive and reliable chemiluminescence immunoassay suitable for the quantitative determination of prostaglandin E₂ (PGE₂) has been developed using 96 well microtiter plates (MTP). The assay is based on a competitive reaction between a highly specific monoclonal anti-PGE₂ antibody (mouse), free antigen and solid phase bound antigen. The MTP was first coated with a bovine serum albumin (BSA)-PGE₂ conjugate. Then, after preincubating, the anti-PGE₂ antibody (Ab) and the analyte were added. The remaining amount of free antibody was captured by the solid phase bound BSA-PGE₂ conjugate. The monoclonal antibody captured on the MTP was determined using biotinylated antimouse-Ab and a complex of avidin and biotin-labelled horseradish peroxidase (HRP). Substrate for HRP was the cyclic diacyl hydrazide compound luminol, enhanced by p-iodophenol. Photons emitted during the reaction were measured using a photomultiplier tube. The assay has been validated with assay buffer and human plasma over a concentration range of 10–50,000 pglml. The lower limit of quantification is 100 pglml (2 pglwell) and 150 pglml (3 pglwell) for buffer and plasma, respectively. The intea-day coefficients of variation (CV) for the range of 100–50,000 pglml are 3.2–8.9% (buffer) and 4.2–17.7% (plasma) and inter-day CV are 2.9–19.8% (buffer) and 3.6–21.2% (plasma). The method can be used for quantification of PGE2 in biological fluids like plasma and suction blister fluid.

The acute effects of beraprost sodium (sodium (±)-(1R^∗, 2R, 3aS^∗, 8bS^∗)-2, 3, 3a 8b-tetrahydro-2-hydroxy-l-((E)-(3S^∗)-3-hydroxy-4-methylI-octen-6-yny1] -1H-cyclopenta [b] bensofuran-5-butyrate), a stable analogue of prostaglandin I₂ which works as a vasodilator and anti-platelet agent, were investigated in patients with non-insulin dependent diabetes mellitus. Its effects on the dorsal pedis artery were examined using a new real-time two-dimensional Doppler ultrasonographic technique and by laser blood flowmetry. Before and 60 min after oral administration of beraprost sodium (Dolner^® 40 μg) and elastase (Elaszym^® 1800 U), the cross-sectional area (CSA) of the dorsal pedis artery and its blood flow index (BFI), calculated from the maximum flow velocity and area, were determined. Dermal microcirculatory blood volume (MBV) was also measured by laser blood flowmetry. In the beraprost sodium group, the CSA, BFI and MBV were significantly increased, while in the elastase group, no significant changes were observed. These result suggest that beraprost sodium has a beneficial effect on diabetic macro- and microangiopathy.

We examined the contribution of prostaglandins to altered reactivity to norepinephrine in rat cremaster third order arterioles of streptozotocin (STZ) treated rats and age-matched controls. NE was applied topically to the cremaster muscle of pentobarbital (35 mg/kg) anesthetized rats before and during topical administration of indomethacin (IND: 10 μg/ml) four and eight weeks after i.v. injection with of 50 mg/kg STZ (STZ-4W; STZ-8W) or vehicle (C-4W; C-8W), and before and during topical administration of 5,8,11,14 eicosatetraynoic acid (ETYA; 20 μg/ml) in STZ-8W and C-8W. Plasma glucose was elevated significantly in STZ-treated rats. Blood pressures and resting arteriolar diameters did not differ. However, vasoconstrictor responses to NE were depressed in STZ-4W and to a greater degree in STZ-8W. IND normalized reactivity to the low doses of NE and partially restored reactivity to the higher doses. ETYA enhanced reactivity to all doses of NE to a greater extent than did IND. These data are consistent with a role for locally produced vasomodulatory arachidonic acid metabolites, including prostaglandins, in the decreased reactivity to NE in diabetic rat cremaster muscle arterioles.

Unlike cyclooxygenase 2 (COX-2), COX-1 has never been identified, purified or cloned in a non-mammalian species. Here we report the RT-PCR cloning of a chicken cDNA that encodes the amphipathic membrane binding region and parts of the dimerization and catalytic domains of COX1-like enzyme. Sequence comparison showed this putative COX-1 to be evolutionarily less conserved than COX-2. Furthermore, whereas COX-1 in mammals is broadly expressed in tissues as a constitutive enzyme, the mRNA detected by our clone in chicken was almost absent in tissues and embryo fibroblasts (CEF). Highest expression was in brain and seminal vesicle. This transcript was not detectable during chick embryogenesis and, as is the case for mammalian COX-1, was not induced above background by mitogen stimulation. The identification of an avian COX-1 shows that COX-1 and COX-2 existed as separate catalysts for prostaglandin synthesis before the divergence of birds and mammals.

The pharmacological profile of a novel dual inhibitor, tepoxalin and of its carboxylic acid metabolite on cyclooxygenase and lipoxygenase pathways was evaluated by in vitro incubation with synovial tissue. Tissue specimens obtained at surgery in rheumatoid arthitis (RA, n=10) or osteoarthritis (OA, n=11) patients were incubated. Tepoxalin (10⁻⁷, 10⁻⁶, 10⁻⁵ M) decreased eicosanoid release calculated in % of tyrode control for OA: LTC₄ to 71−33%, 6-keto-PGF_1a to 37−20%, PGE₂ to 29−6%. For RA: LTC₄ to 56−22%, 6-keto-PGF_a to 43−22%, PGE₂ to 57−32%. Similarly, its metabolite (10⁻⁷, 10⁻⁵ M) decreased release in OA: LTC₄ to 99 and 60%, PGE₂ to 42 and 20%, 6-keto-PGF_1a to 54 and 25%. In RA: LTC₄ to 81 and 45%, PGE₂ to 61 and 30%, 6-keto-PGF_1a to 46 and 18%. Significance (p<0.05) was achieved for all but 1 group (LTC₄, metabolite at 10⁻⁷M vs tyrode).

In summary a marked and dose dependent decrease of LT and PG release was obtained when incubating the dual inhibitor tepoxalin and its active carboxylic acid metabolite with synovial tissue at doses expected to be reached in the joint during therapy.

A high-performance liquid chromatographic (HPLC) method utilizing ultraviolet absorbance coupled with radioisotope detection was developed for the precise and simultaneous determination of iloprost and misoprostol. This assay allows complete resolution of iloprost diastereoisomers and has a total run time of approximately twenty minutes. Samples were prepared for chromatographic analysis by extracting a mixture of tritiated drugs from rat plasma with acetonitrile. The resulting solutions were chromatographed on a reversed phase Zorbax Rx-C8 column using 0.02M potassium phosphate (pH 3.0), acetonitrile, and methanol (46:30:24, $\text{v}\text{v}$) at a flow rate of 1.7 mL/min. 2-Naphthoic acid was employed as an internal standard. The correlation coefficient for varying concentrations of tritiated iloprost (12.7 Ci/mmol specific activity) from 2.18 ng/mL to 21.8 ng/mL was 0.995, and the correlation coefficient for concentrations of tritiated misoprostol (50 Ci/mmol specific activity) from 0.617 ng/mL to 6.17 ng/mL was 0.993. The high selectivity and sensitivity of this assay make it useful for the simultaneous quantitation of iloprost and misoprostol.

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The present studies were performed to evaluate the effects of luteotropic and luteolytic hormones, and also intracellular regulators, on contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. Bovine corpora lutea (CL) from the early, mid and late luteal phases of the estrous cycle were dispersed with collagenase and incubated with no treatment, LH, PGF or LH + PGF (Experiment 1), or with no treatment, or agonists or antagonists of protein kinase C (TPA or H-7) or calcium (A23187 or EGTA; Experiment 2). After incubation, media were collected for determination of progesterone concentrations. Then the rate of GJIC was evaluated for small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells by using the fluorescence recovery after photobleaching technique and laser cytometry. Luteal cells from each stage of the estrous cycle exhibited GJIC, but the rate of GJIC was least (P<0.05) for luteal cells from the late luteal phase. LH increased (P<0.05) GJIC between small luteal cells from the mid and late but not the early luteal phase. PGF increased (P<0.05) GjIC between small luteal cells from the mid luteal phase and diminished (P<0.05) LH-stimulatory effects on GjIC between small luteal cells from the late luteal phase. Throughout the estrous cycle, TPA decreased (P<0.05) the rate of GjIC between large and small, and between small luteal cells, and A23187 decreased (P<0.05) the rate of GJIC between large and small luteal cells. LH and LH + PGF, but not PGF alone increased (P<0.05) progesterone secretion by luteal cells from the mid and late luteal phases. Agonists or antagonists of PKC or calcium did not affect progesterone secretion by luteal cells. These data demonstrate that both luteal cell types communicate with small luteal cells, and the rate of communication depends on the stage of luteal development. LH and PGF affect GjIC between small luteal cells during the fully differentiated (mid-luteal) and regressing (late luteal) stages of the estrous cycle. In contrast, at all stages of luteal development, activation of PKC decreases GjIC between small and between large and small luteal cells, whereas calcium ionophore decreases GjIC only between large and small luteal cells. Luteotropic and luteolytic hormones, and intracellular regulators, may be involved in regulation of cellular interactions within bovine CL which likely is an important mechanism for coordination of luteal function.

To explore the mechanism(s) by which antiestrogens may protect against the development of cardiovascular disorders, we measured the production of vasodilatory, antiaggregatory prostacyclin (PGI₂ and that of vasoconstrictive, proaggregatory thromboxane A₂ (TxA₂) before and after 6 months' use of antiestrogens in postmenopausal patients after operation for stage II breast cancer (n = 38). Urine samples were assayed by high performance liquid chromatography and radioimmunoassays for 2,3-dinor-6-ketoprostaglandin F1α (=metabolite of PGI₂, dinor-6-keto) and for 2,3-dinor-thromboxane B₂ (=metabolite of TxA₂, dinor-TxB₂). In addition, in 35 of these 38 patients we assayed the capacity of platelets to produce thromboxane A2 during standardized blood clotting. The 4 patients using low-dose aspirin had low thromboxane production, and were excluded from further analysis of the data. An antiestrogen regimen consisting either of tamoxifen (n = 15) or of toremifene (n = 19) caused no changes in production of PGI₂ or TxA₂, or in their ratio, and in this regard, these antiestrogens behaved similarly. Hypertensive patients (n = 7) using different antihypertensive agents were characterized by reduced urinary out-put of dinor-6-keto (18.5 ± 6.1 vs 35.5 ± 18.5 ng/mmol, mean ± SD, p < 0.05) and reduced platelet capacity to produce TxA₂ (62.6 ± 67.8 vs 134.6 ± 75.6 ng/mL, p < 0.05). The patients (n = 15) who had used estrogen replacement therapy (ERT) up until diagnosis of breast cancer showed reduced dinor-TxB₂ excretion (15.5 ± 12.7 vs 29.9 ± 20.9 ng/mmol, p < 0.05) before initiation of antiestrogens, and elevated dinor-6-keto output during the antiestrogen regimen (32.4 ± 21.2 vs 22.7 ± 8.7 ng/mmol, p = 0.07). Smokers (n = 6) had elevated dinor-TxB2 output before and during antiestrogen use. Thus we conclude that the cardiovascular protection provided by an antiestrogen regimen is unlikely to be mediated through vaso- and platelet active PGI₂ and TxA₂.

Objective: Prostaglandins production rises dramatically during term and preterm labor. The source of this production is thought to be the fetal membranes and maternal decidua. The enzyme responsible for the conversion of arachidonic acid to the prostaglandins and related endoperoxides is variously known as prostaglandin synthase or cyclooxygenase (COX). An inducible form of this enzyme, COX-2, has been described in several tissues. The purpose of this study was to investigate a possible role for COX-2 in labor by comparing the COX-2 content in amnion and decidua from laboring and non-laboring patients. Study Design: Fetal membranes from seven normal labor and ten elective cesarean sections at term were collected immediately following delivery. The maternal age and gravity were similar between the groups. The amnion and decidua were identified, washed in sterile saline, frozen in liquid nitrogen and stored in −70°C. COX-2 expression was determined using Western Blot analysis with a purified COX-2 antibody. A scanning densitometer was used to quantify the bands. Results were expressed as mean ±S.D. ng/l50μg protein. Results: The concentration of COX-2 in amnion of laboring women showed a twofold increase ( 240.0 ± 17.6 vs. 120.7 ± 5.1) compared to the non-labored group (p<0.05). The concentration in the decidua showed no significant increase during labor (38.1 ± 7.5 vs. 26.4 ± 2.1, p > 0.05).

Conclusion: We evaluated the role of COX-2 in normal labor. Our study demonstrate that COX-2 is significantly induced in the amnion following spontaneous labor. These findings suggest that the induction of amnion COX-2 may be involved in the process of human labor.

We have cloned a rat kidney thromboxane A2 receptor (TP) cDNA. This receptor was shown to be functional in that the thromboxane A₂ mimetics, U46619 and 1-BOP, elicited calcium transients in Xenopus oocytes and HEK293 cells expressing the TP receptor, respectively. Comparison of the affinities of the rat and human TP sites for the agonist radioligand [¹²⁵I]BOP showed that the rat TP site has about a ten-fold higher affinity for this drug (K_D = 0.5 vs. 4.4 nM) while the affinities of the two sites for other compound (U46619, I-PTH-OH) were the same. Our results are significant in that they identify a cloned TP as having a picomolar affinity for [¹²⁵I]BOP.