Inhibition by prostaglandin E2 of neurotransmission in rabbit but not human bronchus — A calcium-related mechanism?

Prostaglandins have been implicated in the development of airway hyperresponsiveness, and this may be mediated via modulation of neurotransmission. We compared the effects of prostaglandin E₂ on the contractile response to electrical field stimulation in rabbit and human bronchus. Prostaglandin E₂ produced marked inhibition in rabbit bronchus (mean % inhibition 35±17, P< 0.05) but was without effect in human bronchus. The inhibition in rabbit bronchus was not the result of a direct effect on muscle tone and the site of action is likely to be pre-synaptic since prostaglandin E₂ had only minor effects on exogenous acetylcholine. Since prostaglandins are known to affect calcium mobilization, we compared the dependence of cholinergic stimulation on the calcium voltage dependent channel (VDC) in the two species. Cholinergic stimulation was dependent on the VDC in rabbit but not human bronchus and this may be an explanation for the different effects of prostaglandin E₂ in the two species.     

Regulation of prostaglandin biosynthesis by human ovarian follicular fluid: a mechanism for ovulation?

We have studied the effect of human ovarian follicular fluid on PG production by bovine seminal vesicles in vitro and found that hFF1 contains a factor of high molecular weight (Mr greater than 30,000) which inhibits PG synthase in a dose-dependent manner. Exposure of this substance to protease activity produced a factor of lower molecular weight (Mr less than 1000) which stimulated PG synthase activity. If this is true of ovarian follicles in vivo, it is possible that increased follicular protease activity stimulates PG synthesis at the time of ovulation.     

Regulation of prostaglandin biosynthesis by human ovarian follicular fluid: A mechanism for ovulation?

We have studied the effect of human ovarian follicular fluid on PG production by bovine seminal vesicles and found that hFF contains a factor of high molecular weight (Mr > 30,000) which inhibits PG synthase in a dose-dependent manner. Exposure of this substance to protease activity produced a factor of lower molecular weight (Mr < 1000) which stimulated PG synthase activity. If this is true of ovarian follicles , it is possible that increased follicular protease activity stimulates PG synthesis at the time of ovulation.     

Mass fragmentographic determination of prostaglandin F2α in human and rabbit urine

Analysis of prostaglandin F_2α (PGF_2α) in urine is a useful indicator of renal prostaglandin synthesis. A mass fragmentographic method for PGF_2α analysis in human urine was developed using [3,3,4,4-²H₄]PGF_2α as an internal standard and carrier. PGF_2α was extracted from urine (20 ml) with chloroform, purified by preparative thin-layer chromatography and converted to the methyl ester trimethylsilyl ether before analysis by gas chromatograph—mass spectrometry. The specificity of the urine analysis was demonstrated by retention time and the use of two pairs of fragments m/e 494/498 and 513/517 with the same results. The coefficient of variation for duplicate analysis averaged 12.6%, n = 17. Urine from recumbent women contained 4.9 ± 2.6 (S.D.) ng/ml or 4.1 ± 1.0 ng PGF_2α per mg creatinine (n = 10) with little diurnal variation. Male urine contained 5.0 ± 2.7 (S.D.) ng/ml or 3.7 ± 2.1 ng/mg creatinine (n = 10). Similar concentrations were found in boys and in girls. These observations indicate that urinary PGF_2α originates from the kidneys with little contribution from the male accessory sexual glands. This method can also be applied to analysis of PGF_2α in rabbit urine.     

β2-Adrenergic regulation of prostaglandin D2 receptor in rabbit platelets

[³H]Prostaglandin D₂ binding to rabbit platelets was increased by about 150% in the presence of β-adrenoceptor agonist, isoproterenol. The isoproterenol-induced potentiation of the [³H]prostaglandin D₂ binding gave a bell-shaped dose-response relationship (maximum response at 3·10⁻⁸ M) in a stereospecific manner. Similar and moderate potentiation was obtained with terbutaline. On the other hand, β-adrenoceptor antagonists such as alprenolol, propranolol and butoxamine (β₂-specific) had no potentiating effect on [³H]prostaglandin D₂ binding; rather, they abolished the isoproterenol-induced increase of [³H]prostaglandin D₂ binding. The β₁-specific antagonist, metoprolol, did not have any effect. Rabbit platelets were found to possess one [³H]prostaglandin D₂ binding site (K_d = 6·10⁻⁷ M, B_max = 787 fmol/mg protein). In the presence of isoproterenol at 3·10⁻⁸ M, B_max was increased with unaltering K_d value. Isoproterenol did not increase [³H]prostaglandin E₁, [³H]prostaglandin E₂ and [³H]prostaglandin F_2α bindings to platelets. The potential effect of isoproterenol was mimicked by forskolin, theophylline, dibutyryl cyclic AMP, prostaglandin E₁ and prostaglandin I₂, but it was abolished by 2′, 5′-dideoxyadenosine, an inhibitor of adenylate cyclase, indicating that elevated level of cyclic AMP may be available for the induction of the increase of [³H]prostaglandin D₂ binding. Prostaglandin D₂-induced cyclic AMP synthesis and antiaggregation activity were also augmented in the presence of isoproterenol. These results suggest a β₂-adrenoceptor-mediated cyclic AMP-dependent mechanism for the regulation of prostaglandin D₂ receptor binding in rabbit platelets.     

Enzymatic formation of prostaglandin F2α in human brain

Prostaglandin (PG)E₂ 9-ketoreductase, which catalyzes the conversion of PGE₂ to PGF₂, was purified from human brain to apparent homogeneity. The molecular weight, isoelectric point, optimum pH, Km value for PGE₂, and turnover number were 34,000, 8.2, 6.5–7.5, 1.0 mM, and 7.6 min^–1, respectively. Among PGs tested, the enzyme also catalyzed the reduction of other PGs such as PGA₂, PGE₁, and 13,14-dihydro-15-keto PGF₂, but not that of PGD₂, 11-PGE₂, PGH₂, PGJ₂, or ¹²-PGJ₂. The reaction product formed from PGE₂ was identified as PGF₂, by TLC combined with HPLC. This enzyme, as is the case for carbonyl reductase, was NADPH-dependent, preferred carbonyl compounds such as 9,10-phenanthrenequinone and menadione as substrates, and was sensitive to indomethacin, ethacrynic acid, and Cibacron blue 3G-A. The reduction of PGE₂ was competitively inhibited by 9,10-phenanthrenequinone, which is a good substrate of this enzyme, indicating that the enzyme catalyzed the reduction of both substrates at the same active site. These results suggest that PGE₂ 9-ketoreductase, which belongs to the family of carbonyl reductases, contributes to the enzymatic formation of PGF₂ in human brain.Special issue dedicated to Dr. Sidney Udenfriend.     

Regulation of prostaglandin availability in human fetal lung by differential localisation of prostaglandin H synthase-1 and prostaglandin dehydrogenase

Specialisation of the respiratory portion of human fetal lung commences around 20-24 weeks gestation. In contrast, human fetal lung in vitro has the capacity to self-differentiate from 12 weeks gestation when grown in media devoid of growth factors or hormones, suggesting activation of autocrine or paracrine factors in vitro, or removal of the fetus from in utero inhibitory mechanisms. Prostaglandins play a key role during in vitro human fetal lung development and are synthesised by prostaglandin H synthase-1 (PGHS-1) and inactivated by 15-hydroxyprostaglandin dehydrogenase (PGDH) with formation of inactive 13,14-dihydro-15-keto-prostaglandins. We have used quantitative immunohistochemistry to determine expression and localisation of PGHS-1, PGDH, PGE2 and 13,14-dihydro-15-keto-PGE2 (PGEM) in human fetal lung with in situ hybridisation to localise PGHS-1 and PGDH mRNA. For the catabolic enzyme PGDH, amounts of mRNA, protein and enzyme product PGEM are increased within epithelium of distal as compared to more proximal airways. For PGHS-1, comparable amounts of mRNA, protein and enzyme product PGE2 are found in proximal and distal lung epithelium. Catabolism by PGDH is a sensitive mechanism for regulating bioavailability of prostaglandins and we propose that active catabolism of prostaglandins within human fetal lung epithelium is an inhibitory mechanism retarding epithelial differentiation in utero.     

Stimulation of HCO3− secretion by the prostaglandin E2 analog enprostil: Studies in human stomach and rat duodenum

This study investigated the action of enprostil, a synthetic analog of PGE₂, on gastric HCO₃⁻ secretion in humans and on duodenal HCO₃⁻ secretion in the anesthetized rat. A previously validated 2-component model was used to calculate gastric HCO₃⁻ and H⁺ secretion in 10 human subjects. Compared to placebo, a single 70 μg oral dose of enprostil increased basal gastric HCO₃⁻ secretion from 1810 +- 340 to 3190 ± 890 μmol/hr (P < 0.05). In addition, enprostil reduced basal gastric H⁺ secretion from 5240 ± 1140 to 1680 ± 530 μmol/hr (P < 0.02). Enprostil also increased HCO₃⁻ and reduced H⁺ secretion during intravenous pentagastrin infusion. In the rat, duodenal HCO₃⁻ secretion was measured by direct titration in situ using perfused segments of duodenum just distal to the Brunner gland area dn devoid of pancreatic and biliary secretions. Addition of enprostil(10 μg/ml) to the duodenal bathing solution increased duodenal HOC₃⁻ secretion from 6.3 ± 1.3 to 15.1 ± 2.0 μmol/cm·hr (P < 0.01, n = 6). The stimulatory action of enprostil on duodenal HCO₃⁻ secretion at 10 μg/ml was comparable in magnitude and duration to that of 10 μg/ml natural PGE₂. In summary, the PGE₂ analog enprostil stimulated gastroduodenal HCO₃⁻ secretion, effects which may be beneficial in protection of the gastroduodenal mucosa against luminal acid.     

Prostaglandin F2α produced by rabbit renal slices is not a metabolite of prostaglandin E2

Slices of rabbit renal medulla and rabbit renal papilla were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15-³H]-arachidonic acid. In both tissues, comparison of the isotope ratios of the radioactive products with the isotope ratio of the added arachidonic indicated that: (a) there was no discernable isotope effect in the biosynthesis of prostaglandin E₂; (b) prostaglandin F₂α was formed by reduction of prostaglandin H₂ and not by reduction of prostaglandin E₂; and (c) most of the radioactive product arose from arachidonic acid that had been incorporated into the tissue and not from the direct action of cyclooxygenase on arachidonic acid in the medium.     

Inhibition of neutrophil apoptosis by acrolein: a mechanism of tobacco-related lung disease?

Cigarette smoking is known to contribute to inflammatory diseases of the respiratory tract by promoting recruitment of inflammatory-immune cells such as neutrophils and perhaps by altering neutrophil functional properties. We investigated whether acrolein, a toxic unsaturated aldehyde found in cigarette smoke, could directly affect neutrophil function. Exposure of freshly isolated human neutrophils to acrolein markedly inhibited spontaneous neutrophil apoptosis as indicated by loss of membrane asymmetry and DNA fragmentation and induced increased neutrophil production of the chemokine interleukin-8 (IL-8). Acrolein (1--50 microM) was found to induce marked activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPKs), and inhibition of p38 MAPK activation by SB-203580 prevented acrolein-induced IL-8 release. However, inhibition of either ERK or p38 MAPK did not affect acrolein-dependent inhibition of apoptosis. Acrolein exposure prevented the activation of caspase-3, a crucial step in the execution of neutrophil apoptosis, presumably by direct inhibition of the enzyme. Our results indicate that acrolein may contribute to smoke-induced inflammatory processes in the lung by increasing neutrophil recruitment and reducing neutrophil clearance by apoptosis.     

Inhibition of ovulation and fertilization by indomethacin and effect of prostaglandin-f2α on early pregnancy in the rabbit

Ovulation induced by HCG in rabbits was blocked by a single subcutaneous injection of 6 mg/kg indomethacin given 6 h after the insemination and HCG treatment. In addition, a time-dependent inhibition in the fertilization rate after indomethacin treatment was also recorded. This suggests that indomethacin, when given at a critical time and at an appropriate dose level, not only blocks ovulation but also interferes with fertilization.Treatment with graded amounts of prostaglandin-F_2α incorporated in a Silastic-polyvinylpyrrolidone-gel (PVP) had marginal to no effect after intravaginal placement on 4- or 6-day pregnancy. However, 5 mg PGF_2α Silastic-PVP tube when placed intravaginally on Day-7 of pregnancy resulted in termination of pregnancy in 66% of the treated does. This implies that young corpora lutea are resistant to PGF_2α treatment and that pregnancy at the time of ovo-implantation can be terminated by PGF_2α incorporated in a Silastic-PVP tube.     

Inhibition of prostaglandin induced uterine activity in the second trimester of pregnancy by β-mimetic adrenergic agents

As a consequence of great individual variations among pregnant women in their response to prostaglandin medication, overstimulation of the uterus might occasionally occur. The need of a drug which could safely prevent excessive and undesirable prostaglandin-induced uterine contractions is therefore apparent.Two β-mimetic adrenergic compounds, orciprenaline and ritodrine, were tested with respect to their ability to reduce prostaglandin-induced uterine activity. Both compounds have successfully depressed the intensity, as well as the frequency of uterine contractions, which were previously induced by intrauterine administration of prostaglandin F_2α. With both drugs the reduction in uterine activity was closely dose-related and accompanied by a considerable increase in the maternal heart rate. While orciprenaline did not alter the maternal blood pressure, ritodrine produced a rise in systolic, and a fall in diastolic pressure. No other side effect was recorded. When the infusion ceased, uterine activity increased in various degrees.On the basis of the present findings the possible role of prostaglandins in the physiological regulation of the uterine contractions during pregnancy is discussed.     

Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ – prostaglandin E2 dependent proliferation of growth plate chondrocytes

Prostaglandin E₂ (PGE₂) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE₂ pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE₂ receptors) is essential. We therefore examined the production of PGE₂ in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE₂ on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE₂ receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE₂ synthesis was determined by mass spectrometry, cell proliferation by DNA [³H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE₂ into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE₂-dependent proliferation. Exogenously added PGE₂ stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10^-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE₂ was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE₂ release, which stimulates cell proliferation via the EP1 receptor.     

Is the spontaneous motility of isolated rat uterus controlled by prostaglandin E?

Variations in the spontaneous contractile activity during 6 hours following isolation of uterine horns from proestrus, metestrus and spayed rats, were explored. In estrus and metestrus preparations the contractions declined during 60 min and between 180--200 min a progressive spontaneous recovery (abolished by indomethacin) was observed up to 360 min. Uteri from proestrus and spayed animals exhibited a continuous depression without recovery during the whole experimental period. At 60 min, uterine horns from estrus animals (which showed a marked contractile decrement) released to the suspending medium significantly less prostaglandin E-like material than at 360 min, i.e. when contractions had almost completely recovered. No modification in the amount of prostaglandin F-like material was detected accompanying these spontaneous contractile variations. In the spayed group at 60 min of functioning (i.e. when the contractile impairment was significantly smaller than at a later time) the release of PGE was greater than at 360 min. These findings suggest a possible control of rat uterine contractions by PGE, rather than by PGF.     

Is the spontaneous motility of isolated rat uterus controlled by prostaglandin E?

Variations in the spontaneous contractile activity during 6 hours following isolation of uterine horns from proestrus, metestrus and spayed rats, were explored. In estrus and metestrus preparations the contractions declined during 60 min and between 180–200 min a progressive spontaneous recovery (abolished by indomethacin) was observed up to 360 min. Uteri from proestrus and spayed animals exhibited a continuous depression without recovery during the whole experimental period. At 60 min, uterine horns from estrus animals (which showed a marked contractile decrement) released to the suspending medium significantly less prostaglandin E-like material than at 360 min, i.e. when contractions had almost completely recovered. No modification in the amount of prostaglandin F-like material was detected accompanying these spontaneous contractile variations. In the spayed group at 60 min of functioning (i.e. when the contractile impairment was significantly smaller than at a later time) the release of PGE was greater than at 360 min. These findings suggest a possible control of rat uterine contractions by PGE, rather than by PGF.     

Inhibition of prostaglandin I2 (PGI2) formation by LDL-cholesterol or LDL-peroxides?

A hypothesis of Gryglewski et al. explains the correlation between increased level of LDL and development of atherosclerosis by inhibition of PGI₂ synthesis by increased peroxide content of LDL. The aim of the present paper was to examine this hypothesis. The major results are: 1) Preparation of LDL in the presence of .02 % butylated hydroxytoluene did not reduce the lipid peroxide content of LDL from men and women and not change the inhibition or stimulation of the in vitro biosynthesis of PGI₂ by LDL isolated from blood of men or women, respectively. 2) In the LDL and HDL, respectively, of healthy men we found nearly the same lipid peroxide levels (nmole malondialdehyde (MDA)/mg lipoprotein-cholesterol) as in the lipoproteins of male patients with hyperlipidemia type IIa or IV, but the peroxide concentration is three times higher in HDL as in LDL. 3) LDL isolated from healthy men inhibited in dose dependent fashion the generation of PGI₂ from PGH₂ by aortic microsomes whereas LDL from premopausal women stimulated PGI₂ formation even calculated as LDL lipid peroxides (in nM MDA/ml). The results call into question the hypothesis that diminished PGI₂ formation by atherosclerotic vessels is related to inhibition of PGI₂ synthetase by lipid peroxides present in LDL in the lesions. A new working hypothesis is presented that also the fatty acid pattern and the lipid class composition in the LDL are important for their influence on the PGI₂ formation.     

DNA-damage tolerance mediated by PCNA?Ub fusions in human cells is dependent on Rev1 but not Polη

In response to replication-blocking lesions, proliferating cell nuclear antigen (PCNA) can be sequentially ubiquitinated at the K164 residue, leading to two modes of DNA-damage tolerance, namely, translesion DNA synthesis (TLS) and error-free lesion bypass. Although the majority of reported data support a model whereby monoubiquitinated PCNA enhances its affinity for TLS polymerases and hence recruits them to the damage sites, this model has also been challenged by several observations. In this study, we expressed the PCNA-164R and ubiquitin (UB) fusion genes in an inducible manner in an attempt to mimic PCNA monoubiquitination in cultured human cells. It was found that expression of both N- and C-terminal PCNA•Ub fusions conferred significant tolerance to ultraviolet (UV)-induced DNA damage. Surprisingly, depletion of Polη, a TLS polymerase dedicated to bypassing UV-induced pyrimidine dimers, did not alter tolerance conferred by PCNA•Ub. In contrast, depletion of Rev1, another TLS polymerase serving as a scaffold for the assembly of the TLS complex, completely abolished PCNA•Ub-mediated damage tolerance. Similar genetic interactions were confirmed when UV-induced monoubiquitination of endogenous PCNA is abolished by RAD18 deletion. Hence, PCNA•Ub fusions bypass the requirement for PCNA monoubiquitination, and UV damage tolerance conferred by these fusions is dependent on Rev1 but independent of Polη.