Influence of chronic antigen exposure on the metabolism of PGH2 by microsomal preparations of airway and parenchymal tissues in the sheep

The effects of repeated antigen exposure on the synthesis of mediators by lung tissues are not well understood. To investigate the influence of antigen challenge on the synthesis of prostaglandins by central airway and peripheral lung tissues, fourteen sensitive sheep underwent biweekly exposure to aerosolized Ascaris suum antigen (7) or saline (7). Following the fifth exposure, microsomal and high speed supernatant fractions were prepared from trachealis muscle and lung parenchyma. Synthesis of thromboxane (TX) A2, prostaglandin (PG) D2 and PGI2 from the PG endoperoxide intermediate, PGH2, was assayed over a range of substrate concentrations from 3-200 microM. Synthesis of PGI2 by trachealis microsomes was approximately 5-fold greater than that of TXA2. PGI2 and TXA2 production was identical in tracheal preparations from Ascaris- and saline-exposed animals. In parenchymal tissues, where TXA2 production predominated over PGI2 by 9-fold, preparations from Ascaris-exposed animals synthesized 50% more TXA2 than controls at PGH2 concentrations of 25 microM and above, whereas synthesis of PGI2 and PGD2 were similar in preparations from both groups of animals. The density of pulmonary mast cells was decreased by 21% in the Ascaris group, whereas polymorphonuclear leukocyte density was unchanged. These results demonstrate the differential synthesis of TXA2 and PGI2 in central airways and peripheral lung regions of the sheep. They further indicate that repeated exposure of the airways to antigen selectively enhances TXA2 synthesis in the lung periphery of sensitized animals. The site of this increased enzymatic activity, whether in resident cells or newly-infiltrated cells, has not been determined.     

Comparison of the actions of 11.9 epoxymethano PGH2 and 9.11 AZO PGH2 on rat aorta and stomach strip preparations

Two synthetic thromboxane A₂ agonists were compared on two isolated smooth muslce preparations, the rat aorta and stomach-strip using a superfusion cascade. 11.9 epoxymethano-PGH₂ contracted both preparations in a concentration dependent manner. Cumulative concentration-response curves showed that azo PGH₂ was twice as active as 11.9 epoxymethano PGH₂ on the aorta and nearly four times as active as 11.9 epoxymethano PGH₂ on the stomach-strip. Statistical analysis of the agonist concentration-response curves showed a significant differences when responses to azo PGH₂ were compared on the two preparations and when responses to azo PGH₂ and 11.9 epoxymethano PGH₂ were compared to the aorta preparation. When EP045, a thromboxane receptor antagonist was used the PA₂ values obtained were not significantly different using the two agonists on the two preparations. The pA₂ values indicate that both agonists act on the same receptor but the comparison of the concentration-response curves suggest that azo PGH₂ may be acting at another receptor as well as the thromboxane receptor in the stomach-strip.     

PGH2, TxA2 and PGI2 have potent and differentiated actions on human uterine contractility

The contractile response to three different prostanoids of the isolated human myometrium and the different layers of the utero-tubal junction (UTJ) was studied $\text{in vitro}$. The prostaglandin endoperoxide, PGH₂, stimulated contractility of both the myometrium and the outer and inner muscle layers of the UTJ, whereas the intermediate layer of the UTJ was inhibited. Thromboxane A₂ generated from PGH₂ and a thromboxane synthase preparation caused a stimulation of both the myometrium and all three layers of the UTJ. The stimulatory response to TxA₂ occurred at concentrations as low as 50–70 pg/ml. The sodium salt of PGI₂ was found to relax both the myometrium and all the layers of the UTJ. Intravenous administration of PGI₂ in repeated doses between 2–8 μg induced facial flushing and headache but had little if any effect on $\text{in vivo}$ uterine contractility. At least under $\text{in vitro}$ conditions, these short-lived prostanoids and/or their metabolites apparently have a specific action on uterine contractility, an action which is manifested at comparatively low concentrations.     

Preparation and biochemical properties of PGH3

PGH₃ was biosynthesised from all-cis-5,8,11,14,17-eicosapentaenoic acid (20:5ω3) by an acetone-pentane powder of ram seminal vesicles and its structure was confirmed by GLC-MS after its reduction to PGF₃α. PGH₃ was transformed by horse platelet microsomes to TXB₃, and by aortic microsomes to Δ¹⁷-6-keto-PGF₁α. The structures of these compounds were confirmed by GLC-MS.     

A comparison of the effects of PGI2, PGE2 and PGH2 on the cyclic nucleotide levels in rat anterior pituitary glands

Rat anterior pituitary explants were incubated with PGI₂, PGH₂ and PGE₂ in the presence of theophylline (1mM) and the production of cyclic AMP was measured. PGE₂ was found to be about 20 times more potent than PGI₂ while PGH₂ was slightly more effective than PGI₂. The results suggest that PGI₂ does not play a physiological role in cyclic AMP mediated events in the rat anterior pituitary.     

Inhibition of GSH-dependent PGH2 isomerase in mammary adenocarcinoma cells by allicin.

We have reported tha allicin, a constituent of garlic oil, has no effect on the activities of platelet cyclooxygenase or thromboxane synthase, or vascular PGI₂ synthase. The effect of allicin on glutathione (GSH) dependent PGH₂ to PGE₂ isomerase is unknown. We therefore studied the effect of allicin on PGE₂ biosynthesis in a murine mammary adenocarcinoma cell line (No 4526). Intact or sonicated cells were incubated with either ¹⁴C-arachidonic acid (AA) or ¹⁴C-PHG₂, respectively. Following metabolism, products were extracted, separated by TLC and analyzed by radiochromatographic scan. PGE₂ was predominantly formed with minimal amounts of PGF_2α and PGD₂. Formation of 6-keto-PGF_1α or TXB₂ was not detected indicating the absence of TXA₂ and PGI₂ synthase activity. Indomethacin and ibuprofen inhibited the PGE₂ formation (p < 0.05). The enzymatic PGE₂ formation in sonicates was blocked by depletion of the cellular non-protein thiols by buthionine sulfoximine and was shown to be dependent on GSH. Allicin, over the range of 10–1000 μM, inhibited the formation of PGE₂ in cells exposed to 2.0 μM ¹⁴C-AA for 20 min. and in sonicated cells incubated with 20.0 μM ¹⁴C-PGH₂ for 2 min (p < 0.05). Allicin did not alter cyclooexygenase-mediated oxygen utilization in ram seminal vessicle microsomes, suggesting that allicin selectively inhibits the GSH-dependent PGH₂ to PGE₂ isomerase in this adenocarcinoma cell line.     

The effect of PGH2 on blood flow in the canine renal and superior mesenteric vascular beds

Effects of the prostaglandin endoperoxide, PGH₂, were investigated in the renal and superior mesenteric vascular beds in anesthetized dogs. Vascular effects of a stable PGH₂ analog were also studied in the intestine. Blood flow was measured with electromagnetic flowmeters and vasoactive hormones were administered by close intra-arterial injection. Authentic PGH₂ increased blood flow in the kidney and intestine in a dose-related manner. Mesenteric blood flow was reduced by the PGH₂ analog in a dose-dependent fashion which was similar to the vasoconstrictor activity of norepinephrine in this organ. PGH₂ is biologically unstable and the type and activity of its metabolic products may vary in different regional vascular beds. Most of the known products of PGH₂ metabolism in the kidney are vasodilators whereas in the intestine both vasodilator and vasoconstrictor metabolites are formed. It has been suggested that the vascular activity of PGH₂ in an organ is dependent on the predominant type and activity of specific terminal enzymes that convert PGH₂ to its various vaso-active products.     

Modulation of the reductive metabolism of halothane by microsomal cytochrome b5 in rat liver

To study the modulation of the reductive metabolism of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) by microsomal cytochrome b₅, formation of 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), major reduced metabolites of halothane, was analyzed in vivo and in vitro. Rats were pretreated with both malotilate (diisopropyl-1,3-dithiol-2-ylidenemalonate) and sodium phenobarbital (malotilate-treated rats) or only with sodium phenobarbital (control rats). The microsomes of malotilate-treated rats had significantly more cytochrome b₅ than the controls, whereas the cytochrome P-450 content was not different between the two groups. At the end of 2-h exposure to 1% halothane in 14% oxygen, the ratio of CDE to CTE in arterial blood was significantly higher in malotilate-treated rats than in the controls. Under anaerobic conditions, the formation of CDE and the ratio of CDE to CTE were significantly greater in microsomal preparations of malotilate-treated rats than those of the controls. In a reconstituted system containing cytochrome P-450_PB purified from rabbit liver, addition of cytochrome b₅ to the system enhanced the formation of CDE and increased the ratio of CDE to CTE. These results suggested that cytochrome b₅ enhances the formation ratio of CDE to CTE by stimulating the supply of a second electron to cytochrome P-450, which might reduce radical reactions in the reductive metabolism of halothane.     

Synthesis of [17,18-3H]trans-13-azaprostanoic acid. A labeled probe for the PGH2/TXA2 receptor

Because of its highly unstable nature, TXA₂, produced by platelet metabolism of arachidonic acid, does not lend itself to use as a receptor probe for its own receptor. As such, the stable TXA₂/PGH₂ antagonist, trans-13-azaprostanoic acid (trans-13-APA, 12b), was prepared as the [17,18 ³H] derivative ([³H] trans-13-APA, 12c) to study this receptor and to better evaluate the mechanism of action of these azaprostanoids. Tritiated trans-13-APA, 12c, was prepared in nearly theoretical specific activity (57 Ci/mmole) from (17z)-trans-13-azaprost-17-enoic acid (11b) by catalytic tritiation. The unsaturated 11b was prepared by condensation of cis-7-amino-3-heptene (8) with 2-(6-carboxyhexyl) cyclopentanone (9), NaBH₄ reduction, chromatography, and hydrolysis of the trans isomer so isolated. The olefins 11a and b were also of biochemical interest because of the unsaturation in the lower side chain. The presence of similar unsaturation in PGH₃ (4) and TXA₃ (3) renders these prostaglandins inactive as proaggregatory agents. Evaluation of the antiaggregatory activity of 11a and b indicated it to be about the same potency in inhibiting human platelet aggregation as the parent cis and trans-13-APAs, suggesting that introduction of a double bond at the 17 position in platelet prostaglandin antagonists is unlikely to result in enhanced antiplatelet activity.     

A comparison of the effects of PGI2, PGE2 and PGH2 on the cyclic nucleotide levels in rat anterior pituitary glands in vitro

Rat anterior pituitary explants were incubated with PGI₂, PGH₂ and PGE₂ in the presence of theophylline (1mM) and the production of cyclic AMP was measured. PGE₂ was found to be about 20 times more potent than PGI₂ while PGH₂ was slightly more effective than PGI₂. The results suggest that PGI₂ does not play a physiological role in cyclic AMP mediated events in the rat anterior pituitary.     

New preparations and molecular structures of cis-MCl2(Ph2PCH2PPh2)2, M = Ru,Os and trans-RuCl2(Ph2PCH2PPh2)2

The title compounds were made by reacting bis(diphenylphosphino)methane (dppm) with reduced solutions of OsCl₆^4? and Ru₂OCl₁₀^4?. The crystal and molecular structures of these compounds have been determined form three-dimensional X-ray study. The cis-isomers crystallize with one CHCl₃ per molecule of the complex. All three compounds crystallize in the monoclinic space group P2₁/n with unit cell dimensions as follows: Cis-OsCl₂(dppm)₂·CHCl₃: a = 13.415(4) Å, b = 22.859(4) Å, c = 16.693(3) Å, β = 105.77(3)°, V = 4926(3) ų, Z = 4. cis-RuCl₂(dppm)₂·CHCl₃: a = 13.442(3) Å, b = 22.833(7) Å, c = 16.750(4) Å, β = 105.53(2)°, V = 4953(3) ų, Z = 4. trans-RuCl₂(dppm)₂: a = 11.368(7) Å, b = 10.656(6) Å, c = 18.832(12) Å; β = 103.90(6)°, V = 2213(7) ų; Z = 2. The structures were refined to R = 0.044 (R_w = 0.055) for cis-OsCl₂(dppm)₂·CHCl₃; R = 0.065 (R_w = 0.079) for cis-RuCl₂(dppm)₂·CHCl₃ and R = 0.028 (R_w = 0.038) for trans-RuCl₂(dppm)₂. The complexes are six coordinate with stable four-membered chelate rings. The PMP angle in the chelate rings is ca. 71° in each case.     

Maintenance of microsomal cytochromes b5 and P-450 in primary cultures of parenchymal liver cells on collagen membranes

In previous reports from various laboratories, the levels of the microsomal cytochromes b₅ and P-450 in hepatocytes in primary culture have been found to be very low and difficult to measure. The studies reported in this paper demonstrate that cytochromes b₅ and P-450 in hepatocytes cultured on floating collagen membranes for periods of at least 10 days are maintained at levels readily measured by conventional techniques and comparable to those of liver $\text{in}$$\text{vivo}$. Addition of high levels of hydrocortisone (10^?4M) to the culture medium for periods up to 10 days resulted in further increases in the levels of these cytochromes. Cells cultured in the presence of hydrocortisone exhibited the appearance of cytochrome P-448, in contrast to the cells cultured in the absence of hydrocortisone, where cytochrome P-450 was maintained.     

The influence of xanthines on the contractile responses of PGF2α and PGD2 in human parenchymal strips

Prostaglandins (PGs) F_2α and D₂ are bronchoconstrictor agents which are released under allergic conditions such as asthma. The efficacy and potency of PGF_2α and PGD₂ differ in some tissues. We compared the effects of these two PGs in sensitized human parenchymal strips. In six experiments, PGF_2α 0.1 and 0.3 μM produced greater contractions than PGD₂ at the same concentrations. There were no significant differences between the contractions from the two PGs at concentrations of 0.01, 0.03, 1.0–10 μM and the two PGs appeared to be equipotent.We studied the effects of the anti-asthmatic drug theophylline, and its analogue enprofylline, on the contraction caused by these PGs. Theophylline 100 μM caused no change to the cumulative concentration response curves. However, enprofylline 100 μM reduced the PGF_2α-induced contractions.     

Thermotropic change in phospholipid packing in microsomal membranes sensed by phospholipase A2

A reversible, temperature-dependent change in phospholipid packing occurring between 0°C and 12°C has been identified in microsomal membranes by the use of phospholipase A₂ from Crotalus atrox. It manifests itself as a drastic increase in susceptibility to the phospholipase and depends on non-lipid (presumably protein) membrane components. It is suggested that this change could underlie the change in transmembrane mobility of phospholipids which occurs in the same temperature range.     

The effects of CO2 and chronic cold exposure on fecundity of female Drosophila melanogaster

Carbon dioxide and chilling are sometimes used to immobilise insects for laboratory research. Both of these methods are known to have short-term effects on behaviour and physiology in Drosophila, but their long-term impacts are unknown. We exposed female D. melanogaster adults to high CO₂ concentrations (4 h at 18,000 ppm) and chronic cold (72 h at 4 °C). The carbon dioxide exposure increased chill coma recovery time, but did not result in changes in offspring number, sex ratio, or size. By contrast, the cold exposure resulted in fewer, smaller offspring, and resulted in a male-biased sex ratio compared to controls. There was no significant interaction between CO₂ and cold. We conclude that although caution must be used in choosing an immobilisation method, CO₂ appears to have less long-term impact than cold.     

Effects of thioredoxin on established airway remodeling in a chronic antigen exposure asthma model

The development and treatment of asthma remains a subject of considerable interest in the medical community. Previous studies implicate an important role of cytokines in the pathology of asthma. In this current study, we examined whether redox-active protein thioredoxin 1 (TRX1) could prevent airway remodeling in an ovalbumin (OVA)-driven mouse chronic antigen exposure asthma model. Balb/c mice were sensitized and then challenged nine times with OVA (days 19-45). In this protocol, airway remodeling was established by day 34. Administration of recombinant human TRX1 during antigen challenge (days 18-32) significantly inhibited airway remodeling, eosinophilic pulmonary inflammation, airway hyperresponsiveness and resulted in decreased lung expression of eotaxin, macrophage inflammatory protein-1alpha and IL-13. Airway remodeling and eosinophilic pulmonary inflammation was also prevented in chronic OVA-exposed Balb/c human TRX1 transgenic mice. Importantly, TRX1-administration, after the establishment of airway remodeling (days 35-45), resulted in improved airway pathology. Our results suggest TRX1 prevents the development of airway remodeling, and also improves established airway remodeling by inhibiting production of chemokines and Th2 cytokines in the lungs.     

Effects of all CIS-5, 8,11,14,17 eicosapentaenoic acid and PGH3 on platelet aggregation

In human platelet-rich plasma (PRP) eicosapentaenoic acid (EPA) inhibited platelet aggregation induced by a stable analogue of PGH₂ (U46619), arachidonic acid, collagen or ADP. EPA was more potent than oleic, linoleic, α-linolenic or γ-linolenic acids. In aspirin-treated platelets, aggregation induced by U46619 was inhibited to a similar extent by arachidonic acid or by EPA over a range of concentrations of 0.05–0.3 mM. EPA incubated with PRP did not induce the generation of a thromboxane (TXA)-like activity; indeed it prevented the formation of TXA₂ induced by arachidonic acid or by collagen. The anti-aggregatory activity of EPA was not influenced by inhibitors of cyclo-oxygenase and lipoxygenase. The anti-aggregatory action of EPA may be caused by a rapid occupancy by EPA of TXA₂/PGH₂ “receptors” on platelet membrane as well as by a slower displacement of arachidonic acid from platelet phospholipids by chemically unchanged molecules of EPA.Not all samples of PRP were irreversibly aggregated by PGH₂, but in those that were, PGH₃ also induced an immediate dose-dependent but reversible aggregation. After a 4 min incubation of non-aggregating doses of PGH₂ or PGH₃ (100–300 nM) with PRP a stable anti-aggregatory compound was detected. The inhibitory activity produced from PGH₃ was apparently more potent (ca 10 times) than that obtained from PGH₂. The anti-aggregating compounds were identified by TLC and GLC-MS as PGD₂ and PGD₃. The apparent difference of potency between PGD₂ and PGD₃ was attributed to the concurrent production of PGE₂ and PGE₃. PGE₂ prevented the inhibitory effect of PGD₂ whereas PGE₃ did not affect the activity of PGD₃.It is concluded that one of the reasons for the low incidence of myocardial infarction in Eskimos could be that the pro-aggregatory arachidonic acid is replaced in their phospholipids by the anti-aggregatory EPA.