Correlation of prostaglandin-induced mitochondrial calcium release with contraction in bovine intrapulmonary vein

The effects of prostaglandins A₂, A₁, F_2α, E₂, E₁, F_1β and an analog of PGH₂ upon calcium release from mitochondria isolated from bovine intrapulmonary vein and contraction of helical strips of the same tissue were determined. The order of activity of the prostaglandins for calcium release was similar to that for contraction with the exception of the PGH₂ analog. It is suggested that prostaglandin A₂, F_2α, E₂ and A₁ induced release of mitochondrial calcium may influence the contractile state of bovine intrapulmonary vein. However, the PGH₂ analog has a subcellular mechanism other than or in addition to mitochondrial calcium release and is different from the other prostaglandins.     

The effects of prostaglandins on aqueous humor dynamics

PGE₂, applied topically to the cornea of enucleated, arterially perfused cat eyes, produced an increase in the rate of aqueous humor (AH) production but only minimal changes in eye pressure, as observed in vivo. In contrast, PGE₁, E₂ and F_2α, administered intra-arterially, induced no change in AH production or arterial perfusate flow rate. In eyes in which the AH inflow rate had been accelerated by prior administration of acetylcholine plus eserine, PGE₁, E₂ and F_2α caused a lowering of the inflow rate as well as vascular dilatation.     

Effects of sex steroids on calling,locomotor activity,and sexual behavior in castrated male Japanese quail

Castrated male Japanese quail were implanted with Silastic capsules containing testosterone (T), estradiol-17β (E₂), 5β-dihydrotestosterone (5β-DHT), Δ₄-androstenedione (Δ₄) 5α-androstanedione (A), 5α-dihydrotestosterone (5α-DHT) or with empty capsules. Calling, monitored continuously and automatically, was induced significantly by T and Δ₄. Locomotor activity, also monitored continuously by floor deflection, was enhanced by T, Δ₄, and E₂. Additional data concerning heterosexual and homosexual behavior were obtained from castrated quails after implantation of T, Δ₄, E₂, or 5α-DHT. T and Δ₄ restored hetero- and homosexual behavior as did E₂ but to a lesser extent. 5α-DHT did not induce either sexual behavior. Growth of the cloacal protrusion was induced in birds implanted with T, Δ₄, A, and 5α-DHT but not with 5β-DHT and E₂. These results indicate that calling and locomotor activity enhancement (including sexual behavior) are two different components of reproductive behavior which require different androgens or their metabolites to be activated.     

Estradiol dependent decrease of binding inhibition by anti-estrogens (a possible test of receptor activation)

The interaction of the uterine estrogen receptor (R) with three anti-estrogens, Dimethylstilbestrol¹, Tamoxifen and Nafoxidine, have been studied either indirectly by competitive experiments or directly using radioactive compounds. The affinity of these anti-estrogens for R was found much higher when determined directly without estradiol (E₂) than when evaluated by competitive experiments. In the presence of E₂, but not in its absence, the inhibitory activity of the anti-estrogens decreased slowly with time. The present report strongly suggests that R is transformed by E₂ into a form less sensitive to anti-estrogen. This “desensitization” of R to the estrogen antagonists is proposed as another $\text{in vitro}$ test for the E₂ induced “activation” of its receptor.     

Improved sensitivity of iodinated histamine-prostaglandin radioimmunoassay by prostaglandin methyl esters

Use of (¹²⁵I)-labeled histamine-prostaglandin tracer increases the sensitivity of the radioimmunoassays of prostaglandin derivatives. Six different antisera were produced for prostaglandins and their derivatives (prostaglandins E₁, E₂, F_1α, F_2α, 13,14-dihydro-15-ketoprostaglandin E₂, and 13,14-dihydro-15-ketoprostaglandin F_2α) and were investigated with the corresponding tritiated and lodinated tracers. Displacement of iodinated tracers by the methyl esters of the prostaglandin compounds resulted, in most cases, in a three- to fivefold increase in sensitivity compared to unesterified inhibitors. Esterification also caused some alteration in the specificities observed. Our results suggest that conformational changes in the esterified prostaglandins (tracer and inhibitor) could explain these charges.     

Prostaglandin stimulation of adenosine 3′,5′-monophosphate accumulation in cultured chondrocytes in the presence or absence of parathyroid hormone

The effect of prostaglandin analogues on the cycle AMP level in cultured chondrocytes were examined. Prostaglandin E₁ at 0.4 to 30 μM, increased the intracellular concentration of cyclic AMP in chondrocytes. Its effect was rapid, being evident within 1 min and reaching a maximum in 10 to 20 min. The maximum level was sustained until 30 min after its addition and then decreased gradually. Prostaglandin D₂ and E₂ also increased the cyclic AMP level in chondrocytes, but they had less effect than prostaglandin E₁. Prostaglandin A₁ had no effect on the nucleotide level in chondrocytes, although they markedly increased the level in fibroblasts. The time course of stimulation of cyclic AMP accumulation in chondrocytes by prostaglandin E₁, D₂ or E₂ was quite different from that by parathyroid hormone (PTH): the effect of prostaglandin was slower and more sustained than that of PTH. PTH potentiated the effect of prostaglandin E₁, E₂, or D₂ on the cyclic AMP level in chondrocytes and that the combined effects of prostaglandin, PTH or both produced a synergistic effect on the accumulation of cyclic AMP in the chondrocytes. These findings suggest that prostaglandin E₁, E₂, and D₂ increase the synthesis of cyclic AMP and that the combined effect of the prostaglandins and PTH on the cyclic AMP level in chondrocytes is partly attributed to the synergistic synthesis of cyclic AMP in the cells.     

A comparison of responses of guinea-pig isolated trachea to six prostaglandins

Effects of prostaglandins (PG) E₁, E₂, F_2α, A₁, A₂ nad B₂ were studied on guinea-pig isolated tracheal chains. PGF_2α, B₂ and A₂ produced contraction, PGE₁ and E₂ relaxation of the chain, but A₁ produced no response. 1) From the cumulative dose response curves, PGF_2α was more active in producing concentration than B₂ or A₂, though its effect was less than that of acetylcholine (ACh). PGE₁-induced relaxation was less than the response to isoproterenol. 2) PGE₁ and E₂ 1 μg/ml caused a 26.1 ± 3.83% (n=5) or a 9.5 ± 3.36 (n=6) decrease of ACh (1 μg/ml)-induced contraction respectively. The degree of relaxation produced by E₁ was greater than that by E₂ (P<0.01). 3) After five minutes preincubation with each of PGA₁, A₂, B₂ and F_2α in concentrations which did not produce any effect, ACh-induced contraction was augmented only after PGA₂ (P<0.05).     

Effect of prostaglandins E1, E2 and F2α on neutrophil aggregation

Recently we have found that chemotactic factors stimulate neutrophils in suspension to aggregate. Because of an obvious analogy to platelet aggregation, we examined the influence of three prostaglandins on this process. Prostaglandins E₁, E₂ and F_2α alone did not cause aggregation of the neutrophils but were able to partially inhibit the aggregation response induced by the synthetic chemotactic tripeptide, formly-methionyl-leucyl-phenylalanine. The minimal inhibitory concentrations for prostaglandins E₁, E₂ and F_2α were 10⁻⁷, 10⁻⁶ and 10⁻⁵M, respectively. These results are similar to those found for the prostaglandin-induced inhibition of platelet aggregation. It may be, therefore, that neutrophil aggregation, like platelet aggregation, is modulated by intracellular prostaglandins and other products of arachidonic acid metabolism.     

Oral Prostaglandins in the Induction of Labour

Prostaglandins E₂ and F₂α were administered by mouth to induce labour in 24 patients at or past term. The drugs were administered at two-hourly intervals in doses ranging from 0·5 to 1·5 mg for prostaglandin E₂ and from 5 to 15 mg for prostaglandin F₂α. Of the 10 cases in which prostaglandin E₂ was used, labour was successfully induced in eight and there were no side effects. With prostaglandin F₂α labour was induced in 12 of 14 patients nine of whom had gastrointestinal disturbance, mostly of mild degree. With both drugs the infant was apparently unaffected and Apgar scores were satisfactory. Uterine hypertonus was not observed and the postpartum blood loss was within normal limits.     

Responsiveness of the lamb ductus arteriosus to prostaglandins and their metabolites

The relative potencies of the prostaglandins A₁, A₂, E₁, E₂, F_2α and their 15-keto-, 15-keto-13,14-dihydro-, and 13,14-dihydro-metabolites were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO₂. All the prostaglandins (except PGF_2α and its 15-keto-metabolites) relaxed the tissue. However, only PGE₁, E₂, and their 13,14-dihydro-metabolites, were effective at concentrations below 10⁻⁸ M. Therefore, events that alter metabolism of circulating PGs in the perinatal period may have significant effects on the relative patency or closure of the ductus arteriosus.     

LTA4-derived 5-oxo-eicosatetraenoic acid: pH-dependent formation and interaction with the LTB4 receptor of human polymorphonuclear leukocytes

5-Oxo-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE) has been identified as a non-enzymatic hydrolysis product of leukotriene A₄ (LTA₄) in addition to 5,12-dihydroxy-(6E,8E,10E,14Z)-eicosatetraenoic acids (5,12-diHETEs) and 5,6-dihydroxy-(7E,9E,11Z,14Z)-eicosatetraenoic acids (5,6-diHETEs). The amount of 5-oxo-ETE detected in the mixture of the hydrolysis products of LTA₄ was found to be pH-dependent. After incubation of LTA₄ in aqueous medium, the ratio of 5-oxo-ETE to 5,12-diHETE was 1:6 at pH 7.5, and 1:1 at pH 9.5. 5-Oxo-ETE was isolated from the alkaline hydrolysis products of LTA₄ in order to evaluate its effects on human polymorphonuclear (PMN) leukocytes. 5-Oxo-ETE induced a rapid and dose-dependent mobilization of calcium in PMN leukocytes with an EC₅₀ of 250 nM, as compared to values of 3.5 nM for leukotriene B₄ (LTB₄) and >500 nM for 5(S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). Pretreatment of the cells with LTB₄ totally abolished the calcium response induced by 5-oxo-ETE. In contrast, the preincubation with 5-oxo-ETE did not affect the calcium mobilization induced by LTB₄. The calcium response induced by 5-oxo-ETE was totally inhibited by the specific LTB₄ receptor antagonist LY223982. These data demonstrate that 5-oxo-ETE can induce calcium mobilization in PMN leukocyte via the LTB₄ receptor in contrast to the closely related analog 5-oxo-(6E,8Z,11Z,14Z)-eicosatetraenoic acid which is known to activate human neutrophils by a mechanism independent of the receptor for LTB₄.     

Effect of prostaglandins on cyclic nucleotide levels in cultured keratinocytes

Guinea pig ear epidermal cells (keratinocytes) were established in primary cultures using trypsin, and treated in their proliferative phase of growth with prostaglandins E₁, D₁, F_1α, E₂, D₂, or F_2α. This phase is induced by the addition of retinoic acid during cell plating. Intracellular content of cAMP and cGMP was measured by radioimmunoassay at various times after treatment.Maximum stimulation of cAMP levels was observed with PGD₂, smaller increases with PGE₂ and relatively transient rises with PGF_2α which were of low significance, but confirm earlier data. Similar results were observed with PGD₁, PGE₁, and PGF_1α with smaller increases. The effects of D and E PGs were biphasic. Significant increases in cGMP were immediately observed with PGD₂ and PGE₂. With PGF_2α, maximum cGMP levels were noted after some delay.All PGs tested showed some effect in elevating cyclic nucleotides in keratinocytes. The most striking result was the increase in cAMP on PGD₂ treatment.     

Effects of prostaglandins and arachidonic acid on baboon cerebral and mesenteric arteries

Effects of prostaglandins (PGs) E₁, E₂, F_2α and I₂ in a wide range of concentration were examined in mesenteric and cerebral arteries isolated from mature baboons. PGs E₁, E₂ and F_2α at low concentrations (10⁻¹⁰ to 10⁻⁷ M) elicited relaxation in helically cut strips of cerebral arteries precontracted with phenylephrine. In contrast, the PGs did not cause relaxation in the mesentric artery. PGI₂ (10⁻⁹ to 10⁻⁶ M) produced marked relaxation in both arteries. The EC₂₅ for PGI₂ in the mesenteric artery was significantly lower than that in the cerebral artery. During baseline conditions, cerebral arteries contracted in response to high concentrations (greater than 10⁻⁷ M) of PGs E₁, E₂ and F_2α. In mesentric arteries, a large contraction was induced by PGs F_2α and E₂ but not by PGE₁. Arachidonic acid (10⁻⁶ M) produced an aspirin-inhibitable relaxation in both arteries to a similar extent, so that the vasodilator PG(s) formed in the two different arterial walls appear to exert a similar relaxant action. Thus, the baboon mesenteric artery was more sensitive to PGI₂ for the relaxant effect than was the cerebral artery, while PGs F_2α, E₁ and E₂ caused only a contraction in the mesenteric artery but both relaxation and contraction in the cerebral artery.     

Activity of prostaglandin E, F, A and B on sphincter, dilator and ciliary muscle preparations of the cat eye

Both sphincter and dilator muscle preparations of the cat iris contract to prostaglandins; F_2α and E₂ are the most potent and A₁ and B₁ the least. Ciliary muscle strips relax to PG's provided that the strips are precontracted. E₁, E₂ and often F_2α are more potent relaxants than the remaining PG's. The effects of PG's are not altered by α or β blockade nor by atropine; however, propranolol blocks the PG induced relaxation of the ciliary muscle. The effects of PG's on the sphincter are antagonized by catecholamines; but the latter act synergistically in contracting the dilator and in relaxing the ciliary muscle. Indomethacin markedly potentiates the effects of PG's on all three muscle preparations.     

Studies of prostaglandins and prostaglandin antagonists on bovine pulmonary vein in vitro

Acetylsalicylic acid (ASA), indomethacin, sodium meclofenamate (FEN), phenylbutazone (PB), phloretin phosphates (PP), SC-19220, and diethylcarbamazine citrate (DECC) were screened against histamine, 5-hydroxytryptamine (5-HT), bradykinin, acetylcholine, and prostaglandins (PG) E₁, E₂, and F_2α to determine their specificity in antagonizing PG's on the bovine pulmonary vein. PG E₂ relaxed the smooth muscle preparation at low concentrations and induced contraction at higher concentrations. PG E₁ consistently evoked dose-related relaxations, whereas PG F_2α contracted the bovine pulmonary vein. Studies with inhibitors suggest that the different actions of prostaglandins could be mediated through different receptors. Sodium meclofenamate and PP dimer blocked PG E₂-induced contractions, whereas relaxations were not blocked. DECC inhibited the relaxant effect of PG E₂. DECC also antagonized histamine, 5-HT, and PG F_2α, suggesting the drug is rather non-specific. Phenylbutazone antagonized the actions of both PG E₂ and PG F_2α on the bovine pulmonary vein. By classifying receptors by antagonism the bovine pulmonary vein appears to contain PG E₂ (PP-type), PG E₂ (FEN-type), PG E₂ (PB-type), and PG F_2α (PB-type) receptors. An absence of SC-type PG-receptors is noted.     

The Influences of Jasmonic Acid Methyl Ester on Microtubules in Potato Cells and Formation of Potato Tubers

Amides in a CH₂Cl₂ extract from the fruits of Piper retrofractum were detected by HPLC/APCI-MS. Seven new unsaturated amides, together with six known ones, were isolated, and their structures were determined to be N-isobutyl-2E,4E,12Z-octadecatrienamide (1), N-isobutyl-2E,4E,14Z-eicosatrienamide (2), 1-(octadeca-2E,4E,12Z-trienoyl)piperidine (3), 1-(eicosa-2E,4E,14Z-trienoyl)piperidine (4), 1-(octadeca-2E,4E-dienoyl)piperidine (5), 1-(eicosa-2E,4E-dienoyl)piperidine (6), and 1-(eicosa-2E,14Z-dienoyl)piperidine (7) on the basis of chemical and spectroscopic evidence.     

Stimulative effect of prostaglandins on production of hexosamine-containing substances by cultured fibroblasts (2) early effect of various prostaglandins at various doses

Early effects of various prostaglandins on the production of hexosamine-containing substances by cultured fibroblasts, which were derived from a rat carrageenin granuloma, were studied. At the stationary phase, the cells were exposed for 6 h to one of the prostaglandin A₁ (PGA₁), A₂, B₁, B₂, D₂, F_1α, F_2α, E₁, E₂ or arachidonic acid in various concentrations ranging from 0.01 to 10 μg/ml for all the stimuli and from 10 pg to 10 μg/ml for PGF_2α. The activity of the cells in incorporating ³H-glucosamine into hexosamine-containing substances (acidic) glycosaminoglycans and glycoproteins) during this period was compared with that of control cells. All the stimuli tested showed more or less stimulative effect on the synthesis of hexosamine-containing substances at their specific concentrations. PGF_2α was found to be the most potent stimulant and its stimulative effect was found significant even at the low concentration of 100 pg/ml. PGD₂, F_1α and E₂ were the next potent stimuli. Their optimum dose were around 1 μg/ml but they still had significant stimulation at the concentration of 0.01 μg/ml. Effect of PGE₂ was rather mild. Stimulation by PGA₁, A₂, B₁ and B₂ or arachidonic acid was seen at high dose, and its seemed to be non-specific. The results suggested that these prostaglandins such as PGF_2α, D₂, F_1α and E₂ play some important role on regulating the production of intercellular ground substances.     

Cooperative Interactions of the Oligodeoxyribonucleotides on the Complementary Template. The Influence of Chemical Groups and Mismatched Nucleotides at the 5′- and 3′-ends of Oligonucleotides on the Parameters of Cooperativity

Abstract

Parameters of cooperative interactions of two or three oligodeoxyribonucleotides or their derivatives bound with the adjacent sites of the complementary template were measured using method of “complementary addressed modification titration” (CAMT). Complementary template (target) were modified with the reactive oligonucleotide derivatives (reagents) bearing covalently attached alkylating 4-[N-(2-chloroethyl)-N-methylaminojbenzylamino- group (C1RCH₂NH)- at 5′-terminal phosphate. The targets had only one binding site for the reagent and either no (T10), or one (T'22 and T22) or two sites (T26) for the oligonucleotides (effectors) cooperatively bound with the adjacent sites on the template. Both unmodified oligonucleotides E₁, E₂ and their derivatives E₁ ^phn, E₂ ^phn bearing N- (2-hydroxyethyl)-phenazinium residues Phn- both at 5′- and 3′- ends covalently linked via ethylenediamine linker were used as effectors. Effectors E₁ and E₂ (E₁ ^Phn and E₂ ^Phn) bind, respectively, upstream or downstream from the reagent. Hexameric (X6) or octameric (X8 or X8^m) reagents were used for the target modification. The reagent X8^m formed one TT-mismatch with the target at the end opposite to location of the reactive moiety. The cooperativity parameter values characterizing the mutual interactions between the reagents X6, X8, X8^m and effectors E₁, E₂, E₁ ^phn, E₂ ^Phn have been found as the ratio of the association constants of the reagents in the presence of effectors. The association constants were calculated from the dependencies of the target modification extent on initial concentrations of the reagents. The use of T26 existing both in linear and hairpin conformations permitted us to estimate additionally the role of indirect cooperativity originating from the induction of the target conformational change by the effectors. The following conclusions were done from the quantitative results. The efficiency of direct cooperativity is independent on the length of oligonucleotide for the same nature of the contact. The cooperativity parameter increases by factor about 3 in the presence of Phn-group covalently attached to oligonucleotides and located at the junctions. The presence of either alkylating group CIRCH₂NH- or TT-mismatch at the junctions eliminates cooperative interaction between the bases. In the same time sufficiently effective cooperative interaction takes place in the case of simultaneous presence of both Phn- and either CIRCH₂NH- group or TT-mismatch at the junction.     

The Cytoprotective Effects of E-α-(4-Methoxyphenyl)-2’,3,4,4'-Tetramethoxychalcone (E-α-p-OMe-C6H4-TMC)—A Novel and Non-Cytotoxic HO-1 Inducer

Cell protection against different noxious stimuli like oxidative stress or chemical toxins plays a central role in the treatment of many diseases. The inducible heme oxygenase isoform, heme oxygenase-1 (HO-1), is known to protect cells against a variety of harmful conditions including apoptosis. Because a number of medium strong electrophiles from a series of α-X-substituted 2’,3,4,4’-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO₂-C₆H₄, Ph, p-OMe-C₆H₄, NO₂, CF₃, COOEt, COOH) had proven to activate Nrf2 resulting in HO-1 induction and inhibit NF-κB downstream target genes, their protective effect against staurosporine induced apoptosis and reactive oxygen species (ROS) production was investigated. RAW264.7 macrophages treated with 19 different chalcones (15 α-X-TMCs, chalcone, 2’-hydroxychalcone, calythropsin and 2’-hydroxy-3,4,4’-trimethoxychalcone) prior to staurosporine treatment were analyzed for apoptosis and ROS production, as well as HO-1 protein expression and enzyme activity. Additionally, Nrf2 and NF-κB activity was assessed. We found that amongst all tested chalcones only E-α-(4-methoxyphenyl)-2’,3,4,4''-tetramethoxychalcone (E-α-p-OMe-C₆H₄-TMC) demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C₆H₄-TMC displayed no significant activity. Also, E-α-p-OMe-C₆H₄-TMC induced HO-1 protein expression and increased HO-1 activity, whilst inhibition of HO-1 by SnPP-IX abolished its antiapoptotic effect. The only weakly electrophilic chalcone E-α-p-OMe-C₆H₄-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus. Furthermore, staurosporine induced NF-κB activity was attenuated following E-α-p-OMe-C₆H₄-TMC treatment. Overall, E-α-p-OMe-C₆H₄-TMC demonstrated its effective cytoprotective potential via a non-toxic induction of HO-1 in RAW264.7 macrophages. The observed cytoprotective effect may partly be related to both, the activation of the Nrf2- and inhibition of the NF-κB pathway.