Uterine vein prostaglandin levels in late pregnant dogs

We studied the uterine venous plasma concentrations of prostaglandins E₂, F_2α, 15 keto 13,14 dihydro E₂ and 15 keto 13,14 dihydro F_2α in late pregnant dogs in order to evaluate the rates of production and metabolism of prostaglandin E₂ and F_2α in pregnancy $\text{in vivo}$. We used a very specific and sensitive gas chromatography-mass spectrometry assay to measure these prostaglandins. The uterine venous concentrations of prostaglandin E₂ and 15 keto 13,14 dihydro E₂ were 1.35±.27 ng/ml and 1.89±.37 ng/ml, respectively; however, we could not find any prostaglandin F_2α and very little of its plasma metabolite in uterine venous plasma. Since uterine microsomes can generate prostaglandin F_2α and E₂ from endoperoxides, prostaglandin F_2α production $\text{in vivo}$ must be regulated through an enzymatic step after endoperoxide formation. Prostaglandin E₂ is produced by pregnant canine uterus in quantities high enough to have a biological effect in late pregnancy; however, prostaglandin F_2α does not appear to play a role at this stage of pregnancy.     

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.     

Properties of solubilized prostaglandin synthetase from sheep vesicular glands.

Prostaglandin synthetase activity associated with the microsomal fraction from sheep vesicular glands has been solubilized by treatment with the non-ionic detergents Tween 20, Lubrol Px and Lubrol Wx. Approx.8 fold purification from microsomes is obtained and over 90% of the activity is recovered in the detergent solubilized fraction. The solubilized synthetase activity is stable at pH 5.0 but is gradually lost at pH 8.0; it is also heat and acid labile. The relative amounts of prostaglandins E₂, D₂ and F_2α formed by the microsomal-bound synthetase and by the solubilized synthetase are similar. Also similar are the pH optima (7.9–8.5) of the two synthetase preparations. The solubilization process appears to yield a fully active enzymatic preparation which could be employed for further purification and characterization of the prostaglandin synthetase complex.     

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.     

Melittin-stimulated arachidonic acid metabolism by cultured malignant human epidermal keratinocytes

Upon melittin stimulation, cultured SCC-13 keratinocytes release prostaglandins E₂, F_2α, 6-keto-F_1α, thromboxane B₂, leukotriene B₄, and 6-sulfido-peptide-containing leukotrienes (SRS) into serum free medium. Release of prostaglandins E₂, F_2α, and SRS, normalized to cell protein, is 3- to 10-fold higher from rapidly growing than confluent cultures. Cells growing with hydrocortisone in the medium produce approximately twice the level of the cyclooxygenase-mediated metabolites PGE₂ and PGF_2α as those without hydrocortisone, but similar levels of the lipoxygenase-mediated metabolite SRS. The results demonstrate the potential utility of squamous carcinoma lines for investigating biochemical pathways of arachidonic acid metabolism in keratinocytes.     

Prostaglandins in the isolated testicular capsule of immature and young adult rats

The presence of prostaglandin (PGs) E₁, E₂, F_1α, F_2α and the metabolite 13, 14-Dihydro-15-keto PG Fa (1+2) has been studied in the isolated testicular capsule of Wistar rats (22 to 90 days of age). Handling and homogenization of tissues were controlled and the in vitro synthesis and degradation were prevented by immediated freezing of samples and homogenization in a solution containing of PG synthetase inhibitor. The PGs were measured by specific and sensitive radioimmuoassay. The isolated rat testicular capsule was found to contain mainly PG E₂ and PG_2α at concentrations about 100 times higher than those in decapsuled testes (ng/g of tissue).     

High-affinity binding sites for prostaglandin E on human lymphocytes.

Binding sites on human lymphocytes for prostaglandins were examined by incubating cells with [³H]prostaglandin (PG) A₁, E₁, E₂, F_1α, and F_2α. Specific reversible binding for [³H]PGE₁ and E₂ was found with a K_d of ~2 × 10^?9M and a B max of ~200 binding sites per cell, assuming uniform distribution. We detected no specific binding of [³H]PGA₁, F_1α, or F_2α to lymphocytes. Also, the addition of 10- to 1000-fold greater amounts of unlabeled PGA, F_1α, or F_2α did not inhibit the binding of [³H]PGE. The time course of [³H]PGE binding appeared to be bimodal with one component complete within 5 min at 37 °C and another component of binding increasing over a 40-min incubation. We feel that the rapid component of binding may represent cell surface receptors for PGE while the slower component may represent a specific uptake mechanism for PGE into the cell. Glass adherent cells had fewer binding sites than nonadherent cells. Preincubation of the cells overnight resulted in a loss of binding sites.     

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.     

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).     

hCG-induced prostaglandin E2 and F2 alpha release in adult rat testis: role in Leydig cell desensitization to hCG.

Testicular levels of prostaglandin E₂ and F_2α were measured in decapsulated adult rat testis following hCG stimulation. Basal levels were, respectively, 342 ± 74 and 502 ± 89 pg/testis. Following hCG administration these basal values are not significantly modified up to 2 hours. From 2 to 24 hours the concentrations are clearly increased above the basal level: at 12 hrs they are 1925 ± 165 for E₂ and 3200 ± 190 for F_2α. Levels are back to normal at 48 hrs and remain so until 144 hrs. An identical pattern of prostaglandin release is observed in vitro in Leydig cell preparations isolated at different times following in vivo hCG injection. This suggests that prostaglandins are secreted by Leydig cells. In hypophysectomized animals the release of both prostaglandins E₂ and F_2α is similar to controls indicating that prostaglandin secretion is not directly linked to testosterone production. alternatively testosterone injections (10 mg) does not modify prostaglandin levels. Binding sites for prostaglandins E₁, E₂ and F_2α are present on the Leydig cells and consequently Leydig cell function may be modulated by endogenous or exogenous prostaglandins. Their level is slightly increased at 24 hrs following hCG stimulation. Since the acute changes in prostaglandin E₂ and F_2α secretion occur during the period of “desensitization” and of acute “down regulation” of the LH-hCG receptor in the Leydig cells it is suggested that prostaglandins are involved in both phenomena.     

A rapid gas chromatographic-mass spectrometric method for prostaglandin analysis at picomole levels

A rapid method for the qualitative and quantitative determinations of PGE₁, E₂, F_1α, and F_2α is described. Tris-TMS-PGF and mono-TMS-PGB methyl esters are obtained by the reaction of the corresponding PGF and PGE methyl esters with N-trimethylsilylimidazole (TSIM) and piperidine. The reaction is instantaneous, and a single derivative with excellent gas chromatographic properties is obtained for each prostaglandin tested. The presence of very prominent peaks in the mass spectra of the derivatives allows the determination of prostaglandins at picomole levels using multiple ion detection (MID).     

Effects of prostaglandins E1, E2 and F2α on the cardiac chronotropism by affecting the cardiac ganglionic transmission in spinal dogs

The effects of several prostaglandins (PGs) injected through the subclavian artery toward the cardiac sympathetic ganglia of spinal dogs were studied by utilizing changes of the heart rate as indicator of ganglionic function. PGF_2α (10–270 μg) administered intra-arterially in the presence or absence of preganglionic stimulation produced weak positive chronotropic effects, which were increased by physostigmine. This positive chronotropic effect of F_2α after physostigmine was inhibited by hexamethonium plus atropine, and depressed after hemicholinium-3 except for the response elicited by the first dose of F_2α. PGE₁ and E₂ injected during preganglionic stimulation did not affect the heart rate. Intra-arterially administered epinephrine and dopamine depressed dose-dependently transmission in the cardiac ganglia, the effect being inhibited by E₁ and E₂ but not by F_2α. These results suggest that F_2α facilitates the release of acetylcholine from preganglionic nerve ending, whereas E₁ and E₂ antagonize the inhibitory actions of catecholamine in the cardiac ganglia.     

Effect of prostaglandins on α-aminoisobutylic acid uptake in cultured fibroblasts

Effect of various prostaglandins on the uptake of α-aminoisobutylic acid by cultured fibroblasts was studied. All the prostaglandins having an OH functional group in an intramolecular 5-membered ring showed an inhibitory effect on the amino acid uptake. The active compounds can be ranked in potency according to the values for the inhibition of the amino acid uptake per cent of control: prostaglandin F_2α(53 %) >F_1α(54 %) >D₂(56 %) >E₂(62 %) >thromboxane B₂ (66 %). Thus, prostaglandin F_2α was found to be the most potent inhibitor to membrane permeability and the inhibitory effect was dose dependent. The inhibition was maximal after 1 hour of exposure to prostaglandin F_2α, persisted at least up to 6 hours in the presence of prostaglandin F_2α.     

Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2alpha receptors.

Pretreatment of membranes for 1 hr at 4° with up to 0.1% Triton X-100 (TX-100) and sodium desoxycholate (SDC), resulted in a greater loss of [³H] prostaglandin (PG)F₂α binding compared to E₁ binding. Lubrol WX (LWX) tended to cause a greater loss of [³H]PGF₂α than E₁ binding. However, the differential loss was not as marked as with TX-100 or SDC. Triton X-305 was relatively ineffective, but loss of [³H]PGE₁ binding was greater than for PGF₂α. Increasing concentrations of dimethylsulfoxide (DMSO) progressively inhibited PGF₂α binding without affecting PGE₁ binding. The detergent, but not DMSO, induced losses of membrane PG binding were due to solubilization of the receptors. Greater amounts of membrane protein and phospholipids were solubilized at detergent (TX-100 and SDC) concentrations that solubilized 100% of PGE₁ receptors compared to 100% solubilization of F₂α receptors. Neither the duration of preincubation nor the amount of membrane protein chosen were responsible for differential PGE₁ and F₂α receptor losses. These differential membrane PG receptor losses raise the possibility of differences in PGE₁ and F₂α receptors association with the membrane structure.     

Prostaglandis show marked differences when tested against early ischemic dysrhythmias in rat

The effects of prostaglandins (PGs) F_1α, I₂, F_2β, and E₂, together with acetylsalicylic acid (ASA) and quinidine were compared on arrhythmias following ligation of the left anterior descending coronary artery in anesthetized rats. An objective method of assessing arrhythmias based on the severity and duration of a sudden fall in blood pressure (BP) was used together with changes in the electrocardiogram (ECG) to quantify the data. When infused at 2 mcg/kg/min pre-ligation, PGs F_1α and I₂ increased the severity of the arrhythmias while F_2β and E₂ decreased the severity. Only the results with PGE₂ were statistically significant. The most effective PG, E₂, produced a 69 per cent decrease in arrhythmic score compared to a 93 per cent decrease with quinidine. ASA produced a non statistically significant 43 per cent decrease in arrhythmic score. PGE₂ had no significant effect when infused post-ligation. None of the PGs had any marked effect on maximum following frequency in $\text{in vivo}$ rat heart when infused 1–10 mcg/kg/min. These data indicate that PGs show marked specificity depending on the prostaglandin in their action on early ischemic arrhythmias. The site of PG antiarrhythmic activity may be in the occluded zone, and the antiarrhythmic action of PGs cannot be explained by their effects on electrical refractoriness in the heart.     

Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Saline washed red blood cells of the toadfish convert [1-¹⁴C] arachidonic acid to products that cochromatograph with prostaglandin E₂ and prostaglandin F_2α. This synthesis is inhibited by indomethacin (10 μg/ml). Conversion of arachidonic acid to prostaglandin E₂ was confirmed by mass spectrometry. When saline washed toadfish red blood cells were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15,-³H]-arachidonic acid, comparison of the isotope ratios of the radioactive products indicated that prostaglandin F_2α was produced by reduction of prostaglandin E₂. The capacity of toadfish red blood cells to reduce prostaglandin E₂ to prostaglandin F_2α was confirmed by incubation of the cells with [1-¹⁴C] prostaglandin E₂.     

Myocardial actions of prostaglandins in isolated cat cardiac tissue.

The inotropic responses to prostaglandins (PG) A₁, E₁, E₂ and F_2α were studied in isolated cat myocardial tissue. PGA₁ and F_2α exhibited no significant inotropic effects, whereas, PGE₂ and PGE₁ produced negative inotropic effects at concentrations of 2.8 × 10⁻⁷ and 2.8 × 10⁻⁶ M in isolated cat papillary muscles.In isolated perfused cat hearts, PGE₁ (2.8 × 10⁻⁶M) produced a negative inotropic effect along with a significant increase in coronary flow. As flow declined, the negative inotropic effect became more severe. PGE₁ at 2.8 × 10⁻⁹ M produced a sustained increase in coronary flow and oxygen consumption with no inotropic effect. PGE₂ and F_2α did not exert significant changes in coronary flow or contractile force.Thus prostaglandins do not appear to exert significant positive inotropic effects at physiologic or at generally accepted pharmacologic concentrations in isolated cat heart preparations. At extremely high concentrations, prostaglandins E₁ and E₂ exert a negative inotropic effect; however, this would not explain the protective effect of these prostaglandins in circulatory shock.     

Prostaglandin stimulation of ovarian ornithine decarboxylase in vitro.

Prostaglandins E₁ and E₂ caused a 5–10 fold stimulation of ornithine decarboxylase activity in granulosa cells isolated from porcine ovarian follicles. The minimally effective concentration of prostaglandin E₂ was 10 ng/ml and the plateau of activity was reached at 500 ng/ml. Prostaglandin F_2α was ineffective. 1-Methyl,3-isobutyl-xanthine, a phosphodiesterase inhibitor, potentiated the effect of both submaximal and maximal effective doses of prostaglandin E₂, suggesting that the effect of prostaglandin E₂ is mediated by cAMP. The effect of prostaglandin E₂ was similar to that of luteinizing hormone and a cAMP analogue, 8-Bromo-cAMP.     

Effects of prostaglandins Fα on dog thyroid cyclic AMP level and function

Prostaglandins F_1α and F_2α, at high concentrations (≥28 μM) enhanced cyclic AMP accumulation in dog thyroid slices. At lower concentrations, they inhibited the cyclic AMP accumulation induced by thyrotropin (TSH), prostaglandin E₁, and cholera toxin. This effect was rapid in onset and of short duration, calcium-dependent and suppressed by methylxanthines. Prostaglandin F_α also inhibited TSH-induced secretion and activated iodine binding to proteins. These characteristics are similar to those of carbamylcholine action, except that prostaglandins F did not enhance cyclic GMP accumulation. The effect of prostaglandin F_α was not inhibited by atropine, phentolamine and adenosine deaminase and can therefore not be ascribed to an induced secretion of acetylcholine, norepinephrine or adenosine. It is suggested that prostaglandins F act by increasing influx of extracellular Ca²⁺. Arachidonic acid also inhibited the TSH-induced cyclic AMP accumulation. However this effect was specific for TSH, it was enhanced in the absence of calcium and was not inhibited by methylxanthines or by indomethacin at concentrations which completely block its conversion to prostaglandin F_α. Arachidonic acid action is sustained. This suggests that arachidonic acid inhibits thyroid adenylate cyclase at the level of its TSH receptor and that this effect is not mediated by prostaglandin F_α or any other cyclooxygenase product.     

Effects of Prostaglandins F<Subscript>2α</Subscript>, E<Subscript>2</Subscript> and E<Subscript>1</Subscript> on Fertility in Mice

PROSTAGLANDIN (PG) F_2αhas antifertility effects in many species^1–3 but there are conflicting suggestions as to its mechanism of action. For example, it may cause the degeneration of the corpus luteum by decreasing blood flow in the uteroovarian vein⁴; alternatively, its action may be due to a hypersecretion of luteinizing hormone (LH) by the pituitary^3,5. I have investigated the effects of PGF_2α, E₂ and E₁ on pregnancy in mice and examined the mechanism of action of PGF_2α.