Prostaglandin biosynthesis by lapine articular chondrocytes in culture

Secondary monolayer and spinner cultures of rabbit articular chondrocytes released into the culture medium prostaglandins the synthesis of which was inhibited by sodium meclofenamate. The prostaglandins measured by radioimmunoassay were, in order of decreasing abundance, prostaglandin E₂, 6-oxoprostaglandin F_1α, (the stable metabolite of prostacyclin) and prostaglandin F_2α. Several lines of evidence indicated that chondrocytes synthesize little if any thromboxane B₂ (the stable metabolite of thromboxane A₂). The presence of prostaglandins was confirmed by radiometric thin-layer chromatography of extracts of culture media incubated with [³H]arachidonic acid-labeled cells. In monolayer culture, chondrocytes synthesized immunoreactive prostaglandins in serum-free as well as serum-containing medium. Monolayer chondrocytes produced higher levels of prostaglandin E₂ relative to 6-oxo-prostaglandin F_1α than did spinner cells, but the latter synthesized more total prostaglandins. The identity of endogenous prostaglandins as well as those synthesized in short-term culture by rabbit cartilage slices was compared to those produced by chondrocytes in long-term culture. Chondrocytes synthesized all of the prosta-glandins found in articular cartilage. Minimal quantities of thromboxane B₂ were detected in cartilage. A higher percentage of 6-oxo-prostaglandin F_1α relative to other prostaglandins was found in cartilage than in either monolayer or spinner chondrocyte cultures. These results demonstrate that articular chondrocytes synthesize prostaglandins and prostacyclin. These prostaglandins may exert significant physiological effects on cartilage, since exogenous prosta-glandins depress chondrocyte sulfated-proteoglycan synthesis and may even promote proteoglycan degradation.     

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.     

Influence of prostaglandins on vasoconstrictor responses in the hindquarters vascular bed of the cat

The effects of prostaglandins (PG) A₁, A₂, B₂, E₁, E₂, 6-keto-E₁, F_2α and indomethacin on vascular resistance and vasoconstrictor responses were investigated in the feline hindquarters vascular bed under conditions of controlled flow so that changes in perfusion pressure directly reflect changes in vascular resistance. Infusion of PGE₁ , PGE₂ and 6-keto-PGE₁ (3 μg/min) into the abdominal aorta significantly dilated the hindquarters vascular bed and inhibited vasoconstrictor responses to sympathetic nerve stimulation and intra-arterial injections of angiotensin, whereas hindquarters vasoconstrictor responses to tyramine and exogenous norepinephrine were unaffected. Infusion of PGA₁, A₂, B₂ and F_2α at a similar rate produced transient changes in hindquarters vascular resistance and did not consistently alter vasoconstrictor responses to sympathetic nerve stimulation, angiotensin, norepinephrine and tyramine. Indomethacin in a dose which greatly attenuates the response to intravenous administration of arachidonic acid enhanced responses to nerve stimulation and norepinephrine. In addition, indomethacin had little or no effect on hindquarters perfusion pressure and systemic arterial pressure. These data suggest that E series prostaglandins possess the ability to modulate the actions of the sympathetic nervous system and angiotensin in the feline hindquarters vascular bed. In addition, these data suggest that PGEs, upon enzymatic conversion and dehydration to A and B series prostaglandins, lose their ability to consistently affect vasoconstrictor responses. Experiments with indomethacin further suggest that locally formed prostaglandins do modulate the effects of the sympathetic nervous system of the feline hindquarters.     

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.     

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).     

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.     

Evidence for enhanced venous smooth muscle turnover of prostaglandin-like substance in portal veins from spontaneously hypertensive rats

The sensitivity of portal veins from 14 to 18 week-old Okamoto-Aoki spontaneously hypertensive rats to prostaglandins A₂, B₂, D₂ and F_2α were enhanced whereas the sensitivity to prostaglandin E₂ was diminished when compared with responses of veins from normotensive Wistar-Kyoto rats. Inhibition of prostaglandin synthesis with both eicosotetraynoic acid (ETYA) and indomethacin (INDO) abolished the observed differences in sensitivity to prostaglandins. Synthesis of prostaglandin-like substance (with arachidonic acid as precursor) was significantly enhanced in portal veins from spontaneously hypertensive rats. Metabolism of prostaglandins E₂ and F_2α, employing the oil-immersion technique of Kalsner and Nickerson, appeared to be similar in veins from normotensive and hypertensive rats. These findings suggest that prostaglandin synthesis is enhanced in venous smooth muscle from hypertensive rats. The increased concentration of endogenous prostaglandin at the venous smooth muscle cell may modify the responses to exogenously administered prostaglandins thus accounting, in part, for the altered sensitivity to these fatty acids.     

In vitro prostaglandin synthesis by various rat renal preparations

Prostaglandin synthesis by eight different structures from the rat kidney (whole cortex, cortical tubules, glomeruli, outer medulla, papilla, glomerular cultured epithelial and mesangial cells, cultured interstitial medullary cells) was measured in vitro after incubation with [¹⁴C]arachidonic acid using high-performance liquid chromatography followed by RIA with four specific anti-prostaglandin antibodies (prostaglandin E₂, prostaglandin F_2α, 6 keto-prostaglandin F_1α, thromboxane B₂). Prostaglandin production by the whole cortex and cortical tubules was very low. The order of abundance for isolated glomeruli was thromboxane B₂ > prostaglandin E₂ > prostaglandin F_2α > 6 keto-prostaglandin F_1α. Mesangial cells synthesized prostaglandin E₂ at a markedly high rate, and in decreasing order: prostaglandin F_2α, thromboxane B₂ and 6 keto-prostaglandin F_1α. The same order of abundance was observed for epithelial cells. The papilla synthesized essentially prostaglandin E₂ and prostaglandin F_2α, whereas the main product for the outer medulla was 6 keto-prostaglandin F_α. Cultured interstitial cells synthesized mainly prostaglandin E₂ and to a lesser extent prostaglandin F_2α. Unidentified peaks eluting between 6 keto-prostaglandin F_α and thromboxane B₂ were also observed chiefly with glomeruli but they were absent with the medullary preparations. They disappeared after incubation with indomethacin or aspirin and represented for glomeruli the greatest percentage of conversion of [¹⁴C]arachidonic acid. These results show that the prostanoid profile varies markedly with the different regions and cells of the rat kidney.     

A comparative study of thromboxane and prostacyclin release from ex vivo cultured bovine vascular endothelium

Thromboxane B₂, 6-keto-Prostaglandin F_1α, and Prostaglandin E₂ release have been quantitated from cultured adult by bovine endothelial cell monolayers and from $\text{ex Vivo}$ vascular segments employing specific radioimmunoassay and thin layer chromatography. Release of all three prostaglandins was demonstrable from both endothelial cell systems under basal conditions and following exposure to the ionophore A23187 and arachidonic acid. In culture, the quantity of 6-keto-PGF_1α released was diminished compared to amounts released from the vessel segments while thromboxane B₂ and prostaglandin E₂ release were similar in the two endothelial model systems. However, the amount of thromboxane B₂ assayed was small and the quantity of thromboxane A₂ it represents is probably of little $\text{in Vivo}$ significance to prostacyclin.     

Characterization and localization of prostaglandin E1 receptors in rat liver plasma membranes

Methodology for measurement and characterization of prostaglandin binding to membranes has been developed. The binding assay was used to study the presence of prostaglandin receptors in high purified cell fractions derived from rat liver. High affinity binding receptors which have a saturation value of 1.0 pmole/mg protein and a dissociation constant of 1.2 nM were found exclusively in the plasma membrane. High affinity receptors were not found in cell fractions containing nuclei, rough microsomes. Golgi complex or mitochondria. The binding by other prostaglandins was competitive with prostaglandin E₁. Competitive binding studies were used to obtain dissociation constants for prostaglandins F_1α, F_2α, B₁, B₂, A₁, A₂, and 15-keto prostaglandin E₂ which were 1100, 100, 300, 180, 16. 16 and 700 nM, respectively. Eicosa-5.8.11.19-tetraynoic acid, an inhibitor of prostaglandin synthesis did not bind appreciably to the prostaglandin E receptor, whereas two prostaglandin analogues, which have high physiological activity compete effectively with prostaglandin E₁ for the receptor. Thus, the binding receptor for the E-type prostaglandins is highly specific both with respect to cell localization as well as the type of substrate. Numerical routines for the fitting of the data and a procedure for the determination of the specific activity of the labelled prostaglandin are provided.     

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.     

Solubilized myocardial adenylate cyclase: activation by prostaglandins

Prostaglandins E₁, E₂, F_2α, and F_2α activated solubilized myocardial adenylate cyclase from guinea pigs and cats. The activation did not require the presence of added phospholipids in contrast to stimulation of the solubilized enzyme by catecholamines, glucagon, and histamine. The data may provide insight into the mechanism and cellular site of action of the prostaglandins.     

Prostaglandin production by type II alveolar epithelial cells

Prostaglandin production was studied in fetal and adult type II alveolar epithelial cells. Two culture systems were employed, fetal rat lung organotypic cultures consisting of fetal type II cells and monolayer cultures of adult lung type II cells. Dexamethasone, thyroxine, prolactin and insulin, hormones which influence lung development, each reduced the production of prostaglandin E and F_α by the organotypic cultures. The fetal cultures produced relatively large quantities of prostaglandin E and F_α and smaller quantities of 6-keto-prosta-glandin F_1α and thromboxane B₂. However, prostaglandin E₂ production was predominant. In contrast, the adult type II cells in monolayer culture produced predominantly prostacyclin (6-keto-prostaglandin F_1a) along with smaller quantities of prostaglandin E₂ and F_2α. The type II cells were relatively unresponsive to prostaglandins. Exogenously added prostaglandin E₂ had no effect on cell growth, and only a minimal effect on cyclic AMP levels in the monolayer cultures.     

The influence of some prostaglandins on DNA synthesis and DNA excision repair in mouse spleen cells in vitro

$\text{In vitro}$ experiments were performed on mouse spleen cells to establish possible influences of some naturally occurring prostaglandins on DNA synthesis and DNA excision repair. The prostaglandins A₁, B₁, E₁, E₂ and F₂ were tested in concentrations of lopg, 5ng and 2.5μg per ml cell suspension. DNA synthesis was significantly increased by pgF_2α in all the three concentrations tested, while the other tested prostaglandins were essentially ineffective. DNA excision repair was significantly inhibited by PgF₁ and PgF₂ at 5ng/'ml and at 2.5μg/ml but increased by PgF_2α in the two lower concentrations. The rejoining of DNA-strand breaks after γ-irradiation was slightly reduced by PgF₁, PgE₂ and PgF_2α at 2.5μg/ml.     

The effect of acute hypoxia on prostaglandin release in perfused human fetal placenta

The release of prostaglandin E₂ and F_2α, thromboxane B₂ and 6-keto-prostaglandin F_1α was measured in isolated human placental cotyledons perfused under high- and low-oxygen conditions. Also the effect of reoxygenation on prostaglandin production was studied. During the high-oxygen period, prostaglandin E₂ accounted for 44 % and 6-keto-prostaglandin F_1α for 28 % of all prostaglandin release, and the rank order of prostaglandin release was E₂ > 6-keto-prostaglandin F_1α > thromboxane B₂ > prostaglandin F_2α. Hypoxia had no significant effect on quantitative prostaglandin release, but the ration of prostaglandin E₂ to prostaglandin F_2α was significantly increased. After the hypoxic period during reoxygenation the release of 6-keto-prostaglandin F_1α was significantly decreased, as was the ratio of 6-keto-prostaglandin F_1α to thromboxane B₂. Also the ratio of the vasodilating prostaglandins (E₂, 6-keto-prostaglandin F_1α) to the vasocontricting prostaglandins (thromboxane B₂, prostaglandin F_2α) was decreased during reoxygenation period. With the constant flow rate, the perfusion pressure increased during hypoxia in six and was unchanged in three preparation. The results indicate that changes in the tissue oxygenation in the placenta affect prostaglandin release in the fetal placental circulation. This may also have circulatory consequences.     

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.     

Rat osteogenic sarcoma cells: comparison of the effects of prostaglandins E1, E2, I2 (prostacyclin), 6-keto F1alpha and thromboxane B2 on cyclic AMP production and adenylate cyclase activity.

The effects of prostaglandins (PG's) E₁, E₂, I₂ (prostacyclin), 6-keto F_1α and thromboxane (Tx) B₂ were compared in freshly isolated cells from a rat osteogenic sarcoma and in membranes from cultured cells of the same tumour. Cyclic AMP production was measured in cells and adenylate cyclase activity was measured in cell membranes. In both systems PGI₂ was less potent than either of the PGE's, and both TxB₂ and 6-keto PGF_1α were only weak agonists. These effects on bone-derived cells suggest that PGI₂ is unlikely to be a potent bone resorbing agent. Resitance experiments suggested that all the PG's share the same receptor site which appears distinct from the site of action of parathyroid hormone.     

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.     

Stimulation of arachidonic acid metabolism in rat kidney mesangial cells by bradykinin,antidiuretic hormone,and their analogues

Kinins, antidiuretic hormone, and several of their analogues stimulated contractile rat kidney mesangial cells growing in culture to produce prostaglandins E₂, F_2α, and I₂. Analogues that are known to be pharmacologically active stimulated arachidonic acid metabolism in these cells, whereas compounds that have little activity were ineffective. Stimulation of arachidonic acid metabolism by these agonists appeared to be receptor-mediated. It is possible that arachidonic acid metabolism is stimulated by contraction of mesangial cells and the released prostaglandins regulate glomerular function.     

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.