The effect of prostaglandins on cultured lapine articular chondrocytes

The effect of 8 prostaglandins (PG) on growth and sulfate incorporation by monolayer and spinner-cultured rabbit articular chondrocytes has been measured. PGA₁, PGB₁, PGE₁ and PGE₂ reduced synthesis of sulfated glycosaminoglycans (GAG) but the PGF series did not. PGA₁ was the most potent, being effective at a concentration of 2.5 μg/ml [6.8 μM] while the others required 25 μg/ml. These compounds had no effect on degradation of GAG. All 8 PGs augmented growth slightly but significantly at 2.5 μg/ml. At the higher concentration, PGA₁ was highly cytotoxic, and PGB₁ as well as PGE₂ reduced cell growth. The cytotoxicity of PGA₁ was also observed in two additional types of cultured connective tissue cells, but the inhibition of sulfated-GAG synthesis by PGA₁ and PGB₁ was confined to the chondrocytes. The response of cultured chondrocytes to exogenous PGs, albeit at apparently unphysiologically high concentrations, together with other evidence, suggests that these compounds may conceivably play a direct role in cartilage metabolism in vivo.     

Prostaglandin inhibition of cartilage chondromucoprotein synthesis: Concept of “intrinsic activity”

We have recently reported that cartilage has two sites for prostaglandin (PG) action. One site (S₁) is stimulated by PGA₁, PGE₁ and PGF_1α and elevates tissue cyclic 3′5′adenosine monophosphate (cAMP). A second site (S₂) is activated by PGA₁ (but not PGE₁ or PGF_1α) and inhibits the synthesis of cartilage macromolecules. The present study is an investigation of the effects of PGB₁ on embryonic chicken cartilage chondromucoprotein synthesis in vitro. PGB₁ was found to inhibit chondromucoprotein synthesis with an apparent affinity for S₂ which was similar to that of PGA₁. The maximal inhibition produced by PGB₁ was, however, approximately one-half the maximal inhibition caused by PGA₁. Studies of the combined effects of PGB₁ and PGA₁ were consistent with the hypothesis that both classes of prostaglandins act at a common site (S₂) with about equal affinity but that PGB₁ has a lower intrinsic activity than PGA₁. Similar studies of the combined effects of PGE₁ or PGF_1α with PGA₁ indicate that neither PGE₁ nor PGF_1α binds significantly to S₂. An independent effect of PGB₁ to activate S₁ and elevate tissue cAMP was also found.     

Prostaglandin induced shape changes in fibroblasts grown in cell culture

PGE₂ induced shape changes in porcine adventitial fibroblasts grown on glass in low density monolayer cell cultures. Incubation of the cells with PGE₂ at concentrations of 100 ng/ml and 1000 ng/ml induced rounding of flat fibroblasts within one hour. The rounded cells had a small rim of cytoplasm around the nucleus and from one to several long thin arborizing cytoplasmic processes extending outward along the substratum. Removal of the PGE₂ resulted in transient blebbing of the cell membrane of both the cell body and the processes as the cells returned to their flat normal morphology within one hour. The effect could be inhibited by 1% fetal calf serum. PGF_2α did not however induce similar changes. This difference between PGE₂ and PGF_2α is similar to a report on spreading and migration of mouse peritoneal macrophages, and suggests that under certain conditions PGE₂ may have the ability to induce shape changes in cells.     

Effects of A and B prostaglandins on cAMP, cortisol, and aldosterone production by the human adrenal.

PGA₁ and PGA₂ (10, 100 μg/ml) significantly increased human adrenal cAMP levels and cortisol output but low doses (1 μg/ml) depressed both parameters. Only 1 μg/ml PGA₁ significantly increased aldosterone output while higher doses depressed same. The low PGA₂ dose (1 μg/ml) depressed aldosterone output. The glucocorticoid and mineralocorticoid outputs appear to be inversely modulated by prostaglandins. PGB₁ and PGB₂ behaved similarly to E type prostaglandins. However, like PGA₁, 1 μg/ml of PGB₁ or PGB₂ significantly increased aldosterone output. Higher doses were ineffective. The present findings reveal an increased complexity of prostaglandin modulation of cyclic nucleotides and steroid output.     

Further studies on prostaglandin E2 (PGE2)-induced gonadotropin release: Comparison with the effect of 15-methyl PGE2, PGAs, PGBs and prostaglandin endoperoxide analogs

It is known that PGE₂ is a potent stimulus of LH release. To determine if the effect of PGE₂ could be enhanced and/or prolonged by retarding its metabolic degradation, a derivative, 15-methyl PGE₂ (15-E₂) which is more slowly degraded than the natural compound was injected intravenously (i.v.) at various dose levels or into the third ventricle (3rd V) of ether-anesthetized, ovariectomized, estrogen (OVX, Eb)-treated rats and its effect on gonadotropin release was compared with that of PGE₂. Both PGs injected i.v. were equally effective in increasing plasma LH and maintaining the elevated levels, although 15-E₂ induced a larger and more sustained increase in plasma FSH than PGE₂. By contrast, 3rd V PGE₂ was clearly more effective than 3rd V 15-E₂ in releasing LH and to a lesser extent, FSH. The effect of 15-E₂ on LH was similar to that produced by 3rd V PGE₁ injected at a similar dose. However, its effect on FSH was greater than that of PGE₁.To evaluate the effect(s) of prostaglandins of the A and B series on gonadotropin release, PGA₁, PGA₂, PGB₁ or PGB₂ were injected intraventricularly in OVX, Eb-treated rats. PGBs were injected into conscious, free-moving rats. PGA₂ or PGB₂ increased plasma LH concnetrations although much less effectively than PGE₂. Third V PGA₁ or PGB₁ were ineffective. The 3rd V injection of two cyclic esters (U-44069 and U-46619), stable analogs of the PG endoperoxide PGG₂ and PGH₂, induced a small, transient increase in LH levels and did not alter plasma FSH in conscious, free-moving animals. PGE₂ injected intraventricularly at a similar dose was demonstrated to be much more potent than the analogs in stimulating LH and FSH release. The results indicate that: 1) 15-E₂, in spite of its described long-lasting activity, does not appear to be more potent than the natural compound in releasing LH, although when injected i.v., it appeared to induce a more sustained increase in plasma FSH; 2) although PGA₂ and PGB₂ can also act centrally to stimulate LH release, their low potency suggests that this is a pharmacological effect; and 3) the two analogs of PG endoperoxides tested proved to be poor stimuli for gonadotropin release. The significance of these findings is discussed.     

Uptake and metabolism of prostaglandins by isolated perfused lung: Species comparison and the role of plasma protein binding

We have investigated the uptake and subsequent metabolism of the prostaglandins (PGs) PGE₁, PGA₁, and PGB₁ by rat, guinea pig and rabbit isolated perfused lungs (IPL). Significant species differences were not observed in the uptake or metabolism of any PG on passage through the IPL. However, differences in the uptake of PGA₁ and PGB₁ and in the metabolism of PGA₁ were observed with a given species when the composition of the perfusion medium was varied. The IPL removed minimal amounts (<20% of the supply rate) of PGA₁ and PGB₁ from the circulation when the perfusate contained 4.5% bovine serum albumin (BSA). In the absence of BSA, however, both PGA₁ and PGB₁ were substantially removed from circulation (～53% of the supply rate) and PGA₁ was also metabolized. The composition of the perfusate had no effect on the uptake and metabolism of PGE₁ which was always taken up and metabolized to a greater extent than was PGA₁ and PGB₁. Thus, the apparent species differences previously reported for the pulmonary biotransformation of PGA can result from differences in the perfusion medium used. Our data suggest that both plasma protein binding and a transport system play important roles in determining the selectivity of the uptake of PGs by the lung.     

High performance liquid chromatography and UV detection for the separation and quantitation of prostaglandins

A fast and reliable method for the separation and quantitation of arachidonic acid metabolites PGF_1α, PGF_2α, PGD₂, PGE₁, PGE₂, PGB₂, PGA₂, 6-keto PGE₁, 6-keto PGF_1α, T×B₂ and 15-keto PGE₂ by high-performance liquid chromatography has been developed. Utilizing a single reverse-phase column and a UV spectrophotometer, sensitivity as little as 30 nanograms of each of these prostaglandins can be separated and subsequently detected. Although this study was performed using standards, it is highly promising for future application to biological fluids.     

Interaction of ouabain and diphenylhydantoin (DPH) with the cardiovascular actions of prostaglandin B2 and prostaglandin A2

Several reports have appeared indicating that ouabain may interact at sites on smooth muscle susceptible to activation by prostaglandins. This study reports on the interaction between ouabain (0) and diphenylhydantoin (DPH) with the cardiovascular actions of prostaglandin B₂ (PGB₂) and prostaglandin A₂ (PGA₂). Fifteen μg/kg i.v. of 0 enhanced the pressor response of the canine hindpaw to norepinephrine and tyramine but did not affect the pressor responses to sympathetic nerve stimulation (SNS), PGB₂ or PGA₂. PGB₂-induced bronchoconstriction (mediated solely by stimulation of smooth muscle) was reduced by ouabain. In a separate group of animals not receiving PG before 0, 0 reduced (p<0.05) the pressor responses to PGB₂. DPH enhanced the cutaneous pressor responses to SNS, NE, PGB₂ and PGA₂ but did not affect the bronchoconstrictor response to PGB₂. These data are consistent with the following conclusions: 1) Ouabain antagonizes the smooth muscle contractions produced by PGB₂. 2) The presence of PGB₂ antagonizes the prostaglandin inhibitory effects of ouabain suggesting that PGB₂ may compete for similar sites or allosterically interact with ouabain in smooth muscle. 3) DPH induced enhancement of PGB₂ and PGA₂ induced vasoconstriction may reflect DPH induced enhancement of adrenergic neurotransmitter release or inhibition of transmitter reuptake.     

Regulations of macrophage-mediated tumor cell killing by prostaglandins: Comparison of the effects of PGE2 and PGI2

Mouse resident peritoneal macrophages stimulated by purified bacterial lipopolysaccharide (LPS) produced both prostaglandin E₂ (PGE₂) and prostaglandin I₂ (PGI₂), the latter detected as its stable metabolite, 6-keto PGF_1α. Maximum production, induced in each case by 1 ng/ml purified LPS, was in the range of 10⁻⁷M for PGI₂ and 3 × 10⁻⁸M for PGE₂. A quantitatively similar increase in intracellular levels of macrophage cyclic AMP (measured on a whole cell basis), with a similar duration of effect, was stimulated by PGE₂ and PGI₂; however, only PGE₂ had a negative regulatory effect on macrophage activation for tumor cell killing. These data confirm that more than a whole cell increase in the concentration of cyclic AMP is needed to shut off nonspecific tumor cell killing mediated by LPS-activated resident peritoneal macrophages.     

Effect of prostaglandins on H-thymidine uptake into human epidermal cells

Prostaglandins A₂, B₁, E₁, E₂, F_1α and F_2α were added to cultures of human epidermal cells (keratinocytes) for 24 hours at 37°C, and the effects on ³H-thymidine uptake into DNA was measured. At 70 μg/ml all prostaglandins tested except PGF_2α inhibited the uptake of ³H-thymidine greater than 50%. However, at 35 μg/ml, PGA₂ and PGB₁ were the only two prostaglandins to show significant inhibition, 96% and 51% respectively. At 17.5 μg/ml only PGA₂ caused substantial inhibition, 68%. In order to determine if the PGA₂ action was mediated by membrane receptors propranolol, phentolamine, metiamide and prostynoic acid were added in conjunction with PGA₂. None of the above receptor antagonists were able to reduce the PGA₂-induced inhibition of ³H-thymidine uptake. These results indicate that the pre-incubation of human keratinocytes with prostaglandins for 24 hours results in a decrease of ³H-thymidine incorporation in DNA. The precise mechanism of action is unknown at this time.     

Prostaglandin removal and metabolism by isolated perfused rat lung

We have investigated the mechanism(s) involved in the removal of prostaglandins (PG) from the pulmonary circulation by the lung. Unidirectional fluxes of PG from the circulation into the lung are measured in an isolated perfused rat lung preparation. Evidence is presented which suggests that a transport system for PG exists in lung tissue. This transport system is responsible for the removal of some PG from the circulation by the lung. PGE₁ and PGF_2α are substrates for this system, whereas PGB₁, PGA₁, and 15-keto-PGF_2α are not. Since PGA₁ is a substrate for the intracellular PG dehydrogenase, the selectivity of the lung's metabolism system for circulating PG is probably due to the selectivity of the transport system for PG. It is shown that the percentage of the pulmonary arterial concentration (C_A) of PGE₁ or PGF_2α that is metabolized on passage through the pulmonary circulation decreases rapidly as C_A increases. When the lungs were perfused with PGE₁ (PGF_2α), the metabolites detected in the venous effluent were 15-keto-PGE₁ (PGF_2α) and 15-keto-13,14-dihydro-PGE₁ (PGF_2α). The time course pattern of the appearance of metabolites in the venous effluent after the initiation of a constant C_A, and the relative concentrations of the metabolites in the venous effluent, were examined as a function of C_A.     

High-performance liquid chromatography of p-nitrophenacyl esters of selected prostaglandins on silver ion-loaded microparticulate cation-exchange resin

A silver ion-loaded microparticulate cation-exchange resin column has been used for high-performance liquid chromatographic (HPLC) separation of the p-nitrophenacyl esters of several series of closely related prostaglandins: 8-iso-PGE₂, 11-epi-PGE₂, 5-trans-PGE₂, PGE₂, PGF_1α, PGE₁, and PGF_2α, PGA₂ and PGB₂; 15 (R)-methyl-PGE₂ and 15 (S)-methyl-PGE₂; 5-trans-PGA₂ and PGA₂; and 5-trans-PGF_2α and PGF_2α. The properties of this column are compared with those of silica-gel and reversed-phase columns.     

Influence of prostaglandins and thyrotropin releasing hormone (TRH) on hormone secretion and growth in wether lambs

A series of experiments were conducted in ewes and wether (castrate male) lambs to evaluate the influence of prostaglandins on secretion of anabolic hormones and to determine if repeated injections of prostaglandin (PG) F₂α would chronically influence the secretion of these hormones and perhaps growth rate as well.A single intravenous injection of PGA₁ and PGB₁ (100 μg/kg) exerted no significant (P > .10) influence on plasma concentrations of prolactin (PRL), growth hormone (GH) or thyrotropin (TSH) in ewes. PGA₁, but not PGB₁, stimulated an increase in the plasma concentration of insulin. Infusion of PGF₂α for 5.5 hr into ewes resulted in increased (P < .05) plasma concentrations of both GH and PRL while TSH and insulin were not significantly influenced. Prostaglandin F₂α, when injected subcutaneously into wether lambs (10 mg twice weekly) stimulated (P < .05) plasma GH concentrations after the first injection, but not after 3 weeks of treatment. Changes in plasma PRL or TSH were not observed consistently in the lambs treated chronically with PGF₂α or TRH.Prostaglandin F₂α, in the present studies, and PGE₁ in previously reported studies (1–3), has been demonstrated to be stimulatory to the secretion of PRL and GH. In contrast, PGA₁ and PGB₁, which lack an 11-hydroxyl group, failed to influence the secretion of either PRL or GH. It would, therefore, appear that the 11-hydroxyl group is a structural requirement for prostaglandins to influence the secretion of these two hormones in sheep.Treatment with thyrotropin releasing hormone (TRH), alone or in combination with PGF₂α, significantly (P < .05) increased growth rate (average daily gains) while PGF₂α did not, despite the fact that both compounds exerted similar effects on plasma GH.     

Resolution of prostaglandin p-nitrophenacyl esters by liquid chromatography and conditions for rapid, quantitative p-nitrophenacylation

The p-nitrophenacyl esters of a number of closely related and isomeric prostaglandins were resolved by HPLC on a microparticulate silica gel column (Zorbax-Sil®, DuPont). Ten F-series prostaglandin analogs, eight E-series prostaglandin analogs, the isomeric 15(R)- and 15(S)- methyl prostaglandins of the E- and F-series and, lastly, PGA₂ and PGB₂ were chromatographed under conditions generating 2,000 to 7,000 theoretical plates. Conditions are described for quantitative conversion of prostaglandins to p-nitrophenacyl esters in less than 6 minutes at room temperature. Linear peak height and peak area plots were obtained for esterified PGE₂ p-nitrophenacyl ester over the range of 0.4 – 3.1 μg. The lower limit of detection of this ester is about 1 ng. A linear relationship is observed between silica gel TLC 1/R_f values and HPLC retention times as predicted by theory.     

Biophysical studies on molecular mechanism of abortificient action of prostaglandins—IV. Conformation energy calculations on PGA1, PGB1 and PGE1

This paper reports the conformation energy (CE) calculations on three forms of prostaglandins (PGs) PGA₁, PGB₁ and PGE₁ on the basis of the empirical potential energy functions, for the simultaneous rotations around C₇–C₈ (θ), C₁₂–C₁₃ (β) and C₁₄–C₁₅ (β) bonds [Fig. 1(a)]. The isoenergy contours plotted for θβ rotations for the different β values show the existence of two low energy regions for thg equal to about 90° and 240° in all the three cases. The absolute minimum was obtained for thg = 240° and almost coincided with the crystallographic conformation for PGE₁ and PGA₁. In the case of PGB₁ series of low energy conformations were obtained with the thg values equal to about 90° and 270°, but none of them coincided with the observed crystallographic conformation. The paper discusses the comparison of the different low energy conformations in these three molecules, their biological relevance and the cause of disagreement in the case of PGB₁ with the crystallographic data.     

PGE2 and PGF2α release by human peritoneal macrophages in endometriosis

Objective: To test for differences in the amount and activity of peritoneal macrophages present in the peritoneal fluid of women with, and without endometriosis using prostaglandin release by macrophages in culture as a marker.Patients: Women of reproductive age undergoing laparoscopy for infertility or chronic pelvic pain with postoperative diagnosis of endometriosis and women undergoing laparoscopy for sterilization.Methods: Peritoneal fluid was aspirated during laparoscopy, volume was recorded, macrophages were isolated via a Ficoll Paque gradient and kept in primary culture. PGE₂ and PGF_2α release of the cells were measured before and after stimulation with zymosan.Results: Women with endometriosis had significantly more peritoneal macrophages than controls. Peritoneal macrophages of women with endometriosis released significantly more PGE₂ than those of the control group: 8.4 ± 2.0 versus 1.4 ± 0.4 ng/ml/10⁶cells (mean ± SEM, p=0.0005) and PGF_2α : 10 ± 4.3 (endometriosis) versus 1.8 ± 0.4 (control) ng/ml/10⁶cells (mean ± SEM, p = 0.045).Conclusion: There is a significant increase in the amount of prostaglandins released by peritoneal macrophages from women with endometriosis. These prostaglandins might alter uterine and tubal contractility, thereby affecting fertility.     

Identification and quantification of prostaglandins in Antarctic krill (Euphausia superba Dana)

Summary The amount and types of prostaglandins present in Antarctic krill (Euphausia superba Dana.) were estimated. Samples of fresh krill were collected during III Antarctic Cruise of RV Polarstern in November 1984. Prostaglandins were extracted, separated by column and thin-layer chromatography and identified as PGA₂, PGB₂, PGE₂, PGF₂. Quantitative measurements were made by a biological method (Vane cascade), concentrations of the most abundant prostaglandins PGE₂ and PGF₂ being 1.6 and 4 ng/1 g of fresh tissue, respectively. Such low level of prostaglandin would not be harmful when using krill as a food supplement.     

Resolution of prostaglandin p-nitrophenacyl esters by liquid chromatography and conditions for rapid, quantitative p-nitrophenacylation

The p-nitrophenacyl esters of a number of closely related and isomeric prostaglandins were resolved by HPLC on a microparticulate silica gel column (Zorbax-Sil ®, DuPont). Ten F-series prostaglandin analogs, eight E-series prostaglandin analogs, the isomeric 15(R)- and 15(S)-methyl prostaglandins of the E- and F-series and, lastly, PGA₂ and PGB₂ were chromatographed under conditions generating 2,000 to 7,000 theoretical plates. Conditions are described for quantitative conversion of prostaglandins to p-nitrophenacyl esters in less than 6 minutes at room temperature. Linear peak height and peak area plots were obtained for in-situ esterified PGE₂ p-nitrophenacyl ester over the range of 0.4 – 3.1 μg. The lower limit of detection of this ester is about 1 ng. A linear relationship is observed between silica gel TLC 1/R_f values and HPLC retention times as predicted by theory.     

Prostaglandin-induced enhancement of the anamnestic response

The ability of various prostaglandins (PGs) to affect the anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA₁, PGE₂ and PGF_2α), PGE₁ was found to have a stimulatory effect, whereas PGA₁, PGE₂ and PGF_2α were ineffective in stimulating or inhibiting the anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE₁-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 μg of PGE₁ were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.     

The synthesis and biological activities of some 12-Aza-prostaglandin analogues

12-Aza-prostaglandin (PG) analogues containing the pyrrolidine-2,4-dione ring system have been synthesized from ,2-disubstituted glycine esters via cyclisation of their -ethoxycarbonylacetyl derivatives. 5-(6-Carboxyhexyl)-1-octylpyrrolidine-2,4-dione (5) had little or no PG-like activity on superfused intestinal or vascular smooth muscle preparations but it selectively antagonised smooth muscle responses to PGE₂, PGE₁, PGF_2α and PGA₂in vitro. At a concentration of 10⁻⁵ g/ml it reduced responses of the rat stomach strip to PGE₂ by over 80% but did not affect responses of this tissue to acetylcholine, 5-hydroxytryptamine (5-HT) or bradykinin. Polyphloretin phosphate (PPP), the known PG antagonist, had a similar effect at the same concentration (10⁻⁵ g/ml).5-(6-Carboxyhexyl)-1-(3-hydroxyoctyl)pyrrolidine-2,4-dione (12) had the same profile of activity on superfused smooth muscle preparations as PGE₂ or PGA₂. On intravenous injection into anaesthetised rats it caused dose-dependent falls in arterial blood pressure with associated tachycardias, which is typical of the response to PGE₂. The smooth muscle activity of (12) was not reduced by passage through isolated perfused guinea-pig lungs nor was its potency as a vasodepressor increased when given intra-arterially to rats. These results suggest that, unlike PGE₂, this analogue is not removed by the pulmonary circulation.