Biological activities of prostaglandin H1 analogs

The influences of epoxymethano and epoxycarbonyl analogs of PGH₁ on washed rabbit platelets, isolated smooth muscles and perfused heart preparations were investigated. On washed rabbit platelets, 11,9-epoxy-methano and 11,9-epoxycarbonyl PGH₁ produced a platelet aggregation whereas 9,11-epoxymethano and 9,11-epoxy-carbonyl PGH₁ produced an inhibition of arachidonic acid-induced platelet aggregation. On isolated rabbit thoracic aorta strips, 9,11-epoxycarbonyl PGH₁ showed strong contracting activity (5 times as active as 11,9-epoxy-methano PGH₂ and 31 times as active as PGH₂). All the analogs of PGH₁ caused contraction of guinea pig tracheal muscle and caused an increase of perfusion pressure in guinea pig heart, though 11,9-epoxymethano and epoxy-carbonyl PGH₁ were far more active than 9,11-epoxymethano and epoxycarbonyl PGH₁. Differences in biological activities between 11,9-epoxymethano and epoxycarbonyl PGH₁, and 9,11-epoxymethano and epoxycarbonyl PGH₁ indicate that the orientation of functional groups at C₉ and C₁₁ influences biological activities.     

Arterial walls are protected against deposition of platelet thrombi by a substance (prostaglandin X) which they make from prostaglandin endoperoxides

Prostaglandin (PG) endoperoxides (PGG₂ and PGH₂) contract arterial smooth muscle and cause platelet aggregation. Microsomes from pig aorta, pig mesenteric arteries, rabbit aorta and rat stomach fundus enzymically transform PG endoperoxides to an unstable product (PGX) which relaxes arterial strips and prevents platelet aggregation. Microsomes from rat stomach corpus, rat liver, rabbit lungs, rabbit spleen, rabbit brain, rabbit kidney medulla, ram seminal vesicles as well as particulate fractions of rat skin homogenates transform PG endoperoxides to PGE- and PGF- rather than to PGX-like activity.PGX differs from the products of enzymic transformation of prostaglandin endoperoxides so far identified, including PGE₂, F_2α, D₂, thromboxane A₂ and their metabolites.PGX is less active in contracting rat fundic strip, chick rectum, guinea pig ileum and guinea pig trachea than are PGG₂ and PGH₂. PGX does not contract the rat colon.PGX is unstable in aqueous solution and its anti-aggregating activity disappears within 0.25 min on boiling or within 10 min at 37° C.As an inhibitor of human platelet aggregation induced $\text{in vitro}$ by arachidonic acid PGX was 30 times more potent than PGE₁. The enzymic formation of PGX is inhibited by 15-hydroperoxy arachidonic acid (IC₅₀ = 0.48 μg/ml), by spontaneously oxidised arachidonic acid (IC₅₀ <100 μg/ml) and by tranylcypromine (IC₅₀ = 160 μg/ml).We conclude that a balance between formation by arterial walls of PGX which prevents platelet aggregation and release by blood platelets of prostaglandin endoperoxides which induce aggregation is of the utmost importance for the control of thrombus formation in vessels.     

Prostaglandin endoperoxides IV. Effects on smooth muscle

The effect on smooth muscle of the endoperoxides PGG₂ and PGH₂, which are intermediates in prostaglandin biosynthesis, was studied in different systems $\text{in vitro}$ and $\text{in vivo}$. On gastrointestinal smooth muscle (gerbil colon, rat stomach) PGG₂ and PGH₂ produced contractions comparable to those of PGE₂ and PGF_2a whereas contractions elicited on vascular (rabbit aorta) and airway (guinea-pig trachea) smooth muscle were considerably greater than those of PGE₂ and PGF_2a respectively. On intravenous injection into guinea-pigs PGG₂ and PGH₂ caused a triphasic change in blood pressure and were 8–10 times more effective than PGF_2a in producing an increase in tracheal insufflation pressure. When given as aerosols the unstable endoperoxides were less effective than PGF_2a. It is concluded that the endoperoxides are potent smooth muscle stimulants and that they are more effective than their degradation products (PGD₂, PGE₂, PGF_2a) in some systems.     

Effects of prostaglandin endoperoxide analogs on contractile elements in lung and gastrointestinal tract

Prostaglandin endoperoxides are formed in the lung as intermediate compounds in the biosynthesis of prostaglandins and thromboxanes. The effects of different doses of two analogs of prostaglandin endoperoxide PGH₂ were compared with those of PGF_2α and PGE₂ on superfused preparations of isolated trachea, bronchiole, peripheral lung, pulmonary artery and gastrointestinal smooth-muscle tissues. Endoperoxide analogs induced contraction of all smooth-muscle structures in the lung and airways. Compared to PGF_2α, analog I was approximately 71 times as potent on guinea-pig trachea, 214 times as potent on guinea-pig lung, and 57 times as potent on guinea-pig polmunary artery. Analog II was moderately less potent on all tissues than analog I. On gastrointestinal smooth-muscle organs, the PGH₂ analogs were generally closer in activity to PGF_2α and PGE₂, or even weaker. The findings show that PG endoperoxide vessels, and suggest that the naturally occurring compounds may participate in the mediation of bronchoconstriction and pulmonary vasoconstriction in disease states.     

Accelerative autoactivation of prostaglandin biosynthesis by PGG2.

Cyclooxygenase catalysis is stimulated by its product, PGG₂, and by other lipid hydroperoxides. The endoperoxide, PGH₂, was not stimulatory. The results provide a direct demonstration of an essential role for lipid hydroperoxides in prostaglandin biosynthesis, and show how the biosynthetic intermediate PGG₂ has a positive accelerative effect.     

The effect of precursors, products, and product analogs of prostaglandin cyclooxygenase upon iris sphincter muscle.

Contractions of isolated iris sphincter muscles were measured in response to several free fatty acids, hydroperoxy and hydroxy derivatives of 20:3(n-3), 20:3(n-6) and 20:4, PGH₂, and the epoxymethano methano analogs of PGH₂. The free acids of prostaglandin precursors elicited comparatively strong contractions, hydroperoxy and hydroxy acids gave intermediate and nonspecific response whereas nonprostaglandin precursor acids elicited little response. PGH₂ was 100 to 1000 times more effective than arachidonic acid or the epoxymethano analogs. The latter compounds inhibited the production of contractions by PGH₂. These results allow an interpretation that the iris sphincter muscle contains an active thromboxane synthase and receptors for endoperoxide and thromboxane that initiate contraction.     

Enzymatic preparation of prostaglandin endoperoxides

A simple and reliable method is described for the preparation of the endoperoxide intermediates (PGG₂ and PGH₂) in the biosynthesis of prostaglandins.The endoperoxides are thermolabile and easily decomposed by water ( min at 37°C). Because of this, special precautions must be taken to work at low temperature and to minimize contact with moisture.Milligram quantities of PGG₂ and PGH₂ were obtained by running several reactions successively and pooling the extracts before chromatographic fractionation. The method is now being developed further to scale up the procedure.     

Polymorphonuclear leukocytes produce thromboxane A2-like activity during phagocytosis

Homogenates of phagocytosing polymorphonuclear leukocytes obtained from rabbit peritoneum were incubated with the prostaglandin endoperoxides PGG₂ or PGH₂. After 2 min at 0°C, incubation mixtures contained an increased rabbit aorta contracting activity. Ether extracts of incubation mixtures contained a substance which contracted the superfused strips of rabbit aorta and coeliac artery and had a half life which was similar to thromboxane A₂. The generation of thromboxane A₂-like activity from PG endoperoxides was prevented by boiling the homogenate prior to incubation, or by pretreatment with benzydamine, a drug which blocks thromboxane formation in platelets. Production of thromboxane A₂-like material by leukocyte homogenates was compared with platelet microsomal thromboxane synthetase.     

Arterial walls generate from prostaglandin endoperoxides a substance (prostaglandin X) which relaxes strips of mesenteric and coeliac arteries and inhibits platelet aggregation

Fresh arterial tissue generates an unstable substance (prostaglandin X) which relaxes vascular smooth muscle and potently inhibits platelet aggregation. The release of prostaglandin (PG) X can be stimulated by incubation with arachidonic acid or prostaglandin endoperoxides PGG₂ or PGH₂. The basal release of PGX or the release stimulated with arachidonic acid can be inhibited by previous treatment with indomethacin or by washing the tissue with a solution containing indomethacin. The formation of PGX from prostaglandin endoperoxides PGG₂ or PGH₂ is not inhibited by indomethacin. 15-hydro-peroxy arachidonic acid (15-HPAA) inhibits the basal release of PGX as well as the release stimulated by arachidonic acid or prostaglandin endoperoxides (PGG₂ or PGH₂). Fresh arterial tissue obtained from control or indomethacin treated rabbits, when incubated with platelet rich plasma (PRP) generates PGX. This generation is inhibited by treating the tissue with 15-HPAA. A biochemical interaction between platelets and vessel wall is postulated by which platelets feed the vessel wall with prostaglandin endoperoxides which are utilized to form PGX. Formation of PGX could be the underlying mechanism which actively prevents, under normal conditions, the accumulation of platelets on the vessel wall.     

Investigation of the vascular actions of arachidonate lipoxygenase and cyclo-oxygenase products on the isolated perfused stomach of rat and rabbit

The vascular actions of several prostanoids and arachidonate lipoxygenase products were investigated on the gastric circulation of rat and rabbit perfused with Kreb's solution. Under resting conditions, prostacyclin and PGE₂ produced small decreases in perfusion pressure with prostacyclin being the more potent. During vasoconstriction induced by infusion of noradrenaline, vasopressin or angiotensin II, prostacyclin was 20–40 times as active as PGE₂ as a gastric vasodilator in rat or rabbit stomach. PGF_2α was a less potent vasoconstrictor than noradrenaline, while the epoxy-methano endoperoxide analogue produced a long-lasting vasoconstriction. The putative metabolite, 6-oxo-PGE₁ was less active than prostacyclin as a vasodilator, having comparable activity to PGE₁, whereas 6-oxo-PGF_1α had very little activity. The endoperoxide, PGH₂ reduced perfusion pressure, this effect being inhibited by concurrent infusion of 15-HPETE. The vasodilation induced by arachidonic acid was likewise reduced by 15-HPETE, and abolished by indomethacin infusion. The arachidonate lipoxygenase hydroperoxides were vasodilator in the gastric circulation, the rank order of potency being 12-HPETE > 11-HPETE > 5-HPETE > 15-HPETE in both rat and rabbit stomach. It is possible that such vasoactive lipoxygenase products, may play modulator roles in the gastric mucosa.     

Metabolism of prostaglandin endoperoxide in animal tissues

The endoperoxide PGH₂ serves as a common intermediate for the enzymatic production of prostaglandins (PGEs and PGFs), thromboxanes (Tx) and prostacyclin (PGI₂). These compounds have quite different physiological activities and apparently perform important regulatory functions in various tissues and organs. We have obtained information on the distribution of individual enzymes responsible for the bioconversion of PGH₂ into these compounds in various tissue preparations. [1-C¹⁴] PGH₂ was incubated with a membrane fraction from each tissue homogenate. The products were isolated and identified by radiometric TLC and gas chromatography-mass spectrometry. Short life intermediates were detected by their specific biological activities. With this approach, we have demonstrated the formation of thromboxanes in rhesus monkey platelets, spleen and bone marrow, guinea pig lung and spleen, rabbit lung, human platelets and thioglycollate stimulated peritoneal macrophage from rat. On the other hand, the membrane preparation of bovine and mare corpus luteum, uteri from rabbit, monkey and human, rat stomach and small intestine, and rabbit lung produced predominantly prostacyclin. In addition, a PGH₂ to PGD₂ isomerase was found in the homogenate of rat brain and polymorphonuclear leukocytes. In those tissues which possess more than one enzyme catalyzing the metabolism of prostaglandin endoperoxide, substrate availability appeared to be one factor controlling the metabolic fate of the endoperoxide. The wide occurrence of thromboxane and prostacyclin synthetases suggests that their biological roles are not limited to the cardiovascular system.     

Concerning the biosynthesis of prostaglandin I2

Two diastereoisomers, 5R,6R-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (7) and 5S,6S-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (10) were synthesized for evaluation as possible biosynthetic intermediates in the enzymatic transformation of PGH₂ or PGG₂ into PGI₂. The synthetic sequence entails the stereospecific reduction of the 9-keto function in PGE₂ methyl ester after protecting the C-11 and C-15 hydroxyls as tbutyldimethylsilyl ethers. The resulting PGF_2α derivative was epoxidized exclusively at the C-5 (6) double bond to yield a mixture of epoxides, which underwent facile rearrangement with SiO₂ to yield the 5S,6S and 5R,6R-5-hydroxy-6(9α)-oxido cyclic ethers. It was found that dog aortic microsomes were unable to transform radioactive 9β-5S,6S[³H] or 9β-5R,6R[³H]-5-hydroxy-6(9α)-oxido cyclic ethers into PGI₂. Also, when either diastereoisomer was included in the incubation mixture, neither isomer diluted the conversion of [1-¹⁴C]arachidonic acid into [1-¹⁴C]PGI₂.     

Differential response of articular chondrocyte populations to thromboxane B2 and analogs of prostaglandin cyclic endoperoxides

The effects of two unsaturated fatty acids, prostaglandin E₂, thromboxane B₂ (TxB₂) and 2 analogs of PG endoperoxide on monolayer cultures of rabbit articular chondrocytes have been studied. Arachidonic and linoleic acids had no effect on either DNA or sulfated-glycosaminoglycan biosynthesis, while 13,14 dihydro-PGE₂ and PGE₂ markedly inhibited the former. Two epoxymethano analogs of endoperoxide PGH₂ (Em-PGH₂) at concentrations of 2.5 and 25 μg/ml stimulated cell proliferation while reducing ³⁵SO₄ incorporation. By contrast, Em-PGH₂ at lower concentrations (0.25 – 250 ng/ml) inhibited DNA synthesis in a dose-dependent manner. TxB₂ at 2.5 μg/ml did not alter cellular proliferation. At lower concentrations, 2.5 and 25 ng/ml, TxB₂ significantly stimulated sulfated-glycosaminoglycan biosynthesis in at least one of the chondrocyte populations tested. The results also demonstrated marked differences in the effects of TxB₂ and the Em-PGH₂ analogs on the partitioning of newly synthesized sulfated-proteoglycan between the cells and medium of these cell cultures.     

Conversions of prostaglandin endoperoxides by prostacyclin synthase from pig aorta

Partially purified prostacyclin synthase from pig aorta converted the prostaglandin (PG) endoperoxide PGH₂ to prostacyclin (PGI₂), and PGH₁ to 12-hydroxy-8,10-heptadecadienoic acid (HHD). Both reactions were inhibited by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HP) in a dose-dependent fashion. However, the reactions PGH₂ → PGI₂ and PGH₁ → HHD appeared to differ: substrate availability was rate limiting in the latter reaction, while the enzyme became rapidly saturated with PGH₂ and a steady rate of prostacyclin formation was observed at higher substrate levels.     

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

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

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

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

Quantitative determination of 6-keto-PGF1α released by rat aorta: Comparison of mass fragmentography and high resolution gas chromatography with electron capture detection

Mass fragmentography (MF) and high resolution gas chromatography with electron capture detection (HRGC-ECD) were used for measuring 6-keto-PGF_1α, the stable hydrolysis product of prostacylin (PGI₂) released by fresh rings of rat aorta, incubated in the absence of the precursors arachidonic acid or prostaglandin endoperoxide (PGH₂). The incubation medium was acidified, extracted, chromatographed on silicic acid column and derivatized. Comparable results were obtained analyzing each sample by MF and HRGC-ECD. Both methods proved to be suitable in terms of sensitivity and specificity for the measurement of 6-keto-PGF_1α produced by individual rat aortae.     

Modulation of human platelet adenylate cyclase by prostacyclin (PGX)

Prostacyclin (PGX) ($\text{5Z}\text{)-9-}\text{deoxy}\text{-6,9α-}\text{epoxy}\text{-Δ}{}^5\text{-}\text{PGF}{}_{\mathrm{1\alpha }}$ has been found to be a potent stimulator of cAMP accumulation in human platelet rich plasma (PRP), and a direct stimulator of platelet microsome adenylate cyclase. Prostacyclin is, on a molar basis, at least 10 times more potent a stimulator of cAMP accumulation in platelets than PGE₁. The prostacyclin stimulation of platelet cAMP accumulation can be antagonized by the prostaglandin endoperoxide PGH₂, and a PGH₂-induced platelet aggregation is antagonized by prostacyclin. A model of platelet homeostasis is proposed that suggests platelet aggregation is controlled by a balance between the adenylate cyclase stimulating activity of prostacyclin, and the cAMP lowering activity of PGH₂.     

PGD2 formation in the vasculature: Characteristics of rat tail vein prostaglandin endorperoxide-D isomerase

Rat tail vein homogenates, microsome and high speed supernatant fractions were incubated with [1-¹⁴C]prostaglandin endoperoxide (PGH₂) and products separated and identified by radio-thinlayer chromatography. PGI₂ synthase was localized to the microsomal fraction, but exhibited low activity compared to rat tail arteries prepared in the same manner. PGH-D isomerase was maximally active in the presence of reduced glutathione at pH 7.5–8.0, exhibited a K_m for PGH₂ of 33 μM, and was inhibited sulfhydryl-directed reagents. The similarities of this enzyme to PGD synthase of the rat cerebral microvasculature are discussed.     

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.