Prostaglandin receptor-adenylate cyclase system in plasma membranes of rat liver and ascites hepatomas, and the effect of GTP upon it.

1. Adenylate cyclase in plasma membranes from rat liver was stimulated by prostaglandin E1, and to a lesser extent by prostaglandin E2. Prostaglandin F1alpha and A1 did not stimulate the cyclase. The prostaglandin E1-mediated activation was found to require GTP when the substrate ATP concentration was reduced from 3 mM to 0.3 mM in the reaction mixture. Adenylate cyclase of the plasma membranes from rat ascites hepatomas AH-130 and AH-7974 was not stimulated by prostaglandin E1 in the presence or the absence of GTP, although the basal activity of adenylate cyclase as well as its stimulation by GTP alone were similar to normal liver plasma membranes. 2. Liver plasma membranes were found to have two specific binders for [3H] prostaglandin E1 with dissociation constants of 17.6-10(-9) M and 13.6-10(8) M (37 degrees C) and one specific binder for [3H]prostaglandin F2alpha with a dissociation constant of 2.31-10(8) M (37 degrees C). The specific binders for prostaglandin E1 could not be detected in the hepatoma plasma membranes. 3. Binding of [3H] prostaglandin E1 to the liver plasma membranes was exchange by, GTP dGPT, GDP, ATP and GMP-P(N)P, but not by GMP, CGMP, DTTP, UTP or CTP. The increase in the binding of [3H] prostaglandin E1 was found to be due to the increased affinity of the specific binders to prostaglandin F2alpha was not affected by GTP. 4. GTP alone was found to increase V of adenylate cyclase of liver plasma membranes, while GTP plus prostaglandin E1 was found to decrease Km of adenylate cyclase in addition to the increase of V to a further extent.     

Purification and properties of prostaglandin D synthetase from rat brain.

The prostaglandin D synthetase system was isolated from rat brain. Prostaglandin endoperoxide synthetase solubilized from a microsomal fraction catalyzed the conversion of arachidonic acid to prostaglandin H2 in the presence of heme and tryptophan. Prostaglandin D synthetase (prostaglandin endoperoxidase-D isomerase) catalyzing the isomerization of prostaglandin H2 to prostaglandin D2 was found predominantly in a cytosol fraction and was purified to apparent homogeneity with a specific activity of 1.7 mumol/min/mg of protein at 24 degrees C. The enzyme also acted upon prostaglandin G2 and produced a compound presumed to be 15-hydroperoxy-prostaglandin D2. Glutathione was not required for the enzyme reaction, but the enzyme was stabilized by thiol compounds including glutathione. The enzyme was inhibited by p-chloromercuribenzoic acid in a reversible manner. The purified enzyme was essentially free of the glutathione S-transferase activity which was found in the cytosol of brain.     

Sensitization of alveolar macrophages to lipopolysaccharide-induced prostaglandin synthesis by exogenous prostaglandins

Rabbit alveolar macrophages were found to produce extraordinary amounts of prostaglandin E2 and F2 alpha with the stimulation of lipopolysaccharide or lipid A. Exogenous prostaglandin E2 greatly enhanced the lipopolysaccharide action on rabbit alveolar macrophages for the induction of prostaglandin F2 alpha release (3-5 fold), while prostaglandin E2 alone did not cause any effect. The enhancement expressed was especially strong when prostaglandin E2 was administered to the cells simultaneously with lipopolysaccharide. The effect of prostaglandin E2 was observed neither with a nonstimulating dose of lipopolysaccharide nor with a stimulating dose of zymosan. This phenomenon was even more pronounced when prostaglandin I2 was used instead of prostaglandin E2, while no sensitization was demonstrated by prostaglandin F2 alpha. These observations suggest that prostaglandins can modulate the activation of the cyclooxygenase pathway of arachidonate metabolism in the activated macrophages by lipopolysaccharide.     

Ethacrynic acid induced release of prostaglandin E to increase renal blood flow

Ethacrynic acid administered to anesthetized dogs was found to increase the level of prostaglandin E as determined by radioimmunoassay in renal venous blood at the time when renal blood flow was increased by this agent. No change was found in the renal venous level of prostaglandin F. When ethacrynic acid was administered after treatment with indomethacin, which blocks the increase in renal blood flow induced by the natriuretic agent, no increase in the renal venous level of prostaglandin E was seen. Thus, the dilation of the renal vasculature would appear to be caused by a stimulation of synthesis and release of prostaglandin E by ethacrynic acid.     

Peroxisomal chain-shortening of prostaglandin F2 alpha

We have recently reported that prostaglandin F2 alpha can be chain-shortened by isolated rat liver peroxisomes. In the present study it is further established by cell fractionation experiments that the enzymes involved in this reaction are localized to peroxisomes. Under the conditions employed, the highest activity was found in the light mitochondrial fraction. Further fractionation of the light mitochondrial fraction by sucrose density gradient centrifugation showed that the prostaglandin oxidation activity comigrated with peroxisomal marker enzymes. Di(2-ethylhexyl)phthalate treatment resulted in a tenfold increased capacity for the conversion of prostaglandin F2 alpha into tetranorprostaglandin F1 alpha. The reaction was not inhibited by KCN. The reaction was further characterized with respect to cofactor requirements. The prostaglandin oxidation was found to be completely dependent on NAD, CoA, ATP, Mg2+ and was stimulated by FAD. Incubation of prostaglandin E2 with peroxisomes resulted in conversion into several products. After alkaline hydrolysis, one of these was identified as tetranorprostaglandin B1.     

Metabolism of prostaglandins E, A, and C in serum.

Three prostaglandin-metabolizing enzymes were detected in human serum. One enzyme was a dehydrase that converted prostaglandin E to prostaglandin A, the second was a prostaglandin A isomerase that converted prostaglandin A to prostaglandin C and the third was a prostaglandin C isomerase that converted prostaglandin C to prostaglandin B. All three were inactivated by sulfhydryl blocking agents. In human serum, only prostaglandin C isomerase had high activity, whereas the two other enzymes had very low activity. In rabbit serum both isomerases were very active, but dehydrase activity could not be detected. Prostaglandin C isomerase activity was also found in crystallized human serum albumin and rabbit serum albumin.     

Metabolism of prostaglandin D2 in the monkey.

[3H7]Prostaglandin D2 was biosynthesized and infused into an unanesthetized monkey. The urinary metabolites were isolated and subsequently identified by gas chromatography-mass spectrometry. Two pathways of prostaglandin D2 metabolism were identified and resulted in metabolites with prostaglandin D (3-hydroxycyclopentanone) and prostaglandin F (cyclopentane-1,3-diol) ring structures. The major prostaglandin D ring metabolite was identified as 9,20-dihydroxy-11,15-dioxo-2,3-dinorprost-5-en-1-oic acid. Nine other prostaglandin D ring metabolites were identified reflecting various combinations of metabolism by beta and omega oxidation, 15 dehydrogenation, and 13-14 reduction. In greater abundance were those prostaglandin D2 metabolites which had the prostaglandin F ring structure. The major prostaglandin D2 metabolite which had the prostaglandin F ring structure was identified as 9,11,15-trihydroxy-2,3-dinorprosta-5,13-dien-1-oic acid (dinor prostaglandin F2 alpha). Nine other metabolites with the prostaglandin F ring structure were identified, including prostaglandin F2 alpha itself. These, for the most part, were the structural counterparts of the metabolites with the prostaglandin D ring. Since many prostaglandin D2 metabolites were found to be identical with the metabolites of prostaglandin F2 alpha, quantitative determinations of prostaglandin F ring metabolites may not be a specific indicator of prostaglandin F2 alpha biosynthesis. Likewise, data involving the measurement of a biological effect of prostaglandin D2 must be re-examined to account for the possible contribution of prostaglandin F2 alpha, a metabolite of prostaglandin D2, to the biological response.     

Study of the physico-chemical properties of prostaglandin F2 alpha and its complexes

To study the intracellular action mechanisms of prostaglandin F2 alpha its interaction with lipid components of biological membranes was investigated. It has been found that prostaglandin forms a complex with phosphatidylcholine and cholesterol. Immobilization ability of phospholipids is changed in the course of complex formation. Ability of prostaglandin F2 alpha for complex formation with insulin was also observed. Combination of monolayer technique and electron microscopy made possible to discover molecular reconstructions when prostaglandin is incorporated into monolayer of biologically active compounds.     

Single shot prostaglandin gel for labor induction

A viscous suspension of prostaglandin E₂ was introduced endocervically to induce labor in patients with favourable induction features. The method was found to be effective and compared favourably with the conventional practices of amniotomy and intravenous oxytocin infusion or oral prostaglandin therapy. Several advantages were found including a high degree of patient acceptability.     

Use of lysed human platelets to study prostaglandin E2 production

The conversion of 1-¹⁴C-arachidonic acid into prostaglandin E₂ was studied in lysed human platelets. Optimum production of the labeled reaction product was obtained when reduced glutathione and hydroquinone were included in the incubations. The labeled product was characterized by silicic acid column chromatography, thin-layer chromatography, and gas-liquid chromatography and was found to behave as standard prostaglandin E₂. The results indicate that the prostaglandin synthetase in the human blood platelet is similar to prostaglandin synthetases found in other tissues.     

Characterization of the biosynthetic pathway of prostaglandin D2 in human platelet-rich plasma

The biosynthetic mechanism of prostaglandin D2 in human platelet-rich plasma has been investigated. Platelet-rich plasma was separated into washed platelets and platelet-poor plasma, and [1-14C]prostaglandin H2 was incubated with each fraction. The enzymatic conversion of the endoperoxide to prostaglandin D2 was found only in platelet-poor plasma and not in washed platelets or platelet lysate. This prostaglandin D synthetase activity was purified to homogeneity and identified as serum albumin by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focusing, and immunoelectrophoresis. The optimal pH and Km value for prostaglandin H2 were 9.0 and 6 microM, respectively. Glutathione was not required for the activity. Although prostaglandin H2 ws converted to prostaglandin D2 and E2 in the reaction, only the prostaglandin D2 formation was dependent on the protein amount and abolished by prior boiling. The action of this activity under physiological conditions was examined in a model system constituted of serum albumin and washed platelets. Prostaglandin D2 formation was observed in association with thrombin-evoked platelet aggregation in this system and was proportional to the number of platelets and the concentration of serum albumin, suggesting that thrombin-stimulated platelets released prostaglandin H2, and the latter compound was then converted to prostaglandin D2 by the action of serum albumin. Consistent with this interpretation, prostaglandin H2 added to platelet-rich plasma was converted in part to prostaglandin D2, and the aggregation caused by this endoperoxide was greatly enhanced by neutralizing the action of prostaglandin D2 with anti-prostaglandin D2 antiserum.     

Metabolic fate of endogenously synthesized prostaglandin D2 in a human female with mastocytosis

Increased production of prostaglandin D2 was recently demonstrated in patients with systemic mastocytosis. One female patient investigated with mastocytosis was found to have overproduction of prostaglandin D2 of such magnitude (150-fold above normal) that it provided the unique opportunity to delineate the metabolic fate of endogenously synthesized prostaglandin D2. A five percent aliquot of a twenty-four hour urine collection from this patient was extracted, purified by silicic acid chromatography, methylated, and finally subjected to high pressure liquid chromatography. Column fractions collected were derivatized and analyzed by gas chromatography-mass spectrometry. Increased quantities of sixteen urinary metabolites were identified and included a series of metabolites retaining the PGD-ring as well as a series of metabolites with a PGF-ring. PGF-ring metabolites were excreted in approximately 4-fold greater relative abundance than PGD-ring metabolites. More than one apparent isomeric form of some PGF-ring metabolites were found. The predominant urinary metabolite was 2,3-dinor-prostaglandin F2. This study provides evidence that endogenously synthesized prostaglandin D2 is converted in substantial part to prostaglandin F2 metabolites in vivo in humans.     

The effect of i-mu-ts'ao on a partially purified prostaglandin E 9-ketoreductase from swine kidney

The enzyme system, prostaglandin E 9-ketoreductase, which catalyzes the reduction of prostaglandin E to form prostaglandin F has been partially purified from swine kidney. This NADPH-linked enzyme is studied spectrophotometrically. The KM of this enzyme for prostaglandin E2 was found to be 180 microM. Studies with the partially purified enzyme indicate that prostaglandin E 9-ketoreductase is affected by a Chinese herbal medicine i-mu-ts'ao (leonurus heterophyllus sweet). An increase in the concentration of i-mu-ts'ao aqueous extract may influence the conversion of prostaglandin E2 into prostaglandin F2 alpha. This finding offers a possible explanation for the physiological role of i-mu-ts'ao when it is treated as a medicine.     

Stimulation of anterior pituitary prostaglandin E content and somatotropin (growth hormone) synthesis by phospholipase A.

The prostaglandin E content of dispersed rat anterior pituitary glands was found to increase in the presence of phospholipase A or arachidonic acid. The increases were abolished by the addition of indomethacin. Similarly, the rate of somatotropin (growth hormone) synthesis was increased by these two agents, and the increases were again abolished by indomethacin. Phospholipase A also stimulated somatotropin release. The stimulation of prostaglandin E accumulation was a specific response to those fatty acids that are precursors for prostaglandin synthesis. One such precursor, [3H]arachidonic acid, was incorporated by rat anterior pituitary glands in vitro, and found to be associated mainly with phosphatidylethanolamine-like material. It is concluded that the intracellular concentration of prostaglandin E is limited by the availability of precursor fatty acids and that this can be increased by the addition of exogenous precursors or by the action of exogenous phospholipase A on the cellular phospholipid. Factors that increased prostaglandin E concentrations also increase the rate of synthesis of somatotropin, providing further evidence for the concept that prostaglandin E is involved in modulation of the rate of synthesis of this hormone.     

Metabolism of prostaglandin E1 and of glutathione conjugate of prostaglandin A1 (GSH-prostaglandin A1) by prostaglandin 9-ketoreductase from rabbit kidney.

Rabbit kidney prostaglandin 9-ketoreductase was found to metabolize the glutathione conjugate of prostaglandin A1 (GSH-prostaglandin A1). Apparent Km (GSH-prostaglandin A1) 13 microM and apparent Km (prostaglandin E1) 200 microM. The cytosolic preparation was subjected to gelfiltration and isoelectric focusing, which revealed that metabolism of prostaglandin E1 and GSH-prostaglandin A1 occurs by means of the same fractions. Furthermore, prostaglandin E1 and GSH-prostaglandin A1 are competitive inhibitors of the enzyme, when GSH-prostaglandin A1 and prostaglandin E1 are tested as substrates, respectively. It si concluded, that GSH-prostaglandin A1 is a much better substrate for prostaglandin 9-ketoreductase from rabbit kidney than is prostaglandin E1.     

Rupture of the uterus following treatment with 16-16-dimethyl E 2 prostaglandin vagitories

Rupture of the uterine body was found after induction of therapeutic abortion with vaginal suppositories containing 16,16-dimethyl prostaglandin E 2 in a 20-year-old primigravida. A short discussion is given on the cervical complications that can occur after prostaglandin induction of abortion, stating that rupture of the uterine body also can be seen. So far, no prostaglandin compound seems to avoid such complications.     

Endogenous prostaglandin release contributes directly to coronary artery tone.

An in vitro coronary artery preparation of beef heart was found to synthesize and release continuously large amounts of a prostaglandin of the E type. Inhibition of prostaglandin synthesis with aspirin, indomethacin, or eicosa-5,8,11,14-tetraynoic acid induced a sustained contraction of the coronary artery, and pretreatment with indomethacin diminished markedly the output of prostaglandin into the bathing medium. It appears that prostaglandin E1, generated from within the vessel wall itself, may act as an intrinsic regulator of coronary artery tone in the beef heart, and that blockade of this function leads to vasospasm.     

Specific prostaglandine E1 and A1 binding sites in rat a dipocyte plasma membranes

Rat adipocyte plasma membranes sacs have been shown to be a sensitive and specific system for studying prostaglandin binding. The binding of prostaglandin E₁ and prostaglandin A₁ increases linearly with increasing protein concentration, and is a temperature-sensitive process. Prostaglandin E₁ binding is not ion dependent, but is enhanced by GTP. Prostaglandin A₁ binding is stimulated by ions, but is not affected by GTP.Discrete binding sites for prostaglandin E₁ and A₁ were found. Scatchard plot analysis showed that the binding of both prostaglandins was biphasic, indicating two types of binding sites. Prostaglandin E₁ had association constants of 4.9 · 10⁹ 1/mole and 4 · 10⁸ 1/mole, while the prostaglandin A₁ association constants and binding capacities varied according to the ionic composition of the buffer. In Tris-HCl buffer, the prostaglandin A₁ association constants were 8.3 · 10⁸ 1/mole and 5.7 · 10⁷ 1/mole, while in the Krebs—Ringer Tris buffer, the results were 1.2 · 10⁹ 1/mole and 8.6 · 10⁶ 1/mole.Some cross-reactivity between prostaglandin E₁ and A₁ was found for their respective binding sites. Using Scatchard plot analysis, it was found that a 10-fold excess of prostaglandin E₁ inhibited prostaglandin A₁ binding by 1–20% depending upon the concentration of prostaglandin A₁ used. Prostaglandin E₁ competes primarily for the A prostaglandin high-affinity binding site. Similar Scatchard analysis using a 20-fold excess of prostaglandin A₁ inhibited prostaglandin E₁ binding by 10–40%. Prostaglandin A₁ was found to compete primarily for the E prostaglandin low-affinity receptor.All of the bound [³H]prostaglandin E₁, but only 64% of the bound [³H]-prostaglandin A₁ can be recovered unmetabolized from the fat cell membrane. There is no non-specific binding of prostaglandin E₁, but 10–15% of prostaglandin A₁ binding to adipocyte membranes is non-specific. Using a parallel line assay to measure relative affinities for the E binding site, prostaglandin E₁ > prostaglandin A₂ > prostaglandin F_2α. Prostaglandin E₂ and 16,16-dimethyl prostaglandin E₂ were equipotent with prostaglandin E₁, while other prostaglandins had lower relative affinities. 7-Oxa-13-prostynoic acid does not appear to antagonize prostaglandin activity in adipocytes at the level of the receptor.     

Metabolic fate of endogenously synthesized prostaglandin D2 in a human female with mastocytosis

Increased production of prostaglandin D₂ was recently demonstrated in patients with systemic mastocytosis. One female patient investigated with mastocytosis was found to have overproduction of prostaglandin D₂ of such magnitude (150-fold above normal) that it provided the unique opportunity to delineate the metabolic fate of endogenously synthesized prostaglandin D₂. A five percent aliquot of a twenty-four hour urine collection from the patient was extracted, purified by silicic acid chromatography, methylated, and finally subjected to high pressure liquid chromatography. Column fractions collected were derivatized and analyzed by gas chromatography-mass spectrometry. Increased quantities of sixteen urinary metabolites were identified and included a series of metabolites retaining the PGD-ring as well as series of metabolites with a PGF-ring. PGF-ring metabolites were excreted in approximately 4-fold greater relative abundance than PGD-ring metabolites. More than one apparent isomeric form of some PGF-ring metabolites were found. The predominant urinary metabolite was 2,3-dinor-prostaglandin F₂. This study provides evidence that endogenously synthesized prostaglandin D₂ is converted in substantial part to prostaglandin F₂ metabolites in humans.     

Rupture of the uterus following treatment with 16-16-dimethyl E 2 prostaglandin vagitories.

Rupture of the uterine body was found after induction of therapeutic abortion with vaginal suppositories containing 16, 16-dimethyl prostaglandin E 2 in a 20-year-old primigravida. A short discussion is given on the cervical complications that can occur after prostaglandin induction of abortion, stating that rupture of the uterine body also can be seen. So far, no prostaglandin compound seems to avoid such complications.