Identification of prostaglandin D2 as a major prostaglandin in homogenates of rat brain

Prostaglandin (PG) E2, D2, F2alpha and thromboxane B2 (TxB2) were determined in homogenates of rat brain by gas-chromatography--mass spectrometry. The level of PGD2 was 735 +/- 19 ng/g, of PGF2alpha 150 +/- 13 ng/g, of TxB2 112 ng/g and of PGE2 86 +/- 8 ng/g. The same relative proportions of cyclooxygenase products were found in incubates of unstimulated sliced rat brain. 14C-PGH2 was converted in high yield into PGD2 by enzyme(s) present in the soluble fraction of the homogenate. These results indicate that PGD2 is the major cyclooxygenase product in the central nervous system of the rat.     

Characterization of brain D2 dopamine receptors

In order to characterize and partially purify solubilized dopamine receptors, canine brain striatum microsomes were solubilized with 1% digitonin, and enriched by either gel permeation chromatography, preparative vertical column isoelectric focusing, or sucrose gradient ultracentrifugation. Chromatography on Sephacryl S-300 in buffer (contaning 0.05% Triton X-100) yielded a Stokes radius of 5.8 nm. Isoelectric focusing of the solubilized, radiolabelled receptor produced peaks of [³H]spiperone radioactivity corresponding to isoelectric values of 5.0 and 7.8, of which the former has been shown elsewhere to be the intact D₂ dopamine receptor. Sucrose density gradient ultracentrifugation, again in buffer containing 0.05% Triton X-100, indicated a hydrodynamic mol. wt of 136,000 Daltons, which corresponds closely to the value of 123,000 Daltons estimated using radiation inactivation. Such molecular characterization will aid in the distinction of dopamine receptor subtypes.     

Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay

A solid-phase enzyme immunoassay for prostaglandin D₂ (PGD₂) was developed in which PGD₂ was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labelled and free PGD₂, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)- propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3 – 100 pg for PGD₂. The IC₅₀ value for PGD₂ in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enyzme immunoassay was applied to the measurement of PGD₂ content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD₂. PGD₂ contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay (r = 0.966) after raising its contents by intravenous administration of PGD₂. The level of PGD₂ in rat brain was extremely low but determined to be 0.11 ± 0.03 ng/g tissue (mean ± S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change.     

Development of a radioimmunoassay for prostaglandin D2 using an antiserum against 11-methoxime prostaglandin D2

A sensitive and specific radioimmunoassay for prostaglandin D₂ has been developed using its stabilized 11-methoxime derivative, which was obtained after treatment of prostaglandin D₂ with methoxamine-HCl. The antiserum was obtained after injection of prostaglandin D₂-methoxamine coupled to bovine serum albumin. A (¹²⁵I)-Histamide prostaglandin D₂-methoxamine tracer was prepared by iodination of the corresponding histamide, followed by thin layer chromatography purification. The sensitivity of the assay was 280 femtomoles per ml at 50% displacement. The cross reactivities were 15% with prostaglandin D₁-methoxamine and less than 0.20% with other prostaglandins. Determination of the half-life of prostaglandin D₂ in a solution containing albumin was also carried out, since it has been shown to catalyze prostaglandin D₂ destruction. The unstability of this prostaglandin is due to the presence of a β-hydroxy ketone group, and all prostaglandins possessing this labile moiety could be stabilized by such a derivatization before developing a radioimmunoassay.     

Biosynthesis of prostaglandin D2 1. Formation of prostaglandin D2 by human platelets

Formation of prostaglandin D₂ (PGD₂) during the aggregation of platelets was determined, employing a specific bioassay. PGD₂ was synthesized in human platelet rich plasma (PRP) in response to thrombin, collagen and epinephrine. Indomethacin pretreatment abolished the biosynthesis of PGD₂. When thrombin treated PRP was incubated for different periods of time and denatured in the presence of SnCl₂ to prevent the formation of PGD₂ from endoperoxides during the extraction procedure, PGD₂ formation was noted within the first minute of incubation and reached a peak level after 4 minutes. PGD₂ from thrombin stimulated PRP was conclusively identified by gas chromatography-mass spectrometry.The formation of PGD₂ during platelet aggregation could represent a mechanism of feedback inhibition of aggregation.     

Cardiovascular effects of prostaglandin D3 and D2 in anesthetized dogs

Prostaglandin (PG) D₃ has been identified as an inhibitor of human platelet aggregation, but little is known of the hemodynamic activity of this material. In morphine pretreated, chloralose-urethan anesthetized dogs, bolus intravenous injections (1, 3.2 and 10 μg/kg) of PGD₃ and also PGD₂ were associated with marked, dose-related increases in pulmonary arterial pressure. Cardiac index and rate increased, while peripheral vascular resistance decreased in response to injections of PGD₃. A biphasic (depressor followed by a pressor phase) effect on systemic arterial pressure was observed after PGD₂, while PGD₃ was associated with dose-related depressor responses. Graded intravenous infusions (0.25, 0.50 and 1.0 μg/kg/min) of PGD₃ and PGD₂ were associated with qualitatively similar cardiovascular responses. Quantitatively, PGD₃ infusions were associated with greater decreases in peripheral vascular resistance and greater increases in cardiac output, heart rate, and peak left ventricular dp/dt than were infusions of PGD₂. In contrast, PGD₃ was less potent than PGD₂ as a pulmonary pressor material. Systemic arterial pressure responses to infusions of the prostaglandins were variable. In these experiments, PGD₃ and PGD₂ were associated with qualitatively similar cardiovascular responses characterized by peripheral vasodilatation.     

Prostaglandin D2 is the major prostaglandin of arachidonic acid metabolism in rat bone marrow homogenate

Homogenate of bone marrow of male rat was incubated with ¹⁴C-arachidonic acid and with ¹⁴C-prostaglandin H₂. The major prostaglandin produced during each incubation was prostaglandin D₂, which was identified by thin-layer chromatography, autoradiography and chemical reduction.     

Molecular conformation of prostaglandin E2

The crystal and molecular structure of prostaglandin E₂ (PGE₂) has been determined by X-ray diffraction. The compound crystallizes in the triclinic space group P1 with Z = 1 and , , , α = 87.347°, β = 94.042°, and γ = 91.010°. Gauche-gauche interactions appear in both side chains. The efficient molecular packing and hydrogen bonding network appears to stabilize the observed molecular conformation.     

The enzymatic conversion of prostaglandin D2 to prostaglandin F2α

Prostaglandins E₂ and D₂ were both converted to prostaglandin F_2α (9α, 11α) by an enzyme present in sheep blood. Neither the 9β, 11α epimer nor the 9α, 11β epimer was produced from PGE₂ or PGD₂ respectively. The rate of reduction was measured using isotope dilution (D₄ PGF_2α) and multiple-ion detection gas chromatography-mass spectrometry.     

Effects of intravenous infusions of prostaglandin D2 in man

Prostaglandin D₂ (PGD₂) was infused intravenously into normal male volunteers. Seven subjects received infusions of 16, 32, 64 ng/kg/min and six of these a further dose of 128 ng/kg/min. Each individual's maximum dose was limited by discomfort caused by intense facial flushing and nasal congestion. At these doses there was no significant effect on systolic or diastolic blood pressure nor on spirometric measurements. There was a small but statistically significant tachycardia at 64 and 128 ng/kg/min. Collagen- and adenosine diphosphate (ADP)-induced platele aggregation was not affected at any of the infusion rates. Infused PGD₂ is unlikely to be a useful antithrombotic agent.     

Potent bronchoconstrictor effects of aerosolized prostaglandin D2 in dogs

Previously, we demonstrated that prostaglandin D₂ (PGD₂), a natural product of the endoperoxide PGH₂, evoked bronchoconstriction when given I.V. to dogs (PROSTAGLANDINS $\text{13}$:255–269, 1977). The present investigation in anesthetized dogs demonstrated that aerosols of PGD₂ (0.001–0.1%) produced concentration-dependent increases in pulmonary resistance (R_L) and decreases in dynamic lung compliance (C_DYN) which were short-lived and equipotent to PGF_2α. These alterations in pulmonary mechanics were partially, yet significantly, inhibited by atropine, thereby suggesting that at least a portion of the bronchoconstriction may be cholinergically mediated. Concomitant cardiovascular depressant effects of both PGD₂ and PGF_2α aerosols were much less and more variable than their bronchopulmonary effects.These results demonstrate a potent bronchoconstrictor effect of aerosolized PGD₂ in dogs. PGD₂ warrants further attention as a possible mediator of the bronchospasm seen in acute, reversible airways obstruction.     

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.     

Prostaglandin D2 as a potential antithrombotic agent

Prostaglandin D₂ was found to be a potent inhibitor of platelet aggregation. Aggregation of human platelets by ADP, collagen and prostaglandin G₂ was inhibited more strongly by PGD₂ than by PGE₁. Although ADP-induced aggregation of rabbit platelets was inhibited more strongly by PGE₁ than by PGD₂ the latter prostaglandin gave a more long-lasting inhibitory effect on platelet aggregation following intravenous or oral administration. These results coupled with the finding that PGD₂ has less hypotensive effects on the cardiovascular system than PGE₁ suggest the possible use of PGD₂ as an antithrombotic agent.     

Effect of acetaminophen on prostaglandin E2 and prostaglandin F2α synthesis in the renal inner medulla of rat

Effects of acetaminophen on the renal inner medullary production of prostaglandin E₂ and F_2α were compared with the well-known effects of aspirin on this process. Acetaminophen was found to elicit a dose-dependent inhibition of both prostaglandin E₂ and F_2α accumulation in media with a K_i of 100–200 μM. This inhibition could not be accounted for by increased accumulation of prostaglandins within slices. Acetaminophen inhibition was reversed by removal of acetaminophen during the incubation or by addition of arachidonic acid. Similar manipulations did not reverse aspirin or indomethacin-mediated inhibition of prostaglandin synthesis. Thin-layer and gas chromatographic analysis of acetaminophen following incubation with slices demonstrated that this material was identical to authentic acetaminophen. This, in addition to the lack of an effect of glutathione on inhibition, suggests that acetaminophen does not have to be metabolized to exert this inhibition. Arachidonic acid did not alter the metabolism or increase the efflux of acetaminophen. Lower levels of prostaglandin E₂ observed with 5 mM acetaminophen and 1 mM aspirin caused a corresponding decrease in cyclic AMP content. Removal of acetaminophen from the second incubation or addition of arachidonic acid caused increases in both prostaglandin E₂ and cyclic AMP. Aspirin inhibition of cyclic AMP content was not reversed by similar manipulations. In vivo inhibition of inner medullary prostaglandin E₂ and prostaglandin F_2α synthesis was observed 2 h after a 375 mg/kg, intraperitoneal injection of acetaminophen. These data suggest that acetaminophen, like aspirin, is capable of reducing tissue prostaglandin synthesis. However, the mechanisms by which these two analgesic and antipyretic agents elicit their inhibition of prostaglandin synthesis are quite different.     

Production of prostaglandin D2 by murine macrophage cell lines

Several tumor-derived murine macrophage cell lines were evaluated as cloned prototypes of tissue macrophages for their ability to metabolize arachidonic acid. Unexpectedly, two cell lines, J774A.1 and WR19M.1, rapidly converted exogenous ¹⁴C-arachidonic acid (AA) to a single major prostaglandin metabolite. The compound, PGD₂, was positively identified by TLC, HPLC, and GC-MS. The enzymatic formation of the PGD₂ was shown by inhibition of its formation by indomethacin and reduced formation of ¹⁴C-PGD₂ and ¹⁴C-PGD₂ in boiled cells. When J774A.1 cells were prelabeled with ³H-AA, cultured for 24 hours, and stimulated with lipopolysaccharide (LPS), PGD₂ was again the predominant product. No other tumor derived cell lines, including several other murine macrophage lines, produced significant amounts of PGD₂. Elicited and activated murine peritoneal macrophages produced only small amounts of PGD₂, but resident peritoneal macrophages produced modest amounts of PGD₂. Exaggerated formation of PGD₂ by J774A.1 and WR19M.1 cells may be a consequence of neoplastic transformation or the clonal expansion of a minor subpopulation of normal tissue macrophages.     

Bronchopulmonary and cardiovascular effects of prostaglandin D2 in the dog

The effects of intravenously administered prostaglandin D₂ (PGD₂) on bronchopulmonary and cardiovascular functions were examined in the dog. PGD₂ (0.03–1.0 μg/kg) was shown to be more active than PGF_2α, a known bronchoconstrictor, in decreasing dynamic lung compliance, tidal volume, and expiratory airflow rate, as well as in elevating lung resistance. PGD₂ demonstrated a potency approximately 4–6 times that of PGF_2α on pulmonary mechanics. Atropine sulfate infusions reduced significantly the resistance and compliance responses to PGF_2α, but only the resistance responses to PGD₂, thereby suggesting that part of the bronchoconstrictor activities of these agents involved a cholinergic component.In another series of anesthetized dogs, PGD₂ (0.1–10.0 μg/kg) increased pulmonary arterial pressure (comparable to PGF_2α) and heart rate (greater than PGF_2α, but less than PGE₂), while concomitantly decreasing systemic arterial pressure in a dose-related manner ( that of PGE₂). Qualitatively similar alterations in cardiovascular parameters were obtained for PGD₂ in conscious dogs.Therefore, potent biologica activity of PGD₂ has been shown in the dog. No physiologic or pathologic role for PGD₂ has yet been demonstrated, but nonetheless, since it is a naturally occurring PG derived from arachidonic acid, further studies are warranted.     

Metabolic pathway to 25-hydroxyvitamin D3-26,23-lactone from 25-hydroxyvitamin D3

mRNA was prepared from autopsy liver samples from a homozygote for α₁-antitrypsin deficiency (PiZZ) and from a normal (PiMM) subject. Both preparation gave equivalent synthesis of α₁-antitrypsin in a wheat germ cell-free system. This suggests that the deficiency of plasma α₁-antitrypsin associated with the Z variant is due to a failure of processing and secretion of the protein rather than of its synthesis. It is likely that it is the resultant intracellular accumulation of the Z protein rather than a deficiency of protease inhibitor that is the primary cause of the liver pathology associated with this variant.     

Biphasic action of prostaglandin E2 on conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 in chick renal tubules

The effect of PGE₂ on the conversion of 25-hydroxyvitamin D₃ (25 OH D₃) to 1,25-dihydroxyvitamin D₃ (1,25- (OH) ₂D₃) by isolated renal tubules from vitamin D deficient chicks was studied under a variety of experimental conditions. In the absence of added vitamin D metabolites, PGE₂ (2 × 10⁻⁶M) caused an immediate inhibition of formation of 1,25-(OH) ₂D₃, followed by a delayed stimulation, apparent after 15 h exposure to PGE₂. Pretreatment of the tubules with 1,25-(OH) ₂D₃ prevented the immediate inhibitory action of PGE₂, and allowed the stimulation to be apparent after 4 h exposure to PGE₂. The cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (IBMX) significantly stimulated the formation of 1,25-(OH) ₂D₃. PGE₂ significantly inhibited 1,25-(OH) ₂D₃ formation in tubules which had been stimulated by IBMX. PGE₂ stimulated the adenylate cyclase activity in a crude particulate fraction from the chick kidney, and raised cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) levels in the renal tubules.It is concluded that PGE₂ can either stimulate or inhibit 1,25-(OH) ₂D₃ formation in chick renal tubules. The stimulatory effect may be partly due to elevation of cyclic AMP. The mechanism of the inhibitory effect requires further investigation.     

The clearance of human fibrinogen fragments D1, D2, D3 and fibrin fragment D1 dimer in mice

The clearance of human fibrinogen fragments D₁, D₂, D₃ and fibrin fragment D₁ dimer were studied in the mouse model. Clearance of these fragments is a complex process involving clearance from blood into three other compartments. The overall clearance of fragment D₁ and its dimer were essentially identical. Fragments D₂ and D₃ cleared at a progressively slower rate. Competition studies were performed between ¹²⁵I-labeled fragment D₁ and large molar excesses of unlabelled human fragments D₁, D₂, D₃, D₁ dimer, fragment E, fibrinogen, macroalbumin, mannan and asialooroscomucoid. Of these ligands only the fragment D variants competed for the clearance of ¹²⁵I-labeled fragment D₁. Cross-competition was observed when ¹²⁵I-labeled fragment D₁ dimer was cleared in the presence of large molar excesses of fragment D₁. Autopsies demonstrated that injected fragments D₁, D₂, D₃ and D₁ dimer cleared primarily in liver and kidneys. In some clearance studies, livers were perfused with tissue culture fluid, subjected to light microscopic autoradiography, and silver grain counts performed to localize cleared fragment D₁. These experiments indicated that 80% of the liver uptake was in hepatocytes. However, when silver grain counts were normalized for the number of parenchymal and nonparenchymal cells, the distribution of silver grains was essentially identical (1.8 and 1.6 grains per cell, respectively). It is concluded that fragments D₁, D₂, D₃ and D₁ dimer are recognized by a similar clearance pathway. Since neither fibrinogen nor fragment E competed for the clearance of fragment D₁, it is suggested that determinants present in the fragment D domain become exposed after plasmin attack on fibrinogen and are responsible for clearance.     

Neuroeffector actions of prostaglandin D2 on isolated dog mesenteric arteries

Treatment with prostaglandin (PG) D₂ in concentrations (10⁻⁸ to 10⁻⁷ M) insufficient to alter the basal tone potentiated the contractile response of helical strips of dog mesenteric arteries to transmural electrical stimulation but did not influence the response to norepinephrine. The potentiating effect of PGD₂ was not prevented by treatment with diphloretin phosphate, a PG antagonist, whereas contractions of dog cerebral arteries induced by PGD₂ were suppressed. The ³H-overflow evoked by transmural stimulation in superfused mesenteric arterial strips previously soaked in ³H-norepinephrine containing media was significantly increased by PGD₂. It is concluded that PGD₂ increases the stimulation-evoked release of norepinephrine from adrenergic nerves innervating the arterial wall. PGD₂ appears to act differently on receptive sites responsible for increasing the release of norepinephrine and for producing arterial contraction.