Prostaglandin D2 inhibits the proliferation of human malignant tumor cells

The cytotoxic effect of prostaglandin (PG) D₂, PGE₁ and PGF_2α was examined on human osteosarcoma cells (KSu cell line) , and PGD₂ was most effective. DNA, RNA and protein syntheses of KSu cells were also found to be inhibited by PGD₂ at a concentration of 5μg/ml. Furthermore, the proliferation of various human malignant tumor cells was inhibited by PGD₂ without exception so far. These results suggest that PGD₂ shows an antineoplastic effect on a variety of human malignant tumor cells.     

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.     

Prostaglandin D2 stimulates calcification of human osteoblastic cells

Studies on prostaglandin (PG) regulation of bone formation and resorption metabolism have been complicated by the heterogeneity of the tissue, which involves the interaction between and the activities of two bone cell types, osteoblasts and osteoclasts. In a simplified assay system using a cultured human osteoblastic cell line which has the capacity to form calcified tissue, we determined the effects of PGs on calcification. Of the PGs tested, PGD2 has a remarkable stimulatory activity on osteoblast calcification, but that the effective form is probably a metabolite, delta 12-PGJ2. This calcification function is not cAMP-mediated. PGD2 acts directly on osteoblast to cause stimulation of calcification.     

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.     

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.     

Prostaglandin F2α produced by rabbit renal slices is not a metabolite of prostaglandin E2

Slices of rabbit renal medulla and rabbit renal papilla were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15-³H]-arachidonic acid. In both tissues, comparison of the isotope ratios of the radioactive products with the isotope ratio of the added arachidonic indicated that: (a) there was no discernable isotope effect in the biosynthesis of prostaglandin E₂; (b) prostaglandin F₂α was formed by reduction of prostaglandin H₂ and not by reduction of prostaglandin E₂; and (c) most of the radioactive product arose from arachidonic acid that had been incorporated into the tissue and not from the direct action of cyclooxygenase on arachidonic acid in the medium.     

Prostaglandin D2 lowers nuclear DNA polymerase activity in cultured mastocytoma cells

Prostaglandin D₂ strongly inhibited growth of cultured mastocytoma P-815, 2-E-6 cells, which were established and cloned from mouse mast tumor cells. The inhibition was dose-dependent (IC₅₀ = 2.09 × 10⁻⁵ M). Prostaglandin D₂ also inhibited the DNA synthesizing activity of the cells dose-dependently. We next measured the activities of endogenous DNA polymerases extracted from untreated and prostaglandin D₂-treated cells. Prostaglandin D₂ pretreatment reduced DNA polymerase α activity by 52%. The sedimentation coefficients of the enzymes from untreated and prostaglandin D₂-treated cells were the same suggesting there was no gross change in the size of the enzyme. Prostaglandin D₂ pretreatment of the cells reduced endogenous DNA polymerase β activity to 68% of the control value; the sedimentation coefficients of the enzymes from treated and untreated cells were both 3.5 S. Interestingly, prostaglandin D₂ had no direct inhibitory effect on the activity of either DNA polymerase α or β. Our results indicate that the activities of DNA polymerase α and β are lower in prostaglandin D₂-treated mastocytoma cells. This finding account for the lower level of DNA synthesis in these cells.     

Prostaglandin E1 metabolism by human plasma

An enzymic prostaglandin E₁ metabolising system in human plasma is described. Various properties of the system have been investigated. Metabolism of prostaglandin E₁ added to whole human blood or plasma, particularly in low concentrations such as those found physiologically, can be extremely rapid and extensive. The importance of these findings in relation to the extraction of prostaglandins from human blood or plasma is discussed.     

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 E2 levels and human periodontal disease

PGE₂ levels in human gingiva from healthy patients and patients with periodontal disease was measured by radioimmunoassay. There was a tenfold elevation of PGE₂ levels in diseased as compared with healthy tissue. PGE₂ levels of 10⁻⁶M or greater were found in purulent exudates from periodontal infections. The results suggest that local PGE₂ production may contribute to the inflammatory changes and bone resorption seen in periodontal disease.     

Involvement of protein kinase C in prostaglandin D2 synthesis by cultured astrocytes

The role of protein kinase C (PKC) and calcium in the stimulation of prostaglandin D₂ (PGD₂) synthesis was investigated in primary rat astroglial cultures using the phorbol esters phorbol 12-myristate, 13-acetate (PMA), phorbol 12,13-dibutyrate (PDB) and the calcium ionophore A₂₃₁₈₇. Both phorbol esters and the ionophore were able to stimulate PGD₂ synthesis in a concentration dependent manner. The inactive stereoisomers of PMA and PDB had no significant effect. Combinations of subthreshold concentrations of phorbol esters (10 nM PMA or 10 nM PBD) potentiated PG formation induced by 100 nM A₂₃₁₈₇. An even more pronounced effect was observed when phorbol ester concentrations were increased to 100nM. The contribution of extra- and intracellular calcium in phorbol ester or A₂₃₁₈₇ stimulated PGD₂ synthesis was evaluated by carrying out experiments with calcium-free media plus EGTA or with the intracellular calcium-chelating agent TMB-8. Ionophore stimulated PGD₂ release was shut down to basal values upon removal of extracellular calcium, whereas phorbol ester stimulated PGD₂ formation persisted at a reduced level. It was unabated also upon further addition of EGTA. In the presence of TMB-8, however, phorbol ester stimulated PGD₂ synthesis was completely suppressed. These data strongly suggest that PKC has an additional effect on the activation of phospholipase A₂ and subsequent prostanoid synthesis, which is independent from extracellular calcium and, thus, support the concept of more than one metabolic pathway in astrocytes that synergistically regulate phospholipase A₂ activity.     

Use of vitamin D4 analogs to investigate differences in hepatic and target cell metabolism of vitamins D2 and D3

In this study, we used molecules with either of the structural differences in the side chains of vitamin D₂ and vitamin D₃ to investigate which feature is responsible for the significant differences in their respective metabolism, pharmacokinetics and toxicity. We used two cell model systems—HepG2 and HPK1A-ras—to study hepatic and target cell metabolism, respectively. Studies with HepG2 revealed that the pattern of 24- and 26-hydroxylation of the side chain reported for 1α-hydroxyvitamin D₂ (1α-OH-D₂) but not for 1α-OH-D₃ is also observed in both 1α-OH-D₄ and Δ²²-1α-OH-D₃ metabolism. This suggests that the structural feature responsible for targeting the enzyme to the C24 or C26 site could be either the C24 methyl group or the 22–23 double bond. In HPK1A-ras cells, the pattern of metabolism observed for the 24-methylated derivative, 1α,25-(OH)₂D₄, was the same pattern of multiple hydroxylations at C24, C26 and C28 seen for vitamin D₂ compounds without evidence of side chain cleavage observed for vitamin D₃ derivatives, suggesting that the C24 methyl group plays a major role in this difference in target cell metabolism of D₂ and D₃ compounds. Novel vitamin D₄ compounds were tested and found to be active in a variety of in vitro biological assays. We conclude that vitamin D₄ analogs and their metabolites offer valuable insights into vitamin D analog design, metabolic enzymes and maybe useful clinically.     

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.     

Measurement of prostaglandin D2 and identification of metabolites in human plasma during intravenous fusion

A stable isotope dilution assay has been developed for the quantification of prostaglandin D₂4 (PGD₂) in plasma. Samples are analysed by capillary column gas chromatography/ negative ion chemical ionisation mass spectrometry (GC/NICIMS). The methods employs an internal standard of [²H₆]PGD₂, prepared biosynthetically by incubation of rat peritoneal mast cells with deuterated arachidonic acid.No PGD₂ was detected in peripheral venous plasma samples obtained from 10 healthy male volunteers (detection limit = 5 pg ml^?1). Following intravenous infusion of PGD₂ at increasing incremental infusion rates ranging from 16–256 ng kg^?1 min^?1, a dose related increase in the plasma concentration of PGD₂ was observed. Plasma levels at 128 ng kg^?1 min^?1 ranged from 724–1444 pg ml^?1. The major circulating metabolites of PGD₂, during infusion, were identified as 13,14-dihydro-15-oxo-PGF_2α and 15-oxo-PGF_2α.     

Ternary solvent mixtures for improved resolution of hydroxylated metabolites of vitamin D2 and vitamin D3 during high-performance liquid chromatography

This paper reports the development of three new ternary solvent mixtures for the liquid-chromatographic separation of metabolites of vitamin D on microparticulate silica. All solvent systems offer reduced peak tailing and improved resolution of vitamin D compounds, particularly of 24(R),25-(OH)₂D₃, when compared to the commonly used hexane—isopropanol mixture. The new mixtures can be substituted for hexane—isopropanol systems presently used for preparative liquid-chromatographic steps prior to radioimmunoassay or competitive protein-binding assay of 24,25-(OH)₂D and 1,25-(OH)₂D in human plasma. Hexane—isopropanol—methanol (87:10:3) mixtures are recommended where the lipid content of samples is high, whereas hexane—ethanol—chloroform (80:10:10) promises to be a useful mixture for differentiating vitamin D₃ metabolites from their vitamin D₂ analogs. A combination of the two solvent systems permits the separate assay of both 24(R),25-(OH)₂D₃ and 24(R),25-(OH)₂D₂ as well as 1,25-(OH)₂D₃ and 1,25-(OH)₂D₂.     

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.     

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.     

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.     

Direct modulation of 25-hydroxyvitamin D3 hydroxylation in kidney tubules by 1,25-dihydroxyvitamin D3

Kidney tubules obtained from chicks fed a high-calcium low-phosphorus diet retained 25-hydroxyvitamin D₃-1-hydroxylase activity after a 10 h incubation in serum-free minimum essential medium. Inclusion of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) in the medium prompted a suppression of 25-hydroxyvitamin D₃-1-hydroxylase and the induction of 25-hydroxyvitamin D₃-24-hydroxylase activities. The enzyme switch-over response could be prompted by 1.6 × 10^?7 M 1,25-dihydroxyvitamin D₃ and occurred within 6 h following treatment. Medium calcium appeared to augment the metabolite's switch-over action.