Olga V. Miller

Modulation of human platelet adenylate cyclase by prostacyclin (PGX)

Prostacyclin (PGX) (57)-9-deoxy-6,9alpha-epoxy-delta5-PGF1alpha has been found to be a potent 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.

Inhibition of PGE1-stimulated cAMP accumulation in human platelets by thromboxane A2

The prostaglandin endoperoxide PGH2, HHT, HETE, thromboxane A2, and thromboxane B2, which are all products of arachidonic acid metabolism of human platelets, were tested for their ability to modulate platelet cyclic nucleotide levels. None of the compounds tested altered the basal level of cAMP or cGMP, and only PGH2 and thromboxane A2 inhibited PGE1-stimulated cAMP accumulation. Thromboxane A2 was found to be a more potent inhibitor of PGE1-stimulated cAMP accumulation and inducer of platelet aggregation than PGH2.

Prostaglandins H1 and H2. Convenient biochemical synthesis and isolation. Further biological and spectroscopic characterization

An easy biochemical preparation of the prostaglandin endoperoxides, PGH1 and PGH2, is described. Both of the endoperoxides are potent contractors of isolated gerbil colon smooth muscle. Contracture with PGH2 is about equal to that caused by the standard, PGE1, while contracture with PGH1 is about half of that caused by PGE1. PGH1 was found to inhibit platelet aggregation induced by PGH2 and is about 1/10 as potent a stimulator of cAMP accumulation as is PGE1. The mass spectra of the methyl esters of both PGH1 and PGH2 were obtained, as were the infrared spectra of the two compounds. The nuclear magnetic resonance spectrum of PGH2 is characterized by signals at 4.58 delta and 4.47 delta for the C-9 and C-11 protons, respectively.

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

Abstract Rat adipocyte plasma membranes sacs have been shown to be a sensitive and specific system for studying prostaglandin binding. The binding of prostaglandin E 1 and prostaglandin A 1 increases linearly with increasing protein concentration, and is a temperature-sensitive process. Prostaglandin E 1 binding is not ion dependent, but is enhanced by GTP. Prostaglandin A 1 binding is stimulated by ions, but is not affected by GTP. Discrete binding sites for prostaglandin E 1 and A 1 were found. Scatchard plot analysis showed that the binding of both prostaglandins was biphasic, indicating two types of binding sites. Prostaglandin E 1 had association constants of 4.9 · 10 9 1/mole and 4 · 10 8 1/mole, while the prostaglandin A 1 association constants and binding capacities varied according to the ionic composition of the buffer. In Tris-HCl buffer, the prostaglandin A 1 association constants were 8.3 · 10 8 1/mole and 5.7 · 10 7 1/mole, while in the Krebs—Ringer Tris buffer, the results were 1.2 · 10 9 1/mole and 8.6 · 10 6 1/mole. Some cross-reactivity between prostaglandin E 1 and A 1 was found for their respective binding sites. Using Scatchard plot analysis, it was found that a 10-fold excess of prostaglandin E 1 inhibited prostaglandin A 1 binding by 1–20% depending upon the concentration of prostaglandin A 1 used. Prostaglandin E 1 competes primarily for the A prostaglandin high-affinity binding site. Similar Scatchard analysis using a 20-fold excess of prostaglandin A 1 inhibited prostaglandin E 1 binding by 10–40%. Prostaglandin A 1 was found to compete primarily for the E prostaglandin low-affinity receptor. All of the bound [ 3 H]prostaglandin E 1 , but only 64% of the bound [ 3 H]-prostaglandin A 1 can be recovered unmetabolized from the fat cell membrane. There is no non-specific binding of prostaglandin E 1 , but 10–15% of prostaglandin A 1 binding to adipocyte membranes is non-specific. Using a parallel line assay to measure relative affinities for the E binding site, prostaglandin E 1 > prostaglandin A 2 > prostaglandin F 2α . Prostaglandin E 2 and 16,16-dimethyl prostaglandin E 2 were equipotent with prostaglandin E 1 , 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.

6-Keto-prostaglandin E1 is not equipotent to prostacyclin (PGI2) as an antiaggregatory agent

A direct comparison of the relative potencies of the two antiaggregatory prostaglandins PGI2 and 6-keto-PGE1 showed PGI2 was at least 20 times more potent than 6-keto-PGE1 when tested against ADP-induced human platelet aggregation. This marked difference in potency was even more evident when the ability of PGI2 and 6-keto-PGI2 to stimulate platelet cyclic AMP levels was determined. When cyclic AMP levels were measured direct comparisons were difficult because the respective dose response curves were not parallel, but 10 ng of PGI2 was equivalent to 300 ng of 6-keto-PGE1. PGI2 was also more potent (10-20 times) than 6-keto-PGE1 as a disaggregatory agent, and the disaggregatory activity of both prostaglandins was enhanced by the phosphodiesterase inhibitor 1-methyl-3-isobutylmethylxanthine. PGI2 was also more active than 6-keto-PGE1 as an inhibitor of thrombus formation in dog coronary arteries in vivo. In vivo, 6-keto-PGE1 was at least 10 times less potent thatn PGI2, the exact difference could not be determined because 6-keto-PGE1 caused significant falls in blood pressure before anti-platelet activity could be detected. PGI2 is an intrinsically more potent anti-aggregatory molecule than 6-keto-PGE1, but these data do not rule out the possibility that some of the activities attributed to PGI2 could be the result of the conversin of PGI2 and/or 6-keto-PGF1 alpha to 6-keto-PGE1.

Immunological evidence for the appearance of a new DNA-polymerase in cells infected with vaccinia virus☆

Abstract Rabbits were immunized with a partially purified DNA-polymerase from control HeLa cells. γ-Globulin was isolated from the antisera and shown to contain antibodies which inhibited the enzymatic activity. The anti-DNA-polymerase γ-globulin did not inactivate the new DNA-polymerase made in response to vaccinia virus infection. All the enzyme increase after infection was due to the new enzyme which could be detected in particulate fractions, or in solubilized form after high speed centrifugation or sucrose density gradient centrifugation. It was concluded that the DNA-polymerase induced by vaccinia virus infection is immunologically distinct from the normal host enzyme.

Acetyl glycerylphosphorylcholine inhibition of prostaglandin I2-stimulated adenosine 3',5'-cyclic monophosphate levels in human platelets: Evidence for thromboxane A2 dependence

Previous studies with AGEPC (1-O-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) stress the independence of the proaggregatory activity of AGEPC from the platelet cyclooxygenase. However, our dose response analyses in human platelet-rich plasma show distinct primary and secondary waves of aggregation in response to AGEPC. Second wave aggregation is inhibited completely by either 10 micro M indomethacin, a cyclooxygenase inhibitor, or 5.6 micro M 9,11-azoprosta-5,13-dienoic acid, a thromboxane A2 synthetase inhibitor. Simultaneous addition of AGEPC and prostaglandin I2 to platelet-rich plasma results in a marked increase in platelet cyclic AMP, which is not different from the prostaglandin I2 response alone. However, if prostaglandin I2 is added to AGEPC-stimulated platelets at a point where secondary aggregation is just beginning, AGEPC can attenuate prostaglandin I2-stimulated cyclic AMP accumulation. The inhibition by AGEPC is blocked by either cyclooxygenase or thromboxane A2 synthetase inhibitors, and radioimmunoassay of thromboxane B2 confirmed that the inhibition of prostaglandin I2-stimulated cyclic AMP accumulation is due to thromboxane A2 synthesis, and that AGEPC-stimulated secondary aggregation does not start until thromboxane A2 is synthesized. These data suggest that much of the bioactivity of AGEPC is attributable to thromboxane A2.

Absorption of prostaglandins by the intestine and vagina of the rat

Abstract An in situ method was used to determine the rate of absorption of prostaglandins★★ E2, F2α, 15-methyl F2α and 15-methyl F2α methyl ester from the small intestine of the rat. Prostaglandin E2 disappeared from the lumen with a t 1 2 of 37 min. From 2–3 % of the radioactivity of the dose was recovered in the blood at 30–60 min, but the compound was extensively metabolized, and intact prostaglandin E2 did not exceed 0.04–0.08% of the dose. Absorption of the more polar prostaglandin F2α and 15-methylprostaglandin F2α proceeded at a slower rate with a t 1 2 of 60–70 min. Radioactivity in the serum at 30–60 min did not exceed 2 % of the dose for those animals given prostaglandin F2α or 0.8% of the dose for those animals given 15-methylprostaglandin F2α. A rapid, initial disappearance of 15-methylprostaglandin F2α methyl ester from the small intestine was observed, followed by a second, much slower rate of absorption. The rapid phase of the absorption was extended at high dosages and resulted in blood levels of radioactivity up to 7 % of the dose when 40 mg/kg were given. These results correlated with the rate of hydrolysis of the methyl ester in the lumen of the intestine which was slower at higher doses. Little or no methyl ester was detected in serum or intestinal tissue, but up to 1 .8 % of the dose of intact 15-methylprostaglandin F2α was estimated to be in the serum at 30 min. Additional experiments showed that 15-methylprostaglandin F2α methyl ester also was absorbed through the large intestine and the vagina. In each case, 15-methylprostaglandin F2α was found in the blood. Metabolites consisted of more polar acids and a component which migrated close to 15-methylprostaglandin F2α on thinlyer chromatograms. The extent of metabolism of 15-methylprostaglandin F2α methyl ester by the liver was determined in a perfusion experiment. The methyl ester was hydrolyzed within 1 min, and the free acid was taken up almost quantitatively by the circulating prostaglandins E and F occurs via 15-dehydrogenation, our results indicate that the liver is capable of extensively degrading the compounds to more polar meta-boites. Further evidence on this point comes from a liver perfusion experiment carried out as described above but with [5,6-3H2]prostaglandin E1. By 60 min, over 50% of the radioactivity in plasma was recovered in volatile form 3H2O and 17% remained in the aqueous phase, indicating extensive carboxyl sidechain cleavage. (An additional 10.3 % chromatographed as prostaglandin E1). Dawson et al.29 have reached similar conclusions. These observations may explain the lack of any prolongation in cardiovascular responses in vivo by the 15-methyl analogs over that observed with the parent compounds17 but does not offer an explanation for the enhanced antifertility activity of the analogs in hamsters, monkeys and humans17,30–32. It would, of course, be possible to speculate that cardiovascular activity relies on continuing levels of compound in the circulation while duration of antifertility activity is controlled by the local action of 15-hydroxyprostaglandin dehydrogenase on prostaglandins at target tissues17. Uterine tissue was found to contain relatively little dehydrogenase33, but high levels have been reported in human placenta34. A number of orally active drugs have been found to have t 1 2 values between 8 and 50 min using the present technique18. Therefore, barriers to successful oral dosage forms for the prostaglandins do not lie in their rate of uptake, but rather in the extensive metabolism occurring during absorption. The extremely low amounts of intact prostaglandin E2 that survived intestinal transport, and the detection of a metabolite in the lumen of the intestine as well as in serum with the correct RF for the 15-keto compound suggest that the 15-dehydrogenation reaction poses the major metabolic block to successful intestinal absorption. In addition, dehydrogenase activity was readily detected in intestinal washes (unpublished observations). It is not possible to completely evaluate the relative role of the various degradative pathways in the intestine versus those in liver or lung during absorption on the basis of the present experiments. However, it is apparent that appreciable blood levels of intact drug could be obtained during intestinal absorption by employing an analog in which only the action of the dehydrogenase was blocked.

A selective thromboxane synthetase inhibitor blocks the cAMP lowering activity of PGH2

Abstract The thromboxane synthetase inhibitor, 9,11-azoprosta-5,13-dienoic acid, blocks both platelet aggregation and the cyclic AMP lowering activity of the prostaglandin endoperoxide PGH 2 . These data indicate PGH 2 must be converted into thromboxane A 2 in order to lower cAMP or induce platelet aggregation.

Inhibition by interferon of the uncoating of vaccinia virus

Abstract The effect of interferon was determined on those steps in vaccinia virus infection that precede release of viral DNA into the cytoplasm. These include loss of the outer protein coat of the particle which exposes the “core” and release of the DNA from the core (uncoating). Primary cultures of chicken embryo fibroblasts were treated with interferon and infected with radioactive vaccinia virus prepared with thymidine- 3 H in the DNA. At various times after infection the cells were ruptured, and the amounts of virus, cores, and viral DNA were determined after separation of these components by sucrose density centrifugation. Uncoating also was measured by DNase sensitivity of the viral DNA. Interferon did not alter either the rate of disappearance of virus particles or the rate of formation of cores. However, uncoating was inhibited strongly so that very little viral DNA was liberated and cores tended to accumulate. The response of uncoating to increasing concentrations of interferon was similar to that determined previously for the synthesis of viral DNA-polymerase and viral DNA. These observations suggest that uncoating is a viral function. Not enough cores accumulated in the interferon-treated cells to account for all the virus that disappeared. Experiments with heat-inactivated virus, and with normal virus in the presence of cycloheximide, showed that chicken embryo fibroblasts can digest both virus and cores to acid-soluble materials without accumulating acid-insoluble intermediates. The inhibition of uncoating obtained with cycloheximide closely resembled that seen with interferon.