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D L Venton

Publications and source records attributed to D L Venton.

At least 37 records · Page 2Linked to original sources
PubMed1987

Preparation and biological evaluation of a potential photoaffinity label for the prostaglandin H2/thromboxane A2 receptor.

Two aromatic azides (24 and 26) were prepared as potential photoaffinity probes for the PGH2/TXA2 receptor. The compounds are based on the well-characterized PGH2/TXA2 receptor antagonist 13-azaprostanoic acid, with the terminus of its lower side chain replaced with phenoxy (24) or benzyl (26) azide functionality. The two compounds were shown to irreversibly inhibit platelet function after photolysis and resuspension. However, of the two aromatic azides, only the benzyl derivative 26 appeared to be selective for the prostaglandin pathway. The latter compound was also prepared as the aromatic 125I (29) derivative, which may ultimately prove useful as a labeled probe for the identification and isolation of the putative TXA2/PGH2 receptor.

Adenosine Diphosphate↗
PubMed1986

Characterization of U46619 binding in unactivated, intact human platelets and determination of binding site affinities of four TXA2/PGH2 receptor antagonists (13-APA, BM 13.177, ONO 3708 and SQ 29,548).

The binding of U46619 and the inhibition of this binding by four TXA2/PGH2 receptor antagonists (13-APA, BM 13.177, ONO 3708 and SQ 29,548) were studied in unactivated, intact human platelets. Washed platelets were equilibrated with [3H]-U46619 (5 nM) and the time course of binding determined. The receptor-specific binding reached equilibrium within 2-4 minutes, and could be displaced by addition of excess unlabelled ligand. Saturation of this binding was achieved at 750 nM. Scatchard transformation of the saturation binding curve yielded a single class of binding site with a Kd of 108 nM and Bmax of 360 fmole/10(8) platelets. When [3H]-U46619 (4 nM) was incubated with platelets in the presence of increasing concentrations of the antagonists, binding of U46619 was inhibited in a dose dependent manner. The potency series for inhibition of U46619 binding was: SQ 29,548 (IC50 = 7.9 nM) greater than ONO 3708 (IC50 = 38 nM) greater than BM 13.177 (IC50 = 0.91 microM) greater than 13-APA (IC50 = 6.2 microM). These findings are consistent with the notion that these compounds all act as competitive antagonists at the level of the platelet TXA2/PGH2 receptor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1986

Production of platelet thromboxane A2 inactivates purified human platelet thromboxane synthase.

Human platelet thromboxane synthase was partially purified by DEAE-cellulose, Affi-Gel Blue, and Sephacryl S-300 chromatography to a specific activity of 259 nmol of thromboxane B2/min per mg. Thromboxane synthase retained 75-90% of its enzymic activity when bound to phenyl-Sepharose. The immobilized enzyme was inactivated at pH 3.0 and inhibited by 1-benzylimidazole and U-63,557A. The ability of the enzyme to produce thromboxane A2 from prostaglandin H2 was dramatically reduced by multiple additions of prostaglandin H2. Our data suggest that the production of thromboxane A2 by the enzyme is self-limiting and that the enzyme is inactivated during the reaction.

Blood Platelets↗
PubMed1986

Ammonia (NH3 and N2H3) direct chemical ionization mass spectrometry of underivatized prostaglandin-H2 and other selected stable prostaglandins.

A study of the positive and negative ion, ammonia (NH3 and N2H3) direct chemical ionization mass spectrometry of highly purified prostaglandin endoperoxide (PGH2) is presented. The positive ion spectra were characterized by an intense [M + NH4]+ adduct at m/z 370 and several fragment ions, most notably a [M + NH4-H2O]+ ion at m/z 352 and an ion at m/z 298, assigned as the [M + NH4-72]+ ion of 12-hydroxy-5,8,10-heptadecatrienoic acid formed form PGH2 in the spectrometer. The negative ion spectra of PGH2 were characterized by a base peak at m/z 352 [M]- and by an ion at m/z 334 corresponding to the loss of water from the parent ion. A combination of negative ion and deuterated ammonia reagent gas was used in making assignments and in demonstrating that the spectra observed were due to intact PGH2 and its stable PGE2 and PGD2 isomers formed in the spectrometer. In addition, use of the latter reagent gas was shown to clearly distinguish between several arachidonic acid metabolites, differing in their number of exchangeable protons. Furthermore, preliminary results with several stable prostaglandins indicate that the spectra are sensitive to different functional groups that are present. Consequently, it would appear that negative and positive ion, NH3 (N2H3) direct chemical ionization mass spectrometry would be useful in the analysis of labile arachidonic acid metabolites, without the need for prior derivatization.

Ammonia↗
PubMed1986

A fast, nondestructive purification scheme for prostaglandin H2 using a nonaqueous, bonded-phase high-performance liquid chromatography system.

Arachidonic acid metabolism produces several biologically important compounds including the leukotrienes and prostaglandins. Prostaglandin H2 (PGH2) is the first metabolite in the arachidonic acid cascade leading to all other prostaglandins. Pivotal to our understanding of PGH2's biology is the ability to separate it in pure form from the numerous other arachidonic acid metabolites produced in a biological milieu. The extensive literature on PGH2 biology and metabolism has relied almost exclusively on the traditional method of separation using gravity flow silicic acid columns. In our hands, such PGH2 preparations were found to contain varying amounts of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), PGE2, PGF2 alpha and other minor impurities as determined by further chromatographic and mass spectral analyses. Analytical separation of PGH2 and other arachidonic acid metabolites has been accomplished using reversed-phase HPLC. However, the labile nature of this molecule in aqueous systems makes such techniques unacceptable for preparative isolation of high purity PGH2 and has necessitated the development of a totally nonaqueous separation. To this end, we attempted several stationary phases and found that the cyano-bonded phase showed the best selectivity for resolving PGH2 from its major contaminants. Separations were performed on self-packed columns using a hexane-isopropanol gradient. Peaks were detected both by liquid scintillation counting and uv spectrophotometry (214 nm). Structure assignments were made by chromatographic comparison with authentic standards (PGF2 alpha, PGE2), biological activity (PGH2--platelet aggregation), and by ammonia direct chemical ionization mass spectrometry (HHT, hydroxy-5,8,10,14-eicosatetraenoic acid, PGH2, PGE2, PGF2 alpha). The latter technique, which by its very nature volatilizes all organic material in the sample, was particularly useful in determining not only that the PGH2 preparations were free from the aforementioned side products, but that they were also free from lipid, protein, and other potential residues frequently found in biological preparations.

Animals↗
PubMed1985

Separation of nucleosides and nucleotides by reversed-phase high-performance liquid chromatography with volatile buffers allowing sample recovery.

Reversed-phase high-performance liquid chromatography using a C18 column with volatile buffers as the eluant was applied to the separation of a number of nucleosides and nucleotides. Groups of seven nucleosides and five nucleoside monophosphates were separated isocratically employing 0.1 M trimethylammonium acetate and 2% acetonitrile at pH 7.0. Groups of seven nucleoside diphosphates and seven nucleoside triphosphates were separated with 0.1 M triethylammonium bicarbonate and 2% acetonitrile titrated to a pH of 7.1 with acetic acid. The techniques described give resolution and separations comparable to nonvolatile buffers. Moreover, the eluant trimethylammonium acetate or triethylammonium bicarbonate buffer can easily be removed in vacuo from the column effluent, making the technique useful for preparative separations of these compounds. The observed elution pattern of nucleoside phosphates suggests that "paired-ion" chromatography is involved in the separation.

Buffers↗
PubMed1985

Thromboxane A2/prostaglandin H2 mobilizes calcium in human blood platelets.

The present study investigated the mechanism by which thromboxane A2/prostaglandin H2 (TXA2/PGH2) stimulates platelet activation. Previous studies in isolated platelet vesicles have suggested that TXA2/PGH2 functions to release calcium from intraplatelet stores. On this basis, we investigated whether TXA2/PGH2 causes mobilization of calcium in intact platelets. Calcium redistribution was measured using the fluorescent probe, chlortetracycline (CTC), and a photon-counting microspectrofluorometer. Human platelet-rich plasma was incubated with CTC (50 microM) for 40 min at 25 degrees C. Shape change was induced with arachidonic acid (AA, 100 microM) or ADP (0.75-1.0 microM). It was found that AA addition resulted in a significant release of intraplatelet calcium. This release was blocked by inhibition of the cyclooxygenase with indomethacin (20 microM) or the specific TXA2/PGH2 antagonist, 13-azaprostanoic acid (13-APA, 100 microM). On the other hand, neither indomethacin nor 13-APA had any effect on calcium release stimulated by ADP. However, prostacyclin (13 nM) inhibited both AA- and ADP-induced calcium release. These findings provide evidence that cyclooxygenase products of AA, i.e., TXA2 and/or PGH2 directly caused the mobilization of intraplatelet calcium. Furthermore, this calcium mobilization appears to be mediated through a specific TXA2/PGH2 receptor interaction.

Adenosine Diphosphate↗
PubMed1985

Reversal of thromboxane A2/prostaglandin H2 and ADP-induced calcium release in intact platelets.

We previously demonstrated that thromboxane A2 and/or prostaglandin H2 (TXA2/PGH2), ADP, and A23187 cause calcium mobilization in intact human platelets. Other studies have also shown that platelet shape change and aggregation induced by a variety of platelet agonists can be reversed by specific antagonists. In the present study, we used the fluorescent calcium probe chlortetracycline to evaluate whether the reversal of platelet activation involves a resequestration of intraplatelet calcium. It was found that the TXA2/PGH2 receptor antagonist 13-azaprostanoic acid (13-APA) reversed calcium mobilization and shape change induced by AA but not that induced by ADP. A similar specificity of action was observed using the specific ADP receptor antagonist, ATP, in that ATP only reversed ADP-induced calcium release and shape change. In contrast, prostacyclin reversed both AA and ADP-induced calcium redistribution and shape change. In the latter experiments, a net calcium sequestration was actually observed on prostacyclin addition. These findings indicate that the resequestration of released calcium leads to platelet deactivation. Furthermore, there appear to be at least two mechanisms by which a reduction in cytosolic calcium can be produced: specific interruption of the agonist-receptor interaction, for example, 13-APA antagonism of TXA2/PGH2; and stimulation of platelet adenosine 3',5'-cyclic monophosphate production by prostacyclin and consequent calcium sequestration.

Adenosine Diphosphate↗
PubMed1984

Entrapment of a highly specific antiprogesterone antiserum using polysiloxane copolymers.

Antiprogesterone antiserum was entrapped within a polysiloxane copolymer prepared from a 3:1 mixture of tetraethoxysilane and 3-aminopropyltriethoxysilane monomers. 400 microliters of this monomer mixture entrapped 70 mg of the 140 mg of immunoglobulins which were added, and the protein could not be washed from the highly stable copolymer which formed. Approximately half of the entrapped antiprogesterone antiserum was found to retain progesterone binding capacity with an apparent Ka equal to that of free antiserum in solution and was insensitive to effects of pH between 3 and 7. These preliminary observations and the unique chemistry of polysiloxane polymer formation suggest that such polymers may be useful in the entrapment of proteins for a variety of applications.

Animals↗
PubMed1984

2-(6-carboxyhexyl)cyclopentanone hexylhydrazone: a potent inhibitor of the blood platelet cyclo-oxygenase.

Previous studies have demonstrated that 13-azaprostanoic acid (13-APA) is a potent and specific antagonist of thromboxane A2/prostaglandin H2 (TXA2/PGH2) at the platelet receptor level. In the present study we evaluated the effects of a new azaprostanoid , 2-(6- carboxyhexyl ) cyclopentanone hexylhydrazone (CPH), on human platelet function. This hydrazone was found to completely inhibit arachidonic acid (AA)-induced platelet aggregation at 1 microM CPH. On the other hand, CPH was not an effective inhibitor of PGH2-induced aggregation. Furthermore, 100 microM CPH was completely ineffective in blocking platelet aggregation stimulated by adenosine diphosphate (ADP) or the stable prostaglandin endoperoxide analog U46619 (which presumably acts at the TXA2/PGH2 receptor). Measurement of platelet thromboxane B2 (TXB2) production demonstrated tha the primary site-of-action of CPH is at the cyclo-oxygenase level. Thus, CP inhibited TXB2 formation from AA in a dose-dependent manner (0.1 microM-100 microM CPH)2. In contrast, CPH blocked TXB2 production from PGH2 only at the highest CPH concentration tested, i.e., 100 microM. These results indicate that relative to 13-APA, addition of a second nitrogen at C14 and a double bond between the 12- and 13- positions results in a loss of receptor activity but produces a high affinity for the platelet cyclo-oxygenase.

Arachidonic Acid↗
PubMed1984

Positive-negative, chemical-ionization, direct exposure mass spectrometry of underivatized prostaglandins.

Nine underivatized prostaglandins were examined using direct exposure, ammonia, chemical-ionization, pulsed positive-negative ion mass spectrometry. The positive ion spectra were characterized by (M+18)+ ion adducts. The negative ion spectra were characterized by ions which depended upon the functionality present in the cyclopentane ring system (acetal for TXB2). The E and D series prostaglandins gave (M-18)- as the major negative ion, while the F series and TXB2 were characterized by negative ions corresponding to (M-1)-, and PGA2 by the parent (M)- ion. Prostaglandin 6-keto-PGF1 alpha was anomalous in this respect showing apparent dehydration, interpreted as an overall (M-18+1)+ and (M-18-1)- in the positive and negative ion spectra, respectively. All major ion types were shown to give essentially a linear response with respect to concentration in the 10-1000 ng range. Although these initial studies were conducted under ideal conditions, it would appear that direct chemical ionization techniques show promise for providing direct structural information on prostaglandins without the need for prior chemical derivatization.

6-Ketoprostaglandin F1 alpha↗
PubMed1984

Synthesis of [17,18-3H] trans-13-azaprostanoic acid. A labeled probe for the PGH2/TXA2 receptor.

Because of its highly unstable nature, TXA2, produced by platelet metabolism of arachidonic acid, does not lend itself to use as a receptor probe for its own receptor. As such, the stable TXA2/PGH2 antagonist, trans-13-azaprostanoic acid (trans-13-APA, 12b), was prepared as the [17, 18 3H] derivative [( 3H] trans-13-APA, 12c) to study this receptor and to better evaluate the mechanism of action of these azaprostanoids. Tritiated trans-13-APA, 12c, was prepared in nearly theoretical specific activity (57 Ci/mmole) from (17Z)-trans-13-azaprost-17-enoic acid (11b) by catalytic tritiation. The unsaturated 11b was prepared by condensation of cis-7-amino-3-heptene (8) with 2-(6-carboxyhexyl) cyclopentanone (9), NaBH4 reduction, chromatography, and hydrolysis of the trans isomer so isolated. The olefins 11a and b were also of biochemical interest because of the unsaturation in the lower side chain. The presence of similar unsaturation in PGH3(4) and TXA3 (3) renders these prostaglandins inactive as proaggregatory agents. Evaluation of the antiaggregatory activity of 11a and b indicated it to be about the same potency in inhibiting human platelet aggregation as the parent cis and trans-13-APAs, suggesting that introduction of a double bond at the 17 position in platelet prostaglandin antagonists is unlikely to result in enhanced antiplatelet activity.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1984

Arachidonic acid-induced platelet aggregation is mediated by a thromboxane A2/prostaglandin H2 receptor interaction.

The mechanism by which the active metabolites of arachidonic acid (AA), i.e., thromboxane A2 and/or prostaglandin H2 (TXA2/PGH2) induce platelet aggregation is not understood. Several reports have suggested that AA-stimulated aggregation is mediated by secreted ADP, whereas other studies have proposed that this response is ADP-independent. In the present report, we used the specific TXA2/PGH2 receptor antagonist, 13-azaprostanoic acid (13-APA), and the ADP antagonist, ATP, to examine the contribution of TXA2/PGH2 or secreted ADP to aggregation. We found that 13-APA, but not ATP, deaggregates platelets stimulated by AA or U46619 (a TXA2/PGH2 mimetic). In contrast, ADP-induced aggregation was reversed in response to ATP but not to 13-APA. These results suggest that TXA2/PGH2-stimulated aggregation is mediated through TXA2/PGH2 receptor occupation. Furthermore, secreted ADP does not appear to be required for maintenance of the AA-aggregation response.

Adenosine Diphosphate↗
PubMed1984

Antagonism of thromboxane A2/prostaglandin H2 by 13-azaprostanoic acid prevents platelet deposition to the de-endothelialized rabbit aorta in vivo.

The present study evaluated the direct involvement of thromboxane A2/prostaglandin H2 (TXA2/PGH2) in the process of thrombus formation at a site of vascular damage. De-endothelialization of the rabbit aorta was performed by a balloon catheter technique. Platelet deposition to the injured vessel was measured using 111Indium-labeled autologous platelets. Studies using the radiolabeled TXA2/PGH2 antagonist, 13-azaprostanoic acid (13-APA), indicated that 13-APA has an in vivo half-life of approximately 35 min and is excreted by the kidney in the metabolized form. Addition of 13-APA to rabbit plasma samples in vitro produced a dose-dependent inhibition of arachidonic acid-induced platelet aggregation. When comparable plasma levels of 13-APA were achieved by infusion of 13-APA (300 micrograms/kg/min for 90 min), a similar dose-dependency of inhibition of ex vivo aggregation was observed. Furthermore, at a plasma concentration of 40 microM, 13-APA was found to inhibit platelet deposition to the de-endothelialized rabbit aorta by 45%. Because 13-APA does not interfere with arachidonic acid metabolism, the ability of 13-APA to suppress thrombus formation is presumably due to direct antagonism of TXA2/PGH2 at the platelet receptor level. These findings, therefore, provide evidence that TXA2 and/or PGH2 have a major role in platelet deposition at a site of vascular damage.

Animals↗
PubMed1983

The thromboxane antagonist, 13-azaprostanoic acid, inhibits arachidonic acid-induced Ca2+ release from isolated platelet membrane vesicles.

In the present study we investigated the ability of the arachidonic acid metabolites, prostaglandin H2 and thromboxane A2, to release Ca2+ from isolated platelet vesicles. The vesicles were prepared through modification of previously described procedures. 45Ca uptake and release were determined by Millipore filtration and isotope counting of the filter paper. Incubation of the vesicles (25 degrees C) with 50 microM CaCl2 (plus 45Ca) resulted in the accumulation of 13 nmol Ca2+ per mg of protein under steady-state conditions. Addition of arachidonic acid (25 microM) resulted in a 42% release of the accumulated Ca2+ and the production of 150 ng thromboxane B2/mg protein. Pretreatment of the vesicles with indomethacin (4 microM) completely inhibited arachidonic acid-induced Ca2+ release and reduced thromboxane B2 synthesis by 82%. Pretreatment of the vesicles with the specific thromboxane A2/prostaglandin H2 antagonist, 13-azaprostanoic acid (20 microM), also resulted in complete inhibition of Ca2+ release but no inhibition of thromboxane B2 production. Addition of prostaglandin H2 (0.3 microM) to the platelet vesicles produced a significant release of Ca2+ only in the presence of the adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (100 microM). This Ca2+ release was totally blocked by 13-azaprostanoic acid (20 microM). The thromboxane synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I, 3.6 microM), in the presence of 2',5'-dideoxyadenosine, only slightly inhibited Ca2+ release in response to added prostaglandin H2, even though thromboxane B2 production was blocked by 95%.

Adenosine Triphosphate↗
PubMed1983

Specific binding of the thromboxane A2 antagonist 13-azaprostanoic acid to human platelet membranes.

In the present study we characterized the interaction between the thromboxane A2/prostaglandin H2 antagonist, trans-13-azaprostanoic acid (13-APA), and isolated human platelet membranes. In these studies, we developed a binding assay using trans [3H] 13-APA as the ligand. It was found that trans [3H] 13-APA specific binding was rapid, reversible, saturable and temperature dependent. Scatchard analysis of the binding data yielded a curvilinear plot which indicated the existence of two classes of binding sites: a high-affinity binding site with an estimated dissociation constant (Kd) of 100 nM; and a low-affinity binding site with an estimated Kd of 3.5 microM. At saturation, approximately 1 pmol/mg protein of [3H] 13-APA was bound to the high affinity site. In order to further characterize the nature of the [3H] 13-APA binding site, we evaluated competitive binding by cis 13-APA, cis 15-APA, prostaglandin F2 alpha, U46619, 6-ketoprostaglandin F1 alpha and thromboxane B2. It was found that the [3H] 13-APA binding site was stereospecific and structurally specific. Thus, the cis isomer of 13-APA exhibited substantially reduced affinity for binding. Furthermore, the prostaglandin derivatives, thromboxane B2 and 6-ketoprostaglandin F1 alpha, which do not possess biological activity, also did not compete for [3H] 13-APA binding. On the other hand, U46619 which acts as a thromboxane A2/prostaglandin H2 mimetic, and prostaglandin F2 alpha which acts as a thromboxane A2/prostaglandin H2 antagonist, both effectively competed for [3H] 13-APA binding. These findings indicate that trans 13-APA binds to a specific site on the platelet membrane which presumably represents the thromboxane A2/prostaglandin H2 receptor.

Binding Sites↗
PubMed1983

In vivo effects of human dialyzable leukocyte lysate. III. Modulation of the spleen cell proliferative response to antigen by components of leukocyte dialysates and an initial characterization of an ampliative nucleoside.

The augmentative effects of isolated components of human dialyzable leukocyte lysates upon the proliferative response to antigen were investigated. Sequential Sephadex G-25 and Bio-Gel P-4 chromatography separated five distinct fractions which, 24 hr after injection into Keyhole limpet hemacyanin (KLH)-sensitive mice, either augmented or suppressed the in vitro spleen cell proliferative response to KLH. An amplifier molecule was isolated from one of the augmentative fractions by high-pressure, reverse-phase liquid chromatography. Preliminary structural analysis of the amplifier component indicated a nucleoside structure, similar to--but possibly distinct from--thymidine.

Animals↗