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D E Mais

Publications and source records attributed to D E Mais.

89 records · Page 5Linked to original sources
PubMed1986

Photoaffinity labelling of a thromboxane A2/prostaglandin H2 antagonist binding site in human platelets.

The diazonium salt of 9,11-dimethylmethano-11,12-methano-16-(4-aminophenoxy)13,14- dihydro-13-aza-15 alpha beta-omega-tetranor TXA2 (PTA-POA) was synthesized and used as a photoaffinity ligand for the putative human platelet TXA2/PGH2 receptor. Incubation of human platelet membranes with the diazonium salt of PTA-POA followed by photolysis at 290 nm(hv) resulted in a 40% decrease in the specific binding of [125I]PTA-OH as measured in the radioligand binding assay. Co-incubation with a TXA2/PGH2 agonist followed by photolysis resulted in no decrease in specific binding. Incubation of the diazonium salt of PTA-POA with solubilized platelet membranes without photolysis followed by Scatchard analysis resulted in no change in the Kd for [125I]PTA-OH (38 nM) and the preparation which was incubated with the diazonium salt (42 nM). However, the Bmax for [125I]PTA-OH binding was reduced from 2.4 pmole/mg protein for control to 1.4 pmole/mg protein. These studies show that the diazonium salt of PTA-POA may be a useful photoaffinity ligand for human platelet TXA2/PGH2 receptors.

Affinity Labels↗
PubMed1986

Alpha 1-adrenergic stimulation of arachidonic acid release and metabolism in a rat thyroid cell line. Mediation of cell replication by prostaglandin E2.

The rat thyroid cell line, FRTL-5, expresses an alpha 1-adrenergic receptor when exposed to thyrotropin. We have found that occupation of this alpha 1-adrenergic receptor by norepinephrine stimulated the release of [3H]arachidonic acid from prelabeled cells. Arachidonic acid was metabolized primarily to prostaglandin E2 and to much smaller amounts of 11-hydroxy-5,8,11,13-eicosatetraenoic acid, 15-hydroxy-5,8,11,13-eicosatetraenoic acid, prostaglandin D2, and thromboxane B2. Synthesis of all these metabolites was inhibited by the cyclooxygenase inhibitor indomethacin. When FRTL-5 cells were starved of thyrotropin for 24 h, norepinephrine nearly doubled [3H]thymidine uptake into DNA. Cyclooxygenase inhibitors inhibited norepinephrine-stimulated thymidine uptake by 60-70%. Of several arachidonic acid metabolites tested, none was able to stimulate thymidine uptake directly in the presence of indomethacin. Prostaglandin E2, however, was able to restore [3H]thymidine uptake when added together with norepinephrine in the presence of indomethacin. Thus, occupation of an alpha 1-adrenergic receptor in a functional rat thyroid cell line leads to arachidonic acid release. Subsequent metabolism of the arachidonic acid by the cyclooxygenase pathway leads to synthesis of prostaglandin E2, which mediates a norepinephrine-stimulated activity related to cell replication.

Adrenergic alpha-Agonists↗
PubMed1986

Identification of a putative thromboxane A2/prostaglandin H2 receptor in human platelet membranes.

The binding of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist (9,11-dimethylmethano-11, 12-methano-16-(3-aza-15 alpha beta-omega-tetranor-TXA2) ([125I]PTA-OH) to membranes prepared from human platelets was characterized. [125I]PTA-OH binding to membranes from human platelets was saturable, displaceable, and dependent on protein concentration. Scatchard analysis of equilibrium binding carried out at 30 degrees C revealed one class of binding sites with a Kd of 30 +/- 4 nM and a Bmax of 1.8 +/- 0.3 pmol/mg of protein (n = 5). Kinetic analysis of the binding of [125I]PTA-OH at 0 degrees C yielded a k1 of 1.35 X 10(6) M-1 min-1 and a k-1 of 0.032 min-1, Kd = k-1/k1 = 24 nM. The potencies of a series of TXA2/PGH2 antagonists as inhibitors of [125I]PTA-OH binding was correlated with their potencies as inhibitors of platelet aggregation induced by the TXA2/PGH2 mimetic, U46619 (1 microM) (r = 0.93, p less than 0.01). A series of TXA2/PGH2 mimetics also displaced [125I]PTA-OH from its binding site, and their potencies as inhibitors of [125I]PTA-OH binding were correlated with their potencies as stimulators of platelet aggregation (r = 0.91, p less than 0.05). The IC50 values for displacement of [125I]PTA-OH by PGF2 alpha, PGD2, and the stable PGI2 analog Iloprost were greater than 25 microM, suggesting that [125I]PTA-OH does not bind to other known platelet prostaglandin receptors. These data are consistent with the notion that this binding site may represent the platelet TXA2/PGH2 receptor.

Binding, Competitive↗
PubMed1985

Hydrodynamic properties of a thromboxane A2/prostaglandin H2 antagonist binding site solubilized from human platelets.

The hydrodynamic properties of a binding site for the thromboxane A2/prostaglandin H2 receptor antagonist 9,11-dimethylmethano-11, 12-methano-16-(3-iodo-4-hydroxyphenyl)-13, 14-dihydro-13-aza-15 alpha beta-omega-tetranor-thromboxane A2 (I-PTA-OH) were determined in solubilized membrane proteins from human platelets using the detergent 3-[(3-cholamidopropyl)-dimethylammonio] 1-propane-sulfonate (CHAPS). Gel filtration revealed a Stokes radius of 5.25 +/- 0.37 nm (n=9). Molecular weight determined by gel filtration assuming a spherical protein was 180,000-220,000 Daltons. Sedimentation through sucrose or glycerol gradients revealed a sedimentation coefficient of 6.3 +/- 0.2 Svedberg units (n=5). The molecular weight calculated using the Stokes radius and sedimentation coefficient was 140,000 Daltons. The frictional ratio f/fo was 1.4, corresponding to an axial ratio of 7:1.

Binding Sites↗
PubMed1985

Further studies on the effects of epimers of thromboxane A2 antagonists on platelets and veins.

The C-15 hydroxy epimers of three TXA2/PGH2 antagonists were resolved by HPLC and their pharmacology evaluated in canine saphenous veins and human platelets. For all three compounds, the epimers were equipotent in their ability to antagonize U46619 (1 microM) induced platelet aggregation. In contrast, one of the epimers for all three of the antagonists was significantly more potent than the other in antagonizing U46619 (10 nM) induced saphenous vein contractions. The data support the notion that the TXA2/PGH2 receptors in veins may be different from those in platelets.

Animals↗
PubMed1985

Solubilization of a thromboxane A2/prostaglandin H2 antagonist binding site from human platelets.

A binding site for 9,11-dimethylmethano-11,12-methano-16-(3-[125I]iodo-4-hydroxyph eny l)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranorthromboxane A2 ([125I]-PTA-OH), a thromboxane A2/prostaglandin H2 antagonist, was solubilized into the 200,000 X g supernatant from human platelet membranes by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Binding to the solubilized site was saturable, displaceable, and reversible. Displaceable binding was not affected by sodium, potassium, or phosphate concentrations up to 50 mM or by magnesium to 5 mM but was increased 14% (P less than 0.05) by 5 mM calcium. A pH optimum for displaceable binding occurred between pH 7.0 and 7.5. Scatchard analysis of [125I]-PTA-OH binding to the solubilized binding site revealed a single class of sites, having a dissociation constant (Kd) of 66 +/- 16 nM (n = 3) and a Bmax of 750 +/- 80 fmol/mg of protein. The Kd for the membranes prior to solubilization was 47 +/- 11 nM (n = 3) and the Bmax was 700 +/- 90 fmol sites per mg of protein. The association rate constant, k1, was 1.57 X 10(7) M-1 X min-1 and the dissociation rate constant, k-1, was 0.61 +/- 0.04 min-1 (n = 4), yielding a Kd (k-1/k1) of 39 nM. Several thromboxane A2/prostaglandin H2 agonists and antagonists displaced bound [125I]-PTA-OH at concentrations similar to those at which they affect platelet aggregation. Collectively, these observations suggest that the solubilized protein is the thromboxane A2/prostaglandin H2 binding site that mediates platelet aggregation.

Blood Platelets↗
PubMed1985

Thromboxane A2 and prostaglandin endoperoxide receptors in platelets and vascular smooth muscle.

9,11-Dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13, 14-dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 (I-PTA-OH), a recently synthesized thromboxane (TX) A2/prostaglandin (PG) H2 receptor antagonist, was shown to be a competitive antagonist of human platelet aggregation induced by the stable endoperoxide analog U46619. This antagonism was due to competitive blockade of the platelet TXA2/PGH2 receptor since I-PTA-OH did not antagonize the first phase of ADP-induced aggregation which is TXA2/PGH2 independent, nor did it inhibit TXA2 synthesis. In addition, analysis of dose-response curves to U46619 (0.1 to 40 microM) in the presence of increasing concentrations of I-PTA-OH (0.5 to 10 microM) showed that I-PTA-OH produced a parallel rightward shift of the dose-response curve. Further analysis of the data in the form of a Schild plot yielded a straight line with a slope (m = 1.03) not significantly different from -1. These results are consistent with the notion that I-PTA-OH acts as a competitive antagonist of the TXA2/PGH2 receptor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1985

Pharmacologic characterization of human and canine thromboxane A2/prostaglandin H2 receptors in platelets and blood vessels: evidence for different receptors.

The present study was undertaken to characterize thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors in platelets and blood vessels. Both human and canine platelet aggregation and saphenous vein contractions were induced by the stable TXA2/PGH2 mimetics (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619) and 9,11-epithio-11,12-methano-TXA2 (ONO-11113). ONO-11113 was a more potent agonist than U46619 in the human saphenous vein but less potent in the platelet. These agonists were equipotent in the canine platelet but ONO-11113 was more potent in the saphenous vein. Platelet aggregation and saphenous vein contraction induced by U46619 were blocked in a dose-dependent manner by the TXA2/PGH2 receptor agonists 9,11-dimethylmethano-11,12-methano-16-phenyl-13,14-dehydro-13-aza- 15 alpha beta-omega-tetranor-TXA2 (ONO-11120), 9,11-dimethylmethano-11,12-methano-16-(4-hydroxyphenyl)-13,14-d ihy dro-13-aza-15 alpha beta-omega-tetranor-TXA2 (PTA-OH), 9,11-dimethylmethano-11,12-methano-16-(4-methoxyphenyl)-13,14-d ihy dro-13-aza-15 alpha beta-omega-tetranor-TXA2 (PTA-OM), 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl-13,1 4-dihydro-13- aza-15 alpha beta-omega-tetranor-TXA2 (I-PTA-OH) and 9,11-dimethylmethano-11,12-methano-15-phenyl-13,14-dihydro-13-aza- 15 alpha beta-omega-pentanor-TXA2 [PTA-(omega-1)].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Binding of an 125I-labeled thromboxane A2/prostaglandin H2 receptor antagonist to washed canine platelets.

A binding site for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 125I-PTA-OH (9,11-dimethylmethano-11,12-methano-16-(4-methoxyphenyl)-13,14-dih ydro-13-aza-1 5 alpha beta-w-tetranor-TXA2) to washed canine platelets is described. 127I-PTA-OH competitively antagonized aggregation induced by the TXA2/PGH2 mimetic U46619. A Schild analysis of the pharmacologic study revealed pA2 of 7.97 and a slope of -0.95. The pA2 value yielded a Kd of 11 nM. Specific binding in Tris-NaCl buffer (pH 7.4) is not affected by extracellular Ca2+ or Mg2+ in concentrations up to 750 microM. The pH optimum for binding resides between 7.0 and 7.4. The association rate constant, k1, was 4.5 X 10(6) M-1 min-1, and the dissociation rate constant, k-1, was 1.45 X 10(-1) min-1, yielding a kinetically determined Kd (k-1/k1) of 32 nM. Scatchard analysis of I-PTA-OH binding to washed canine platelets revealed two classes of binding sites, a high affinity site (Kd = 24 nM, Bmax = 71 fmol/10(7) platelets) (4400 binding sites/platelet) and a low affinity site (Kd = 2.1 microM). Several TXA2/PGH2 receptor antagonists competed with specific 125I-PTA-OH binding, and the rank order of potency for displacing the ligand correlated (r = 0.97) with the rank order of potency for their ability to inhibit U46619-induced aggregation in canine platelet-rich plasma. Prostaglandins F2 alpha and E2 also displaced the ligand, but only at much higher concentrations. Binding of I-PTA-OH or the TXA2/PGH2 mimetic U46619 was unaffected by the aggregating agents epinephrine (10 microM) or ADP (5 microM). The similarity in the Kd values obtained kinetically, by equilibrium binding studies for the high affinity site and by Schild analysis, suggests that this high affinity site mediates TXA2/PGH2 induced platelet aggregation. In addition, the close correlation between the abilities of the antagonists to displace the ligand and to inhibit U46619-induced aggregation suggests that this site may represent a TXA2/PGH2 receptor.

Adenosine Diphosphate↗
PubMed1985

Binding of a thromboxane A2/prostaglandin H2 receptor antagonist to washed human platelets.

A binding site for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist [125I]9,11-dimethylmethano-11, 12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 to washed human platelets was studied. 9,11-Dimethylmethano-11, 12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 competitively antagonized aggregation of washed human platelets induced by the TXA2/PGH2 mimetic U46619. A Schild analysis of the pharmacologic study revealed a pA2 of 8.08 and a slope of -1.12. The pA2 value yielded a Kd of 8 nM. The association rate constant (k1) for [125I]PTA-OH was 6.6 X 10(6)M-1 min-1 and the dissociation rate constant (k-1) was 1.82 X 10(-1), yielding a kinetically determined Kd (k-1/k1) of 27 nM. Scatchard analysis of [125I]PTA-OH binding to washed human platelets revealed one class of binding sites with a Kd of 21 +/- 5 nM and maximum binding of 42 +/- 6.4 fmol/10(7) platelets (N = 5) (2530 +/- 380 binding sites/platelet). Several TXA2/PGH2 receptor agonists and antagonists competed with [125I]PTA-OH for binding. For the four antagonists used in this study, the rank order of potency for displacing the ligand from its binding site correlated (r = 0.93) with the rank order of potency for their ability to inhibit U46619-induced aggregation in human platelet-rich plasma. The antiaggregatory prostaglandins prostaglandin F2 alpha, prostaglandin D2, and Iloprost also displaced the ligand, but only at concentrations considerably higher than that required to produce their pharmacologic effects.(ABSTRACT TRUNCATED AT 250 WORDS)

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

A role for serotonin in alpha-naphthylthiourea-induced pulmonary edema.

alpha-Naphthylthiourea (ANTU) damages the pulmonary capillary endothelium producing a marked pulmonary edema. Since the pulmonary microvasculature regulates the circulating levels of serotonin (5-HT), the role of 5-HT in the pathophysiology of ANTU-induced pulmonary edema was examined. Mice treated with ANTU (10 mg/kg, ip) rapidly developed pulmonary edema which was maximal at 3 hr and was resolved by 12 hr. The lung content of both endogenous 5-HT and a tracer dose of 5-[3H]HT paralleled the time course of the development and resolution of the pulmonary edema. ANTU produced a significant thrombocytopenia (58 to 72%) at all time points, and an elevated platelet content of 5-HT and 5-[3H]HT during the resolution phase (6 to 12 hr). Drugs possessing select effects on 5-HT were shown to alter the edematogenic response to ANTU. Fluoxetine, a selective inhibitor of 5-HT uptake, potentiated the pulmonary edema, while clorgyline, an irreversible inhibitor of type A monoamine oxidase, was without effect. Reserpine which depletes 5-HT stores prevented both thrombocytopenia and pulmonary edema in response to ANTU. Reloading the lung and platelet 5-HT stores of reserpinized animals reestablished the normal response to ANTU. Pretreatment with the selective 5-HT2 receptor antagonist, ketanserin, prevented the thrombocytopenia, the increase in lung content of 5-HT and 5-[3H]HT, and prevented the edematogenic response to ANTU by 70%. These data indicate a major role for 5-HT in the pathophysiology of acute lung microvascular injury produced by ANTU.

Animals↗
PubMed1984

Hyperoxia and paraquat alter the kinetics of platelet serotonin uptake.

Platelets and serotonin (5-HT) have been increasingly implicated in the pathophysiological response to lung microvascular injury. These experiments were undertaken to study the effect of agents known to injure the pulmonary microvasculature on platelet function, i.e., 5-HT uptake kinetics. Mice were exposed to 100% normobaric oxygen (24 to 96 hr) or pretreated ip (3 to 24 hr) with paraquat dichloride (50 mg/kg), diquat dibromide (68 mg/kg), or alpha-naphthylthiourea (ANTU, 10 mg/kg). Mouse platelet 5-HT accumulation was described by a saturable uptake system, possessing high affinity and low capacity, acting in conjunction with passive diffusion. The kinetic constants for the saturable uptake system in control mice were Km 3.37 +/- 0.32 X 10(-7) M and Vmax 46.7 +/- 3.5 pmol 10(8) platelets-1 min-1. Exposure to oxygen for 24 hr resulted in an increased affinity, Km 1.91 +/- 0.67 X 10(-7) M, and a decreased Vmax, 20.0 +/- 5.0 pmol 10(8) platelets-1 4 min-1, with no change in the passive diffusion component. Pretreatment with paraquat (3 hr) produced similar changes (Km 1.71 +/- 0.27 X 10(-7) M and Vmax 15.8 +/- 1.6 pmol 10(8) platelets-1 4 min-1), while diquat and ANTU failed to alter the kinetics of platelet 5-HT uptake. These changes appeared to require a pulmonary toxicant capable of generating a flux of oxygen radicals, and were not due to either the presence of residual toxicant, the presence of an endogenous inhibitor in platelet-rich plasma, or changes in the platelet content of endogenous 5-HT. This study has shown that hyperoxia and paraquat affect platelet function, and suggests that this alteration may contribute to the pathophysiological response of the pulmonary microvasculature to injury produced by these agents.

Animals↗
PubMed1984

Hyperoxia alters platelet disposition of serotonin.

Exposure of mice to normobaric 100% oxygen for 24-96 h is known to damage the pulmonary capillary endothelium and alter the disposition of serotonin (5-hydroxytryptamine, 5-HT). We now report that such exposure increased the concentration of platelets and decreased the platelet content of 5-HT and a tracer dose of [3H]5-HT. After 48 h of hyperoxia exposure, the platelet concentration was elevated to 121% of air-exposed control values and the content of 5-HT and [3H]5-HT/10(9) platelets was decreased 76% and 57%, respectively. Kinetic analysis of the platelet 5-HT uptake process indicated that hyperoxia exposure caused an increased affinity and decreased velocity of transport. After 48 h of oxygen exposure the Km for platelet 5-HT uptake was 1.91 +/- 0.67 X 10(-7) M and the Vmax 30.1 +/- 4.6 pmol/10(8) platelets/4 min compared to air-exposed control values of Km 3.37 +/- 0.32 X 10(-7) M and Vmax 46.7 +/- 3.5 pmol/10(8) platelets/4 min, respectively. These changes in platelet 5-HT uptake and disposition may contribute to the pathophysiology of the pulmonary microvascular response to hyperoxia-induced injury.

Animals↗
PubMed1983

Reparative changes following oxygen-induced lung injury: effect on serotonin disposition and metabolism.

Exposure of mice to normobaric 100% oxygen for 72 hr has been shown to damage lung capillary endothelial cells and to markedly alter the pulmonary disposition and metabolism of endogenous serotonin (5-HT) and exogenous [3H]5-HT in a time-dependent manner. We have extended these studies to examine the reparative changes occurring in lung following moderate oxygen-induced injury. Pulmonary angiotensin converting enzyme (ACE) activity, a biochemical marker of endothelial cell injury, was decreased after a 72-hr oxygen exposure and progressively increased above control levels during the recovery period (130%, 168 hr) and paralleled lung protein content. The pulmonary disposition of [3H]5-HT also provided an index of endothelial cell function. Lung levels of [3H]5-HT were elevated 183% (0 hr) and 200% (24 hr) and returned to control values by 72 hr of air recovery. Pulmonary edema followed a similar time-course that corresponded to reported ultrastructural changes. The circulating platelet concentration progressively decreased from control values at 0 hr to 68% of control of 168 hr. In contrast to lung where 5-HT was significantly elevated during the early reparative period, platelet 5-HT content was significantly decreased and, like lung, returned to control values by 72 hr. The two forms of lung monoamine oxidase (MAO) showed different responses during the reparative phase. Type A MAO was slightly elevated throughout the recovery period. In contrast, type B MAO was decreased at 0 hr but increased throughout the reparative phase, being significantly elevated 128% at 72 hr and 139% at 168 hr. These data suggest that the lung is capable of readily recovering from moderate oxygen-induced injury and that certain biochemical parameters described herein provide useful indices of pulmonary microvascular function.

Animals↗
PubMed1981

Determination of serotonin, its precursors, metabolites and [3H] serotonin in lung by high-performance liquid chromatography with fluorescence detection.

A rapid, simple, sensitive method for the determination of serotonin, its precursors, metabolites and [3H] serotonin in lung is described. Tissue preparation requires only homogenization in dilute perchloric acid and centrifugation prior to separation by high performance liquid chromatography using a reversed-phase column. Detection is based on the native fluorescence of indole compounds and detection limits ranged between 30-90 pg injected. The method has been used to determine these compounds in mouse lung and plasma.

Animals↗
PubMed1981

Structural specificity of the inhibition of serotonin accumulation in rat lung slices.

The structural specificity required for the inhibition of 5-HT accumulation in rat lung slices was determined using compounds possessing a single structural modification of the 5-HT molecule. The structural modifications involved the indole ring nitrogen atom, the alpha-carbon atom to the sidechain amine, the sidechain amine, and the substitution pattern of the indole nucleus.

Animals↗