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Biomedical subjects

D E Mais

Publications and source records attributed to D E Mais.

At least 19 recordsLinked to original sources
PubMed1992

Characterization by photoaffinity labelling of the human platelet thromboxane A2/prostaglandin H2 receptor: evidence for N-linked glycosylation.

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) are potent proaggregatory and vasoconstrictor lipids acting through a receptor referred to as the TXA2/PGH2 receptor. The receptor was purified using a modification of a previously described method from human platelet membranes solubilized using the detergent (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) and a combination of affinity chromatography and wheat germ lectin chromatography. This procedure resulted in a 1075 +/- 375-fold purification and a specific activity of 1.45 +/- 0.55 nmol/mg protein (n = 5). Repeating these chromatography steps on this partially purified receptor resulted in a preparation with a specific activity of 21 +/- 3 nmol/mg protein (n = 5). This represents the theoretical specific activity if one assumes a molecular weight of 50,000 for the receptor. The fold purification was 11,750 +/- 1250 based on crude membranes and an overall yield of 24%. To further the characterization of this receptor, we synthesized a new radioiodinated photoaffinity probe, 7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4-azido-3-iodobenzenesulfonylamino++ + )- bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid (I-SAP-N3). [125I]l-SAP-N3 irreversibly incorporated into the purified receptor yielding a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography and indicated a molecular weight for the receptor of 50-51 kDa. The incorporation of the ligand could be inhibited by a variety of TXA2/PGH2 analogues. In addition, photoaffinity labelling was inhibited in a stereoselective manner as demonstrated by the pair of enantiomers (d)- and (l)-S145. Digestion of photoaffinity labelled receptor with N-glycosidase F demonstrated the presence of at least two N-linked glycosylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗
PubMed1992

Synthesis and biochemical properties of an 125I-labelled ryanodine derivative.

The synthesis of a novel radioiodinated ryanodine-O10eq-N-acylamino acylate along with biological data are reported. The affinity of the iodinated product, 7, was comparable to ryanodine, 7.97 nM and 6.47 nM, respectively. Conversion of the non-radioactive iodinated ryanodine analog to the [125I] isotope was accomplished by conversion of 7 to the trimethyltin derivative followed by [125I] exchange using chloramine-T in organic solvent. The radioiodinated ryanodine analog, 9, bound to cardiac membrane preparations in a protein dependent and saturable manner indicating that this analog may represent a useful new tool for the study of ryanodine receptors and that modifications about the C-10 hydroxy group of ryanodine may be carried out without loss in biological activity.

Animals↗
PubMed1992

7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4- iodobenzenesulfonylamino)bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid: a novel high-affinity radiolabeled antagonist for platelet thromboxane A2/prostaglandin H2 receptors.

A high-affinity thromboxane (TX)A2/prostaglandin (PG) H2 receptor antagonist, I-SAP [7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4- iodobenzenesulfonylamino)bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid] and its radiolabeled analog [125I]SAP (Mais et al., 1991) are characterized in the present study. I-SAP antagonized I-BOP ([1S-(1 alpha, 2 beta(5Z),3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4- (4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2y l]-5'heptenoic acid) and U46619 [15S-hydroxy-11 alpha,9 alpha-(epoxymethano)-prosta-5Z,13E-dienoic acid)], two different TXA2/PGH2 mimetics, induced aggregation of washed human platelets in a similar manner (pA2 of 8.11 +/- 0.09, Kd = 7.8 nM, n = 3; pA2 = 8.01 +/- 0.05, Kd = 9.7 nM, n = 8, respectively). I-SAP also had agonistic activity, producing platelet shape change (EC50 = 9.7 nM +/- 0.6 nM at pH 7.4, n = 3) which was blocked by pretreatment of platelets with SQ29548 ([1S-(1 alpha,2 beta(5Z),3 beta,4 alpha)]-7-[3-[[2- [(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept- 2-yl]-5-heptenoic acid), a TXA2/PGH2 receptor antagonist. Radioligand binding studies were performed with [125I]SAP using washed human platelets. Competition of three agonists and four antagonists for binding with [125I]SAP was determined. The compounds showed the appropriate rank order potencies, including stereoselective competition by a pair of stereoisomeric antagonists. In washed human platelets, the Kd for I-SAP was 468 +/- 49 pM and the maximum binding (Bmax) was 2057 +/- 156 sites/platelet at pH 7.4 (n = 6). The Bmax was significantly increased 49% to 3072 +/- 205 sites/platelet at pH 6.5 (P less than .01 but the Kd was unchanged (490 +/- 18 pM, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1992

In vitro characterization of a novel TXA2/PGH2 receptor ligand (S-145) in platelets and vascular and airway smooth muscle.

The stereoisomers of S-145, a novel thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor ligand, were compared to TXA2/PGH2 receptor antagonists, SQ29548 and BM13505 in guinea pig platelets, aortas and trachea. Equilibrium binding assays in platelets yielded Kd values (nanomolar) for (+)-S-145 (0.57 +/- 0.04), (-)-S-145 (9.2 +/- 1.3), SQ29548 (11.1 +/- 0.70) and BM13505 (118 +/- 16). In aortas, the corresponding Kb values (nanomolar) were (0.014 +/- 0.002), (1.90 +/- 0.31), (16.8 +/- 3.3) and (142 +/- 29), respectively, whereas in trachea, the Kd values (nanomolar) were (0.019 +/- 0.004), (1.12 +/- 0.18), (1.94 +/- 0.30) and (18.99 +/- 2.59), respectively. S-145 stereoisomers elicited platelet shape change stereoselectively that was characterized by EC50 values 8 to 16-fold higher than the EC50 values for these ligands to block aggregation induced by TXA2/PGH2 mimetic, U44069. S-145 (+)- and (-)-isomers stereoselectively induced transient aortic contraction at concentrations 214,000- and 16,000-fold higher, respectively, than the corresponding Kb values in this tissue. S-145-induced platelet shape change and aortic contraction were inhibitable by low concentrations of SQ29548. We postulate that S-145 may elicit partial agonist activity in platelets and aorta via lower affinity for the active than inactive state of the TXA2/PGH2 receptor in those tissues. S-145 had no agonist activity in isolated trachea possibly indicating different TXA2/PGH2 recognition sites in aorta and trachea or a smaller preligand ratio of active to inactive TXA2/PGH2 receptors in trachea than in aorta.

Animals↗
PubMed1992

Binding of a novel radioiodinated thromboxane A2/prostaglandin H2 antagonist to guinea pig lung membranes.

The utilization of a novel radioiodinated TXA2/PGH2 receptor antagonist, ISAP (7-[(1R,2S,3S,5R)-6,6-dimethyl-3(4- iodobenzenesulfonylamino)-bicyclo[3.1.1]-hept-2-yl]-5(Z)-heptenoic acid) to characterize TXA2/PGH2 receptors from guinea pig lung parenchymal membranes in radioligand binding assays is described. [125I]ISAP binding was saturable, displaceable, and dependent upon protein concentration. The time course of binding yielded k1 = 2.12 x 10(8) M-1 min-1, k1 = 4.46 x 10(-3) min-1, Kd = k-1/k1 = 17.8 pM. Equilibrium binding studies indicated a single class of high affinity binding sites with a Kd of 52.7 +/- 1.9 pM and a Bmax of 92.7 +/- 7.2 fmoles/mg protein (n = 4). Binding was inhibited by a series of structurally diverse mimetics and antagonists with the rank order of potency IBOP greater than ONO11113 = SQ26655 greater than U46619 (mimetics) and (d)-S-145 greater than ISAP greater than (1)-S-145 greater than SQ29548 greater than BM13505 = I-PTA-OH (antagonists), with entantioselectivity of binding demonstrated by (d) and (1) S-145. Binding was also inhibited by prostanoids (PGD2, PGF2 alpha, and 9 alpha, 11 beta-PGF2) thought to act at the airway TXA2/PHH2 receptor, but not by histamine or carbachol, and only weakly by LTB4 and LTD4, consistent with specific binding to the lung TXA2/PGH2 receptor.

Animals↗
PubMed1991

Differential effect of pH on thromboxane A2/prostaglandin H2 receptor agonist and antagonist binding in human platelets.

The effects of changes in pH on the binding of agonists and antagonists to the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were determined. Competition binding studies were performed with the TXA2/PGH2 mimetic [1S-1 alpha,2 beta (5Z), 3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4'-iodophenoxy)-1-buteny) 7-oxabicyclo-[2.2.1]-heptan-2-yl]-5-heptenoic acid ([125I]BOP). The pH optimum for binding of [125I] BOP to washed human platelets was broad with a range of pH 4-6 in contrast to that of the TXA2/PGH2 receptor antagonist 9,11-dimethyl-methano-11,12-methano-16-(3-iodo-4-hydroxyl)-13-aza-15 alpha,beta-omega-tetranorthromboxane A2 ([125I]PTA-OH) which was 7.4. Scatchard analysis of [125I]BOP binding in washed platelets at pH 7.4, 6.0, and 5.0 revealed an increase in affinity (Kd = 1.16 +/- 0.06, 0.64 +/- 0.09, and 0.48 +/- 0.05 nM, respectively) and an increase in the number of receptors (Bmax = 2807 +/- 415, 5397 +/- 636, and 7265 +/- 753 sites/platelet, respectively). The potency of I-BOP to induce shape change in washed platelets at pH 6.0 was also significantly increased from an EC50 value of 0.34 +/- 0.016 nM at pH 7.4 to 0.174 +/- 0.014 nM at pH 6.0 (n = 6, p less than 0.05). In contrast, the EC50 value for thrombin was unaffected by the change in pH. In competition binding studies with [125I]BOP, the affinity of the agonists U46619 and ONO11113 were increased at pH 6.0 compared to 7.4. In contrast, the affinity of the TXA2/PGH2 receptor antagonists I-PTA-OH, SQ29548, and L657925 were either decreased or unchanged at pH 6.0 compared to 7.4. Diethyl pyrocarbonate and N-bromosuccinimide, reagents used to modify histidine residues, reversed the increase in affinity of [125I]BOP at pH 6.0 to values equivalent to those at pH 7.4. In solubilized platelet membranes, the effects of NBS were blocked by coincubation with the TXA2/PGH2 mimetic U46619. The results suggest that agonist and antagonist binding characteristics are different for the TXA2/PGH2 receptor and that histidine residue(s) may play an important role in the binding of TXA2/PGH2 ligands to the receptor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1991

Platelet and vascular thromboxane A2/prostaglandin H2 receptors. Evidence for different subclasses in the rat.

Thromboxane A2 (TXA2) and its precursor prostaglandin H2 (PGH2) induce platelet aggregation and vascular contraction through shared cell surface receptors commonly referred to as TXA2 or TXA2/PGH2 receptors. Whether different subclasses of TXA2/PGH2 receptors exist in platelets and vascular smooth muscle cells is controversial. In this study, TXA2 receptors on washed rat and human platelets and cultured rat aortic smooth muscle cells (RASMC) were characterized using radioligand competition binding assays with the 125I-labeled TXA2/PGH2 receptor agonist [1S-(1 alpha,2 beta(5Z),3 alpha(1E,3R*),4 alpha)] -7- [3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl) -7- oxabicyclo-[2.2.1]- heptan-2-yl] -5- heptenoic acid (I-BOP) and various agonists and antagonists. Scatchard analyses of equilibrium binding data revealed Kd values of 205 +/- 68 pM (N = 6), 2.2 +/- 0.3 nM (N = 9) and 310 +/- 60 pM (N = 7) and Bmax values of 1.3 +/- 0.45 fmol/10(6) platelets, 2.8 +/- 0.2 fmol/10(6) platelets and 20.9 +/- 2.2 fmol/10(6) cells for rat and human platelets and RASMC, respectively. Concentration-dependent increases in intracellular free Ca2+ concentrations induced by I-BOP were observed in RASMC loaded with the calcium sensitive dye fura-2. The IC50 values for various TXA2/PGH2 analogues in competition binding assays with 125I-BOP were determined. Based on their IC50 values, the rank orders were I-BOP less than L657925 less than ONO11113 less than or equal to SQ29548 less than PTA-TPO less than PTA-NO less than or equal to L657926 less than or equal to I-PTA-OH less than PTA-OH[2] = meta-I-PTA-PO less than or equal to ONO11120[2] = ONO11120[1] less than PTA-OH[1] in rat platelets. I-BOP less than SQ29548 less than PTA-TPO = L657925 less than or equal to ONO11113 less than I-PTA-OH less than PTA-NO less than or equal to meta-I-PTA-PO less than or equal to PTA-OH[2] less than ONO11120[2] less than or equal to ONO11120[1] less than L657926 less than or equal to PTA-OH[1] in human platelets, and I-BOP less than L657925 less than ONO11113 less than or equal to SQ29548 less than ONO11120[2] less than or equal to L657926 less than or equal to PTA-OH[2] less than PTA-TPO less than ONO11120[1] less than I-PTA-OH less than meta-I-PTA-PO less than PTA-NO less than PTA-OH[1] in RASMC.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Interactions of dihydropyridine Ca2+ channel agonists with the human platelet thromboxane A2/prostaglandin H2 receptor.

The specific interactions at the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor by four 1-4 dihydropyridine (DHP) agonists were studied. Using competition equilibrium binding assays with the TXA2/PGH2 receptor agonist [125I]BOP and the antagonist [125I]PTA-OH, the affinities of racemic BAY K 8644 (BAY), racemic CGP 28392 (CGP) and (+) and (-) SDZ 202-791 (SDZ) for the TXA2/PGH2 receptor were determined. The rank order potencies for competition were BAY greater than (-)SDZ greater than CGP greater than or equal to (+)SDZ. Bay, CGP and SDZ (stereoselectively) inhibited specific incorporation of the TXA2/PGH2 receptor photoaffinity probe [125I]PTA Azido into three proteins associated with the TXA2/PGH2 receptor with Mr of 43, 39 and 27 kDa as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. Using the fluorescent Ca2+ probe Fura-2, it was observed that I-BOP failed to stimulate classical divalent cation channels. However, SDZ stereoselectively inhibited the rise in [Ca2+]i induced by I-BOP while not affecting that induced by thrombin. Although DHPs specifically and stereoselectively interact with the TXA2/PGH2 receptor on human platelets, the TXA2/PGH2 receptor-mediated rise in [Ca2+]i is not through stimulation of a classical divalent cation channel.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Characterization of a thromboxane A2/prostaglandin H2 receptor in guinea pig lung membranes using a radioiodinated thromboxane mimetic.

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) are potent constrictors of airway smooth muscle and may mediate some of the pulmonary effects of leukotrienes. To date, the TXA2/PGH2 receptor in lung has not been well characterized. In this report, we describe the evaluation of the TXA2/PGH2 receptor in guinea pig lung membranes using the new radiolabeled TXA2 mimetic [1S(1 alpha,2 beta(5Z),3 alpha(1E,3S*),4 alpha)]-7-[3-(3-hydroxy-4-(4'- iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-h eptenoic acid (IBOP). IBOP elicited a dose-dependent contraction of guinea pig lung parenchymal strips (EC50 = 3.03 +/- 0.97 nM, three experiments), which was blocked by the TXA2/PGH2 antagonists SQ29548 (pKB = 7.44 +/- 0.2, three experiments), BM13505 (pKB = 6.29 +/- 0.26, three experiments), and I-PTA-OH (pKB = 5.82 +/- 0.36, three experiments). In radioligand binding studies, the binding of [125I]IBOP to guinea pig lung membranes prepared from perfused lungs was saturable, displaceable, and dependent upon protein concentration. Binding was optimal at pH 6.5 and was enhanced by the addition of mono- and divalent cations. The standard assay buffer was 25 mM 3-(N-morpholino)propanesulfonic acid, pH 6.5, 100 mM NaCl, 5 mM MgCl2. Binding was inhibited by pretreatment with dithiothreitol, N-ethylmaleimide, or beta-mercaptoethanol. Binding was unaffected by the addition of guanine nucleotide analogs at concentrations up to 300 microM. Analysis of the time course of binding of [125]IBOP at 30 degrees yielded k-1 = 0.0447 min-1, k1 = 2.49 x 10(8) M-1 min-1, and Kd = k-1/k1 = 180 pM. Computer analysis of equilibrium binding studies using nonlinear methods (LUNDON-1) revealed a single class of noninteracting binding sites with a Kd of 86.9 +/- 11.9 pM and a Bmax of 81.8 +/- 7.7 fmol/mg of protein (three experiments). [125I]IBOP binding to guinea pig lung membranes was inhibited by a series of TXA2/PGH2 receptor agonists and antagonists, with a rank order different from that previously determined for washed guinea pig platelets (Spearman's r = 0.686, p greater than 0.05). [125I]IBOP binding to guinea pig lung membranes was also inhibited by the prostanoids prostaglandin D2, prostaglandin E2, prostaglandin F2 alpha, and 9 alpha,11 beta-prostaglandin F2, all of which have been proposed to act at the TXA2/PGH2 receptor in lung.

Animals↗
PubMed1991

Quantitative autoradiography of the binding sites for [125I] iodoglyburide, a novel high-affinity ligand for ATP-sensitive potassium channels in rat brain.

We have developed a high specific activity ligand for localization of ATP-sensitive potassium channels in the brain. When brain sections were incubated with [125I]iodoglyburide (N-[2-[[[(cyclohexylamino)carbonyl]amino]sulfonyl]ethyl]-5-125I-2- methoxybenzamide), the ligand bound to a single site with a KD of 495 pM and a maximum binding site density of 176 fmol/mg of tissue. Glyburide was the most potent inhibitor of specific [125I]iodoglyburide binding to rat forebrain sections whereas iodoglyburide and glipizide were slightly less potent. The binding was also sensitive to ATP which completely inhibited binding at concentrations of 10 mM. Autoradiographic localization of [125I]iodoglyburide binding indicated a broad distribution of the ATP-sensitive potassium channel in the brain. The highest levels of binding were seen in the globus pallidus and ventral pallidum followed by the septohippocampal nucleus, anterior pituitary, the CA2 and CA3 region of the hippocampus, ventral pallidum, the molecular layer of the cerebellum and substantia nigra zona reticulata. The hilus and dorsal subiculum of the hippocampus, molecular layer of the dentate gyrus, cerebral cortex, lateral olfactory tract nucleus, olfactory tubercle and the zona incerta contained relatively high levels of binding. A lower level of binding (approximately 3- to 4-fold) was found throughout the remainder of the brain. These results indicate that the ATP-sensitive potassium channel has a broad presence in the rat brain and that a few select brain regions are enriched in this subtype of neuronal potassium channels.

Affinity Labels↗
PubMed1990

Photoaffinity receptor antagonist for human platelet thromboxane A2/prostaglandin H2 receptors.

9,11-Dimethylmethano -11,12-methano-16-(3-azido-4-iodophenoxy)-13,14- dihydro-13-aza-15 alpha beta-omega-tetranor TXA2 (I-PTA-PON3) was synthesized and evaluated as a potential photoaffinity probe of the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. I-PTA-PON3 inhibited the aggregation of washed human platelets induced by the TXA2 mimetic U46619 [(15S)-hydroxy-11 alpha, 9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid]. Schild analysis of the data revealed a Kd of 9.5 nM and a slope not significantly different from -1. Equilibrium binding studies using [125I]PTA-OH, a TXA2/PGH2 receptor antagonist, showed that I-PTA-PON3 plus photolysis resulted in a 52% reduction in the number of binding sites (1252 +/- 202/platelet) compared to the nonphotolyzed group (2557 +/- 293/platelet) (N = 5, P less than 0.05) with no significant change in the Kd. Repetition of the incubation with I-PTA-PON3 and photolysis a second time resulted in a further 77% (578 +/- 163 binding sites/platelet) reduction in the number of binding sites. Incubation of washed human platelets with I-PTA-PON3 (163 nM) followed by photolysis and removal of the non-covalently bound I-PTA-PON3 resulted in no change in the EC50 value for the TXA2 mimetic, U46619, when compared to controls that were either exposed to I-PTA-PON3 and not photolyzed or exposed only to photolysis. The second photolysis of I-PTA-PON3 resulted in a significant 42% increase in the EC50 value of U46619-induced aggregation compared to the non-photolyzed group (N = 4, P less than 0.05). These results suggest that I-PTA-PON3 is a useful probe for the study of TXA2/PGH2 receptors and that spare TXA2/PGH2 receptors may exist in the platelet.

Affinity Labels↗
PubMed1990

Characterization of thromboxane A2/prostaglandin H2 receptors in human vascular smooth muscle cells.

The present study utilizes a newly synthesized TXA2/PGH2 mimetic, I-BOP, to characterize the TXA2/PGH2 receptor in suspensions of cultured human vascular smooth muscle cells. [125I]-BOP bound in a saturable and specific manner (Kd = 2.6 +/- 0.6 nM; Bmax = 33,540 +/- 6,200 sites/cell; 69 fmoles/mg protein, n = 12). Competition binding assays were performed with [125I]-BOP and the TXA2/PGH2 receptor antagonists SQ29548, L657925 and L657926 and the receptor agonist U46619. I-BOP induced concentration-dependent increases in intracellular free calcium which were inhibited by SQ29548. The results provide radioligand binding evidence for the presence of a TXA2/PGH2 receptor in human vascular smooth muscle cells.

Binding, Competitive↗