2-(Anilinomethyl)imidazolines as alpha(1)-adrenoceptor agonists: the identification of alpha(1A) subtype selective 2'-carboxylic acid esters and amides.

2-(Anilinomethyl)imidazolines with 2'-esters or 2'-amides are potent agonists of the cloned human alpha(1)-adrenoceptors in vitro. The size and shape of the ortho substituent can have significant effects on the potency, efficacy, and subtype selectivity of these 2-(anilinomethyl)imidazolines. alpha(1A)-subtype selective agonists have been identified.

Pharmacologic actions of the second-generation leukotriene B4 receptor antagonist LY293111: in vitro studies.

The in vitro actions were investigated of LY293111, a potent and selective leukotriene B4 (LTB4) receptor antagonist, on human neutrophils, human blood fractions, guinea pig lung membranes, and guinea pig parenchymal and tracheal strips. The IC50 for inhibiting [3H]LTB4 binding to human neutrophils was 17.6 +/- 4.8 nM. LY293111 inhibited LTB4-induced human neutrophil aggregation (IC50 = 32 +/- 5 nM), luminol-dependent chemiluminescence (IC50 = 20 +/- 2 nM), chemotaxis (IC50 = 6.3 +/- 1.7 nM), and superoxide production by adherent cells (IC50 = 0.5 nM). Corresponding responses induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine were inhibited by 100-fold higher concentrations of LY293111. LTB4 binding to guinea pig tissues and subsequent activation were also inhibited. The Ki for inhibition of [3H]LTB4 binding to lung membranes was 7.1 +/- 0.8 nM; IC50 for preventing binding of [3H]LTB4 to spleen membranes was 65 nM. The compound inhibited LTB4-induced contraction of guinea pig lung parenchyma. At 10 nM, LY293111 caused a parallel rightward shift of the LTB4 concentration-response curve. At higher concentrations, plots were shifted in a nonparallel manner, and maximum responses were depressed. LY293111 did not prevent antigen-stimulated contraction of sensitized trachea strips. At micromolar concentrations, LY293111 inhibited production of LTB4 and thromboxane B2 by plasma-depleted human blood stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine and thrombin. In addition, at these higher concentrations, formation of LTB4 by A23187-activated whole blood and conversion of arachidonic acid to LTB4 by a human neutrophil cytosolic fraction were inhibited. In summary, LY293111 is a second-generation LTB4 receptor antagonist with much improved potency in a variety of functional assay systems.

Development of scintillation-proximity assays for alpha adrenoceptors.

Binding assays have long been used to determine compound affinity and selectivity for various seven-transmembrane receptors. Over time, the degree of complexity has significantly reduced, whereas the throughput of the various assays has greatly increased. In this article, we detail the development of a filter-binding assay and a scintillation-proximity assay (SPA) designed to quantify a compound's affinity for the three alpha1-adrenoceptor subtypes, alpha1A, alpha1B, and alpha1D. The various components of the assays such as ease of assay performance, robustness, cost, and generation of radioactive waste are compared and contrasted. On the basis of the results, the SPA offers many advantages of high-throughput assay formats over the traditional filter-binding assay. To follow up on the success of the alpha1-adrenoceptor SPA, SPAs for the three alpha2-adrenoceptors were developed and are detailed in this article. Affinity data generated for a select number of alpha2 compounds agree with reported literature values. These assays, like those for alpha1 subtypes, are very amenable to high-throughput screening campaigns. In conclusion, scintillation-proximity assays offer significant advantages over filter-binding assays.

Phenylalanine in the second membrane-spanning domain of alpha 1A-adrenergic receptor determines subtype selectivity of dihydropyridine antagonists.

The alpha 1-adrenergic receptors (alpha 1-AR) belong to the G-protein coupled seven-transmembrane biogenic amine receptor family. Three subtypes have been successfully cloned in the alpha 1-adrenergic receptor family, and they share 50% identical amino acid sequences and 70% similarity. We have constructed seven chimeric receptors of the alpha 1A-AR. Each of the chimeras contains alpha 1D-subtype amino acid sequences within the membrane-spanning domains. Comparisons of ligand affinities with these chimeras has provided information on the importance of certain amino acid residues in determining receptor subtype specificity in the alpha 1A- and alpha 1D-ARs. With ligands in the dihydropyridine series, the niguldipine analog 1 was found to have respective pKi's of 9.32 +/- 0.17 for alpha 1A-AR; 6.84 +/- 0.24 for alpha 1D-AR; and 6.76 +/- 0.28 for alpha 1A/D(TM2), respectively. This trend was also exhibited by two other niguldipine analogs, 2 and 3, which had similar pKi's toward alpha 1D-AR and alpha 1A/D(TM2). This subtype selectivity was also maintained in the piperdine derivative, 4, and alpha 1A-AR selective ligand, which showed the same parallel trends in binding affinities with alpha 1A-AR and the six chimeras as the niguldipine analogs. Since in considering the second membrane-spanning domain, the alpha 1A- and alpha 1D-ARs only differ at positions 76, 77, 85, and 86, we were able to show through mutational studies that phenylalanine 86 is solely responsible for the selectivity found in the chimeric receptor alpha 1A/D(TM2) exhibited against the ligands 1-4 used in this study. A model based on the rhodopsin structure places the amino acid at position 86 in the final turn toward the extracellular region. This is four helical turns above aspartic acid-79, a conserved amino acid in the second membrane-spanning domain. This is the first report that suggests a significant involvement of the second membrane-spanning domain in antagonist binding in the biogenic amines class of the superfamily of seven-transmembrane receptors.

Structure activity relationships of a series of buspirone analogs at alpha-1 adrenoceptors: further evidence that rat aorta alpha-1 adrenoceptors are of the alpha-1D-subtype.

The activity of a series of busprione analogs at recombinant and rat thoracic aorta alpha-1 adrenoceptors was investigated. Compound affinity for recombinant alpha-1A, alpha-1B and alpha-1D adrenoceptors from human and animal sources was determined by radioligand binding assays using membranes prepared from rat-1 fibroblasts expressing recombinant receptors with ( +/- )-[125l]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone as the radioligand. Compound affinity and functional activity at rat aortic alpha-1 adrenoceptors were determined using endothelium denuded rings contracted with phenylephrine. BMY 7378 ¿8-(2-[4-(2-methoxyphenyl)-1-piperazinyl]-ehtyl)-8-azaspiro [4,5]decane-7,9-dione dihydrochloride¿ and MDL 73005EF ¿8-[2-(1,4-benzodioxan-2-ylmethylamino) ethyl]-8-azaspiro[4,5]decane-7,9-dione hydrochloride¿ were found to have significant selectivity for the alpha-1D-subtype and were high affinity antagonists of the alpha-1 adrenoceptors in the rat aorta. Leverage plot analysis of affinities of the buspirone analogs and a series of structurally diverse alpha-1 antagonists for recombinant alpha-1 adrenoceptors and rat aorta alpha-1 adrenoceptors demonstrate that the alpha-1 adrenoceptors in the rat aorta are predominantly of the alpha-1D subtype.

The specific contribution of the novel alpha-1D adrenoceptor to the contraction of vascular smooth muscle.

With a selective antagonist, the specific contribution of the alpha-1D adrenoceptor (AR) to vascular smooth muscle contraction has been assessed. BMY 7378 bound to membranes expressing the cloned rat alpha-1D AR with a > 100-fold higher affinity (K1 = 2 nM) than binding to either the cloned rat alpha-1A AR (Ki = 800 nM) or the hamster alpha-1B AR (Ki = 600 nM). BMY 7378 exhibited differential potency in inhibiting vascular smooth muscle contraction. In the rat aorta and iliac artery, BMY 7378 was a high-affinity antagonist, producing parallel shifts in the phenylephrine concentration-response curve. The dissociation constants for this compound by Schild analysis were 0.95 and 4 nM for the aorta and iliac artery, respectively. The slopes of these Schild plots were not significantly different from unity. BMY 7378 was a weak antagonist in the rat caudal, mesenteric resistance and renal arteries, with Schild slopes significantly < 1. With ribonuclease protection assays, alpha-1D mRNA was found in all blood vessels examined. These data suggest that (1) BMY 7378 is a selective alpha-1D AR antagonist that can be used in functional systems to assess the contribution of this receptor in vascular smooth muscle contraction; (2) the alpha-1D AR appears to play a major role in the contraction of the aorta and iliac artery; (3) despite the fact that the mRNA for the alpha-1D AR can be detected in the caudal, mesenteric resistance (4) and renal arteries, it does not appear to play a role in mediating contraction of these blood vessels; and (4) expression of alpha-1D mRNA in a particular artery does not ensure that this receptor is involved in regulating the contraction of that artery.

BMY 7378 is a selective antagonist of the D subtype of alpha 1-adrenoceptors.

BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8- azaspiro[4.5]decane-7,9-dione dihydrochloride), a 5-HT1A receptor partial agonist, also binds to alpha 1-adrenoceptors. Competition assays were performed using (+/-)-beta-([125I]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone ([125I]HEAT), and membranes prepared from Rat-1 fibroblasts expressing hamster alpha 1b-, bovine alpha 1c-, or rat alpha 1d-adrenoceptor, or their respective human homologues. Results indicate that BMY 7378 is selective for the alpha 1D-adrenoceptor subtype (pKi: hamster alpha 1b-adrenoceptor 6.2 +/- 0.03, human alpha 1b-adrenoceptor 7.2 +/- 0.05; bovine alpha 1c-adrenoceptor 6.1 +/- 0.02, human alpha 1c-adrenoceptor 6.6 +/- 0.20; rat alpha 1d-adrenoceptor 8.2 +/- 0.06, human alpha 1d-adrenoceptor 9.4 +/- 0.05) and has high affinity (pA2, 8.9 +/- 0.1) for rat aorta alpha 1-adrenoceptor.

Characterization of alpha-1 adrenoceptor subtypes in human and canine prostate membranes.

Experiments were designed to characterize the predominant subtype of alpha-1 adrenoceptors in human and canine prostate tissue. The chemical (+/-)-beta-([125I]iodo-4-hydroxyphenyl)-ethyl- aminomethyl-tetralone bound in a specific, saturable manner to a single class of binding sites in membranes that expressed recombinant hamster alpha-1B, bovine alpha-1C and rat alpha-1D receptors expressed in rat-1 fibroblasts and to those from prostate tissue. Competition assays with human and canine prostate membranes revealed only a single class of binding sites. Binding affinity in both human and canine prostate most significantly correlated with binding affinity for the recombinant bovine alpha-1C receptor (r = .98 human, .95 canine). Further analysis with leverage plots demonstrated that binding affinity in human and canine prostate tissue is best predicted by binding affinity to recombinant bovine alpha-1C (P < .01 human and P < .001 canine). These data are consistent with a single class of alpha-1 adrenoceptors in human and canine prostate tissue, which is best represented as the alpha-1C subtype.

Effect of alkaline phosphatase on thromboxane mimetic induced platelet activation.

Recently it has been reported that alkaline phosphatase selectively inhibited thromboxane mimetic induced platelet aggregation and secretion suggesting that the phosphorylation state on the platelet surface may be important for thromboxane induced platelet activation. We report here studies attempting to elucidate the mechanism of action of alkaline phosphatase. Washed human platelet aggregation induced by the thromboxane mimetic IBOP was completely abolished when incubated with alkaline phosphatase (1 unit/ml) for 5 min. The effect was inhibited by co-incubation with 5mM phosphate. Binding studies using [125I]BOP showed that neither the affinity of IBOP for the receptor (control: 9.2 +/- 2.1 nM, alkaline phosphatase: 7.9 +/- 1.8 nM) nor the Bmax (control: 1780 +/- 320 sites/plt. alkaline phosphatase: 1920 +/- 290 sites/plt) were effected by alkaline phosphatase treatment. GTPase activity was measured in platelet membranes treated with and without alkaline phosphatase as measured by IBOP induced hydrolysis of [gamma-32P]GTP. The EC50 values for IBOP induced GTPase were similar whereas the maximum amount of released Pi in the control membranes was more than two fold greater than in alkaline phosphatase treated membranes. These studies suggest that thromboxane induced platelet activation may be dependent upon the phosphorylation state of the thromboxane receptor and/or closely associated protein.

Characterization of thromboxane A2/prostaglandin H2 binding sites in guinea pig cardiac membrane preparations.

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) induce platelet aggregation and are potent vasoconstrictors, and they have been implicated in coronary vasospasm and myocardial infarction. The 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) was used to characterize binding to microsomal membrane preparations from saline-perfused guinea pig atria (GPA) and ventricles (GPV). [125I]IBOP bound to GPA and GPV in a protein-dependent and saturable manner, although total binding was two-fold greater and non-specific binding was proportionately less in GPA compared to GPV. Analysis of equilibrium binding data indicated one class of binding sites in both GPA and GPV with Kd values of 333 +/- 117 and 645 +/- 187 pM, respectively, which were in close agreement with kinetically determined Kd values of 226 and 882 pM, respectively. Bmax values of GPA and GPV of 57 +/- 5.6 and 24 +/- 4.3 fmol/mg protein were significantly different (P < 0.01). Ki values (from IC50s) were determined for various TXA2/PGH2 analogues and prostaglandins in competition binding assays with [125I]IBOP. The rank order for ability to inhibit binding in GPA was U46619 = SQ29548 > I-PTA-OH > PGF2 alpha = PGE2. In GPV, the rank order was U46619 = SQ29548 > PGF2 alpha = I-PTA-OH = PGE2. [125I]IBOP binding to GPA and GPV was completely displaced by the TXA2/PGH2 agonist U46619 and by the TXA2/PGH2 antagonist SQ29548.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary actions of LY255283, a leukotriene B4 receptor antagonist.

The actions of LY255283, a leukotriene (LT) B4 receptor antagonist, were examined on guinea pig lung. LTB4 and LY255283 displaced [3H]LTB4 from its binding site on lung membranes with pKi values of 9.9 and 7.0, respectively. In the functional correlate of the binding studies, LY255283 competitively reduced contractile responses of lung parenchyma to LTB4 (pA2 = 7.2). LTB4 produced airway obstruction which was reduced by LY255283 administered i.v. (ED50 = 2.8 mg/kg) or orally (ED50 = 11.0 mg/kg). Contractile responses to histamine, LTD4 and the thromboxane mimetic, U46619, were not reduced by LY255283. The compound also did not inhibit cyclooxygenase or 5-lipoxygenase enzymes. We conclude that LY255283 selectively antagonized pharmacologic responses to LTB4 on lung tissue and appears to be a useful tool to investigate the role of LTB4 in pulmonary disease.

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.

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.

Interaction of 5-HETE, 12-HETE, 15-HETE and 5,12-diHETE at the human platelet thromboxane A2/prostaglandin H2 receptor.

A variety of lipoxygenase products such as 12- and 15-hydroxyeicosatetraenoic acid (12- and 15-HETE) inhibit thromboxane A2 (TXA2) mimetic induced human platelet aggregation in a stereoselective manner. The mechanism of this inhibition remains unclear. To determine if this inhibition is due to a receptor level interaction of the lipoxygenase products at the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor, radioligand binding studies were performed using a new [125I]-labelled thromboxane mimetic [125I]BOP. The mono-HETES 5(S), 12(R), 12(S) and 15(S) inhibited binding of the radioligand to the TXA2/PGH2 receptor in washed human platelets with IC50 values of greater than 25, 0.73, 2.06 and 2.0 microM respectively. LTB4 and its positional isomer 5(S), 12(S)-diHETE were less potent with IC50 values greater than 10 microM for LTB4 and 9.38 microM for 5(S), 12(S)-diHETE. Thus, stereoselective inhibition of the binding of the radioligand was demonstrated between 12(R)- and 12(S)-HETE. These lipoxygenase products also inhibited IBOP (10nM) induced platelet aggregation in a concentration dependent fashion with a similar rank order of potency as that obtained in the competition binding assay. These results suggest that, at least in part, the platelet inhibitory properties of these HETEs may be mediated through their interaction at the TXA2/PGH2 receptor.

Mechanisms of leukotriene E4 partial agonist activity at leukotriene D4 receptors in differentiated U-937 cells.

Leukotriene E4 (LTE4) is shown to be a partial agonist of leukotriene D4 (LTD4) in differentiated U-937 cells. The data that support this conclusion are: 1) LTE4 completely displaced [3H]LTD4 from its receptors in U-937 cell membranes. 2) LTE4 induced only 30 +/- 4% of the maximal Ca2+ transient induced by LTD4 in the presence of 1 mM extracellular Ca2+ and 60 +/- 4% of the maximal LTD4 response in the absence of extracellular Ca2+. 3) LTE4 induced only a fraction of the inositol phosphates metabolized by LTD4. Moreover, LTE4 resulted in essentially no production of the inositol 1,4,5-trisphosphate isomer, while LTD4 induced a rapid and substantial transient increase in this isomer. The generation of inositol phosphates by both agonists was unaffected by extracellular Ca2+. 4) The EC50 values for Ca2+ mobilization for LTD4 and LTE4 corresponded with their affinity (Kd values) for the LTD4 receptor. 5) A series of structurally diverse LTD4 receptor antagonists blocked the Ca2+ mobilization responses to LTD4 and LTE4 with identical rank orders of potency. 6) LTE4 acted as an antagonist of LTD4 of potency. 6) LTE4 acted as an antagonist of LTD4 effects when they were coadministered. 7) LTE4 and LTD4 acutely desensitized Ca2+ mobilization to each other. All of the effects of LTE4 are explained by its partial agonist activity at the LTD4 receptor as shown by the following data. 1) Neither LTD4 nor LTE4 had any effect on the agonist activity of fMet-Leu-Phe, LTB4, or platelet-activating factor. 2) None of the above agonists or antagonists to the above receptors affected any of the activities of LTD4 or LTE4. 3) Neither LTD4 nor LTE4 induced desensitization of Ca2+ mobilization to any of the non-LTD4 receptor agonists tested. 4) Under the conditions studied, we have not observed any evidence of multiple subclasses of LTD4 receptors in U-937 cells. LTE4 is a partial agonist of the LTD4 receptor, because it can only couple the LTD4 receptor to a portion of the signaling system available to the receptor when occupied by LTD4. Specifically, LTD4 caused the activation of receptor-operated calcium channels, mobilization of intracellular Ca2+, the activation of phosphatidylinositol-phospholipase C, and the liberation of an additional, as yet undefined, intracellular mediator. To do this, LTD4 receptors couple to at least two and perhaps more guanine nucleotide binding proteins. LTE4 is unable to activate the phosphatidylinositol-phospholipase C but can mimic the other effects of LTD4.(ABSTRACT TRUNCATED AT 400 WORDS)

Subcellular localization of a thromboxane A2/prostaglandin H2 receptor antagonist binding site in human platelets.

The subcellular localization of a binding site for the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist, 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4- hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranor TXA2 ([125I]-PTA-OH), was determined. Subcellular fractions of platelets were prepared by glycerol lysis or nitrogen cavitation, and were characterized by the use of enzymatic markers specific for plasma membranes, endoplasmic reticulum (dense tubular system), mitochondria, granules, and cytosolic constituents. The Kd and density of binding sites in the subcellular fractions were determined by Scatchard analysis of equilibrium binding data. The Kd and Bmax for [125I]-PTA-OH determined in the lysates were 49 +/- 11 nM and 4.1 +/- 1.7 pmol/mg protein respectively (N = 6). The Kd values were not significantly different in any of the fractions assayed. The binding sites were coenriched (4.5 +/- 0.66 fold) with the enzymatic markers for plasma membranes (3.7 +/- 0.5 fold) and dense tubular system (2.4 +/- 0.4 fold). The binding sites were not coenriched with markers for cytoplasmic constituents, mitochondria, or granules. The ability of the TXA2/PGH2 mimetic U46619 to compete with [125I]-PTA-OH for the binding site was also determined for the various subcellular fractions. The IC50 for U46619 was 5.4 +/- 1.2 microM in the lysate, and was not significantly different in the subcellular fractions. These data suggest that the binding site is the TXA2/PGH2 receptor described previously. These data are consistent with the notion that the putative TXA2/PGH2 receptor is localized in the plasma membranes and/or the dense tubular system.