Femtosecond response of polyatomic molecules to ultra-intense hard X-rays.

X-ray free-electron lasers enable the investigation of the structure and dynamics of diverse systems, including atoms, molecules, nanocrystals and single bioparticles, under extreme conditions. Many imaging applications that target biological systems and complex materials use hard X-ray pulses with extremely high peak intensities (exceeding 1020 watts per square centimetre). However, fundamental investigations have focused mainly on the individual response of atoms and small molecules using soft X-rays with much lower intensities. Studies with intense X-ray pulses have shown that irradiated atoms reach a very high degree of ionization, owing to multiphoton absorption, which in a heteronuclear molecular system occurs predominantly locally on a heavy atom (provided that the absorption cross-section of the heavy atom is considerably larger than those of its neighbours) and is followed by efficient redistribution of the induced charge. In serial femtosecond crystallography of biological objects-an application of X-ray free-electron lasers that greatly enhances our ability to determine protein structure-the ionization of heavy atoms increases the local radiation damage that is seen in the diffraction patterns of these objects and has been suggested as a way of phasing the diffraction data. On the basis of experiments using either soft or less-intense hard X-rays, it is thought that the induced charge and associated radiation damage of atoms in polyatomic molecules can be inferred from the charge that is induced in an isolated atom under otherwise comparable irradiation conditions. Here we show that the femtosecond response of small polyatomic molecules that contain one heavy atom to ultra-intense (with intensities approaching 1020 watts per square centimetre), hard (with photon energies of 8.3 kiloelectronvolts) X-ray pulses is qualitatively different: our experimental and modelling results establish that, under these conditions, the ionization of a molecule is considerably enhanced compared to that of an individual heavy atom with the same absorption cross-section. This enhancement is driven by ultrafast charge transfer within the molecule, which refills the core holes that are created in the heavy atom, providing further targets for inner-shell ionization and resulting in the emission of more than 50 electrons during the X-ray pulse. Our results demonstrate that efficient modelling of X-ray-driven processes in complex systems at ultrahigh intensities is feasible.

Pulse Energy and Pulse Duration Effects in the Ionization and Fragmentation of Iodomethane by Ultraintense Hard X Rays.

The interaction of intense femtosecond x-ray pulses with molecules sensitively depends on the interplay between multiple photoabsorptions, Auger decay, charge rearrangement, and nuclear motion. Here, we report on a combined experimental and theoretical study of the ionization and fragmentation of iodomethane (CH_{3}I) by ultraintense (∼10^{19} W/cm^{2}) x-ray pulses at 8.3 keV, demonstrating how these dynamics depend on the x-ray pulse energy and duration. We show that the timing of multiple ionization steps leading to a particular reaction product and, thus, the product's final kinetic energy, is determined by the pulse duration rather than the pulse energy or intensity. While the overall degree of ionization is mainly defined by the pulse energy, our measurement reveals that the yield of the fragments with the highest charge states is enhanced for short pulse durations, in contrast to earlier observations for atoms and small molecules in the soft x-ray domain. We attribute this effect to a decreased charge transfer efficiency at larger internuclear separations, which are reached during longer pulses.

Differential colon cancer cell adhesion to E-, P-, and L-selectin: role of mucin-type glycoproteins.

E-, P-, and L-selectin support the adhesion of leukocytes to the vessel wall through the recognition of specific carbohydrate ligands, which often contain sialylated, fucosylated lactosamines such as sialyl Lewis x [sLex; Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-]. E-selectin expressed by activated endothelium has been shown to support the adhesion of sLex-bearing colon cancer cells. In the present study, we examine the interactions of multiple colon cancer cell lines with all three selectins. Three colon cancer cell lines (LS 180, T84, and COLO 205) bound to recombinant purified E-, P-, and L-selectin. The colon cancer line COLO 320 bound to P- and L-selectin but not E-selectin; conversely, HT-29 cells bound E-selectin but not P- and L-selectin. Caco-2 showed little or no interaction with any of the three selectins. Treatment of the cells with O-sialoglycoprotease from Pasteurella haemolytica, an enzyme that selectively cleaves mucin-type O-linked glycoproteins, reduced binding to purified P- and L-selectin in all cases. In addition, recombinant soluble P- and L-selectin bound to affinity-purified mucins from all adherent tumor cell lines. Of the four tumor cell lines that interacted with E-selectin, O-glycoprotease treatment substantially diminished adhesion of LS 180 and T84, had little effect on COLO 205, and failed to inhibit the binding of HT-29. As predicted by these data, E-selectin showed substantial binding only to mucins purified from LS 180 and T84. These findings suggest that L- and P-selectin interact primarily with mucin-type ligands on colon cancers, whereas E-selectin can recognize both mucin and nonmucin ligands. Binding of the colon cancer lines to purified selectins correlates with their adhesion to activated endothelial cells (E-selectin-dependent), platelets (P-selectin-dependent), and neutrophils (L-selectin-dependent). These differential tumor cell-selectin interactions may influence metastatic spread and may also contribute to the observed variability in host response to tumor progression.

A common binding site for primary prostanoids in vascular smooth muscles: a definitive discrimination of the binding for thromboxane A2/prostaglandin H2 receptor agonist from its antagonist.

Differences in binding characteristics between agonists and antagonists for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were examined in rat cultured vascular smooth muscle cells (VSMC). Scatchard analysis indicated the existence of two binding sites for the TXA2/PGH2 agonist, whereas a single class of recognition sites for the receptor antagonists were observed with approximately the same maximum binding capacity (Bmax) as a high-affinity binding site of the agonist. Weak binding inhibition by approx. 100 nM of primary prostanoids (PGE1, PGF2 alpha and PGD2) was detected only with the TXA2/PGH2 agonist, and not with the antagonist. Primary prostanoids as well as TXA2/PGH2 agonists (U46619 and STA2) suppressed the [3H]PGF2 alpha and [3H]PGE1 binding with almost the same potency, whereas TXA2/PGH2 antagonists (S-145, SQ29,548 and ONO3708) did not. The Bmax value of the binding sites was roughly identical in PGF2 alpha, PGE1 and a low-affinity binding site of U46619. These results suggest the existence of two binding sites for TXA2/PGH2 in VSMC, i.e., a high-affinity binding site corresponding to that of the TXA2/PGH2 antagonists and a low-affinity binding site in common with primary prostanoids.

Purification and characterization of a high-affinity binding protein for pancreatic-type phospholipase A2.

A high-affinity and specific binding site for mammalian group I phospholipase A2 (PLA2-I) was found on the membranes of bovine corpus luteum. Affinity labeling experiments revealed that PLA2-I binds to a single polypeptide with a mass of 190-200 kDa. The PLA2-I binding protein in the membranes was solubilized in an active form with n-octyl beta-D-thioglucoside, and then purified approx. 16,000-fold. The purification procedures consisted of diethylaminoethyl-Sephacel chromatography, PLA2-I-affinity gel chromatography and gel-filtration high-performance liquid chromatography on a TSKgel G3,000SWXL column. The final preparation migrated as a single molecular species of 190 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and identification of the 190 kDa protein as the PLA2-I binding protein was demonstrated by ligand blotting analysis. The purified protein possessed a binding capacity with high affinity and specificity for a mammalian mature type of PLA2-I. Treatment of the purified material with N-glycosidase F resulted in increased mobility of the protein on SDS-PAGE as well as considerable abolition of the PLA2-I binding activity, thus suggesting the requirement of the carbohydrate moiety of the PLA2-I binding protein for receptor-ligand interactions.

Migration of vascular smooth muscle cells by phospholipase A2 via specific binding sites.

Pancreatic-type group I phospholipase A2 (PLA2-I), EC 3.1.1.4, long thought to act as a digestive enzyme, has a specific binding site in several types of tissues and cells and these sites promote PLA2-I-stimulated DNA synthesis. In this study we report a PLA2-I action on the migration of rat embryonic thoracic aorta smooth muscle cells (A7r5). A7r5 cells had a single class of PLA2-I binding site with an equilibrium binding constant (Kd) value of 1.7 nM and a maximum binding capacity (Bmax) of 40,000 sites/cell. The migration activity of PLA2-I for A7r5 cells was examined using modified Boyden chambers. PLA2-I stimulated the migration dose-dependently, and the ED50 value was about 1 nM, which was almost the same as the Kd value for PLA2-I binding. Checkerboard analysis showed that the response of A7r5 cells to PLA2-I was chemokinetic, but not chemotactic. These findings reveal a new aspect of PLA2-I in the modulation of vascular function.

Synergistic effect of thromboxane A2 and N-formylmethionylleucylphenylalanine on platelet-activating factor synthesis in human polymorphonuclear neutrophils.

The effects of thromboxane A2 (TXA2) on the synthesis of platelet-activating factor (PAF) and leukotriene B4 (LTB4) were studied using human polymorphonuclear neutrophils (PMN). Scatchard analysis for binding experiments using [3H]S-145, a specific TXA2/prostaglandin H2 (PGH2) receptor antagonist, revealed the existence of a single class of binding sites (Kd = 83.0 +/- 2.8 nM, Bmax = 113.0 +/- 3.1 fmol/2.10(6) cells) in human PMN. Upon stimulation with a combination of U46619, a TXA2 mimetic agonist, and N-formylmethionylleucylphenylalanine (FMLP, 1 microM), the synthesis of PAF was detected, although this was not significantly enhanced by U46619 or FMLP alone. The maximal production of PAF as well as the maximal activity of acetyl-CoA acetyltransferase was observed at approx. 20 min after addition of both stimuli. The effects of U46619 plus FMLP on PAF synthesis showed dose dependence to different concentrations of U46619 (0.1-10 microM), and were completely inhibited by S-145. Contrarily, no significant amounts of LTB4 were detected by radioimmunoassay during the stimulation with U46619 and FMLP. These results suggest that TXA2 and FMLP synergistically activate human PMN to induce PAF synthesis and this effect of TXA2 is mediated through its specific receptor.

Suppression of murine endotoxic shock by sPLA2 inhibitor, indoxam, through group IIA sPLA2-independent mechanisms.

Endotoxic shock is a systemic inflammatory process, involving a variety of proinflammatory mediators. Two types of secretory phospholipase A2 (sPLA2) have been implicated in this process. Group IB sPLA2 (PLA2-IB) binds to the PLA2 receptor (PLA2R), and PLA2R-deficient mice exhibit resistance to endotoxin-induced lethality with reduced plasma levels of proinflammatory cytokines, such as TNF-alpha. Group IIA sPLA2 (PLA2-IIA) is found in many tissues and cell types, and local and systemic levels are elevated under numerous inflammatory conditions including sepsis. In this study, we investigated the effect of a specific sPLA2 inhibitor, indoxam, on murine endotoxic shock. Indoxam suppressed the elevation of plasma TNF-alpha with a similar potency in PLA2-IIA-expressing and PLA2-IIA-deficient mice after LPS challenge. In PLA2-IIA-deficient mice, indoxam also suppressed the elevation of plasma IL-1beta, IL-6 and NO, and prolonged survival after LPS challenge. Indoxam was found to block the PLA2-IB binding to murine PLA2R with a high potency (Ki=30 nM). The inhibitory effects of indoxam on the LPS-induced elevation of plasma TNF-alpha levels could not be observed in mice deficient in PLA2R. These findings suggest that indoxam blocks the production of proinflammatory cytokines during endotoxemia through PLA2-IIA-independent mechanisms, possibly via blockade of the PLA2R function.

Beta 2-adrenergic regulation of prostaglandin D2 receptor in rabbit platelets.

[3H]Prostaglandin D2 binding to rabbit platelets was increased by about 150% in the presence of beta-adrenoceptor agonist, isoproterenol. The isoproterenol-induced potentiation of the [3H]prostaglandin D2 binding gave a bell-shaped dose-response relationship (maximum response at 3 X 10(-8) M) in a stereospecific manner. Similar and moderate potentiation was obtained with terbutaline. On the other hand, beta-adrenoceptor antagonists such as alprenolol, propranolol and butoxamine (beta 2-specific) had no potentiating effect on [3H]prostaglandin D2 binding; rather, they abolished the isoproterenol-induced increase of [3H]prostaglandin D2 binding. The beta 1-specific antagonist, metoprolol, did not have any effect. Rabbit platelets were found to possess one [3H]prostaglandin D2 binding site (Kd = 6 X 10(-7) M, Bmax = 787 fmol/mg protein). In the presence of isoproterenol at 3 X 10(-8) M, Bmax was increased with unaltering Kd value. Isoproterenol did not increase [3H]prostaglandin E1, [3H]prostaglandin E2 and [3H]prostaglandin F2 alpha bindings to platelets. The potential effect of isoproterenol was mimicked by forskolin, theophylline, dibutyryl cyclic AMP, prostaglandin E1 and prostaglandin I2, but it was abolished by 2', 5'-dideoxyadenosine, an inhibitor of adenylate cyclase, indicating that elevated level of cyclic AMP may be available for the induction of the increase of [3H]prostaglandin D2 binding. Prostaglandin D2-induced cyclic AMP synthesis and antiaggregation activity were also augmented in the presence of isoproterenol. These results suggest a beta 2-adrenoceptor-mediated cyclic AMP-dependent mechanism for the regulation of prostaglandin D2 receptor binding in rabbit platelets.

Group X secretory phospholipase A(2) induces potent productions of various lipid mediators in mouse peritoneal macrophages.

We have previously shown the expression of group X secretory phospholipase A(2) (sPLA(2)-X) in mouse splenic macrophages and its powerful potency for releasing fatty acids from various intact cell membranes. Here, we examined the potency of sPLA(2)-X in the production of lipid mediators in murine peritoneal macrophages. Mouse sPLA(2)-X was found to induce a marked release of fatty acids including arachidonic acid and linoleic acid, which contrasted with little, if any, release by the action of group IB and IIA sPLA(2)s. In resting macrophages, sPLA(2)-X elicited a modest production of prostaglandin E(2) and thromboxane A(2). After the induction of cyclooxygenase-2 (COX-2) by pretreatment with lipopolysaccharide, a dramatic increase in the production of these eicosanoids was observed in sPLA(2)-X-treated macrophages, which was completely blocked by the addition of either the specific sPLA(2) inhibitor indoxam or the COX inhibitor indomethacin. In accordance with its higher hydrolyzing activity toward phosphatidylcholine, mouse sPLA(2)-X induced a potent production of lysophosphatidylcholine. These findings strongly suggest that sPLA(2)-X plays a critical role in the production of various lipid mediators from macrophages. These events might be relevant to the progression of various pathological states, including chronic inflammation and atherosclerosis.

Different effects of two thromboxane A2/prostaglandin H2 receptor ligands, U46619 and S-145, on rabbit platelets.

Stimulation of rabbit platelets with U46619 induced platelet shape change, aggregation and secretion of ATP. However, S-145, which specifically binds to the thromboxane A2/prostaglandin H2 receptor like U46619, induced only shape change. Both compounds rapidly elevated cytoplasmic Ca2+ concentration although only U46619 evoked the formation of inositol phosphates. Chelating external Ca2+ with EGTA did not affect the S-145-induced platelet shape change while intracellular Ca2+ movement was severely reduced. These results suggest an essential role of phospholipase C in the induction of platelet aggregation and secretion and that some factor other than Ca2+ and phospholipase C participates in platelet shape change.

Endothelin-induced intracellular Ca2+ mobilization through its specific receptors in murine peritoneal macrophages.

We studied the presence of specific binding sites for endothelin (ET) and the effect of ET on cytosolic free Ca2+ concentration ([Ca2+]i) in murine thioglycolate-activated peritoneal macrophages. Scatchard analysis for binding experiments using [125I]ET-1 or [125I]ET-3 revealed the existence of a single class of binding sites. The binding parameters (Kd and Bmax) for [125I]ET-1 were almost identical to those for [125I]ET-3. In addition, unlabeled 3 ET isopeptides (ET-1, ET-2 and ET-3) inhibited the specific binding of both ET-1 and ET-3 with similar inhibitory potencies. All 3 ET isopeptides caused an increase in [Ca2+]i in the same dose-dependent manner (0.01-100 nM). These results demonstrate the existence of an ET receptor with the same affinity for all isoforms that mediates the ET-induced intracellular Ca2+ mobilization in murine peritoneal macrophages.

Effect of pancreatic type phospholipase A2 on isolated porcine cerebral arteries via its specific binding sites.

The addition of porcine pancreatic group I phospholipase A2 (PLA2-I) produced a transient contraction followed by a relaxation in helical strips of porcine cerebral arteries. Its ED50 value (2.3 nM) was almost identical to the Kd value (3.9 nM) calculated from the specific binding of 125I-labeled porcine PLA2-I in cultured porcine cerebral arterial smooth muscle cells. Type-specific action of PLA2s and homologous desensitization strongly implicated the involvement of PLA2-I-specific sites in the response. The transient contraction was abolished by treatment with indomethacin as well as by the removal of endothelium, indicating the dependence of vasoconstrictor prostaglandins synthesized by PLA2-I in endothelium. The PLA2-I-induced relaxation response was also observed in bovine and cat cerebral arteries, thus providing a new aspect of PLA2-I as a vasoactive substance.

Identification of group X secretory phospholipase A(2) as a natural ligand for mouse phospholipase A(2) receptor.

Phospholipase A(2) receptor (PLA(2)R) mediates various biological responses elicited by group IB secretory phospholipase A(2) (sPLA(2)-IB). The recently cloned group X sPLA(2) (sPLA(2)-X) possesses several structural features characteristic of sPLA(2)-IB. Here, we detected a specific binding site of sPLA(2)-X in mouse osteoblastic MC3T3-E(1) cells. Cross-linking experiments demonstrated its molecular weight (180 kDa) to be similar to that of PLA(2)R. In fact, sPLA(2)-X was found to bind the recombinant PLA(2)R expressed in COS-7 cells, and its specific binding detected in mouse lung membranes was abolished by the deficiency of PLA(2)R. These findings demonstrate sPLA(2)-X to be one of the high-affinity ligands for mouse PLA(2)R.

Clearance of group X secretory phospholipase A(2) via mouse phospholipase A(2) receptor.

Given the potent hydrolyzing activity toward phosphatidylcholine, group X secretory phospholipase A(2) (sPLA(2)-X) elicits a marked release of arachidonic acid linked to the potent production of lipid mediators in various cell types. We have recently shown that sPLA(2)-X can also act as a ligand for mouse phospholipase A(2) receptor (PLA(2)R). Here, we found that sPLA(2)-X was internalized and degraded via binding to PLA(2)R associated with the diminished prostaglandin E(2) (PGE(2)) formation in PLA(2)R-expressing Chinese hamster ovary (CHO) cells compared to CHO cells. Indirect immunocytochemical analysis revealed that internalized sPLA(2)-X was co-localized with PLA(2)R in the punctate structures in PLA(2)R-expressing CHO cells. Moreover, in mouse osteoblastic MC3T3-E(1) cells that endogenously express the PLA(2)R, the internalized sPLA(2)-X was localized in lysosomes. These findings demonstrate that PLA(2)R acts as a clearance receptor for sPLA(2)-X to suppress its strong enzymatic activity.

Potential role of group X secretory phospholipase A(2) in cyclooxygenase-2-dependent PGE(2) formation during colon tumorigenesis.

Although the cyclooxygenase-2 (COX-2) pathway of the arachidonic acid cascade has been suggested to play an important role in colon carcinogenesis, there is little information concerning the identity of phospholipase A(2) (PLA(2)) involved in the arachidonic acid release in colon tumors. Here, we compared the potencies of three types of secretory PLA(2)s (group IB, IIA and X sPLA(2)s) for the arachidonic acid release from cultured human colon adenocarcinoma cells, and found that group X sPLA(2) has the most powerful potency in the release of arachidonic acid leading to COX-2-dependent prostaglandin E(2) (PGE(2)) formation. Furthermore, immunohistological analysis revealed the elevated expression of group X sPLA(2) in human colon adenocarcinoma neoplastic cells in concert with augmented expression of COX-2. These findings suggest a critical role of group X sPLA(2) in the PGE(2) biosynthesis during colon tumorigenesis.

Synthesis of thromboxane receptor antagonists with bicyclo[3.1.0]hexane ring systems.

Thromboxane A2 receptor antagonists 11a, 15a, 26a, 30a, 34a, 36a, 46a, 52a, 61a, 72a, and 82a, which contain 6-oxabicyclo[3.1.0]hexane, 6-thiabicyclo[3.1.0]hexane, bicyclo[3.1.0]hexane, or 6,6-dimethylbicyclo[3.1.0]hexane ring systems with heptenoic and (phenylsulfonyl)amino side chains, and their corresponding sodium salts and methyl esters were synthesized. This study then examined the inhibitory effects of their sodium salts for the platelet aggregation induced by arachidonic acid with rabbit platelet-rich plasma and platelet aggregation induced by collagen with rat washed platelets.

Synthesis and in vitro activity of various derivatives of a novel thromboxane receptor antagonist, (+/-)-(5Z)-7-[3-endo-[(phenylsulfonyl)amino]bicyclo[2.2.1] hept-2-exo-yl]heptenoic acid.

Several sulfonyl derivatives (13a-t) of (+/-)-(5Z)-7-(3-endo-aminobicyclo[2.2.1]hept-2-exo-yl)heptenoic acid (VI) were synthesized via its methyl ester 10. Sulfonylation of 10 with 11a-t followed by saponification yielded 13a-t. Inhibitory concentrations (IC50) of the corresponding sodium salts 14a-t for platelet aggregation were measured with rat washed platelets (WP) and rabbit platelet-rich plasma (PRP). IC50 values of some derivatives for contraction of the rat aorta were also measured. The IC50 values for rat WP increased from 2.9 to 26 nM in the order of 14a, 14c, 14d, and 14b for derivatives with an arylsulfonyl residue, depending on the number of of intervening methylene groups. Methyl derivative 14e exhibited a higher IC50 value than n-hexyl derivative 14f. Substitution with a p-methyl, p-fluoro-, or p-chloro group in 14a retained or slightly reduced its IC50 value, while a p-n-pentyl or p-oxycarbonyl group augmented it significantly. The representative 14a suppressed (15S)-15-hydroxy-11,9-(epoxymethano)prosta-5(Z),13(E)-dienoic acid (U-46619) induced aggregation of human WP with an IC50 value of 7.7 nM, which corresponds well to the IC50 value of 3 nM obtained for each displacement by 14a of [3H]-U-46619 or (5Z,15 xi)-9 alpha, 11 alpha-(dimethylmethano)-15-hydroxy-16-(3-[125I]iodo- 4-hydroxyphenyl)-17,18,19,20-tetranor-13-aza-11a-carbathrombo-5-en oic acid [( 125I]-PTA-OH) bound to human WP. Synthesis of thromboxane A2 (TxA2) in human WP stimulated by thrombin was not inhibited by 14a at a concentration up to 10 microM. From these observations, the corresponding acid 13a (S-145) was concluded to be a potent TxA2 receptor antagonist.

1,3-Disubstituted benzazepines as novel, potent, selective neuropeptide Y Y1 receptor antagonists.

A novel series of potent and selective non-peptide neuropeptide Y (NPY) Y1 receptor antagonists, having benzazepine nuclei, have been designed, synthesized, and evaluated for activity. Chemical modification of the R(1) and R(3) substituents in structure 1 (Chart 1) yields several compounds that show high affinity for the Y1 receptor (K(i) values of less than 10 nM). SAR studies revealed that introduction of an isopropylurea group at R(1) and a 3-(benzo-condensed-urea) group, 3-(fluorophenylurea) group, or a 3-(N-(4-hydroxyphenyl)guanidine) group at R(3) in structure 1 afforded potent and subtype-selective NPY Y1 receptor antagonists. 3-(3-(Benzothiazol-6-yl)ureido)-1-N-(3-(N'-(3-isopropylureido++ +))benzyl )-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (21), which was one of the most potent derivatives, competitively inhibited specific [(125)I]peptide YY (PYY) binding to Y1 receptors in human neuroblastoma SK-N-MC cells (K(i) = 5.1 nM). 21 not only inhibited the Y1 receptor-mediated increase in cytosolic free Ca(2+) concentration in SK-N-MC cells but also antagonized the Y1 receptor-mediated inhibitory effect of peptide YY on gastrin-induced histamine release in rat enterochromaffin-like cells. 21 showed no significant affinity in 17 receptor binding assays including Y2, Y4, and Y5 receptors.

Kinetic studies on stereospecific recognition by the thromboxane A2/prostaglandin H2 receptor of the antagonist, S-145.

1. The mechanism for the stereospecific recognition of the antagonist S-145 by the thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor was examined by ligand-binding techniques in rat vascular smooth muscle cells (VSMCs) and in human platelet membranes. 2. Scatchard analysis revealed the existence of a single class of binding sites with the same maximum number for both [3H]-(+)-S-145 and [3H]-(-)-S-145 in both cell types. The dissociation constants (Kd) for the binding of the (+)-isomer in rat VSMCs and human platelet membranes were, respectively, 0.40 +/- 0.03 and 0.20 +/- 0.02 nM, each value being lower than that for the (-)-isomer (3.57 +/- 0.74 and 2.87 +/- 0.08 nM, respectively). 3. The rank orders of potency (Ki) for a series of TXA2/PGH2 ligands at inhibiting [3H]-(+)-S-145 binding were highly correlated with those determined for [3H]-(-)-S-145 binding in both cell preparations. 4. Kinetic analysis of the binding of both radioligands revealed a much lower dissociation rate constant (k-1) and a slightly greater association rate constant (k1) for the (+)-isomer compared to those for the (-)-isomer. 5. These results suggest that it is at the stage of dissociation from the TXA2/PGH2 receptor that the stereochemistry of the optical isomers of S-145 confers their difference in affinity for these receptors in rat VSMCs and human platelet membranes.