Blockade of thromboxane responses in the airway of the cat by SQ 29,548

The effects of SQ 29,548, a thromboxane receptor antagonist, on airway responses were investigated in paralyzed, anesthetized, mechanically ventilated cats. Intravenous injections of the thromboxane and prostaglandin precursor, arachidonic acid (AA), and the thromboxane mimic, U 46619, produced dose-related increases in transpulmonary pressure and lung resistance and decreases in dynamic compliance. After administration of SQ 29,548 (0.5 mg/kg iv), bronchoconstrictor responses to AA were reduced by approximately 50%, whereas responses to U 46619 were reduced by approximately 90%. The cyclooxygenase inhibitor, sodium meclofenamate (2.5 mg/kg iv), blocked the component of the airway response to AA remaining after treatment with SQ 29,548. The thromboxane receptor antagonist had no significant effect on bronchoconstrictor responses to prostaglandins F2 alpha, and D2, methacholine, 5-hydroxytryptamine, histamine, or BAY K 8644, an agent that promotes calcium entry. Reductions in systemic arterial pressure in response to AA were enhanced by the thromboxane receptor antagonist and abolished by meclofenamate. SQ 29,548 had no effect on terminal enzyme activity in microsomal fractions from cat lung. These data support the hypothesis that AA-induced bronchoconstriction in the cat is mediated in large part by the actions of thromboxane A2. These data also suggest that U 46619 and U 44069 stimulate the same airway receptor as thromboxane A2 and mimic the bronchomotor effects of this hormone, which has not yet been isolated as a pure substance. These data demonstrate that SQ 29,548 is a selective thromboxane receptor antagonist in the airways of the closed-chest cat and may be a useful probe for studying responses to thromboxane A2 in physiological and pathophysiological processes in the lung.     

Identification of residues important for ligand binding of thromboxane A2 receptor in the second extracellular loop using the NMR experiment-guided mutagenesis approach

The second extracellular loop (eLP2) of the thromboxane A(2) receptor (TP) had been proposed to be involved in ligand binding. Through two-dimensional (1)H NMR experiments, the overall three-dimensional structure of a constrained synthetic peptide mimicking the eLP2 had been determined by our group (Ruan, K.-H., So, S.-P., Wu, J., Li, D., Huang, A., and Kung, J. (2001) Biochemistry 40, 275-280). To further identify the residues involved in ligand binding, a TP receptor antagonist, SQ29,548 was used to interact with the synthetic peptide. High resolution two-dimensional (1)H NMR experiments, NOESY, and TOCSY were performed for the peptide, SQ29,548, and peptide with SQ29,548, respectively. Through completed (1)H NMR assignment and by comparing the different spectra, extra peaks were observed on the NOESY spectrum of the peptide with SQ29,548, which implied the contacts between residues of eLP2 at Val(176), Leu(185), Thr(186), and Leu(187) with SQ29,548 at position H2, H7, and H8. Site-directed mutagenesis was used to confirm the possible ligand-binding sites on native human TP receptor. Each of the four residues was mutated to the residues either in the same group, with different structure or different charged. The mutated receptors were then tested for their ligand binding activity. The receptor with V176L mutant retained binding activity to SQ29,548. All other mutations resulted in decreased or lost binding activity to SQ29,548. These mutagenesis results supported the prediction from NMR experiments in which Val(176), Leu(185), Thr(186), and Leu(187) are the possible residues involved in ligand binding. This information facilitates the understanding of the molecular mechanism of thromboxane A(2) binding to the important receptor and its signal transduction.     

Identification of thromboxane A2 receptor in cultured vascular endothelial cells of rat aorta

The binding site for [3H]SQ29,548, a potent and selective thromboxane A2 (TXA2) receptor antagonist, was studied in cultured vascular endothelial cells (VEC) of the rat aorta. Specific binding of [3H]SQ29,548 to rat VEC at 24 degrees C was saturable, displaceable and of high affinity. Scatchard analysis of equilibrium binding studies indicated that rat VEC contain a single class of binding sites with a Kd of 2.7 nM. The number of maximum binding sites (25.8 fmol/10(6) cells) for [3H]SQ29,548 on rat VEC was respectively 23 and 3.2 times more than that on rat platelets and rat vascular smooth muscle cells. Four TXA2 receptor antagonists and U46619 completely suppressed [3H]SQ29,548 binding to rat VEC, whereas other prostanoids, such as PGD2, PGF2 alpha, PGE1 and Iloprost, displaced the ligand binding only at considerably higher concentrations. These results suggest that the specific receptor for TXA2 is present in rat vascular endothelial cells.     

Specific receptors for thromboxane A2 in cultured vascular smooth muscle cells of rat aorta

The specific binding site for thromboxane A2 (TXA2) was studied in cultured vascular smooth muscle cells (VSMC) of the rat aorta. [3H]SQ29,548, a potent and selective TXA2 receptor antagonist, displayed high-affinity and specificity, as well as saturable and displaceable binding to rat VSMC in culture. Scatchard analysis of equilibrium binding at 24 degrees C revealed a single class of binding sites with a Kd of 1.7 nM and a Bmax of 8.0 fmol/10(6) cells. A series of TXA2 receptor antagonists completely suppressed [3H]SQ29,548 binding to rat VSMC, and the rank order of their inhibitory potencies (Ki) correlated well with the potencies for suppression of the U46619-induced contraction of rat thoracic aorta. These results suggest that specific binding sites for [3H]SQ29,548 represent the TXA2 receptor in rat VSMC.     

Selective modulation of the human platelet thromboxane A2/prostaglandin H2 receptor by eicosapentaenoic and docosahexaenoic acids in intact platelets and solubilized platelet membranes.

We previously demonstrated that nonesterified as well as esterified eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) inhibit U46619-induced platelet aggregation and [3H]U46619 specific binding to washed human platelets. It was also demonstrated that esterification of these fatty acids resulted in a decrease in the affinity of [3H]U46619 for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. In order to investigate the specificity of this inhibition, the effects of 20:5n-3 and 22:6n-3 on the function and binding of the platelet alpha 2-adrenergic receptor were studied. It was found that neither 20:5n-3 nor 22:6n-3 (nonesterified or esterified) altered epinephrine-induced aggregation or [3H]yohimbine specific binding. Moreover, Scatchard analysis revealed that esterification with either 20:5n-3 or 22:6n-3 did not alter the dissociation constant for [3H]yohimbine binding. Modulation of the TXA2/PGH2 receptor by 20:5n-3 and 22:6n-3 was next evaluated using CHAPS- and digitonin-solubilized platelet membranes. [3H]SQ29,548 dissociation constants of 26.5 nM and 20.8 nM were measured for CHAPS and digitonin-solubilized membranes, respectively. Competitive binding experiments in these solubilized preparations revealed that 20:5n-3 or 22:6n-3 blocked [3H] SQ29,548 binding with IC50 values in the range of 6-15 microM, while concentrations of these fatty acids of up to 100 microM showed no effect on [3H]yohimbine binding. On the other hand, the IC50 values for inhibition of [3H] SQ29,548 binding by linoleic acid (18:2n-6) and gamma-linolenic acid (18:3n-6) were in the range of 150 microM. Furthermore, 18:2n-6 and 18:3n-6 showed similar inhibitory effects on [3H]yohimbine binding. Finally, competition binding studies performed in a partially purified TXA2/PGH2 receptor preparation also demonstrated inhibition of [3H]SQ29,548 binding by 20:5n-3 and 22:6n-3. Collectively, these findings support the notion that 20:5n-3 and 22:6n-3 can selectively and directly modulate TXA2/PGH2 receptor function, and that this mechanism of action may contribute to the antiplatelet activity associated with diets rich in these fatty acids.     

Characterization of binding of a specific antagonist, [3H]-SQ 29,548, to soluble thromboxane A2/prostaglandin H2 (TP) receptors in human platelet membranes.

Binding of [3H]-SQ 29,548 was characterized to soluble thromboxane A2/prostaglandin H2 (TP) receptors from human platelet membranes as a means of examining ligand-receptor interactions outside the lipophilic environment of the cell membrane. Kinetic determination revealed a rate of ligand-receptor association of 1.4 x 10(7) +/- 0.2 M-1 x min-1 and a rate of dissociation of 0.5 +/- 0.07 min-1. The resultant equilibrium affinity constant was 36.3 +/- 5.8 nM. Saturation binding analysis revealed a single class of [3H]-SQ 29,548 binding sites with an affinity constant of 39.7 +/- 4.3 nM and a B(max) of 1735.7 +/- 69.1 fmol/mg protein. Specific [3H]-SQ 29,548 binding was inhibited by specific TP receptor antagonists and agonists in a rank order of potency similar to that seen in platelet membranes: SQ 33,961 much greater than SQ 29,548 greater than BM 13,505 greater than or equal to U 46619 greater than BM 13,177. PGD2, PGE2 and PGI2 did not appreciably inhibit the specific binding of [3H]-SQ 29,548. These data indicate that [3H]-SQ 29,548 binding to soluble human platelet TP receptors was specific, saturable, and reversible.     

DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors.

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A2/prostaglandin H2 (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [3H]SQ 29,548 (5 nM) in human platelets with kd/slope factor values of 9.6 +/- 1.4 microM/1.1 +/- 0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [3H]SQ 29,548 binding in platelets (Kd/slope factor:200 microM/0.86). [3H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (Kd: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (Bmax: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC50 = 46 microM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

Characterization of the substrate mimic bound to engineered prostacyclin synthase in solution using high-resolution NMR spectroscopy and mutagenesis: implication of the molecular mechanism in biosynthesis of prostacyclin

High-resolution NMR spectroscopy was used to determine the docking of a substrate (prostaglandin H2) mimic (U46619) to the engineered prostacyclin (PGI2) synthase (PGIS) in solution. The binding of U46619 to the PGIS protein was demonstrated by 1D NMR titration, and the significant perturbation of the chemical shifts of protons at C-11, H2C, and H20 of U46619 were observed upon U46619 binding to the engineered PGIS in a concentration-dependent manner. The detailed conformational change and 3D structure of the PGIS-bound U46619 were further demonstrated by 2D 1H NMR experiments using the transferred NOE technique. The distances between the protons H20 and H2, H18 and H2, and H18 and H4 are shorter following their binding to the PGIS in solution-down to within 5 A. These shorter distances resulted in a widely open conformation, where the triangle shape of the unbound U46619 changed to a more compact conformation with an oval shape. The bound conformation of U46619 fits the crystal structure of the PGIS substrate binding pocket considerably better than that of the unbound U46619. The residues important to the substrate binding in the active site pocket of PGIS were also predicted. For example, Trp282 could be one of the most important residues and is suspected to play a role in the determination of specific catalytic function, which has been established by the docking studies using the NMR structure of the PGIS-bound form of U46619 and the PGIS crystal structure. These studies have provided the structural information for the interaction of the PGIS with its substrate mimic. The noted conformational changes where the C-6 position is closer to the C-9 position of U46619 provided the first experimental data for understanding the molecular mechanism of the catalytic function of PGIS in the isomerization of PGH2 to prostacyclin.     

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.     

Endothelial thromboxane receptors: biochemical characterization and functional implications

We have identified thromboxane specific receptors in membrane preparations of bovine pulmonary artery endothelial cells using a potent thromboxane specific antagonist, [125I]-PTA-OH in a binding assay. The binding was specific and saturable. Neither thromboxane B2, prostaglandin D2 nor prostaglandin F2 alpha displaced the ligand (0.1 nM) at concentrations up to 10 microM. However, binding was displaced by IPTA-OH greater than SQ29548 greater than U46619. In addition, we observed that thromboxane mimetic U46619 significantly lowered the basal production of prostacyclin and also markedly suppressed bradykinin-stimulated prostacyclin released by endothelial cells. We propose that an important biological effect of thromboxane on vascular endothelial cells may be the suppression of prostacyclin production.     

Characterization of thromboxane A2/prostaglandin H2 receptors and histamine H1 receptors in cultured guinea-pig tracheal smooth-muscle cells.

We characterized thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors and histamine H1 receptors in Guinea-pig cultured tracheal smooth-muscle cells (TSMC). [3H]SQ 29,548 (a TXA2 antagonist)-binding sites were saturable and a high affinity with a dissociation constant of 6.2 +/- 0.60 nM (mean +/- S.E.) and a receptor density of 46 +/- 4.6 fmol/10(6) cells. [3H]SQ 29548 binding was completely inhibited by TXA2 mimetics or antagonists. Intracellular calcium concentration ([Ca2+]i) in TSMC was increased with U46619 stimulation and the increase was attenuated by TXA2 antagonists, the potencies of which correlated with those inhibiting the activities of the [3H]SQ 29548 binding. [3H]Mepyramine (a H1 antagonist)-binding sites were also present in TSMC. [3H]Mepyramine had a single class of low-affinity-binding sites with a dissociation constant of 2.6 +/- 0.081 microM and a receptor density of 10.6 +/- 0.11 nmol/mg protein. [3H]Mepyramine binding in TSMC membrane was inhibited by H1 antagonists, but not by H2 antagonists. The inhibition constants of mepyramine in TSMC were 910-times lower than those in tracheal membranes. In contrast, the histamine-induced increase in [Ca2+]i in TSMC was inhibited in the presence of low concentrations of H1 antagonists. All these observations provide evidence that TXA2/PGH2 receptors, mepyramine-binding sites and/or H1 receptors are expressed in cultured TSMC.     

Residues within transmembrane helices 2 and 5 of the human gonadotropin-releasing hormone receptor contribute to agonist and antagonist binding

To understand the ligand binding properties of the human GnRH receptor (hGnRH-R), 24 site-specific mutants within transmembrane helices (TMH) 1, 2, and 5 and the extracellular loop 2 (E2) were generated. These mutants were analyzed by using a functional reporter gene assay, monitoring receptor signaling via adenylate cyclase to a cAMP-responsive element fused to Photinus pyralis luciferase. The functional behavior of 14 receptor mutants, capable of G-protein coupling and signaling, was studied in detail with different well described agonistic and antagonistic peptide ligands. Furthermore, the binding constants were determined in displacement binding experiments with the antagonist [125I]Cetrorelix. The substitution of residues K36, Q204, W205, H207, Q208, F20, F213, F216, and S217 for alanine had no or only a marginal effect on ligand binding and signaling. In contrast, substitution of N87, Eg9, D9, R179, W206, Y211, F214, and T215 for alanine resulted in receptor proteins neither capable of ligand binding nor signal transduction. Within those mutants affecting ligand binding and signaling to various degrees, W101A, N102A, and N212Q differentiate between agonists and antagonists. Thus, in addition to N102 already described, the residues W101 in TMH2 and N212 in TMH5 are important for the architecture of the ligand-binding pocket. Based on the experimental data, three-dimensional models for binding of the superagonist D-Trp6-GnRH (Triptorelin) and the antagonist Cetrorelix to the hGnRH-R are proposed. Both decapeptidic ligands are bound to the receptor in a bent conformation with distinct interactions within the binding pocket formed by all TMHs, E2, and E3. The antagonist Cetrorelix with bulky hydrophobic N-terminal amino acids interacts with quite different receptor residues, a hint at the failure to induce an active, G protein-coupling receptor conformation.     

DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A₂/prostaglandin H₂ (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [³H]SQ 29,548 (5 nM) in human platelets with k_d/slope factor values of 9.6±1.4 μM/1.1±0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [³H]SQ 29,548 binding in platelets (K_d/slope factor:200 μM/0.86). [³H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (K_d: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (B_max: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC₅₀=46 μM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

Pharmacologic antagonism of thromboxane A2 receptors by trimetoquinol analogs in vitro and in vivo.

Although (-)-(S)-trimetoquinol [1-(3,4,5-trimethoxy-benzyl)- 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; TMQ] is recognized as a potent bronchodilator, (+)-(R)-TMQ is a selective antagonist of human platelet aggregation and serotonin secretion induced by thromboxane A2 (TXA2) agonists. To confirm the pharmacological actions of TMQ analogs, the interaction of the drugs with TXA2 receptors was examined in human platelets and in a mouse sudden death model. The inhibitory potencies of TMQ analogs (pIC50 values) for displacement of [3H]SQ 29,548 binding to platelets showed excellent correlation with the respective pIC50 (-log IC50) values for U46619-induced aggregation (r = 0.99, P less than 0.01) and serotonin secretion (r = 0.99, P less than 0.01) in human platelet-rich plasma and for whole blood aggregation (r = 0.99, P less than 0.01). In each system, the rank order of inhibitory potencies was rac-iodoTMQ greater than or equal to (+)-(R)-TMQ greater than rac-TMQ much greater than (-)-(S)-TMQ. Antithrombotic effects of TMQ analogs were evaluated in a mouse sudden death model. In vivo antithrombotic potencies of these compounds were consistent with the in vitro potencies as TXA2 receptor antagonists in platelet systems. Administration of rac-iodoTMQ, (+)-(R)-TMQ and rac-TMQ 15 min before the injection of U46619 (800 micrograms/kg, iv) protected mice against U46619-induced sudden death. On the other hand, (-)-(S)-TMQ did not protect animals against death. Protection of U46619-induced cardiopulmonary thrombosis by TMQ analogs was seen at doses of 3-100 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inverse Agonism of SQ 29,548 and Ramatroban on Thromboxane A2 Receptor

G protein-coupled receptors (GPCRs) show some level of basal activity even in the absence of an agonist, a phenomenon referred to as constitutive activity. Such constitutive activity in GPCRs is known to have important pathophysiological roles in human disease. The thromboxane A2 receptor (TP) is a GPCR that promotes thrombosis in response to binding of the prostanoid, thromboxane A2. TP dysfunction is widely implicated in pathophysiological conditions such as bleeding disorders, hypertension and cardiovascular disease. Recently, we reported the characterization of a few constitutively active mutants (CAMs) in TP, including a genetic variant A160T. Using these CAMs as reporters, we now test the inverse agonist properties of known antagonists of TP, SQ 29,548, Ramatroban, L-670596 and Diclofenac, in HEK293T cells. Interestingly, SQ 29,548 reduced the basal activity of both, WT-TP and the CAMs while Ramatroban was able to reduce the basal activity of only the CAMs. Diclofenac and L-670596 showed no statistically significant reduction in basal activity of WT-TP or CAMs. To investigate the role of these compounds on human platelet function, we tested their effects on human megakaryocyte based system for platelet activation. Both SQ 29,548 and Ramatroban reduced the platelet hyperactivity of the A160T genetic variant. Taken together, our results suggest that SQ 29,548 and Ramatroban are inverse agonists for TP, whereas, L-670596 and Diclofenac are neutral antagonists. Our findings have important therapeutic applications in the treatment of TP mediated pathophysiological conditions.     

The function and three-dimensional structure of a thromboxane A2/cysteinyl leukotriene-binding protein from the saliva of a mosquito vector of the malaria parasite

The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A(2) (TXA(2)) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C(4) (LTC(4))-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA(2) analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD7. In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA(2) analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA(2) analog U46619 is stabilized by hydrogen bonding interactions of the ω-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC(4) and occupies a very similar position to LTE(4) in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.     

Characterization of endothelial thromboxane receptors in rabbit aorta

An increased synthesis of thromboxane (TX) A(2) is associated with a number of cardiovascular diseases including atherosclerosis, unstable angina and hypertension. We previously identified a subgroup of NZW rabbits in which isolated arteries failed to contract to the TX agonists, U46619 or I-BOP. In vascular smooth muscle membranes, there was a significant decrease in TX receptors, termed TP. These rabbits are referred to as vTP- and those with the TP receptor are called vTP+. Because TP receptors are expressed in some types of endothelial cells, the present study was designed to determine whether functional TP receptors are present in endothelial cells cultured from aortas of vTP+ and vTP- rabbits. Radioligand binding studies were performed with (125)I-BOP. Aortic endothelial cells from vTP+ rabbits exhibited specific and saturable binding. In contrast, in endothelial preparations from vTP- rabbit aortas, no measurable binding to (125)I-BOP was detected. Using an anti-TP receptor antibody, we compared the amount of receptor expressed in endothelial cell lysates obtained from vTP+ and vTP- rabbits. Consistent with the results observed radioligand binding assays, the expression of TP receptor protein was decreased in vTP- compared to vTP+ endothelial cells. An in vitro wound healing assay was used on confluent monolayers of endothelial cells. In the untreated vTP+ cells, the area of the scratch was completely closed by 30 h. In the vTP+ cells treated with U46619 (3 microM), the rate of closure of the scratch area was reduced with approximately 12% of the scratch area remaining at 30 h. Pretreatment with the TP receptor antagonist, SQ 29548 (10 microM) prevented the inhibitory effect of U46619. The rate of closure of the scratch in the vTP- was not altered by U46619. In a separate study, U46619 (3 microM) increased the release of 6-keto PGF(1alpha), the stable metabolite of prostacyclin, in vTP+ but not vTP- endothelial cells. Pretreatment with SQ29548 (10 microM) or the cyclooxygenase inhibitor, indomethacin (10 microM) blocked the increase in vTP+ endothelial cells. In vascular reactivity studies in aortas from vTP+ rabbits, removal of the endothelium enhanced the vasoconstrictor response to U46619 indicating that activation of endothelial TP receptors may modulate vascular tone via the release of the vasodilator, prostacyclin. The results of this study suggest an important role for endothelial TP receptors in modulating vascular function.     

Amino acid residues 62 and 193 play the key role in regulating the synergism of substrate binding in oyster arginine kinase

The purpose of this study is to clarify the amino acid residues responsible for the synergism in substrate binding of arginine kinase (AK), a key enzyme in invertebrate energy metabolism. AKs contain a pair of highly conserved amino acids (D62 and R193) that form an ion pair, and replacement of these residues can cause a pronounced loss of activity. Interestingly, in the oyster Crassostrea AK, these residues are replaced by an N and a K, respectively. Despite this replacement, the enzyme retains high activity and moderate synergism in substrate binding (Kd/Km=2.3). We replaced the N62 by G or D and the K193 by G or R in Crassostrea AK, and also constructed the double mutants of N62G/K193G and N62D/K193R. All of the mutants retained 50-90% of the wild-type activity. In N62G and N62D mutants, the Kmarg for arginine binding was comparable to that of wild-type enzyme, but the Kdarg was increased 2-5-fold, resulting in a strong synergism (Kd/Km=4.9-11.3). On the other hand, in K193G and K193R mutants, the Kmarg was increased 4-fold, and synergism was lost almost completely (Kd/Km=1.0-1.4). The N62G/K193G double mutant showed similar characteristics to the K193G and K193R mutants. Another double mutant, N62D/K193R, similar to the amino acid pair in the wild-type enzyme, had characteristics similar to those of the wild-type enzyme. These results indicate that the amino acid residues 62 and 193 play the key role in mediating the synergism in substrate binding of oyster arginine kinase.     

Identification of the residues in the helix F/G loop important to catalytic function of membrane-bound prostacyclin synthase

A topological model of prostaglandin I(2) synthase (PGIS) was created by homology modeling. This model, along with site-specific antibodies and other topology studies, has suggested that the residue(s) within helix F/G loop of PGIS may be involved in forming the substrate access channel and located in a position that influences the membrane-bound PGIS catalytic function (1). To test this hypothesis, we have explored an approach to identify the residues of the helix F/G loop important to enzyme activity of the membrane-bound PGIS by a combination of 2-D NMR experiment and mutagenesis methods. Using the distance measured from the model as a guide, the helix F/G loop was mimicked in a synthetic peptide by introducing a spacer to maintain a distance of about 7 A between the N- and the C-termini (PGIS residues 208 and 230). The peptide was used to interact with the enzyme substrate analogue, U46619. High-resolution 2-D NMR experiments were performed to determine the contacts between the peptide and U46619. The interaction between the constrained F/G loop peptide and U46619 was confirmed by the observation of the conformational changes of the peptide and U46619 using the comparison of the cross-peaks between the NOESY spectra of U46619 with the peptide, without the peptide, and the peptide alone. Through the combination of the 2-D NMR experiments, completed (1)H NMR assignments of the F/G loop segment in the presence and absence of U46619 were obtained, and these data were used to predict the contact residues (Leu214 and Pro215) of the F/G loop with PGIS substrate. The predicted influence of residues on enzyme catalytic activity in membrane-bound environments was confirmed by the mutagenesis of the F/G loop residues of human PGIS. These observations support that the F/G loop is involved in forming the substrate access channel for membrane-bound PGIS and suggests that the NMR experiment-based mutagenesis approach may be applied to study structure and function relationships for other proteins.     

Three-dimensional structure and function study on the active region in the extracellular ligand-binding domain of human IL-6 receptor

In this study the three-dimensional (3-D) model of the ligand-binding domain (V106-P322) of human interleukin-6 receptor (hlL-6 R) was constructed by computer-guided ho-mology modeling technique using the crystal structure of the ligand-binding domain (K52-L251) of human growth hormone receptor (hGHR) as templet. Furthermore, the active binding region of the 3-D model of hlL-6R with the ligand (hlL-6) was predicted. In light of the structural characteristics of the active region, a hydrophobic pocket shielded by two hydrophilic residues (E115 and E505) of the region was identified by a combination of molecular modelling and the site-directed or double-site mutation of the twelve crucial residues in the ligand-binding domain of hIL-6R (V106-P322). We observed and analyzed the effects of these mutants on the spatial conformation of the pocket-like region of hlL-6 R. The results indicated that any site-directed mutation of the five Cys residues (four conservative Cys residues: Cyst 21, Cys132, Cys165, Cys1