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

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

Desensitization of platelet thromboxane A2/prostaglandin H2 receptors by the mimetic U46619

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) activate platelets through membrane receptors. This study sought to determine if changes occur in the TXA2/PGH2 receptor during its desensitization induced by exposure to its agonist 11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619). Washed human platelets were incubated for 30 min with U46619 in the presence of an antiaggregatory agent, Iloprost. The platelets were washed and the aggregation response to U46619 was determined. The EC50 increased from 372 +/- 94 nM in the control group to 826 +/- 143 nM for the U46619-pretreated group (n = 7). This desensitization was specific inasmuch as the aggregation responses to thrombin and the calcium ionophore A23187 were not affected by U46619 pretreatment. Desensitization was accompanied by a decrease in the number of binding sites for [3H]U46619 from 789 +/- 189 in the control to 386 +/- 120 sites per platelet in the U46619-treated group (n = 5) and a decrease in the number of binding sites for the TXA2/PGH2 receptor antagonist, [125I]9,11-dimethylmethano-11,12-methano-16(3-iodo-4-hydroxyphenyl )-13,14- dihydro-13-aza-15-alpha beta-omega-tetranor-TXA2 from 3988 +/- 957 to 2443 +/- 553 (n = 8). The Kd for U46619 was 37 +/- 10 nM in the control group and 23 +/- 11 nM for the U46619-treated group (n = 5). The Kd for I-9,11-dimethylmethano-11,12-methano-16(3-iodo-4-hydroxyphenyl)-13 ,14-dihydro- 13-aza-15-alpha beta-omega-tetranor-TXA2 changed from 58 +/- 12 nM in the control to 44 +/- 9 nM in the U46619-treated group (n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct platelet thromboxane A2/prostaglandin H2 receptor subtypes. A radioligand binding study of human platelets.

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) may aggregate platelets via a common membrane receptor(s). To further characterize this receptor, binding of the radiolabeled TXA2/PGH2 mimetic [125I]BOP to washed human platelets (WP) was investigated. [125I]BOP was competitively displaced from its platelet binding site by stable TXA2/PGH2 analogues. Competition curves were shallow with Hill coefficients of -0.73 +/- 0.05 (P less than 0.001 different from unity) (90 +/- 1% specific binding). Scatchard plots were curvilinear and most consistent with two binding sites; a high-affinity site with Kd of 234 +/- 103 pM, Bmax of 0.7 +/- 0.3 pM/mg protein (180 +/- 87 sites/WP), and a lower affinity site with Kd of 2.31 +/- 0.86 nM, Bmax of 2.2 +/- 0.3 pM/mg protein (666 +/- 65 sites/WP). [125I]BOP association and dissociation kinetics gave a Kd of 157 pM without evidence of negative cooperativity. The EC50 for I-BOP-induced initial Ca2+ increase was 209 +/- 24 pM, shape change was 263 +/- 65 pM, and aggregation was 4.4 +/- 0.5 nM. Parallel binding studies using the TXA2/PGH2 receptor antagonist [125I]PTA-OH showed a single binding site. The rank order for TXA2/PGH2 analogues to displace [125I]PTA-OH was identical to that for [125I]BOP. These studies indicate that [125I]BOP binds to two distinct sites on human platelets that may represent platelet TXA2/PGH2 receptor subtypes. The close correlation of IC50 values for I-BOP-induced platelet shape change and aggregation with the two Kds for [125I]BOP binding suggests that these platelet responses may be independently mediated by the two putative receptors.     

Human erythroleukemia cells express functional thromboxane A2/prostaglandin H2 receptors

The human erythroleukemia (HEL) cell line is a cultured hematopoietic cell line reported to express platelet membrane glycoproteins and alpha-2 adrenergic receptors. The present studies were designed to determine if functional thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptors exist in HEL cells. Radioligand binding assays were performed using [125I]PTA-OH, a TXA2/PGH2 receptor antagonist. Scatchard analysis revealed one class of binding sites for 1-PTA-OH with a Kd = 122 +/- 18 nM and maximum binding = 1.7 +/- 0.3 x 10(5) sites/cell. Competition for [125I]PTA-OH binding with the TXA2/PGH2 receptor agonists SQ26655 and U46619 revealed one class of binding sites for SQ26655 with a Kd = 17 nM and two classes of binding sites for for U46619 with a Kd = 45 nM for the high-affinity site and a Kd = 450 nM for the low-affinity site. Competition for [125I]PTA-OH by the steroisomeric TXA2/PGH2 receptor antagonists L657925 and L657926 revealed two classes of binding sites for the more potent L657925 with a Kd = 8 nM for the high-affinity site and a Kd = 400 nM for the low-affinity site whereas L657926 bound to one class of sites with a Kd = 380 nM. Stimulation of the TXA2/PGH2 receptor by SQ26655 and U46619 resulted in concentration-dependent increases in [Ca++], as measured by FURA-2 fluorescence, with EC50 values of 28 +/- 2 and 149 +/- 32 nM, respectively. I-PTA-OH, L657925 and L657926 antagonized this response to U46619 with IC50 values similar in rank potency to that seen in the binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homologous desensitization of human platelet thromboxane A2/prostaglandin H2 receptors.

Desensitization of platelet thromboxane (TX)A2/prostaglandin (PG)H2 receptors was induced by incubating platelet-rich plasma with the stable PGH2 analog 11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) (1 microM). Iloprost, a stable prostacyclin analog, was included in the incubation to prevent platelet activation. 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-heptenoic acid (I-BOP), was used to induce platelet aggregation, shape change and increases in intracellular free calcium. The EC50 values for I-BOP-induced rise in intracellular free calcium (control = 10.2 +/- 1.5 nM; desensitized = 79.4 +/- 22.4 nM, n = 6, P less than .05), aggregation (control = 15.8 +/- 2.4 nM; desensitized = 51.7 +/- 11.9 nM; P less than .05, n = 5) and shape change (control = 172 +/- 37 pM; desensitized = 350 +/- 60 pM; P less than .05, n = 7) were increased by the preincubation with U46619. Aggregation responses to thrombin and the calcium ionophore, ionomycin, were unaltered by the preincubation with U46619. Equilibrium binding studies at pH 7.4 revealed a decrease in the number of binding sites for the receptor 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) (control = 3246 +/- 509 sites/platelet, desensitized = 2198 +/- 324 sites/platelet, n = 6, P less than .05) without a change in affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in vascular responsiveness to a thromboxane mimetic in endotoxin tolerance.

Our previous studies have demonstrated that peritoneal macrophages obtained from endotoxin-tolerant rats exhibit altered cellular activation by endotoxin, possibly involving changes in guanine nucleotide regulatory (G) protein-coupled signal transduction pathways. Endotoxin-tolerant rats also exhibit cross tolerance and altered hemodynamic responses to thromboxane (Tx)A2 mimetics, suggesting potential changes in vascular responsiveness. We tested the hypothesis that endotoxin tolerance results in vascular hyporesponsiveness to a TxA2 mimetic via alterations in the TxA2 receptor, G protein function, and/or second messenger production. Rats were rendered endotoxin tolerant by increasing sublethal consecutive doses of Salmonella enteritidis endotoxin (100 to 5000 micrograms/kg, i.p.) for 4 days. The animals were sacrificed 2 days after the final dose of endotoxin for removal of aortas. Contractile responses of aortic rings to U46619, a TxA2 agonist, were assessed in control and tolerant rats. The EC50 values for U46619 were 14.8 +/- 6.6 nM and 32.3 +/- 3.1 nM (n = 5-7), (P < 0.05) for control and tolerant rats, respectively. Crude membranes were prepared from aortas of control and tolerant rats, and binding of I-BOP TxA2/PGH2 receptor agonist, [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-heptenoic acid (I-BOP), a TxA2 agonist, was assessed by Scatchard analysis. I-BOP binding to the TxA2 receptor was saturable and revealed a single class of TxA2 receptors for both groups. There was no significant difference in control (n = 7) compared with tolerant (n = 5) Kd values (2.1 +/- 0.2 vs. 2.4 +/- 0.9 nM, respectively), or Bmax (31 +/- 6 vs. 28 +/- 12 fmol/mg protein, respectively). To assess potential changes in G protein function, aortic membrane GTpase activity was determined. GTPase activity in tolerant membranes was significantly reduced (P < 0.05) compared with control membranes (309 +/- 23 (n = 5) vs. 440 +/- 32 (n = 7) pmol/mg/protein/min, respectively). However, U46619-stimulated phosphoinositide production was similar in vascular tissue from control and tolerant rats. These observations suggest that the decreased contractile response to TxA2 mimetics in endotoxin tolerance does not result from a change in receptor number, affinity of TxA2 receptors, or changes in phosphatidylinositol metabolism but is associated with decreased vascular G protein function.     

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.     

Mechanism for homologous downregulation of thromboxane A2 receptors in cultured human chronic myelogenous leukemia (K562) cells.

Desensitization of the biologic response to thromboxane A2 (TXA2) mimetics has been observed ex vivo in human platelets due to TXA2 receptor uncoupling and downregulation. To define more clearly the mechanisms of homologous TXA2 receptor downregulation, the effects of the TXA2 mimetics U44069 ([15S)-hydroxy-9,11- (epoxymethano) prostadienoic acid] and I-BOP ([1S-(1 alpha 2 beta(5Z),3 alpha(1E,3S),4 alpha))-7-[3-(3-hydroxy-4- (p-iodophenoxy)-1-butenyl)-7-oxabicyclo[2.2.1]heptan-2-yl]-5 -heptenoic acid) on receptor-mediated calcium fluxes and on ligand binding to TXA2 receptors were studied in the K562 cultured human leukemic cell line which possesses many platelet characteristics. Incubation with U44069 resulted in a time-dependent decrease in the amplitude of TXA2 receptor-mediated intracellular free calcium transients. Under the same conditions, binding of [125I] BOP demonstrated a concurrent loss of K562 plasma membrane binding sites to approximately one-third the original number. The loss of [125I]BOP binding was prevented by coincubation with the TXA2 antagonist SQ29548 ([1S-1 alpha,2 beta (5Z), 3 beta,4 alpha]-7- (3-[2-[phenylamino)-carbonyl) hydrazino) methyl)-7-oxabicyclo-(2.2.1)- heptan-2-yl)-5-heptenoic acid]) and was reversed upon removal of U44069 from the culture medium. SQ29548 alone had no affect on receptor density or affinity. Loss of surface receptors was demonstrated to be mediated by agonist-occupied receptor internalization which was inhibited by incubation at 4 degrees C and did not occur with antagonist occupation. The results indicate that homologous downregulation of TXA2 receptors in K562 cells occurs by agonist-mediated active internalization of plasma membrane TXA2 receptors.     

Castration reduces platelet thromboxane A2 receptor density and aggregability

BACKGROUND: Exogenously administered testosterone upregulates platelet thromboxane A2 (TXA2) receptors and increases aggregation response to thromboxane mimetics in healthy male volunteers. However, the biological impact of endogenous testosterone on platelet TXA2 receptor expression, especially in older men at risk of coronary artery disease, is unclear. AIM: To investigate the impact of reduction in circulating testosterone on platelet TXA2 receptor expression in older men. DESIGN: Cross-sectional case-control study. METHODS: We studied surgically and/or medically castrated men with prostate cancer (group A, n = 8, aged 71 +/- 8 years) and age-matched, uncastrated urology patients (group B, n = 7, aged 67 +/- 9 years). Plasma testosterone was measured by radioimmunoassay. Platelet TXA2 receptor expression was assessed by radioligand binding studies using radioactive 125I-BOP. Platelet aggregation responses to TXA2-mimetic I-BOP, and to thrombin, were also studied. RESULTS: Group A had significantly lower plasma testosterone than group B (16 +/- 5 ng/dl vs. 308 +/- 47 ng/dl, p<0.001). Platelet TXA2 receptor density (B(max)) but not affinity (K(d)) was lower in group A (0.50 +/- 0.12 vs. 1.01 +/- 0.17 pmol/mg protein, p = 0.03). Maximum platelet aggregation response to I-BOP (E(max)), but not sensitivity (EC50) was lower in group A (53 +/- 2% vs. 63 +/- 2%, p = 0.003 ANOVA). In vitro, high concentrations of hydroxyflutamide (100 microM) competitively inhibited U46619-induced platelet aggregation in washed platelets, without affecting the binding of 125I-BOP to platelet TXA2 receptors. DISCUSSION: Endogenous testosterone regulates platelet TXA2 receptor B(max) and the E(max) aggregation response to thromboxane mimetic I-BOP. Blockade of androgen receptors or inhibition of testosterone production may reduce platelet aggregation responses. Preliminary evidence suggests the presence of functional androgen receptors on human platelets, which may regulate TXA2 receptor expression.     

The thromboxane receptor antagonist PBT-3, a hepoxilin stable analog, selectively antagonizes the TPalpha isoform in transfected COS-7 cells

The hepoxilin analog PBT-3 [10(S)-hydroxy-11,12-cyclopropyleicosa-5Z,8Z,14Z-trienoic acid methyl ester] was previously shown to inhibit the aggregation of human platelets and to antagonize the binding of the thromboxane receptor agonist I-BOP [[1S-[1alpha,2alpha (Z),3beta(1E,3S*),4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid] in human platelets (Pace-Asciak et al., 2002). We show herein that PBT-3 inhibits, to different degrees, binding of the TP receptor antagonist [3H]SQ 29,548 [[1S-[1alpha,2alpha (Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2. 1]hept-2-yl]-5-heptenoic acid], to the TP receptor isoforms in TPalpha- and TPbeta-transfected COS-7 cells. These isoforms possess a different tail length, the alpha being shorter than the beta isoform. In contrast, SQ 29,548 shows no selection for the two TP isoforms. The IC50 value for PBT-3 = 2.0 +/- 0.3 x 10-7 M was observed for TPalpha, whereas this was one-sixth less active on the TPbeta isoform (IC50 = 1.2 +/- 0.2 x 10-6 M), suggesting selectivity for the TPalpha isoform. To investigate whether the tail contributes to the difference in competition binding by PBT-3, we investigated the tailless TP isoform expressed in transfected COS-7 cells. Its IC50 was similar to that of the TPalpha isoform. In additional studies, we investigated the effect of PBT-3 on the collagen and I-BOP evoked intracellular calcium release and on the collagen and I-BOP evoked phosphorylation of pleckstrin. PBT-3 blocked both pathways further demonstrating its TP receptor antagonist activity. These results demonstrate that the action of PBT-3 in inhibiting platelet aggregation is mediated via inhibition of the TPalpha isoform of the thromboxane receptor and that the tail may play an important role in recognition of this TP receptor antagonist.     

Characterization of [5,6-3H]SQ 29,548 as a high affinity radioligand, binding to thromboxane A2/prostaglandin H2-receptors in human platelets

The binding of 5,6-3H(1S-[1 alpha, 2 beta(5Z), 3 beta, 4 alpha])-7-[3-([2-[(phenyl amino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2-yl]-5- heptenoic acid to receptors in human washed platelets (WP) and platelet membranes (PM) was characterized with regard to kinetics, saturability and competitive inhibition by putative thromboxane A2/prostaglandin H2 (TP)-receptor ligands. Specific binding of [3H]SQ 29,548 routinely amounted to 90 to 97% of total binding. The rate of association was 1.6 x 10(7) and 2.5 x 10(7) M-1 x min-1 in WP and PM, respectively. The corresponding rate of dissociation was 0.07 and 0.12 min-1, resulting in dissociation constants of 4.1 and 5.8 nM in WP and PM, respectively. Saturable binding to a single class of receptors indicated a receptor density of 2633 fmol/mg of protein in WP (1394 receptors/platelet; kd, 4.5 nM) and 1466 fmol/mg of protein in PM (kd, 11.3 nM). Specific binding of [3H]SQ 29,548 was inhibited by five antagonists (high/low affinity kd values in nanomolar), SQ 29,548 (WP, 5.2; PM, 7.3), SQ 28,668 (WP, 32; PM, 73), SQ 30,741 (WP, 28; PM, 50), BM 13,177 (WP, 140; PM, 4834) and BM 13,505 (WP, 5/379; PM, 11). Two agonists, U 44069 and U 46619, inhibited the binding in a biphasic manner, indicating binding to two receptor sites (approximately 20/80%) with kd values of 4/72 and 4/170 nM, respectively, in WP and 7/136 and 19/502 nM, respectively in PM. The demonstrated high affinity binding of [3H]SQ 29,548 to human platelet TP-receptors should make this radioligand a suitable and potentially useful tool in future studies of function, structure and regulation of TP-receptors.     

Identification of a high-affinity peripheral-type benzodiazepine binding site in rat aortic smooth muscle membranes

The existence of a benzodiazepine binding site in rat aortic smooth muscle membranes was explored employing [3H]Ro5-4864 as radioligand. The binding site was concentrated in the mitochondrial fraction enriched with cytochrome c oxidase and semicarbazide-insensitive monoamine oxidase. [3H]Ro5-4864 binds to the membranes in the mitochondrial fraction with high affinity. The dissociation constant (KD) determined by saturation binding was 2.8 +/- 0.7 nM (n = 5). The association rate constant (k1) was 4.7 +/- 0.8 x 10(6) M1 min-1, and the dissociation rate constant (k-1) was 0.028 +/- 0.005 min-1 (n = 3). The kinetically determined KD was 6.0 +/- 0.8 nM (n = 3) at 0.5 nM [3H]Ro5-4864. The density of binding determined from saturation binding experiments was 14.0 +/- 1.2 pmol/mg protein (n = 5). The Hill coefficient of binding was 0.94 +/- 0.02 (n = 5) indicating that [3H] Ro5-4864 binds to a single site. The [3H]Ro5-4864 binding was inhibited by Ro5-4864 (Ki = 6.1 +/- 1.9 nM), PK 11195 (Ki = 8.9 +/- 1.8 nM), diazepam (Ki = 87.3 +/- 3.4 nM), flunitrazepam (Ki = 94.6 +/- 1.8 nM), clonazepam (Ki = 6.3 +/- 1.3 microM) and Ro15-1788 (Ki = 16.8 +/- 1.5 microM). The rank order of potency of the competitive inhibition of [3H]Ro5-4864 binding (Ro5-4864 = PK 11195 greater than diazepam = flunitrazepam much greater than clonazepam greater than Ro15-1788) is characteristic of the peripheral-type benzodiazepine binding site. The data indicate an abundant high affinity peripheral-type benzodiazepine binding site of unknown function in rat aortic smooth muscle cells.     

Platelet receptor-mediated factor X activation by factor IXa. High-affinity factor IXa receptors induced by factor VIII are deficient on platelets in Scott syndrome.

We have studied factor IXa binding and factor X activation with normal platelets and with platelets obtained from a patient with a bleeding disorder and an isolated deficiency of platelet procoagulant activity termed Scott syndrome. In the absence of factor VIIIa and factor X, normal, thrombin-treated platelets exposed 560 +/- 35 sites for factor IXa with a Kd of 2.75 +/- 0.27 mM, compared with 461 +/- 60 sites per patient platelet with Kd of 3.2 +/- 0.33 nM. The addition of factor VIIIa and factor X resulted in a decrease in the Kd for normal platelets to 0.68 nM but had no effect on the Kd for patient platelets. The concentrations of factor IXa required for half-maximal rates of factor X activation for normal (0.52 nM) and patient platelets (2.5 nM) were similar to those determined from equilibrium binding studies. Kinetic parameters for factor X activation by factor IXa showed that the Km and Kcat were identical for normal and patient platelets in the absence of factor VIIIa. In the presence of factor VIIIa, and kcat for patient platelets (163 min-1) was only 33% of that for normal platelets (491 min-1): This result can be explained by the difference in affinity for factor IXa between normal and patient platelets in the presence of factor VIIIa, suggesting impaired factor VIIIa binding to Scott syndrome platelets.     

Muscarinic cholinergic ligand binding to intact mouse pituitary tumor cells (AtT-20/D16-16) coupling with two biochemical effectors: adenylate cyclase and phosphatidylinositol turnover

(-)-[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors on intact mouse pituitary tumor cells (AtT-20/D16-16) was characterized in an attempt to correlate radioligand binding properties with receptor-coupled biochemical responses. Performing rinse time studies for 2 hr produced a remarkably improved ratio of specific/total (+)-[3H]QNB binding (85%). Kinetic experiments yielded association (k+1) and dissociation (k-1) rate constants of 2.2 X 10(8) M-1 min-1 and 6.8 X 10(-3) min-1, respectively. Receptor occupancy curves demonstrated a uniform population of specific, saturable (-)-[3H]QNB binding sites with a Hill coefficient equal to 1.0 and an apparent dissociation constant (Kd) equal to 34 pM under our conditions. Stereoselectivity was observed with the enantiomers (dexetimide and levetimide) of benzetimide (a factor of 4300). Concentrations of carbachol that produced a half-maximal inhibition of cyclic AMP formation and a concentration of carbachol for producing half-maximal stimulation of phosphatidylinositol turnover in the intact cells were 0.45 and 170 microM, respectively. Schild analysis revealed that pirenzepine, a nonclassical muscarinic antagonist, had a 40-fold greater affinity for reversing carbachol-stimulated phosphatidylinositol turnover (inhibition constant or Ki = 7 nM), compared to its antagonism of the carbachol-mediated inhibition of isoproterenol-stimulated cyclic AMP formation (Ki = 280 nM). Interestingly, pirenzepine inhibited (-)-[3H]QNB binding with a Ki value of 72 nM. In contrast, atropine was nearly equipotent (Ki = 0.3-0.5 nM) in binding studies and in both effector systems.     

3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H]CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain

The cyclic, conformationally restricted octapeptide [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H]CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k+1) of 1.25 x 10(8) M-1 min-1 and 2.49 x 10(8) M-1 min-1 at 25 and 37 degrees C, respectively, were obtained, whereas dissociation rates (k-1) at the same temperatures were 1.93 x 10(-2) min-1 and 1.03 x 10(-1) min-1 at 25 and 37 degrees C, respectively. Saturation isotherms of [3H]CTOP binding to rat brain membranes gave apparent Kd values of 0.16 and 0.41 nM at 25 and 37 degrees C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37 degrees C, respectively. [3H]CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on [3H]CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide [3H]-[D-Pen2,D-Pen5]enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific [3H]CTOP binding was inhibited by a large number of opioid or opiate ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular heterogeneity of leukotriene receptors: correlation of smooth muscle contraction and radioligand binding in guinea-pig lung

The [3H]leukotriene C4 ([3H]LTC4) and [3H]leukotriene D4 ([3H] LTD4) specific binding sites in guinea-pig lung membranes were characterized and correlated with smooth muscle contractile activities of a series of LTC-, D- and E-type analogs. [3H]LTC4 bound to the specific sites with high affinity (dissociation constant Kd = 15 +/- 5 nM), saturable capacity (maximum binding = 68 +/- 15 pmol/mg of membrane protein), stereoselectivity and specificity. The [3H]LTC4 specific binding sites were detected in the membranes isolated from leukotriene sensitive (e.g., lung and heart) or insensitive (e.g., brain and red blood cells) tissues. [3H] LTD4 also bound to specific sites with high affinity (Kd = 0.20 +/- 0.05 nM), low capacity (maximum binding = 1.1 +/- 0.2 pmol/mg of membrane protein) stereoselectivity and specificity. The [3H] LTD4 specific binding sites were detected in the membranes isolated from lung and trachea. [3H]LTC4 specific binding was inhibited by treatment of the membranes with the sulfhydryl alkylating agent N-ethylmaleimide. [3H]LTD4 specific binding was more sensitive to heat treatment and p-hydroxymercuribenzoate than the [3H]LTC4 specific binding. Radioligand competition activities of the LTD- and LTE-type analogs correlated well with the agonist and antagonist smooth muscle contractile activities. In contrast, the radioligand competition activity of the LTC-type analogs did not correlate with smooth muscle contractile activities. These results indicate that the [3H]LTC4 and [3H]LTD4 specific binding sites in guinea-pig lung membranes are chemically and physically distinct. The [3H]LTD4 specific binding sites represent physiologically and pharmacologically important receptors, and the smooth muscle contraction induced by LTD-, and possible LTE-, type analogs are mediated through the LTD4 receptors.     

3H]TA-3090, a selective benzothiazepine-type calcium channel receptor antagonist: in vitro characterization

Binding of the new benzothiazepine calcium channel blocker, (+)-(2S,3S)-3-acetoxy-8-chloro-5-(2-(dimethylamino)ethyl)-2,3-dihydro-2- (4- methoxyphenyl)-1,5-benzothiazepine-4-(5H)-one maleate, [3H]TA-3090), was characterized and its specificity for rat myocardial benzothiazepine receptors described. Scatchard plots and nonlinear regression analysis of specific [3H]TA-3090 binding best fit a one-site binding model (Kd = 8.8 +/- 2.7 nM, Bmax = 132 +/- 38 fmol/mg protein). Kinetically derived affinity constants were in close agreement (Kd = 7.86 nM) with those obtained from analysis of equilibrium binding data. In comparison, under identical conditions [3H]diltiazem exhibited a Kd of 38 nM and Bmax, 106 fmol/mg protein. Specific binding was saturable, reversible and stereoselective (d-cis-TA-3090 Ki = 14 nM; 1-cis-TA-3090 Ki = 2700 nM). Competitions for [3H]TA-3090 binding were conducted with nifedipine, propranolol, prazosin, quinuclidinyl benzilate, verapamil and yohimbine. Only the calcium channel blockers nifedipine and verapamil inhibited specific [3H]TA-3090 binding. Nifedipine could maximally inhibit only 52% of specifically bound [3H]TA-3090 at 10 microM. In contrast, however, 10 microM verapamil completely inhibited specific radioligand binding (Ki = 93 +/- 28 nM) but with six times less efficacy than TA-3090. Thus, these data demonstrate that [3H]TA-3090 is a potent radioligand selective for the benzothiazepine binding site and is consistent with the hypothesis that [3H]TA-3090 interacts with a myocardial benzothiazepine receptor site.     

Beta-1 receptor is the predominant beta-adrenoreceptor on rat brown adipose tissue

A new in vitro radioligand binding assay is described for brown adipose tissue using the beta adrenergic antagonist [3H]CGP 12177 (4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazol-2-one). Binding was saturable and stereoselectively inhibited by propranolol. There was 60 to 80% specific binding using either 30 microM l-isoproterenol or 10 microM l-propranolol to define nonsaturable binding. [3H]CGP 12177 was bound to partially purified membranes from collagenase-separated brown adipocytes with a Kd of 0.84 nM, as determined from kinetic studies, and 1.24 +/- 0.13 nM as found by equilibrium binding studies; maximum binding was 14.2 +/- 0.9 fmol/mg of protein. Membranes from whole-pad homogenates had a similar Kd of 1.17 +/- 0.14 nM but twice the maximum number of binding sites (28.5 +/- 4.4 fmol/mg of protein). Intact brown adipocytes had a Kd of 0.55 nM and a maximum binding of 29.4 +/- 1.5 fmol X 10(-6)/cell or 17,700 sites per cell. Competitive binding studies showed about 80% of the binding sites to be of the beta-1 and 20% of the beta-2 subtype. The pA2 values derived from inhibition of isoproterenol-stimulated in vitro oxygen consumption in intact brown adipocytes by the beta-1 selective antagonist metoprolol and beta-2 selective lCl 118551 were in close agreement with their respective K1 values at the beta-1 receptor as derived from competitive binding studies. These data strongly suggest that the beta-1 adrenoreceptor on brown adipose tissue is primarily responsible for the initiation of thermogenesis in this tissue.     

D-Pen2,4'-125I-Phe4,D-Pen5]enkephalin: a selective high affinity radioligand for delta opioid receptors with exceptional specific activity.

[D-Pen2,4'-125I-Phe4,D-Pen5]enkephalin ([125I]DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. [125I]DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 +/- 67 pM and a receptor density (Bmax) value of 36.4 +/- 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM [125I] DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 +/- 0.88 X 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM [3H DPDPE and 0.8 nM [3H] [D-Pen2,4'-Cl-Phe4, D-Pen5]enkephalin, respectively. The dissociation rate of [125I]DPDPE (0.917 +/- 0.117 X 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of [125I]DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes [125I]DPDPE a valuable new radioligand for studies of delta opioid receptors.