Prostaglandin moieties that determine receptor binding specificity in the bovine corpus luteum.

This study provided a pharmacological evaluation of prostaglandin binding to bovine luteal plasma membrane. It was found that [3H]PGF2 alpha' [3H]PGE2' [3H]PGE1 and [3H]PGD2 all bound with high affinity to luteal plasma membrane but had different specificities. Binding of [3H]PGF2 alpha and [3H]PGD2 was inhibited by non-radioactive PGF2 alpha (IC50 values of 21 and 9 nmol l-1, respectively), PGD2 (35 and 21 nmol l-1), and PGE2 (223 and 81 nmol l-1), but not by PGE1 (> 10,000 and 5616 nmol l-1). In contrast, [3H]PGE1 was inhibited by non-radioactive PGE1 (14 nmol l-1) and PGE2 (7 nmol l-1), but minimally by PGD2 (2316 nmol l-1) and PGF2 alpha (595 nmol l-1). Binding of [3H]PGE2 was inhibited by all four prostaglandins, but slopes of the dissociation curves indicated two binding sites. Binding of [3H]PGE1 was inhibited, resulting in low IC50 values, by pharmacological agonists that are specific for EP3 receptor and possibly EP2 receptor. High affinity binding of [3H]PGF2 alpha required a C15 hydroxyl group and a C1 carboxylic acid that are present on all physiological prostaglandins. Specificity of binding for the FP receptor depended on the C9 hydroxyl group and the C5/C6 double bond. Alteration of the C11 position had little effect on affinity for the FP receptor. In conclusion, there is a luteal EP receptor with high affinity for PGE1' PGE2' agonists of EP3 receptors, and some agonists of EP2 receptors. The luteal FP receptor binds PGF2 alpha' PGD2 (high affinity), and PGE2 (moderate affinity) but not PGE1 due to affinity determination by the C9 and C5/C6 moieties, but not the C11 moiety.     

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.     

M&B 28,767: a potent anti-secretory and anti-ulcer PG analogue. A comparative study with 16, 16' dimethyl PGE2 methylester

M&B 28,767 [(+/-)11-deoxy-16-phenoxy-omega-tetranor PGE1] and 16, 16'-dimethyl PGE2 methylester (DMPG) were compared for their effects on gastric acid secretion (GAS) and gastric ulceration (GU), employing various laboratory models. In anaesthetised rats, GAS was stimulated by a continuous i.v. infusion of pentagastrin (30 micrograms/kg/h), and PG analogues were perfused through the stomach for 1 h. M&B 28,767 (3-15 micrograms/kg/h) and DMPG (3-60 micrograms/kg/h) reduced GAS in a dose-related manner, the ED50 values being 4 and 15 micrograms/kg/h respectively. In conscious rats possessing indwelling gastric cannulae, oral doses of M&B 28,767 (0.025-0.1 microgram/kg) and DMPG (0.50-1.0 microgram/kg) caused a prolonged inhibition of pentagastrin-stimulated GAS. M&B 28,767 was 17 times more potent than DMPG; the respective ED50 values were 0.036 and 0.6 microgram/kg. Indomethacin-induced ulceration in rats, was reduced by both M&B 28,767 and DMPG; the respective ED50 values being 3.0 and 0.8 micrograms/kg. Both compounds given orally increased gastrointestinal motility in mice; M&B 28,767 (1-3 mg/kg) and DMPG (0.1-0.3 mg/kg) caused diarrhoea, the former being about 0.1 times as potent as the latter. In another test, M&B 28,767 (0.5-5.0 mg/kg) and DMPG (10-40 micrograms/kg) overcame morphine-induced constipation in a dose-related manner, the respective ED50s being 0.9-1.4 mg/kg and 20-40 micrograms/kg. Thus, M&B 28,767 had a better profile of activity than DMPG as an antisecretory and antiulcer agent.     

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.     

A prostaglandin E receptor coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rabbit cortical collecting tubule cells

At different concentrations, prostaglandin E2 (PGE2) can either stimulate or inhibit cAMP formation in freshly isolated rabbit cortical collecting tubule (RCCT) cells, but in cultured RCCT cells PGE2 can only stimulate cAMP synthesis (Sonnenburg, W. K., and Smith W. L. (1989) J. Biol. Chem. 263, 6155-6160). Here, we report characteristics of [3H]PGE2 binding to membrane receptor preparations from both freshly isolated and cultured RCCT cells. [3H]PGE2 binding to membranes from freshly isolated RCCT cells was saturable and partially reversible. Equilibrium binding analyses indicated that in the absence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) there is a single class of PGE2 binding sites (KD = 4.2 +/- 0.4 nM; Bmax = 583 +/- 28 fmol/mg); in the presence of 100 microM GTP gamma S, there is also only one class of binding sites but with a somewhat lower KD = 1.2 +/- 0.5 nM (Bmax = 370 +/- 40 fmol/mg). This stimulatory effect of GTP gamma S was blocked by pretreatment of the freshly isolated RCCT cells with pertussis toxin. The relative affinities of prostanoids for the [3H]PGE2-binding site were determined to be 17,18,19,20-tetranor-16-phenoxy-PGE2-methylsulfonylamide (sulprostone) approximately PGE2 approximately PGE1 approximately 16,16-dimethyl-PGE2 greater than carbacyclin approximately PGF2 alpha greater than PGD2. This is the order of potency with which prostaglandins inhibit arginine vasopressin-induced cAMP formation in fresh RCCT cells. Interestingly, [3H]PGE2 binding to membranes from cultured cells, which, unlike fresh cells, fail to show an inhibitory response to PGE2, was only 10-20% of that observed with membranes from fresh cells; moreover, binding of [3H]PGE2 to membranes from cultured cells was neither stimulated by GTP gamma S nor inhibited by sulprostone. The prostanoid binding specificities and the unusual pertussis toxin-sensitive, stimulatory effect of GTP gamma S on binding of [3H]PGE2 to membranes from freshly isolated RCCT cells are characteristics shared by a Gi-linked PGE receptor from renal medulla (Watanabe, T., Umegaki, K., and Smith, W. L. (1986) J. Biol. Chem. 261, 14340-14349). Our results suggest that the [3H]PGE2 binding site of freshly isolated RCCT cells is the PGE receptor which is coupled to a Gi to attenuate arginine vasopressin-induced cAMP synthesis in the renal collecting tubule.     

Characterization of Adenosine Receptors in the PC 12 Pheochromocytoma Cell Line Using Radioligand Binding: Evidence for A-2 Selectivity

Examination of the binding characteristics of the adenosine agonist radioligands [3H]N6-cyclohexyladenosine [( 3H]CHA), [3H]cyclopentyladenosine [( 3H]CPA), and [3H]5'-N-ethylcarboxamido adenosine [( 3H]NECA) to membranes prepared from PC12 cells showed that the A-1-selective ligands (CHA and CPA) had minimal binding, which was not amenable to analysis using curve-fitting programs. However, [3H]NECA, a nonselective A-1/A-2 agonist, gave reproducible binding, which was enhanced by removal of endogenous adenosine, using the catabolic enzyme adenosine deaminase. This binding was of high affinity (KD = 4.7 nM) with limited capacity (263 fmol/mg of protein). Specific binding of [3H]NECA was unaffected by the presence of either CPA (50 nM) or MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 microM), a finding suggesting involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. Binding of [3H]NECA to PC12 cell membranes was stereo-selective, with the R isomer of N6-phenylisopropyladenosine (PIA) being approximately 12 times more active than S-PIA. The A-1-selective agonist CPA was a weak inhibitor of [3H]NECA binding (Ki = 251 nM). The rank order of activity of adenosine agonists in displacing specific [3H]NECA binding was NECA greater than or equal to 2-chloroadenosine greater than CHA greater than or equal to 5'-N-methylcarboxamido adenosine greater than or equal to R-PIA greater than CPA greater than S-PIA. Binding was also displaced by the marine adenosine agonist 1-methylisoguanosine and by a series of xanthine antagonists with the activity order being 1,3-dipropyl-8-(2-amino-4-chloro)phenylxanthine greater than 8-phenyltheophylline greater than 8-p-sulfophenyltheophylline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a prostacyclin receptor coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mouse mastocytoma P-815 cells.

A stable analogue of prostacyclin, iloprost, specifically bound to 30,000 x g pellet (the membrane fraction) prepared from mouse mastocytoma P-815 cells. The binding was dependent on time, temperature and pH, and absolutely required a divalent cation. The equilibrium dissociation constant and the maximal concentration of the binding site as determined by Scatchard plot analysis were 10.4 nM and 1.12 pmol/mg of protein, respectively. The Hill coefficient was 1.0, indicating a single entity of binding site and no cooperativity. The binding site was highly specific for iloprost among PGs tested (iloprost much greater than PGE1 greater than carbacyclin greater than PGE2). In contrast, the membrane fraction had the binding site specific for PGE2 and PGE1, which was distinct from the prostacyclin receptor. The dissociation of bound [3H]iloprost from the membrane fraction was specifically enhanced by guanine nucleotides. Furthermore, iloprost dose-dependently enhanced the activity of adenylate cyclase in a GTP-dependent manner. These results indicate that a specific prostacyclin receptor is coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mastocytoma cells.     

Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ--prostaglandin E2 dependent proliferation of growth plate chondrocytes

Prostaglandin E2 (PGE2) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE2 pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE2 receptors) is essential. We therefore examined the production of PGE2 in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE2 on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE2 receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE2 synthesis was determined by mass spectrometry, cell proliferation by DNA [3H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE2 into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE2-dependent proliferation. Exogenously added PGE2 stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE2 was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE2 release, which stimulates cell proliferation via the EP1 receptor.     

Prostaglandin E(2) receptors, EP2 and EP4, differentially modulate TNF-alpha and IL-6 production induced by lipopolysaccharide in mouse peritoneal neutrophils

The expression and function of prostaglandin (PG) E(2) receptors were examined in mouse neutrophils exudated into the peritoneal cavity by casein treatment. Expressions of the EP2 and EP4 receptors were detected in neutrophils by Northern blot, but those of EP1 and EP3 receptors were not detected by RT-PCR. EP2-selective agonist, ONO-AE1-259, and EP4-selective agonist, ONO-AE1-329, stimulated cAMP formation in the cells. PGE(2) affected the TNF-alpha and IL-6 production in lipopolysaccharide (LPS)-treated neutrophils; it suppressed the TNF-alpha production and enhanced the IL-6 production. The PGE(2) effects were mimicked by dibutyryl cAMP. This is the first study of the enhancement of IL-6 production by cAMP-elevating reagents in neutrophils. Using neutrophils from EP2- and EP4-deficient mice in combination with EP2- and EP4-selective agonists, it was found that the augmentation of IL-6 was mediated mainly by the EP2 receptor and the suppression of TNF-alpha by the EP4 receptor and partially by the EP2 receptor. These findings indicate that casein-induced peritoneal neutrophils express Gs-coupled PGE(2) receptors, EP2 and EP4, which might differentially regulate the LPS-induced production of TNF-alpha and IL-6.     

Decrease of prostaglandin E2 receptor binding is accompanied by reduced antilipolytic effects of prostaglandin E2 in isolated rat adipocytes

The effect of treatment of isolated rat adipocytes with prostaglandin E2 (PGE2) on subsequent [3H]PGE2 binding was studied. In addition, the antilipolytic effects of was studied. In addition, the antilipolytic effects of PGE2, adenosine, and insulin were studied in control and PGE2-treated adipocytes. Treatment of adipocytes with PGE2 (1 microM) decreased the binding of [3H]PGE2 by 61% (from 11.0 to 4.6 fmol/10(6) cells, P less than 0.005). Scatchard analysis of the binding data demonstrated that the decrease of PGE2 receptor binding was due to a decrease in the apparent number of PGE2 receptors while the apparent receptor affinity was unaltered. Reduction of the PGE2 receptor binding was specifically regulated inasmuch as structurally related compounds such as PGF2 alpha and arachidonic acid had only minor effects on subsequent [3H]PGE2 receptor binding. Reduction of the available receptor number was associated with a significant decrease in the antilipolytic effect of PGE2 on the isoproterenol-stimulated lipolysis (P less than 0.05). The maximal antilipolytic effect of PGE2 was decreased by 45%. Desensitization of the biological effect of PGE2 (antilipolysis) was only partially specifically regulated inasmuch as the antilipolytic compound phenylisopropyladenosine also had reduced antilipolytic effect in PGE2-treated cells. However, the antilipolytic effect of insulin was similar in control and PGE2-treated cells. It was found that the PGE2-induced decrease of [3H]PGE2 receptor binding may be due to a very tight coupling between the PGE2 molecule and its specific receptor. This tight coupling may then represent an occupancy of the receptor rather than a true loss of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the alpha 1-adrenergic receptor subtype in a smooth muscle cell line

We have identified in the DDT1 smooth muscle cell line a [3H]dihydroergocryptine-binding site having the characteristics of an alpha 1-adrenergic receptor. Specific binding of [3H]dihydroergocryptine to DDT1 cells grown either in monolayer or suspension culture was reversible, saturable, and of high affinity, and the binding site demonstrated stereoselectivity. [3H]Dihydroergocryptine dissociation constants of 1.4 +/- 0.2 nM and 1.4 +/- 0.3 nM were observed for suspension and monolayer cells, respectively. However, the concentration of binding sites in suspension-cultured cells (65,100 +/- 8,300 sites/cell) was significantly greater (p less than 0.001) than that found in monolayer cells (27,900 +/- 4,300 sites/cell). The order of agonist competition for the binding site was epinephrine (Ki = 0.92 +/- 0.32 microM) greater than or equal to norepinephrine (Ki = 2.2 +/- 1.0 microM) greater than isoproterenol (Ki = 137 +/- 17 microM), consistent with an alpha-adrenergic interaction. Results of competition experiments with specific antagonists prazosin (alpha 1-selective) or yohimbine (alpha 2-selective) and a computer modeling technique indicated that the alpha-adrenergic receptor of the DDT1 cell was predominantly (greater than 95%) the alpha 1-subtype.     

Solubilization and purification of the prostaglandin E2 receptor from cardiac sarcolemma.

A prostaglandin E2 (PGE2) receptor was solubilized and isolated from cardiac sarcolemma membranes. Its binding characteristics are almost identical to those of the membrane bound receptor. [3H]PGE2 binding to solubilized and membrane bound receptor was sensitive to elevated temperature and no binding was observed in the absence of NaCl. No significant effects of DTT, ATP, Mg2+, Ca2+ or of changes in buffer pH were observed on [3H]PGE2 binding to either solubilized or membrane-bound receptor. Unlabelled PGE1 displaced over 90% of [3H]PGE2 from the CHAPS-solubilized receptor. PGD2, PGI2, PGF2 alpha and 6-keto-PGF1 alpha were not effective in displacing [3H]PGE2 from the receptor. Scatchard analysis of [3H]PGE2 binding to CHAPS-solubilized receptor revealed the presence of two types of PGE2 binding sites with Kd of 0.33 +/- 0.05 nM and 3.00 +/- 0.27 nM and Bmax of 0.5 +/- 0.04 and 2.0 +/- 0.1 pmol/mg of protein. The functional PGE2 receptor was isolated from CHAPS-solubilized SL membrane using two independent methods: first by a WGA-Sepharose chromatography and second by sucrose gradient density centrifugation. Receptor isolated by these two methods bound [3H]PGE2. Unlabelled PGE1 and PGE2 displaced [3H]PGE2 from the purified receptor. Scatchard analysis of [3H]PGE2 binding to purified receptor revealed the presence of the two binding sites as observed for the membrane bound and CHAPS-solubilized receptor. SDS-polyacrylamide gel electrophoresis of the purified receptor fractions revealed the presence of a protein band of M(r) of approx. 100,000. This 100-kDa was photolabelled with [3H]azido-PGE2, a photoactive derivative of PGE2. We propose that this 100-kDa protein is a cardiac PGE2 receptor.     

In vitro pharmacological characterization of CJ-042794, a novel, potent, and selective prostaglandin EP(4) receptor antagonist

Activation of the prostaglandin E(2) (PGE(2)) EP(4) receptor, a G-protein-coupled receptor (GPCR), results in increases in intracellular cyclic AMP (cAMP) levels via stimulation of adenylate cyclase. Here we describe the in vitro pharmacological characterization of a novel EP(4) receptor antagonist, CJ-042794 (4-{(1S)-1-[({5-chloro-2-[(4-fluorophenyl)oxy]phenyl}carbonyl)amino]ethyl}benzoic acid). CJ-042794 inhibited [(3)H]-PGE(2) binding to the human EP(4) receptor with a mean pK(i) of 8.5, a binding affinity that was at least 200-fold more selective for the human EP(4) receptor than other human EP receptor subtypes (EP(1), EP(2), and EP(3)). CJ-042794 did not exhibit any remarkable binding to 65 additional proteins, including GPCRs, enzymes, and ion channels, suggesting that CJ-042794 is highly selective for the EP(4) receptor. CJ-042794 competitively inhibited PGE(2)-evoked elevations of intracellular cAMP levels in HEK293 cells overexpressing human EP(4) receptor with a mean pA(2) value of 8.6. PGE(2) inhibited the lipopolysaccharide (LPS)-induced production of tumor necrosis factor alpha (TNFalpha) in human whole blood (HWB); CJ-042794 reversed the inhibitory effects of PGE(2) on LPS-induced TNFalpha production in a concentration-dependent manner. These results suggest that CJ-042794, a novel, potent, and selective EP(4) receptor antagonist, has excellent pharmacological properties that make it a useful tool for exploring the physiological role of EP(4) receptors.     

Prostaglandin E1 receptor from mouse mastocytoma P-815 cells couples to 60 kDa GTP-binding protein.

The stable [3H]prostaglandin E1 (PGE1)-bound receptor, which couples to 60 kDa GTP-binding protein, from membranes of mouse mastocytoma P-815 cells has been purified and characterized. When the membranes were preincubated with [3H]PGE1 for 60 min at 37 degrees C, the dissociation of the ligand from the receptor was remarkably decreased, even in the presence of GTP gamma S. The stable [3H]PGE1-bound receptor complex was solubilized with 6% digitonin. The solubilized [3H]PGE1 receptor was eluted with [35S]GTP gamma S bindings activity from an Ultrogel AcA44 column. The fractions containing activities of both [3H]PGE1 and [35S]GTP gamma S bindings were further purified by column chromatographies on wheat germ agglutinin (WGA)-agarose and phenyl-Sepharose CL-4B. The partially purified [3H]PGE1-bound receptor was affinity-labeled with [14C]5'-p-fluorosulfonylbenzoylguanosine and a protein with a molecular mass of 60 kDa was detected. These results suggest that the ligand-bound PGE1 receptor of P-815 cells associates with a novel GTP-binding protein with a molecular mass of 60 kDa.     

Specific binding of the thromboxane A2 antagonist 13-azaprostanoic acid to human platelet membranes

In the present study we characterized the interaction between the thromboxane A2/prostaglandin H2 antagonist, trans-13-azaprostanoic acid (13-APA), and isolated human platelet membranes. In these studies, we developed a binding assay using trans [3H] 13-APA as the ligand. It was found that trans [3H] 13-APA specific binding was rapid, reversible, saturable and temperature dependent. Scatchard analysis of the binding data yielded a curvilinear plot which indicated the existence of two classes of binding sites: a high-affinity binding site with an estimated dissociation constant (Kd) of 100 nM; and a low-affinity binding site with an estimated Kd of 3.5 microM. At saturation, approximately 1 pmol/mg protein of [3H] 13-APA was bound to the high affinity site. In order to further characterize the nature of the [3H] 13-APA binding site, we evaluated competitive binding by cis 13-APA, cis 15-APA, prostaglandin F2 alpha, U46619, 6-ketoprostaglandin F1 alpha and thromboxane B2. It was found that the [3H] 13-APA binding site was stereospecific and structurally specific. Thus, the cis isomer of 13-APA exhibited substantially reduced affinity for binding. Furthermore, the prostaglandin derivatives, thromboxane B2 and 6-ketoprostaglandin F1 alpha, which do not possess biological activity, also did not compete for [3H] 13-APA binding. On the other hand, U46619 which acts as a thromboxane A2/prostaglandin H2 mimetic, and prostaglandin F2 alpha which acts as a thromboxane A2/prostaglandin H2 antagonist, both effectively competed for [3H] 13-APA binding. These findings indicate that trans 13-APA binds to a specific site on the platelet membrane which presumably represents the thromboxane A2/prostaglandin H2 receptor.     

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.     

Characterization of monoclonal antibodies to the beta-adrenergic antagonist alprenolol as models of the receptor binding site

Antibodies to receptor ligands have been valuable in understanding the nature of receptor-ligand interactions. We have developed four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol by immunizing A/J mice with (-)-alprenolol coupled to keyhole limpet hemocyanin. The antisera from these mice displayed specific [3H]dihydroalprenolol ([3H]DHA) binding that was inhibited by alprenolol, propranolol, and isoproterenol. Somatic cell fusion of spleen cells from the immunized mice to SP2/0 myeloma cells, followed by limited dilution subcloning, resulted in the isolation of four hybridomas (1B7, 5B7, 5D9, and 2G9) demonstrating three different classes of ligand binding characteristics. 1B7 had the highest binding affinity for antagonists based on Scatchard analysis (Kd [125I]- CYP = 1.4 X 10(-10) M; Kd [3H]DHA = 6.5 X 10(-9) M), and was the only antibody to demonstrate agonist-inhibition of [3H]DHA binding. Ki values computed from competitive inhibition curves of [3H]DHA binding to 1B7 resulted in a rank order of potency similar to that of beta-2-adrenergic receptors: (-)-propranolol greater than acebutolol amine greater than isoproterenol greater than (+)-propranolol greater than epinephrine greater than norepinephrine. 5B7 and 5D9 exemplified a second class of antibody. This pair had lower antagonist binding affinities (Kd [3H]DHA = 2 X 10(-8) M and 2.5 X 10(-7) M, respectively) and was stereoselective in binding receptor antagonists: (-)-propranolol greater than (+)-propranolol greater than acebutolol amine. Agonist inhibition of [3H]DHA binding to these antibodies could only be observed at very high concentrations (greater than 10(-4) M agonist), and was not dose-dependent. Finally, the class of anti-alprenolol monoclonal antibodies represented by 2G9 had the lowest antagonist binding affinity of all (IC50 alprenolol = 1 X 10(-5) M), did not demonstrate ligand stereoselectivity, and did not recognize agonists. We propose that antibodies raised against beta-adrenergic receptor ligands demonstrating stereoselective agonist binding will also demonstrate high affinity antagonist binding, and that they will closely parallel the binding characteristics of the receptor. According to this "agonist best-fit hypothesis," anti-idiotypic antibodies raised against the binding site of these idiotypes might contain true mirror images of the beta-adrenergic receptor binding site.     

Identification of Adenosine Receptor in Rat Pineal Gland: Evidence for A-2 Selectivity

We have examined the binding of the adenosine agonist radioligands [3H]cyclohexyladenosine [( 3H]CHA), R-N6-[3H]phenylisopropyladenosine [( 3H]R-PIA), and 5'-N-ethylcarboxamido[3H]adenosine [( 3H]NECA) to membranes prepared from rat pineal gland. The results showed that the A-1-selective ligands (CHA and R-PIA) had less than or equal to 10% specific binding. By contrast, [3H]NECA, a nonselective A-1/A-2 ligand, gave 72% specific binding of the total binding. This specific binding was insensitive to cyclopentyladenosine (50 nM) or R-PIA (50 microM). To characterize this binding, we used the N-ethylmaleimide pretreatment method. Under these conditions, this binding was of high affinity with a KD of 51 +/- 10 nM and an apparent Bmax of 1,060 +/- 239 fmol/mg of protein. Specific binding was unaffected by the presence of MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 microM) (-25%), a result suggesting the involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. The rank of activity of adenosine analogues in displacing specific [3H]NECA binding was NECA greater than 2-chloroadenosine greater than S-adenosyl-L-homocysteine greater than CHA. Binding was also displaced by 3-isobutyl-1-methylxanthine (IC50 = 23.6 microM). These findings are consistent with the selective labeling by [3H]NECA of an A-2-type adenosine receptor in rat pineal membranes.     

Characterization and Regulation of β1-Adrenergic Receptors in a Human Neuroepithelioma Cell Line

Intact human neuroepithelioma SK-N-MC cells bound the beta-adrenergic antagonist (-)-[3H]-CGP 12177 with a KD of 0.13 nM and a Bmax of 17,500 sites/cell. When the cells were exposed to beta-adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol much greater than norepinephrine greater than epinephrine, which is indicative of a beta 1-subtype receptor. Membranes prepared from the cells bound (-)-3-[125I]iodocyanopindolol with a KD of 11.5 pM. Inhibition of agonist-stimulated cyclic AMP production and competition binding experiments indicated that the beta 1-selective antagonists CGP 20712A and ICI 89,406 were much more potent than the beta 2-selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only beta 1-adrenergic receptors. Northern blot analysis of SK-N-MC mRNA using cDNA probes for the beta 1- and beta 2-adrenergic receptors revealed the presence of a very strong beta 1-adrenergic receptor mRNA signal, while under the same conditions no beta 2-adrenergic receptor mRNA was observed. Thus, SK-N-MC cells appear to express a pure population of beta 1-adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down-regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.