Characteristics of endothelin receptors in the central nervous system of spontaneously hypertensive rats.

The binding of [125I]sarafotoxin 6b (SRT 6b) and [125I]endothelin-1 (ET-1) to endothelin (ET) receptors of neuronal membranes prepared, from regions of the brain and spinal cord of 8 week-old, spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. Spontaneously hypertensive rats had significantly higher blood pressure as compared to WKY rats. Heart rate was similar in SHR and WKY rats. [125I]SRT 6b and [125I]ET-1 bound to the membranes of the cerebral cortex, hypothalamus, ventrolateral medulla, dorsomedial medulla and spinal cord at a single, high affinity site. The Kd and Bmax values of the binding of [125I]SRT 6b were found to be similar to binding of [125I]ET-1 in all the regions. The concentration-dependent inhibition of binding of [125I]ET-1 by unlabeled ET-1, in spinal cord membranes showed an IC50 value of 2.66 +/- 0.59 nM and a Ki value of 2.35 +/- 0.52 nM in WKY rats and an IC50 value of 2.82 +/- 0.76 nM and a Ki value of 2.43 +/- 0.70 nM in SHR rats. On the other hand, the concentration-dependent inhibition of the binding of [125I]SRT 6b by unlabeled ET-1, in spinal cord membranes showed an IC50 value of 10.31 +/- 1.82 pM and a Ki value of 8.44 +/- 1.41 pM in WKY rats, while SHR rats showed an IC50 value of 10.28 +/- 1.94 pM and a Ki value of 8.89 +/- 2.00 pM. The binding of [125I]SRT 6b and [125I]ET-1 in the cerebral cortex, dorsomedial medulla and spinal cord membranes was found to be similar in SHR and WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS) [corrected]     

Molecular cloning and radioligand binding characterization of the chemokine receptor CCR5 from rhesus macaque and human

The aim of this study was to determine if macaque represents a suitable species for the pre-clinical evaluation of novel CCR5 antagonists, such as maraviroc (UK-427,857). To do this we cloned and expressed CCR5 from rhesus macaque and compared the binding properties of [125I]-MIP-1beta and [3H]-maraviroc with human recombinant CCR5. [125I]-MIP-1beta bound with similar high affinity to CCR5 from macaque (K(d) = 0.24 +/- 0.05 nM) and human (K(d) = 0.23 +/- 0.05 nM) and with similar kinetic properties. In competition binding studies the affinity of a range of human chemokines for macaque CCR5 was also similar to human CCR5. Maraviroc inhibited binding of [125I]-MIP-1beta to CCR5 from macaque and human with similar potency (IC50 = 17.50 +/- 1.24 nM and 7.18 +/- 0.93 nM, respectively) and antagonised MIP-1beta induced intracellular calcium release mediated through CCR5 from macaque and human with similar potency (IC50 = 17.50 +/- 3.30 nM and 12.07 +/- 1.89, respectively). [3H]-maraviroc bound with high affinity to CCR5 from macaque (K(d) = 1.36+/-0.07 nM) and human (K(d) = 0.86 +/- 0.08 nM), but was found to dissociate approximately 10-fold more quickly from macaque CCR5. However, as with the human receptor, maraviroc was shown to be a high affinity, potent functional antagonist of macaque CCR5 thereby indicating that the macaque should be a suitable species in which to evaluate the pharmacology, safety and potential mechanism-related toxicology of novel CCR5 antagonists.     

Characterization of two new ETB selective radioligands, [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1 in human heart.

Two new endothelin receptor radioligands, [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1, were characterized in tissue sections of human right atrium and left ventricle. Both radioligands had high affinity ([125I]-BQ3020 right atrium: KD = 0.145 +/- 0.037 nM, left ventricle: KD = 0.107 +/- 0.004 nM; [125I]-[Ala1,3,11,15]ET-1 right atrium: KD = 0.239 +/- 0.036 nM, left ventricle: KD = 0.199 +/- 0.027 nM). Competition binding experiments were performed in the left ventricle. The selective ETA receptor compound BQ123 competed with low affinity against [125I]-BQ3020 (KD = 28.7 +/- 2.7 microM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 28.5 +/- 4.2 microM). The selective ETB receptor compound BQ3020 competed with high affinity against [125I]-BQ3020 (KD = 40.8 +/- 6.6 pM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 0.276 +/- 0.099 nM). Another selective ETB receptor compound, [Ala1,3,11,15]ET-1 also competed with high affinity against [125I]-BQ3020 (KD = 0.663 +/- 0.120 nM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 0.643 +/- 0.124 nM). These results indicate that [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1 are selective ETB receptor radioligands. [Ala1,3,11,15]ET-1 competed with the non-selective radioligand [125I]-ET-1 in left ventricle and revealed the presence of ETA and ETB receptors in the proportions of 76:24% respectively in the human left ventricle.     

Beta-adrenergic receptors in guinea-pig liver plasma membranes and thermal lability of [3H]dihydroalprenolol binding sites

beta-Adrenergic receptors in guinea-pig liver plasma membranes were characterized by radioligand binding, using l-[3H]dihydroalprenolol ([3H]DHA), l-3-[125I]iodocyanopindolol ([125I]CYP) and dl-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2- one hydrochloride [( 3H]CGP-12177). The binding of both [125I]CYP and [3H]CGP-12177 to membranes exhibited high affinity (Kd = 3.5 +/- 0.2 pM for [125I]CYP and 0.75 +/- 0.10 nM for [3H]CGP-12177) and stereospecificity; the maximal binding sites were 130 +/- 15 and 137 +/- 8 fmoles/mg protein respectively. Catecholaminergic agonists competed for these binding sites in the order l-isoproterenol greater than l-epinephrine greater than l-norepinephrine, which is typical for beta 2-adrenergic receptors. The binding data are supported by parallel experiments on adenylate cyclase activation by catecholamines, and on antagonism of this activation by beta 1- and beta 2-selective blockers. The binding of [3H]DHA was excessive (Bmax = 21.4 pmoles/mg protein), exhibited low affinity (Kd = 34.6 nM), and lacked stereospecificity. When liver membranes were incubated at 50 degrees for 40 min in the presence of an agonist, l-isoproterenol, the binding of [3H]DHA to the heat-treated membranes exhibited high affinity (Kd = 1.07 +/- 0.17 nM) and the Bmax was reduced to 139 +/- 22 fmoles/mg protein. In such membranes, as opposed to native membranes, stereospecificity was evident and catecholaminergic agonists competed for the binding sites in the order typical for beta 2-adrenergic receptors. However, agonist competition of the binding to the heat-treated membranes could not be modulated by guanine nucleotides, indicating a loss of communication between the receptor and the guanine nucleotide regulatory protein.     

Failure of BQ123, a more potent antagonist of sarafotoxin 6b than of endothelin-1, to distinguish between these agonists in binding experiments.

1. In homogenates of human saphenous vein, [125I]-ET-1 and [125I]-S6b each labelled a single population of high affinity binding sites with K(D) values of 0.64 +/- 0.11 nM and 0.55 +/- 0.08 nM respectively. Hill slopes were close to one. However, the density of receptors labelled by [125I]-ET-1 was significantly greater than that by [125I]-S6b (187.6 +/- 23.0 compared to 91.7 +/- 23.6 fmol mg-1 protein, P < 0.02). 2. BQ123, an ET(A-)selective antagonist, inhibited specific [125I]-ET-1 and [125I]-S6b binding with equal affinity. BQ123 competed in a biphasic manner for both [125I]-ET-1 (0.1 nM) and [125I]-S6b (0.1 nM) with ET(A) K(D) values of 0.55 +/- 0.17 nM and 0.52 +/- 0.02 nM and ET(B) K(D) values of 14.4 +/- 2.60 microM and 11.2 +/- 0.31 microM respectively. S6b monophasically inhibited 0.1 nM [125I]-ET-1 (K(D) 1.16 +/- 0.9 nM) but competed for 0.25 nM [125I]-ET-1 in a biphasic manner (K(D) high affinity site 1.99 +/- 0.84 nM, K(D) low affinity site 0.68 +/- 0.63 microM, ratio 67% : 33%). 3. BQ123 antagonized the vasoconstrictor responses of ET-1 with a pK(B) value of 6.47 whereas BQ123 exhibited 50 fold higher affinity against S6b-mediated vasoconstriction with a pK(B) value of 8.18. Regression slopes were 0.80 +/- 0.13 and 1.08 +/- 0.11 respectively. 4. In desensitization experiments, S6b (300 nM) did not contract preparations which were no longer responsive to ET-1 whereas a small contraction to ET-1 (300 nM) was obtained in preparations rendered unresponsive to S6b. 5. Medial sections of non-diseased human aorta, which express only ET(A) receptors, were used to compare dissociation rates of the two agonists. The time course for the dissociation of [125I]-ET-1 and [125I]-S6b was similar with 20-30% of each ligand dissociating at 4 h. 6. These data suggest that whilst BQ123, in common with other endothelin antagonists, is a much more potent blocker of S6b contractile responses than of ET-1 contractile responses, this is not reflected by the equal affinity of BQ123 determined in competition binding experiments against both [125I]-ET-1 and [125I]-S6b. This discrepancy in antagonist potency is probably not due to a marked difference in the rate of dissociation of [125I]-ET-1 and [125I]-S6b from endothelin receptors. One possible explanation is that ET-1 is activating an additional population of receptors which may have lower affinity for BQ123. This is suggested by the discrepancy in receptor density identified by [125I]-ET-1 and [125I]-S6b.     

p-[125I]iodoclonidine is a partial agonist at the alpha 2-adrenergic receptor

The binding properties of p-[125I]iodoclonidine [( 125I]PIC) to human platelet membranes and the functional characteristics of PIC are reported. [125I]PIC bound rapidly and reversibly to platelet membranes, with a first-order association rate constant (kon) at room temperature of 8.0 +/- 2.7 x 10(6) M-1 sec-1 and a dissociation rate constant (koff) of 2.0 +/- 0.8 x 10(-3) sec-1. Scatchard plots of specific [125I]PIC binding (0.1-5 nM) were linear, with a Kd of 1.2 +/- 0.1 nM. [125I]PIC bound to the same number of high affinity sites as the alpha 2-adrenergic receptor (alpha 2-AR) full agonist [3H] bromoxidine (UK14,304), which represented approximately 40% of the sites bound by the antagonist [3H]yohimbine. Guanosine 5'-(beta, gamma-imido)triphosphate greatly reduced the amount of [125I]PIC bound (greater than 80%), without changing the Kd of the residual binding. In competition experiments, the alpha 2-AR-selective ligands yohimbine, bromoxidine, oxymetazoline, clonidine, p-aminoclonidine, (-)-epinephrine, and idazoxan all had Ki values in the low nanomolar range, whereas prazosin, propranolol, and serotonin yielded Ki values in the micromolar range. Epinephrine competition for [125I]PIC binding was stereoselective. Competition for [3H]bromoxidine binding by PIC gave a Ki of 1.0 nM (nH = 1.0), whereas competition for [3H]yohimbine could be resolved into high and low affinity components, with Ki values of 3.7 and 84 nM, respectively. PIC had minimal agonist activity in inhibiting adenylate cyclase in platelet membranes, but it potentiated platelet aggregation induced by ADP with an EC50 of 1.5 microM. PIC also inhibited epinephrine-induced aggregation, with an IC50 of 5.1 microM. Thus, PIC behaves as a partial agonist in a human platelet aggregation assay. [125I]PIC binds to the alpha 2B-AR in NG-10815 cell membranes with a Kd of 0.5 +/- 0.1 nM. [125I]PIC should prove useful in binding assays involving tissues with a low receptor density or in small tissue samples and in studies of cloned and expressed alpha 2-AR.     

Endothelin receptors in human coronary artery and aorta.

1. ETA and ETB-selective and non-selective ligands were used to define the endothelin receptors in the media (vascular smooth muscle layer) of human aorta and coronary artery. Saturation experiments with iodinated endothelin-1 (ET-1), endothelin-2 and sarafotoxin 6b (S6b) identified high affinity binding sites in aorta (KD [125I]-ET-1 0.33 +/- 0.02 nM (n = 9), KD [125I]-ET-2 1.04 +/- 0.23 nM (n = 5), KD [125I]-S6b 0.15 +/- 0.01 nM (n = 9 +/- s.e.mean)) and coronary artery (KD [125I]-ET-1 0.43 +/- 0.10 nM, KD [125I]-ET-2 0.71 +/- 0.17 nM, KD [125I]-S6b 0.27 +/- 0.03 nM (n = 3 +/- s.e.mean)). Hill coefficients (nH) approached unity in each case. 2. No specific binding was detectable with [125I]-ET-3 (4 pM-4 nM) in aorta. Unlabelled ET-3 competed monophasically with [125I]-ET-1 in aorta (KD, 8.21 +/- 1.62 nM, compared to unlabelled ET-1 KD, 0.60 +/- 0.20 nM) (n = 3 +/- s.e.mean). In coronary artery, the KD and Bmax values calculated from [125I]-ET-3 saturation experiments were 2.13 +/- 1.39 nM and 20.6 +/- 12.9 fmol mg-1 protein, respectively (n = 3 +/- s.e.mean). 3. ETA antagonists competed monophasically for [125I]-ET-1 (100 pM) binding sites with nanomolar or subnanomolar affinity in the aorta (KD BQ123, 0.47 +/- 0.13 nM; KD FR139317, 0.40 +/- 0.10 nM; KD PD151242, 2.09 +/- 0.48 nM) and coronary artery (KD FR139317, 0.41 +/- 0.13 nM; KD PD151242, 3.60 +/- 0.74 nM) (n = 3 +/- s.e.mean). However, two site fits were preferred on analysis of competition experiments with ETB-selective agonists versus [125I]-ET-1 in coronary artery (BQ3020: KDETA 0.96 +/- 0.14 microM, KD ETB 1.34 +/- 1.08 nM and sarafotoxin 6c: KD ETA 1.15 +/- 0.14 microM, KD ETB 1.77 +/- 0.72 nM) (n = 3 +/- s.e.mean). The selectivity of the agonists for ETB receptors (700 fold) was lower than reported in other species. 4. Sarafotoxin 6b (2 pM-2 microM) completely inhibited [125I]-ET-1 (100 pM) binding in aorta (KD 1.36 +/- 0.22 nM) (n = 3 +/- s.e.mean). The non-peptide compounds Ro462005 and bosentan, competed with [125I]-ET-1 binding in coronary artery with KD values of 0.19 +/- 0.04 microM and 2.94 +/- 0.95 nM, respectively (n = 3 +/- s.e.mean). 5. Inhibition of [125I]-ET-2 and [125I]-S6b binding by FR139317 was similar to the inhibition of [125I]-ET-1 binding in both arteries, being monophasic with KD values in the same range. 6. ETA receptors in coronary artery media were detected by [125I]-PD151242 (KD 0.23 +/- 0.04 nM, Bmax 10.1 +/- 1.2 fmol mg-1 protein) (n = 3 +/- s.e.mean). [125I]-BQ3020, an ETB-selective radioligand, indicated the presence of a smaller population of ETB receptors in this tissue (KD 0.60 +/- 0.31 nM, Bmax 4.5 +/- 2.1 fmol mg-1 protein) (n = 3 +/- s.e.mean). 7. Autoradiography with [125I]-PD151242 and [125I]-BQ3020 confirmed the predominance of ETA receptors in the media of both arteries. 8. The results of this study indicate that ETA receptors predominate in the vascular smooth muscle of human cardiac arteries, with a small and variable population of ETB receptors detectable in the coronary artery.     

Identification of selective, high affinity [125I]-angiotensin and [125I]-bradykinin binding sites in rat intestinal epithelia.

Specific [125I]-angiotensin II (AII) and [125I]-bradykinin (Bk) binding sites have been identified within epithelial membranes from rat jejunum and descending colon. These high affinity intestinal sites exhibited KD values of 0.64 +/- 0.16 nM for [125I]-AII and 0.69 +/- 0.13 nM for [125I]-Bk, which were similar to those for [125I]-AII (0.85 nM) and [125I]-Bk binding sites (1.03 nM) previously identified in renal cortex epithelia. Specific [125I]-AII binding capacity was only 19.77 +/- 2.74 fmol mg-1 in small intestine and 11.31 +/- 2.66 fmol mg-1 in descending colon epithelia while a larger population, 332.0 +/- 72.9 fmol-mg-1 of specific [125I]-Bk sites were identified in epithelial membranes from small intestine. Significant hydrolysis of both free [125I]-AII and [125I]-Bk was observed while membrane bound peptides remained relatively resistant to degradation. Whilst no corrections have been made to the observed values of KD and Bmax quoted above, one may assume that the calculated reductions in the free hormone concentration will result in a decrease of the KD value for both peptides. Loss of membrane bound peptide, particularly of [125I]-AII, may indicate that the calculated Bmax value is an underestimation. Despite the rapid degradation of unbound [125I]-AII and [125I]-Bk during incubations the kinetics of specific peptide binding were reversible and highly selective. The order of potency for specific [125I]-AII binding was [Sar1, Leu8]-AII greater than or equal to [Sar1, Thr8]-AII greater than or equal to AII greater than [Sar1, Ile8]-AII greater than or equal to [Des, Asp1, Ile8] AII greater than AIII. Specific [125I]-Bk binding was also highly selective, the order of potency being Phe8-Bk greater than or equal to Tyr8-Bk greater than or equal to Lys-Bk much greater than Des, Arg1-Bk. AII exhibited an IC50 of greater than 1mM for specific [125I]-Bk binding and likewise Phe8-Bk for specific [125I]-AII binding.     

Binding of [3H]dihydrotetrabenazine and [125I]azidoiodoketanserin photoaffinity labeling of the monoamine transporter of platelet 5-HT organelles

The carrier for 5-hydroxytryptamine (5-HT) of the 5-HT storage organelles of blood platelets was characterized by [3H]dihydrotetrabenazine binding and [125I]azidoiodokentanserin photoaffinity labeling. [3H]Dihydrotetrabenazine bound with high affinity to membrane preparations from different animal species. The [3H]dihydrotetrabenazine Bmax value was about 10-fold higher in rabbit (9.4 +/- 1.3 pmol/mg protein) than in human, rat and guinea-pig preparations (Bmax values = 1.1 +/- 0.2, 1.2 +/- 0.1 and 0.52 +/- 0.06 pmol/mg protein, respectively). After rabbit platelet subcellular fractionation, [3H]dihydrotetrabenazine binding was highly enriched in the fraction corresponding to pure 5-HT organelles, whereas ligand binding was much lower in the other subcellular fractions. Conversely, [3H]paroxetine binding sites were more concentrated in the lower density fractions, with no binding to the 5-HT granules. In competition experiments, [3H]dihydrotetrabenazine binding to human platelet membranes and rabbit platelet 5-HT organelles was markedly inhibited by the benzo[a]quinolizine derivatives, tetrabenazine and Ro 4-1284, and by ketanserin. In isolated rabbit platelet 5-HT organelles, reserpine showed a relatively high IC50 (930 nM), but the presence of ATP increased its potency about 10-fold. Paroxetine, methysergide and carrier substrates had little or no effect. After photoaffinity labeling of rabbit 5-HT granules with [125I]azidoiodoketanserin, the radioactivity was incorporated into several polypeptides. The presence of Ro 4-1284, reserpine and ketanserin prevented the labeling of a polypeptide of 85 kDa. The data obtained suggest that this protein represents a component of the granular carrier which binds [3H]dihydrotetrabenazine.     

Epithelial modulation of the relaxant activity of atriopeptides in rat and guinea-pig tracheal smooth muscle.

Three peptide components of atrial natriuretic factor (ANF) caused relaxation of carbachol-contracted guinea-pig isolated tracheal smooth muscle. These were the 1-28, 5-28 and 5-27 peptide sequences (ANF(1-28), ANF-(5-28) and ANF-(5-27)). The peptides were 10-30 times more potent in epithelium-denuded than in epithelium-intact preparations. In the absence of airway epithelium, ANF-(1-28) was the most potent relaxant (mean pD2 = 7.40 +/- 0.08), with ANF-(5-27) and ANF-(5-28) 2-3 fold less potent. The neutral endopeptidase inhibitor phosphoramidon (1 microM) increased the potency of ANF-(5-27) in both epithelium-intact and epithelium-denuded guinea-pig tracheal rings. In contrast, removal of the epithelium from rat trachea, or pretreatment with phosphoramidon (1 microM) decreased relaxant responsiveness to ANF-(5-27). Thus, in rat trachea, epithelial endopeptidase may convert ANF-(5-27) to a more active relaxant peptide. Human bronchial preparations with or without epithelium, obtained from non-diseased lung samples and from a single sample of asthmatic lung, were virtually unresponsive to ANF-(5-27). Consistent with the spasmolytic effects of ANF in guinea-pig trachea, autoradiographic analysis revealed the presence of a sparse population of specific binding sites for [125I]ANF-(1-28) over both tracheal smooth muscle and epithelium. The present study shows that the relaxant effects of atriopeptins in rat and guinea-pig airway smooth muscle were modulated by the epithelium and the activity of neutral endopeptidase. However, marked species differences in airway smooth muscle responsiveness to ANF and in the modulatory role of the airway epithelium were evident.     

Characterization of rat glomerular thromboxane A2 receptors: comparison to rat platelets.

This study was designed to characterize rat glomerular thromboxane A2 (TxA2) receptors and compare them to rat platelet TxA2 receptors. The radioligand binding characteristics of the receptors were characterized using [125I][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-2yl]-5-heptenoic acid ([125I]BOP), a TxA2 agonist. Equilibrium binding with [125I]BOP, as well as competitive binding assays between [125I]BOP and 13-azapinane TxA2 receptors antagonists, were performed in rat glomerular membranes (RGM) and washed rat platelets (WRP). [125I]BOP identified a single class of TxA2 receptor sites in glomerular membranes with a Kd of 318 +/- 55 pM and a Bmax of 260 +/- 62 fmol/mg protein (n = 14). [125I]BOP was displaced by the TxA2 agonist 15S-hydroxy-11 alpha,9 alpha(epoxymethano)-prosta-5Z,13E-dienoic acid (U-46,619) (IC50 = 22 +/- 6 nM, n = 3), the antagonist SQ-29,548 (IC50 = 41 +/- 7 nM, n = 4), and stereoselectively by the antagonists (-)-9-chlorobenzyl-6-fluoro-1,2,3,4-tetrahydrocarbazol-1-yl acetic acid (L-657,925) (IC50 = 0.27 +/- 0.04 nM, n = 3) and (+)-9-chlorobenzyl-6-fluoro-1,2,3,4-tetrahydrocarbazol-1-yl acetic acid (L-657,926) (IC50 = 124 +/- 0 nM, n = 2). The ability of six 13-azapinane TxA2 antagonists to compete with [125I]BOP was evaluated. The rank orders for the 13-azapinanes showed no significant correlation between RGM and WRP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the binding of a potent, selective, radioiodinated antagonist to the human neurokinin-1 receptor.

We have synthesized a potent, selective, radioiodinated antagonist of the human neurokinin-1 (NK1) receptor and have characterized its binding to the cloned receptor expressed in Chinese hamster ovary cells. (cis)-2-(Diphenylmethyl)-N-[(2-iodophenyl)-methyl]-1- azabicyclo[2.2.2]octan-3-amine (L-703606) inhibits binding of 125I-Tyr8-substance P to the human NK1 receptor with an IC50 of 2 nM. This compound is a competitive antagonist of substance P-induced inositol phosphate generation, with a Kb of 29 nM. [125I]L-703606 binds to a single class of high affinity binding sites in human NK1/Chinese hamster ovary cell membranes (Kd = 0.3 nM). Substance P inhibits the binding of [125I]L-703606 to 65% of the NK1 receptor sites with a Kd of 0.04 +/- 0.03 nM and to the remaining 35% of the sites with a Kd of 1.5 +/- 0.7 nM. Addition of the nonhydrolyzable GTP analog guanylyl-5'-(beta, gamma-imido)diphosphate [Gpp(NH)p] shifts greater than 90% of the binding sites to the lower affinity state. In addition, Gpp(NH)p markedly alters the dissociation of substance P from the NK1 receptor by increasing the number of sites in the low affinity, rapidly dissociating state. However, Gpp(NH)p does not affect the rate of dissociation of [125I]L-703606. These data suggest that the pharmacological properties of [125I]L-703606 binding to the human NK1 receptor are similar to those of antagonists of nonpeptide guanine nucleotide-binding protein-coupled receptors and that this ligand will be useful for the biochemical and pharmacological characterization of the human NK1 receptor.     

Characteristics of endothelin binding sites in the spinal cord of spontaneously hypertensive rats.

The binding of [125I]sarafotoxin 6b (SRT 6b) and [125I]endothelin-1 (ET-1) to endothelin (ET) receptors of neuronal membranes prepared from cerebral cortex and spinal cord of 8-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) was determined. SHR had significantly higher blood pressure as compared to WKY. Heart rate was similar in SHR and WKY. [125I]SRT 6b and [125I] ET-1 bound to the membranes of cerebral cortex and spinal cord at a single, high affinity site. The binding of [125I]SRT 6b and [125I]ET-1 in the cerebral cortex and spinal cord membranes was found to be similar in SHR and WKY. Concentration-dependent inhibition of [125I]ET-1 binding in spinal cord membranes by unlabeled ET-1, ET-2 and ET-3 was performed. The Ki values were found to be 2.35 +/- 0.68, 0.29 +/- 0.08 and 24.10 +/- 5.90 nM for ET-1, ET-2 and ET-3, respectively in WKY. The Ki values of ET-1 and ET-2 were found to be similar in WKY and SHR. However, the Ki value of ET-3 were found to be significantly lower (P less than 0.004) in SHR as compared to WKY. Concentration-dependent inhibition of [125I]SRT 6b binding in spinal cord membranes by unlabeled ET-1, ET-2 and ET-3 were also performed. The Ki values were found to be 9.50 +/- 2.10 pM, 0.17 +/- 0.04 nM and 31.20 +/- 6.00 nM for ET-1, ET-2 and ET-3, respectively in WKY. The Ki values of ET-1 and ET-2 were found to be similar in WKY and SHR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and characterization of a high affinity radioiodinated probe for the alpha 2-adrenergic receptor

The availability of radioiodinated probes has facilitated the localization and molecular characterization of cell membrane receptors for hormones and neurotransmitters. However, such probes are not available for the study of the alpha 2-adrenergic receptor. This report describes the synthesis and characterization of functionalized derivatives of the selective alpha 2-adrenergic antagonists, rauwolscine and yohimbine, which can be radiolabeled to high specific activity with 125I. Following demethylation of rauwolscine or yohimbine, the resultant carboxylic acid derivatives were reacted with 4-aminophenethylamine to yield the respective 4-aminophenethyl carboxamides, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-[N-4-amino-phenethyl]carboxamide (rau-pAPC) and 17 alpha-hydroxy-20 beta-yohimban-16 alpha-[N-4-aminophenethyl]carboxamide. In competitive inhibition studies using rat renal membranes and the radioligand [3H]rauwolscine, rau-pAPC (Ki = 11 +/- 1 nM) exhibited a 14-fold greater affinity than the corresponding yohimbine derivative (Ki = 136 +/- 45 nM). The higher affinity compound, rau-pAPC, was radioiodinated by the chloramine T method, and the product, 125I-rau-pAPC [17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-3 -[125I]iodophenethyl)carboxamide], was purified by reverse phase HPLC to high specific activity (2175 Ci/mmol) and its binding characteristics were investigated in rat kidney membranes. Specific binding of 125I-rau-pAPC was saturable and of high affinity as determined by Scatchard analysis (KD = 1.8 +/- 0.3 nM) or from kinetic studies (KD = k2/k1 = 0.056 +/- 0.013 min-1)/4.3 +/- 0.2 X 10(7) M-1 min-1 = 1.3 +/- 0.3 nM). In competition studies, alpha-adrenergic antagonists and agonists inhibited the binding of 125I-rau-pAPC with a potency order consistent with an interaction at alpha 2-adrenergic receptors (rauwolscine greater than phentolamine greater than prazosin; clonidine greater than (-)-epinephrine greater than (-)-norepinephrine greater than dopamine greater than (+)-epinephrine). In rat liver and human platelet membranes, high affinity binding of 125I-rau-pAPC was also observed (liver, KD = 1.2 +/- 0.4 nM; platelet, KD = 3.2 +/- 1.5 nM). In addition, the density of alpha 2-adrenergic receptors identified from binding studies with 125I-rau-pAPC in kidney, liver, and platelet membranes was similar to that observed in parallel studies with [3H]rauwolscine. These findings indicate that 125I-rau-pAPC is a high affinity probe that selectively identifies alpha 2-adrenergic binding sites. Availability of this radioligand should facilitate the localization and biochemical characterization of this alpha-adrenergic receptor subtype.     

Selectivity of [125I]-PD151242 for human, rat and porcine endothelin ETA receptors in the heart.

1. Endothelin-1 binds with high affinity to heart where it acts as a potent positive inotropic agent. Our aim was to characterize the labelled and unlabelled ETA-selective antagonist PD151242 in heart tissues derived from man, rat and pigs by use of radioligand binding techniques. 2. Binding of [125I]-PD151242 to sections of human left ventricle was time-dependent and reached equilibrium after 120 min at 23 degrees C with an association rate constant of 0.0235 min-1 nM-1. The binding was reversible at 23 degrees C with a dissociation rate constant of 0.00144 min-1. 3. Saturation binding assays with [125I]-PD151242 revealed a single population of high affinity ET receptors in human left ventricle (KD = 1.07 +/- 0.08 nM; Bmax = 29.8 +/- 4.2 fmol mg-1 protein), porcine left ventricle (KD = 1.92 +/- 0.27 nM; Bmax = 493 +/- 248 fmol mg-1 protein), and rat heart (KD = 0.64 +/- 0.08 nM; Bmax = 82.34.7 fmol mg-1 protein). 4. Unlabelled PD151242 competed with specific [125I]-ET-1 binding to human left ventricle tissue in a biphasic manner with high affinity binding to the ETA-site (KD = 7.21 +/- 2.83 nM) and lower affinity for the ETB-subtype (KD = 104 +/- 23 microM), indicating a greater than 10000 fold selectivity to the high affinity site. 5. The ETA-selective ligand FR139317 competed for [125I]-PD151242 binding in human left ventricle with nanomolar affinity (KD = 0.37 +/- 0.10 nM), whereas the ETB-selective compound, BQ3020, competed with only micromolar affinity (KD = 1.5 +/- 0.26 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Iodo-resiniferatoxin, a new potent vanilloid receptor antagonist

The highly potent vanilloid receptor (VR) agonist resiniferatoxin has been radiolabeled with 125I, and the pharmacology to the cloned rodent VR, VR1, and the endogenous VR in rat spinal cord membranes has been characterized. [125I]RTX binding to human embryonic kidney 293 cells expressing VR1 was reversible and with high affinity (Kd = 4.3 nM) in an apparent monophasic manner. In rat spinal cord membranes, [125I]RTX bound with a similar high affinity (Kd = 4.2 nM) to a limited number of binding sites (Bmax = 51 +/- 8 fmol/mg of protein). The pharmacology of recombinant rodent VR1 and the endogenous rat VR1 was indistinguishable when measuring displacement of [125I]RTX binding (i.e., the following rank order of affinity was observed: RTX > I-RTX > olvanil > capsaicin > capsazepine). Capsaicin and RTX induced large nondesensitizing currents in Xenopus laevis oocytes expressing VR1 (EC50 values were 1300 nM and 0.2 nM, respectively), whereas I-RTX induced no current per se at concentrations up to 10 microM. However, I-RTX completely blocked capsaicin-induced currents (IC50 = 3.9 nM). In vivo, I-RTX effectively blocked the pain responses elicited by capsaicin (ED50 = 16 ng/mouse, intrathecally). The present study showed that I-RTX is at least 40-fold more potent than the previously known VR antagonist, capsazepine. Thus, I-RTX as well as its radiolabeled form, should be highly useful for further exploring the physiological roles of VRs in the brain and periphery.     

[125I]-PD151242: a selective radioligand for human ETA receptors.

Our aim was to synthesize a new endothelin ETA selective radioligand, [125I]-PD151242 and characterize the compound in human vascular tissue. Binding of [125I]-PD151242 to sections of human aorta was time-dependent and reached equilibrium after 120 min at 23 degrees C with an association rate constant of 1.26 +/- 0.17 x 10(8) M-1 min-1 (n = 3 individuals +/- s.e.mean). The binding was reversible at 23 degrees C with an observed dissociation rate constant of 0.0025 +/- 0.0006 min-1 (n = 3). Saturation binding assays using [125I]-PD151242 revealed a single population of high affinity ET receptors (n = 3) in aorta (KD = 0.76 +/- 0.17 nM; Bmax = 5.98 +/- 1.56 fmol mg-1 protein), pulmonary (KD = 1.75 +/- 0.20 nM; Bmax = 12.78 +/- 1.39 fmol mg-1 protein) and coronary arteries (KD = 0.51 +/- 0.07 nM; Bmax = 44.9 +/- 1.67 fmol mg-1 protein). ETA selective ligands competed for [125I]-PD151242 binding in aorta with nanomolar affinity (BQ123, KD = 0.41 +/- 0.26 nM; FR139317, KD = 0.55 +/- 0.11 nM) whereas the ETB selective compound, BQ3020, competed with micromolar affinity (KD = 1.36 +/- 0.25 microM). In isolated coronary arteries, PD151242 was a functional antagonist and caused a significant, parallel rightward shift of the ET-1 dose-response curve with a pA2 value of 5.92 (n = 5) and a slope of unity. The high affinity and selectivity of [125I]-PD151242 for ETA receptors will facilitate the characterization of this sub-type in human tissues.     

[125I]-PD151242: a selective ligand for endothelin ETA receptors in human kidney which localizes to renal vasculature.

1. The linear tetrapeptide radioligand, [125I]-PD151242 was used to characterize ETA receptors in human kidney which is an ETB-rich tissue. Saturation binding assays with [125I]-PD151242 revealed a single population of high affinity endothelin receptors: KD = 0.75 +/- 0.07 nM and Bmax = 48.4 +/- 1.6 fmol mg-1 protein (n = 3 individuals +/- s.e.mean). Hill slopes were close to unity and a one site fit was preferred to a two site model. 2. ETA-receptor-selective ligands competed for [125I]-PD151242 binding with sub-nanomolar affinity: BQ123 KD = 0.43 +/- 0.10 nM, Bmax = 46.6 +/- 7.9 fmol mg-1 protein; FR139317, KD = 0.37 +/- 0.06 nM, Bmax = 39.5 +/- 6.5 fmol mg-1 protein (n = 3 individuals +/- s.e.mean). In each case, monophasic inhibition curves were obtained and a one site fit was preferred to a two site model. The ETB-selective agonist, BQ3020 at the highest concentration tested (10 microM) inhibited binding by only 50%. The non-selective RO462005 competed for the binding of [125I]-PD151242: KD = 1.31 +/- 1.38 microM, Bmax = 33.0 +/- 9.7 fmol mg-1 protein. Endothelin-2 and sarafotoxin S6B inhibited [125I]-PD151242 binding to renal tissue whereas ET-3 and sarafotoxin S6C were less effective. Non-endothelin and non-sarafotoxin peptides did not compete. 3. No degradation of [125I]-PD151242 was detected following incubation of the ligand with renal tissue under the conditions of the binding assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of a thromboxane A2/prostaglandin H2 receptor antagonist to guinea-pig platelets

The binding of [125I]9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxypheny l)-13,14- dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 [( 125I]PTA-OH), a thromboxane A2/prostaglandin H2 receptor antagonist, to washed guinea-pig platelets was studied. [125I]PTA-OH bound to guinea-pig platelets in a saturable and displaceable manner. The Kd for [125I]PTA-OH was 14.5 +/- 2 nM (n = 4) and the Bmax was 32 +/- 7 fmol/10(7) platelets or 1,927 +/- 422 binding sites/platelet. The IC50 value for a series of 13-azapinane TXA2 analogs to antagonize the TXA2/PGH2 mimetic U46619-induced platelet aggregation and displace [125I]PTA-OH from its binding site was determined. The IC50 values for the series of five antagonists were highly correlated (r = 0.99) in the binding assays and aggregation studies. The ability of a series of five agonists to displace [125I]PTA-OH from its binding site was compared to their ability to induce platelet aggregation. All the agonists completely displaced the ligand from its binding site but their rank order did not correlate well with their ability to induce aggregation (r = 0.37). Collectively, the data are consistent with the notion that [125I]PTA-OH binds to a putative TXA2/PGH2 receptor in guinea-pig platelets.     

Evaluation of the receptor selectivity of the H3 receptor antagonists, iodophenpropit and thioperamide: an interaction with the 5-HT3 receptor revealed.

1. In the present study we evaluated the receptor selectivity of the potent histamine H3 receptor antagonist, iodophenpropit (IPP) in comparison with the prototype antagonist, thioperamide. 2. IPP proved to be a potent competitive H3 receptor antagonist as measured against (R)-alpha-methylhistamine-induced inhibition of electrically-evoked contractions of the guinea-pig jejunum (pA2 = 9.12 +/- 0.06, Schild slope: 1.0 +/- 0.1, n = 8). In the same assay, thioperamide was slightly less potent (pA2 = 8.9 +/- 0.2). 3. In radioligand binding studies, IPP showed a high affinity for the H3 receptor. Displacement of [125I]-IPP binding to rat cortex membranes by unlabelled IPP resulted in a Ki value of 0.97 +/- 0.06 nM (n = 3). In contrast, IPP showed only a weak affinity for the histamine H1- and H2 receptor. Displacement of [3H]-mepyramine and [125I]-iodoaminopotentidine binding to respectively guinea-pig H1- and human H2 receptors by IPP resulted in Ki values of 1.71 +/- 0.32 microM (n = 3) and 2.28 +/- 0.81 microM (n = 3). For thioperamide the affinities for the H1-, H2- and H3 receptor were respectively > 10 microM, > 10 microM and 4.3 +/- 1.6 nM (n = 7). 4. Testing IPP and thioperamide in 39 different receptor binding assays revealed that IPP showed relatively high affinity for the 5-hydroxytryptamine 5-HT3 receptor (Ki = 11 +/- 1 nM, n = 3), the alpha 2-adrenoceptor (Ki = 120 +/- 5 nM, n = 3) and the sigma receptor (Ki = 170 +/- 70 nM, n = 3). Thioperamide showed relatively high affinity for the 5-HT3 receptor (Ki = 120 +/- 30 nM, n = 3) and the sigma receptor (Ki = 180 +/- 90 nM, n = 3). 5. Due to the low density of histamine H3 receptors in the brain, the interaction of IPP with the 5-HT3-, the alpha 2- and the sigma receptor might interfere with [125I]-IPP binding to rat cortex membranes. Yet, in this preparation [125I]-IPP binding was not influenced by ondansetron, yohimbine or haloperidol.(ABSTRACT TRUNCATED AT 250 WORDS)