Binding of an adenosine A1 receptor agonist and adenosine A1 receptor antagonist to sheep pineal membranes

The pineal organ of vertebrates produces melatonin and adenosine. In lower vertebrates, adenosine modulates melatonin production. We report herein that 2-chloro-cyclopentyl-[3H]-adenosine ([3H]CCPA: adenosine A1 receptor agonist) and [3H]-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX: adenosine A1 receptor antagonist), bind specifically to sheep pineal membranes. Binding of [3H]CCPA reached equilibrium at 90 min and dissociation revealed the presence of two components. Saturation analysis suggested the presence of a single population of binding sites (Kd = 1.67 +/- 0.06 nM, Bmax = 2386 fmol/mg protein). Binding was sensitive to GTP and GTPgammaS. Binding of [3H]DPCPX reached equilibrium at 60 min and dissociation was monophasic. Saturation analysis revealed a single population of binding sites (Kd = 5.8 +/- 1.12 nM, Bmax = 1116 fmol/mg protein). The specificity of the [3H]-analogues used and the rank order potency of the competitors tested in the competition experiments suggested the presence of A1 receptors. Future investigations are necessary to elucidate the significance of the differences observed between the binding properties of the adenosine A1 receptor agonist and adenosine A1 receptor antagonist.     

Opposite effects of CCK(B) agonists in grooming behaviour in rats: further evidence for two CCK(B) subsites

Opioid receptor binding properties of [3H]Tyr-D-Ala-Phe-Phe-NH2 (TAPP) were characterized in rat brain and Chinese hamster ovary (CHO) cells expressing the rat mu-receptor. In rat brain, [3H]TAPP labeled a single class of opioid sites with a dissociation constant (Kd) of 0.31 nM and maximal number of binding sites (Bmax) of 119 fmol/mg protein. In CHO-mu/1 cell membranes, the Kd and Bmax values were 0.78 nM and 1806 fmol/mg protein, respectively. Binding to rat brain was demonstrated to be pharmacologically identical to that obtained with CHO-mu/1 cell membranes and modulated by Na+ ions and guanine nucleotides. The high affinity and selectivity of [3H]TAPP together with its low non-specific binding make this radioligand a useful tool for labeling the native and cloned mu-opioid receptor.     

Polyneuropathy due to ethylene oxide, propylene oxide, and butylene oxide

Radioligand exchange was examined for its ability to derive the dissociation constant (Kd) of the rat ventral prostate nuclear androgen receptor. In one 24-h, 12 degrees C incubation, Scatchard plot analysis of [3H]dihydrotestosterone ([3H]DHT) exchange binding produced a Kd of 6.9 x 10(-9) M. Specific binding of [3H]DHT ranged from 114 to 758 pM, and the extrapolated value for the total number of binding sites (n) was 1320 pM. When aliquots from the same receptor pool were incubated with unlabeled DHT, and bound androgen was measured by radioimmunoassay, each titration point held a concentration of specifically bound unlabeled DHT little different from the preincubation value of bound endogenous ligand (1338 pM), suggesting that few, if any, unoccupied sites were created during the incubation. In a second radioligand exchange assay, unoccupied receptor sites were measured at the end of incubation. Virtually no unoccupied sites were found, though the range of predicted values was 124 to 383 pM (n = 425 pM). The data, in toto, suggest that although radioactive ligand exchanges with bound unlabeled ligand, the dynamics of the process do not include the creation of unoccupied sites. Since the Kd is determined by measuring the concentrations of unoccupied sites, free ligand, and receptor sites bound to ligand, the absence of unoccupied sites suggests that radioligand exchange cannot be used to directly determine the Kd of the prostate nuclear androgen receptor. The numerical value obtained from radioligand exchange, therefore, instead of being a Kd, is very likely the result of a graphic plot of the increase in specific activity of bound radioligand as [3H]DHT is titrated to higher levels. In the last phase of the study a technique was developed which allows for the correct determination of the Kd of the rat ventral prostate nuclear androgen receptor. For the determination, data from an experiment measuring uptake binding into unoccupied sites were combined with data obtained from radioligand exchange binding. From this, the Kd of the receptor was calculated to be 1 x 10(-12) M.     

Circannual variations in the binding of [3H]lysergic acid diethylamide to serotonin2A receptors and of [3H]paroxetine to serotonin uptake sites in platelets from healthy volunteers

BACKGROUND: Circannual variations occur in several serotonergic parameters, including platelet serotonin uptake and platelet [3H]imipramine binding. METHODS: Binding of [3H]lysergic acid diethylamide ([3H]LSD) to platelet serotonin (5-HT)2A receptors and binding of [3H]paroxetine to platelet serotonin uptake sites were studied longitudinally for 1 year in 12 healthy volunteers. RESULTS: For [3H]LSD, the number of binding sites (Bmax) showed no significant seasonal variation (two-way analysis of variance), although Bmax was significantly higher during the months October through February than during the months April through August (32.6 vs. 29.8 fmol/mg protein; p = .015). For [3H]paroxetine, Bmax showed a significant seasonal variation (p = .003) with maximum in August (1322 fmol/mg protein) and minimum in February (1168 fmol/mg protein). The affinity constant (Kd) showed a significant seasonal variation for [3H]LSD binding (p = .046), but not for [3H]paroxetine binding. The seasonal fluctuations in [3H]LSD binding and in paroxetine binding tended to be inversely correlated for Bmax (r = -.70; p = .08) and were significantly negatively correlated for Kd (r = -.88; p = .009). CONCLUSIONS: The present study demonstrates a seasonal effect on platelet serotonin uptake site binding and indicates a possible seasonal effect on 5-HT2A receptor binding. The results imply that circannual fluctuations should be taken into account when these platelet serotonin markers are studied.     

Efficacy of captopril on posttransplant erythrocytosis. Long-term follow-up

The presence of both nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive dipyridamole binding sites in postmortem human ependymal tissue is reported. Displacement studies using 15 nM [3H]dipyridamole revealed 50-60% of the sites were sensitive to nitrobenzylthioinosine. Non-linear analysis of binding isotherms to estimate the density of nitrobenzylthioinosine-sensitive and -insensitive sites revealed a maximum number of nitrobenzylthioinosine-sensitive binding sites (Bmax) of 395 +/- 19 fmol/mg protein and a nitrobenzylthioinosine-insensitive Bmax of 3910 +/- 700 fmol/mg protein (corresponding Kd values of 0.1 nM and 114 nM respectively). Thus there are approximately 10 times as many nitrobenzylthioinosine-insensitive sites as nitrobenzylthioinosine-sensitive [3H]dipyridamole binding sites in human ependymal membranes.     

Ligand binding profiles of U-69, 593-sensitive and-insensitive sites in human cerebral cortex membranes: evidence of kappa opioid receptors heterogeneity

Receptor binding studies were performed to characterize the properties of subtypes of kappa opioid receptors in membrane preparations of human cerebral cortex. [3H]U69,593 ([3H]U69), a selective kappa 1-agonist, and [3H]diprenorphine ([3H]DIP), a non-selective opioid antagonist, in the presence of 1 microM each of DAMGO, DPDPE and U-69 to block mu-, delta-, and kappa 1-sites, labeled single population of binding sites, respectively. [3H]U-69 binding sites (KD = 3.8 +/- 0.2 nM, Bmax = 6.3 +/- 0.2 fmol/mg protein) had a binding profile that correspond to kappa 1-receptor. That is, dynorphin A (1-13) (Dyn A), bremazocine (BZC), U50,488H (U50), (-)ethylketocyclazocine (EKC) and nor-binaltorphimine (nor-BNI) bound to this site with high affinities. [3H]DIP labeled binding sites (Kd = 7.3 +/- 0.2 nM, Bmax = 102 +/- 9 fmol/mg protein) that were not sensitive to U-50, but to BZC, EKC and nor-BNI. These results indicate that kappa 1 and Kappa 2 opioid receptors exist in human cerebral cortex with different ligand binding profiles.     

Regulation of opioid receptors in rat sensory neurons in culture

To determine whether opioid receptors in sensory neurons are regulated by chronic exposure to opioids, we assessed the binding of various opioid ligands to membranes derived from isolated rat dorsal root ganglia neurons grown in culture. Equilibrium binding of [3H]diprenorphine onto membranes from cells grown for 13-15 days revealed a saturable binding site with a Kd value of 0.3 +/- 0.2 nM and an approximate Bmax value of 1300 +/- 200 fmol/mg of protein. [3H]Diprenorphine binding increased 3-fold from 1-15 days in culture. The mu receptors represent approximately 70 +/- 11% of the [3H]diprenorphine binding sites, as indicated by saturation binding of [3H]DAMGO. The kappa and delta receptors represent approximately 10 +/- 3% and approximately 5 +/- 2% of the [3H]diprenorphine binding sites, respectively. Preexposure of neurons to 10 microM naloxone for 48 hr up-regulated the receptors by 40%, whereas incubation with 100 nM to 10 microM DAMGO for 48 hr resulted in a significant decrease in the Bmax value of opioid receptors, with a maximum reduction of 70%. The identification of a high level of opioid receptors expressed in isolated sensory neurons and their modulation by opioids demonstrates that cultured sensory neurons are an excellent model with which to study opioid receptor regulation.     

Fetal death in mice lacking 5alpha-reductase type 1 caused by estrogen excess

Female mice deficient in steroid 5alpha-reductase type 1 have a decreased litter size. The average litter in homozygous deficient females is 2.7 pups vs. 8.0 pups in wild type controls. Oogenesis, fertilization, implantation, and placental morphology appear normal in the mutant animals. Fetal loss occurs between gestation days 10.75 and 11.0 commensurate with a midpregnancy surge in placental androgen production and an induction of 5alpha-reductase type 1 expression in the decidua of wild type mice. Plasma levels of androstenedione and testosterone are 2- to 3-fold higher on gestation day 9, and estradiol levels are chronically elevated by 2- to 3-fold throughout early and midgestation in the knockout mice. Administration of an estrogen receptor antagonist or inhibitors of aromatase reverse the high rate of fetal death in the mutant mice, and estradiol treatment of wild type pregnant mice causes fetal wastage. The results suggest that in the deficient mice, a failure to 5alpha-reduce androgens leads to their conversion to estrogens, which in turn causes fetal death in midgestation. These findings indicate that the 5alpha-reduction of androgens in female animals plays a crucial role in guarding against estrogen toxicity during pregnancy.     

Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]-SCH 58261 binding

1. The present study describes for the first time the characterization of the adenosine A2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies. 2. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 0.85 nM and 35 fmol mg-1 protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [3H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments. 3. Thermodynamic data indicate that [3H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A2A-adenosine receptors. 4. It is concluded that in human lymphocyte membranes [3H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A2A-receptor. The presence of A2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.     

Mechanism of endothelium-dependent relaxation induced by thrombin in the pig coronary artery

The present study describes the characterization of the binding properties and autoradiographic distribution of a new nonpeptide antagonist of neurotensin receptors, [3H]SR 142948A (2-[[5-(2,6-dimethoxyphenyl)-1-(4-(N-(3-dimethylaminopropyl)-N-methyl carbamoyl)-2-isopropylphenyl)-1H-pyrazole-3-carbonyl]-amino]-ad amantane-2-carboxylic acid, hydrochloride), in the rat brain. The binding of [3H]SR 142948A in brain membrane homogenates was specific, time-dependent, reversible and saturable. [3H]SR 142948A bound to an apparently homogeneous population of sites, with a Kd of 3.5 nM and a Bmax value of 508 fmol/mg of protein, which was 80% higher than that observed in saturation experiments with [3H]neurotensin. [3H]SR 142948A binding was inhibited by SR 142948A, the related nonpeptide receptor antagonist, SR 48692 (2-[[1-(7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole -3-carbonyl]amino]-adamantane-2-carboxylic acid) and neurotensin. Saturation and competition studies in the presence or absence of the histamine H1 receptor antagonist, levocabastine, revealed that [3H]SR 142948A bound with similar affinities to both the levocabastine-insensitive neurotensin NT1 receptors (20% of the total binding population) and the recently cloned levocabastine-sensitive neurotensin NT2 receptors (80% of the receptors) (Kd = 6.8 and 4.8 nM, respectively). The regional distribution of [3H]SR 142948A binding in the rat brain closely matched the distribution of [125I]neurotensin binding. In conclusion, these findings indicate that [3H]SR 142948A is a new potent antagonist radioligand which recognizes with high affinity both neurotensin NT1 and NT2 receptors and represents thus an excellent tool to study neurotensin receptors in the rat brain.     

Labeling of A2A adenosine receptors in human platelets by use of the new nonxanthine antagonist radioligand [3H]SCH 58261

The present study describes the binding to human platelet A2A adenosine receptors of the new potent and selective antagonist radioligand [3H]5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine ([3H]SCH 58261). Saturation experiments revealed that [3H]SCH 58261 labels a single class of recognition sites with high affinity (Kd = 0.85 nM), limited capacity (apparent Bmax = 85 fmol/mg of protein) and good specific binding (about 60%). [3H]SCH 58261 binding was not modulated by either the divalent cation Mg(+2) or guanine nucleotides. In competition experiments, a series of both adenosine agonists and antagonists inhibited [3H]SCH 58261 binding to A2A platelet receptors with rank order of potency and affinity similar to those observed in rat striatal membranes with the same radioligand. This confirms that the platelet A2A receptor is similar to that labeled in the brain striatum. Binding data were also found to be in good agreement with the results from functional studies such as A2A agonist-induced stimulation of adenylate cyclase or platelet aggregation inhibition. The present findings indicate that [3H]SCH 58261 is the first radioligand available for the characterization of the A2A receptor subtype in platelets.     

Muscarinic cholinergic receptor subtypes expression by human placenta

The presence of a cholinergic system in the placenta is suggested by several data, but no information is available concerning cholinergic receptor expression by placenta. The present study was designed to investigate muscarinic cholinergic receptors in sections of human placenta using a radioligand binding techniques with [3H]N-methyl scopolamine ([3H]NMS) as a ligand. [3H]NMS was bound to sections of human placenta in a manner consistent with the labelling of muscarinic cholinergic receptors. The dissociation constant (Kd) value was 0.1 +/- 0.03 nM and the maximum density of binding site (Bmax) value was 10.82 +/- 0.09 fmol/mg of tissue. The binding was time-, temperature- and concentration-dependent, belonging to one class of high affinity sites. Analysis of [3H]NMS displacement curves by compounds acting on the different subtypes of muscarinic cholinergic receptor subtypes suggests that human placenta expresses the four subtypes (M1-M4) of muscarinic cholinergic receptor assayable with radioligand binding assay techniques. The demonstration of muscarinic cholinergic recognition sites in human placenta may contribute to define the possible significance of placental cholinergic system. Moreover, human placenta can be used as an easily obtainable human source of M1-M4 muscarinic cholinergic receptor subtypes.     

Androgen and estrogen receptors in the developing mouse brain

Specific binding of the androgens, 5alpha-dihydrotestosterone (DHT) and testosterone, and of 17beta-estradiol by brain cytosol from mice at 3-5,9-11, and 18-23 days of age was measured by charcoal assay and glycerol gradient centrifugation and analyzed by Scatchard plots. The immature mouse brain contains putative receptors for these steroids which migrate at 8 S in gradients at low ionic strength and at 5 S in 0.5 M KCl. Investigation of estradiol binding was complicated by the presence in cytosol from 3-5 day-old mice, and to a lesser extent from 9-11 day-old mice, of the high capacity, fetoneonatal estradiol binding protein (FEBP) which is no longer detectable at 3 weeks. The rapid dissociation of the FEBP-estradiol complex under non-equilibrium conditions probably led to over-estimation of free steroid concentration and thus to an apparent increase in the affinity of 8 S receptor for estradiol with age (for female brain cytosol KD=9.5 X 10(-10)M at 3-5 days and 2.7 X 10(-10)M at 18-23 days). The number of estradiol binding sites remains relatively constant during the first 3 weeks at 7-9 fmol/mg protein, while the number of DHT binding sites in female brain increases from 3.2+/-0.3 to 6.6+/-0.9 to 9.6+/-0.3 (mean+/-SE) fmol/mg protein in the 3 age groups. Dissociation constants and numbers of sites for both DHT and estradiol binding are similar in brain cytosol from male and female mice. Testosterone and DHT compete for the same binding site, but its affinity for DHT is about twice that for testosterone. The high affinity of the brain receptor for DHT (KD=4-5 X 10(-10)M) may reflect the slow metabolism of DHT to 5alpha-androstanediols, amounting to less than 10% after 2 h at 0 C. Binding of DHT and estradiol to cytosol from brain regions was also investigated. DHT receptors increase in parallel in various regions with age; the concentration of sites in the hypothalamus-preoptic area (HPOA) is 1.2-3.4 times that in the cerebral cortex (C). The concentration of estradiol binding sites in HPOA to that in C increases about 12-fold from neonatal to adult stages, reflecting both an increase in HPOA sites and a decrease in C sites, while the concentration in the remainder of the brain shows little change. Androgen and estrogen receptors in brain cytosol from immature mice can be distinguished by their different specificities and developmental patterns in whole brain and brain regions. The presence and properties of these receptors in the brain of neonatal mice are discussed with respect to their possible role in sexual differentiation of the brain.     

Relationship of variations in tumor cell kinetics induced by primary chemotherapy to tumor regression and prognosis in locally advanced breast cancer

We have studied binding of isradipine to A7r5 vascular smooth muscle cells as a function of membrane potential and cell proliferation. Consistent with a voltage-modulated receptor model, two classes of binding sites were detected in confluent cultures: high-affinity sites under depolarizing (50 mM K+) conditions (Kd = 45 +/- 3 pM), and lower affinity sites under resting (5 mM K+) conditions (Kd = 181 +/- 20 pM). However, proliferating cells also displayed the high-affinity state at rest (Kd = 29 +/- 9 pM) in addition to a low-affinity site (Kd = 869 +/- 383 pM). Analysis of dissociation rates also revealed two receptor classes during proliferation. Proliferating cells showed a single class of high-affinity sites (Kd = 39 +/- 6 pM) when depolarized, similar to confluent cells. Receptor density in confluent monolayers increased from 15 +/- 3 fmol/10(6) cells at 5 days to 72 +/- 6 fmol/10(6) cells after 10 days. These results suggest (i) that some L-type Ca2+ channels are spontaneously active in proliferating vascular smooth muscle cells, but require depolarization to activate in a confluent monolayer, and (ii) that the density of dihydropyridine receptors increases after a monolayer becomes confluent.     

3H]-SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes

1. The present study describes the direct labelling of A2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[l,5-c]pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays. 2. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 1.34 nM and 75 fmol mg(-1) protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [3H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments. 3. Thermodynamic data indicated that [3H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A2A adenosine receptors. 4. It was concluded that in human neutrophil membranes, [3H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A2A adenosine receptors of other tissues. The receptors labelled by [3H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.     

Identification of a specific binding site for the anabolic steroid stanozolol in male rat liver microsomes

Male rat liver microsomes contain a [3H]dexamethasone binding site, capable of binding glucocorticoids and progesterone. We have shown previously that the 17 alpha-alkylated androgen, stanozolol, can inhibit the [3H]dexamethasone binding to microsomes through a negative allosteric mechanism, which gives rise to the possibility of its interaction with a different binding site. In this study, the existence of a single-saturating binding site, capable of binding the radioactive steroid with a maximum number of the specific binding site of 49 +/- 2 pmol/mg of protein and a Kd of 37 +/- 1.3 nM was demonstrated by using [3H]stanozolol. In competition experiments, only stanozolol and danazol were able to compete with [3H]stanozolol for its binding to microsomes, among more than 60 steroids and other compounds tested. The binding of [3H]stanozolol was depressed after protease treatment of the microsomes, or after the administration of cycloheximide to adult male rats for 24 hr, which suggest its proteic nature. The [3H]stanozolol binding site was detected in many tissues of the rat, with the highest concentrations being found in the liver. It was detected from birth, increasing afterward in concentration and reaching a peak at 2 to 3 months of age. This is the first experimental verification of the existence in liver microsomes of a specific binding site for some 17 alpha-alkylate androgens, such as stanozolol and danazol, different from the androgen receptor or the [3H]dexamethasone binding site.     

Steroid hormone receptor studies in melanoma model systems

The transplantable B-16 melanotic melanoma carried in syngeneic C57Bl/6J female mice and the Syrian hamster melanoma cell line, RPMI 3460, were utilized to determine whether steroid-hormone receptors are present in animal melanomas. In the B-16 melanoma, a cytoplasmic-estrogen receptor is detectable, but there is no evidence for androgen or progestin receptors. Some tumors contain a glucocorticoid-binding macromolecule. Sucrose-density gradient centrifugation of cytosol after incubation with [3H]-estradiol revealed an 8S peak that was suppressed by excess radioinert diethylstilbesterol. Binding varied from 5-35 fmoles per mg cytosol protein. Scatchard analysis of [3H]-estradiol binding in cytosol yielded a single class of high-affinity binding sites; the dissociation constant is 6 x 10(-10) M. The receptor molecule is shown to be estrogen-specific by ligand competition assays. In contrast to B-16 melanoma, no estrogen, androgen, or progestin receptor can be found in the Syrian hamster melanoma cell line. However, a substantial level of specific binding is observed using [3H]-dexamethasone. Sucrose-gradient centrifugation of cytosol from this cell line after incubation with [3H]-dexamethasone revealed a 7S peak that was suppressed by excess radioinert dexamethasone. Scatchard analysis indicated a single class of high-affinity sites with a dissociation constant of 2 x 10(-9) M. Binding levels from 70-610 fmoles per mg cytosol protein were observed. The Syrian hamster melanoma cells also exhibit a biological response to glucocorticoids: Dexamethasone causes both an inhibition of growth and a decrease in final-cell density in these cells.