Selectivity of (3)H-MADAM binding to 5-hydroxytryptamine transporters in vitro and in vivo in mice; correlation with behavioural effects

1. Binding of the novel radioligand (3)H-2-(2-dimethylaminomethyl-phenylsulphanyl)-5-methyl-phenylamine ((3)H-MADAM) to the serotonin transporter (SERT) was used to characterise a range of selective serotonin re-uptake inhibitors (SSRIs) in vitro and in vivo. 2. (3)H-MADAM bound with high affinity in a saturable manner to both human SERT expressed in CHO cells (K(d)=0.20 nm (pK(d)=9.74+/-0.12), B(max)=35+/-4 fmol mg(-1) protein) and mouse cerebral cortex membranes (K(d)=0.21 nm (pK(d)=9.66+/-0.10), B(max)=50+/-24 fmol mg(-1) protein). 3. Binding of (3)H-MADAM was highly selective for SERT in vitro as demonstrated by the in vitro profile of MADAM tested at 75 different receptors, ion channels and transporters. This was further substantiated by the pharmacological profile of the binding. Hence, the binding of (3)H-MADAM was potently inhibited by SSRIs but not by selective inhibitors of noradrenaline transport and dopamine transport. Likewise, a 5-HT(2A/2C) receptor antagonist did not inhibit (3)H-MADAM binding. 4. (3)H-MADAM binding in vivo was inhibited only by compounds which also inhibited the binding of (3)H-MADAM in vitro (the SSRIs, mixed SERT/noradrenaline transport inhibitors and clomipramine), confirming the selectivity of (3)H-MADAM for SERT also in vivo. Moreover, compounds effective in inhibiting (3)H-MADAM binding were the only ones found to be active in the mouse 5-HTP potentiation test confirming the model as a behavioural correlate to in vivo 5-HT uptake. 5. Finally, it was found that a SERT occupancy of 85-95% was necessary to produce 50% of the maximum behavioural response (ED(50)).     

Pharmacological and biochemical characterization of A3 adenosine receptors in Jurkat T cells

1. The present work was devoted to the study of A3 adenosine receptors in Jurkat cells, a human leukemia line. 2. The A3 subtype was found by means of RT-PCR experiments and characterized by using the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with K(D) of 1.9+/-0.2 nM and B(max) of 1.3+/-0.1 pmol mg(-1) of protein. 3. The pharmacological profile of [3H]-MRE 3008F20 binding on Jurkat cells was established using typical adenosine ligands which displayed a rank order of potency typical of the A3 subtype. 4. Thermodynamic data indicated that [3H]-MRE 3008F20 binding to A3 subtype in Jurkat cells was entropy- and enthalpy-driven, according with that found in cells expressing the recombinant human A3 subtype. 5. In functional assays the high affinity A3 agonists Cl-IB-MECA and IB-MECA were able to inhibit cyclic AMP accumulation and stimulate Ca(2+) release from intracellular Ca(2+) pools followed by Ca(2+) influx. 6. The presence of the other adenosine subtypes was investigated in Jurkat cells. A1 receptors were characterized using [3H]-DPCPX binding with a K(D) of 0.9+/-0.1 nM and B(max) of 42+/-3 fmol mg(-1) of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a K(D) of 2.5+/-0.3 nM and a B(max) of 1.4+/-0.2 pmol mg(-1) of protein. 7. In conclusion, by means of the first antagonist radioligand [3H]-MRE 3008F20 we could demonstrate the existence of functional A3 receptors on Jurkat cells.     

Seasonal variations in [3H]citalopram platelet binding between healthy controls and violent offenders in Finland

Monthly binding densities (B(max)) of [3H]citalopram to the platelet serotonin transporter (SERT) was measured longitudinally over 1 year in a control group of 18 healthy Finnish male volunteers. Single platelet samples were also analysed from 33 men who were incarcerated for violent crimes during the same calendar year. A statistically significant seasonal variation in SERT B(max) was observed in both data sets, and bi-monthly floating averages for SERT B(max) were calculated and then fit to an annual sinusoidal curve for both groups. The B(max) for platelet [3H]citalopram binding showed a statistically significant (p = 0.001) seasonal variance between a winter (January-February) maximum of 1590 fmol/mg protein and a summer (July-August) minimum of 1216 fmol/mg protein for the control group, with an R2 of 70% for the annual sinusoidal curve fit. A statistically significant (p = 0.007) seasonal variance was also observed between a winter (January-February) maximum of 1980 fmol/mg protein and an autumnal (August-September) minimum of 1234 fmol/mg protein for the violent offenders, again with an R2 of 70% for the annual sinusoidal curve fit. This observation lends additional support to the idea that violent human behavior and impulsivity may be directly linked to values of SERT B(max), which can be affected by various psychoactive drugs and also varies with the natural change of seasons.     

Irreversible binding of a novel phenylisothiocyanate tropane analog to monoamine transporters in rat brain

Irreversible tropane analogs have been useful in identifying binding sites of cocaine on biogenic amine transporters, including transporters for dopamine (DAT), serotonin (SERT) and norepinephrine (NET). The present study characterizes the properties of the novel phenylisothiocyanate tropane HD-205, synthesized from the highly potent 2-napthyl tropane analog WF-23. In radioligand binding studies in brain membranes, direct IC(50) values of HD-205 were 4.1, 14 and 280nM at DAT, SERT and NET, respectively. Wash-resistant binding was characterized by preincubation of HD-205 with brain membranes, followed by extensive washing before performing transporter radioligand binding. Results for HD-205 showed wash-resistant IC(50) values of 191, 230 and 840nM at DAT, SERT and NET, respectively. Saturation binding studies with [(125)I]RTI-55 in membranes pretreated with 100nM HD-205 showed that HD-205 significantly decreased the B(max) but not K(D) of DAT and SERT binding. To further characterize its irreversible binding, an iodinated analog of HD-205, HD-244, was prepared from a trimethylsilyl precursor. The direct IC(50) of HD-244 at DAT was 20nM. [(125)I]HD-244 was synthesized with chloramine-T, purified on HPLC, reacted with rat striatal membranes, and proteins were separated by SDS-PAGE. Results showed several non-specific labeled bands, but only a single specific band of radioactivity co-migrating with an immunoreactive DAT band at approx. 80 kilodaltons was detected, suggesting that [(125)I]HD-244 covalently labeled DAT protein in striatal membranes. These results demonstrate that phenylisothiocyanate analogs of WF-23 can be used as potential ligands to map distinct binding sites of cocaine analogs at DAT.     

Changes in [(3)H]glibenclamide binding to mouse forebrain membranes during morphine tolerance

The characteristics of specific binding of the ATP-sensitive K(+) (K(ATP)) channel blocker [3H]glibenclamide to forebrain membranes (P(2) fraction, 4 degrees C) obtained from morphine-naive and -tolerant mice were evaluated. Morphine tolerance was induced by osmotic minipumps that released 45 mg/kg/day of morphine subcutaneously for 6 days. This treatment enhanced the antinociceptive ED(50) of morphine without changing its E(max). In morphine-naive animals, (1) both the association and the dissociation of [3H]glibenclamide were biphasic; (2) [3H]glibenclamide was displaced by other sulfonylureas (order of potency: glibenclamide>glipizide&z.Gt;tolbutamide) with pseudo-Hill coefficients lower than unity and biphasic Hofstee plots; and (3) Scatchard plots of saturation experiments were curvilinear, showed a Hill coefficient of 0.81+/-0.04 and suggested the presence of two binding sites with a K(D) of 0.13 and 3.17 nM and a B(max) of 12.30 and 84.47 fmol/mg protein, respectively. By contrast, in membranes obtained from morphine-tolerant animals, (1) the Scatchard plots showed only one population of binding sites with a K(D) of 0.87 nM and a B(max) of 77.99 fmol/mg protein, and the Hill coefficient was very close to unity (0.96+/-0.1); (2) competition experiments (using glibenclamide as displacer) showed a pseudo-Hill coefficient of 0.99+/-0.04; and (3) dissociation experiments showed only one phase of dissociation. These results suggest that [3H]glibenclamide binds to two different sites in membranes obtained from morphine-naive animals, but to only one site in morphine-tolerant animals. Consequently, it seems that morphine tolerance in mice involves adaptive changes in K(ATP) channels.     

(125)I]-(Pyr(1))Apelin-13 is a novel radioligand for localizing the APJ orphan receptor in human and rat tissues with evidence for a vasoconstrictor role in man

1. We have determined the binding characteristics of [(125)I]-(Pyr(1))Apelin-13, a putative ligand for the APJ orphan receptor in human cardiovascular and rat tissue and investigated the functional properties of (Pyr(1))Apelin-13 in human saphenous vein. 2. The binding of [(125)I]-(Pyr(1))Apelin-13 to sections of human heart tissue was time dependent and rapid at 23 degrees C. Data were fitted to a single site model with an association rate constant (k(obs)) of 0.115 min(-1). [(125)I]-(Pyr(1))Apelin-13 also dissociated from a single site with a dissociation rate constant of 0.0105 min(-1). 3. In saturation binding experiments [(125)I]-(Pyr(1))Apelin-13 bound to human left ventricle with a K(D) value of 0.35+/-0.08 nM, B(max) of 4.3+/-0.9 fmol mg(-1) protein with a Hill slope of 0.97+/-0.04 and to the right atria with a K(D) of 0.33+/-0.09 nM, B(max) of 3.1+/-0.6 fmol mg(-1) protein and a Hill slope of 0.93+/-0.05. 4. [(125)I]-(Pyr(1))Apelin-13 binding sites were localized using autoradiography to human cardiovascular tissue, including coronary artery, aorta and saphenous vein grafts. In rat tissue a high density of receptors were localized to the molecular layer of the rat cerebellum, rat lung, rat heart and low levels in the rat kidney cortex. 2. (Pyr(1))Apelin-13 potently contracted human saphenous vein with a pD(2) value of 8.4+/-0.2 (n=8). The maximum response elicited by the peptide was 22.6+/-6% of 100 mM KCl. 6. We provide the first evidence of APJ receptor expression, relative densities and functional properties of (Pyr(1))Apelin-13 in human cardiovascular tissue.     

Characterization and distribution of prolactin releasing peptide (PrRP) binding sites in the rat--evidence for a novel binding site subtype in cardiac and skeletal muscle

Prolactin releasing peptide (PrRP) was recently purified from bovine hypothalamus and binds to the orphan receptor, UHR-1. We examined the distribution and kinetics of (125)I-PrRP binding in rat tissues together with molecular characterization by chemical cross-linking and Northern blotting. In this study (125)I-PrRP binding showed specificity and rapid association and dissociation. Specific binding was found in membranes from rat tissues including brain (hypothalamus, medulla oblongata and cerebellum), pituitary, heart, soleus muscle, adipose tissue, kidney, adrenal gland, testis and small intestine. In hypothalamus, pituitary, heart and soleus competition analysis indicated only one class of binding site in each tissue. Binding affinity for PrRP (IC(50)) and binding site density (B(max)) respectively were 5.2+/-0.9 nM and 674+/-97 fmol mg protein(-1) in hypothalamus (n = 5), 1.4+/-0.6 nM and 541+/-126 fmol mg protein(-1) in pituitary (n = 3), 6.6+/-0.7 nM and 628+/-74 fmol mg protein(-1) in heart (n = 4) and 9.8+/-0.9 nM and 677+/-121 fmol mg protein(-1) in soleus muscle (n = 4). Analysis of (125)I-PrRP-binding site complexes by chemical cross-linking showed a binding site M(r) of 69,000 in hypothalamus and 41,000 in heart and soleus. Northern analysis of polyA(+) RNA from hypothalamus showed a 4.2 kb band as expected for UHR-1, but heart and soleus showed a 4.8 kb band. Taken together these results indicate that there may be different subtypes of PrRP binding sites in rat tissues which may differ from UHR-1.     

Antagonism of nicotinic receptors of rat chromaffin cells by N,N, N-trimethyl-1-(4-trans-stilbenoxy)-2-propylammonium iodide: a patch clamp and ligand binding study

The effect of the oxystilbene derivative F3 was tested on nAChRs of whole-cell patch-clamped rat chromaffin cells in vitro and of rat adrenal gland membranes using (125)I-epibatidine. F3 (30 nM) rapidly and reversibly blocked inward currents generated by pulse applications of nicotine, shifting the dose-response curve to the right in a parallel fashion without changing the maximum response. The action of F3 was voltage insensitive and not due to altered current reversal potential. The R isomer of F3 was more potent (IC(50) = 350+/-30 nM) than its S-enantiomer (IC(50) = 1.5+/-0.3 microM). Nicotine-evoked currents were insensitive to 10 microM alpha-bungarotoxin. Equi-amplitude currents evoked by nicotine or epibatidine were similarly antagonized by R-F3 in a reversible fashion. Epibatidine-evoked currents readily produced receptor desensitization. Adrenal membranes specifically bound (125)I-epibatidine with a single population of binding sites endowed with high affinity (K(D) = 159 pM) and B(max) of 6.5+/-1.3 fmol mg(-1) of protein. (125)I-epibatidine binding was specifically displaced by cytisine (K(i) = 68 nM) or ACh (K(i) = 348 nM). F3 specifically displaced (125)I-epibatidine binding although with lower affinity (K(i) = 29.6 microM) than in electrophysiological experiments. (125)I-epibatidine binding to rat adrenal tissue was insensitive to alpha-bungarotoxin which readily antagonized (125)I-epibatidine binding to bovine adrenal tissue. The present results suggest that F3 is a relatively potent and apparently competitive antagonist of nAChRs on rat chromaffin cells. Since previous studies have indicated that F3 targets different subtypes on chick neuronal tissue, it appears that nAChRs display interspecies differences to be considered for drug development studies.     

Glycosylation of the human prostacyclin receptor: role in ligand binding and signal transduction

Prostacyclin, a potent vasodilator and inhibitor of platelet aggregation, acts through a cell-surface G protein-coupled receptor [prostacyclin (IP)]. The human (h) IP contains two consensus sites for N-linked glycosylation (N(7) and N(78)). However, the role of glycosylation is unknown. Mutant receptors (N(7)-Q(7),N(78)-Q(78) and N(7),N(78)-Q(7),Q(78)) were generated by replacing N(7) and/or N(78) with Q's. Receptor glycosylation was similar in the wild-type and N(7)-Q(7) and was inhibited with tunicamycin. N(78)-Q(78) and N(7),N(78)-Q(7),Q(78) demonstrated little or no glycosylation. Membrane localization was reduced for each mutant concomitant with impaired glycosylation. Partial localization to the plasma membrane allowed direct examination of the effect of glycosylation on IP function. High-affinity binding to N(7)-Q(7) was similar (K(d) = 21.7 +/- 1.7 nM, n = 4) to that of the wild-type receptor (K(d) = 24.3 +/- 3.6 nM, n = 4), despite a reduced value for B(max) (0.35 +/- 0.03 fmol/mg of protein versus 3.34 +/- 0.52 fmol/mg of protein, n = 4). Binding to N(78)-Q(78) (B(max) = 0.27 +/- 0.03 fmol/mg of protein, n = 3; K(d) = 149.1 +/- 11.1, n = 3) and N(7),N(78)-Q(7),Q(78) (no specific binding) was further impaired. Agonist-induced adenylyl cyclase activation was reduced in N(7)-Q(7) cells, whereas N(78)-Q(78) cells responded only to high concentrations of iloprost and N(7),N(78)-Q(7),Q(78) were unresponsive. Inositol phosphate generation was evident only with the wild-type. Only the wild-type and N(7)-Q(7) receptors underwent agonist-induced sequestration. Our findings demonstrate greater glycosylation at N(78) compared with N(7). The extent of N-linked glycosylation of hIP may be important for membrane localization, ligand binding, and signal transduction.     

3H]R214127: a novel high-affinity radioligand for the mGlu1 receptor reveals a common binding site shared by multiple allosteric antagonists

R214127 was shown to be a potent and noncompetitive metabotropic glutamate 1 (mGlu1) receptor-selective antagonist. The kinetics and pharmacology of [(3)H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone (R214127) binding to rat mGlu1a receptor Chinese hamster ovary (CHO)-dhfr(-) membranes was investigated, as well as the distribution of [(3)H]R214127 binding in rat brain tissue and sections. Specific binding to rat mGlu1a receptor CHO-dhfr(-) membranes was approximately 92% of total and was optimal at 4 degrees C. Full association was reached within 5 min, and [(3)H]R214127 bound to a single binding site with an apparent K(D) of 0.90 +/- 0.14 nM and a B(max) of 6512 +/- 1501 fmol/mg of protein. Inhibition experiments showed that [(3)H]R214127 binding was completely blocked by 2-quinoxaline-carboxamide-N-adamantan-1-yl (NPS 2390), (3aS,6aS)-6a-naphtalan-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan-1-on (BAY 36-7620), and 7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), but was not displaced by competitive mGlu1 receptor ligands such as glutamate and quisqualate, suggesting that R214127, NPS 2390, BAY 36-7620, and CPCCOEt bind to the same site or mutually exclusive sites. Experiments using rat cortex, striatum, hippocampus and cerebellum revealed that [(3)H]R214127 labeled a single high-affinity binding site (K(D) approximately 1 nM). B(max) values were highest in the cerebellum (4302 +/- 2042 fmol/mg of protein) and were 741 +/- 48, 688 +/- 125, and 471 +/- 68 fmol/mg of protein in the striatum, hippocampus, and cortex, respectively. The distribution of [(3)H]R214127 binding in rat brain was investigated in more detail by radioligand autoradiography. A high density of binding sites was detected in the molecular layer of the cerebellum. Moderate labeling was seen in the CA3 and dentate gyrus of the hippocampus, thalamus, olfactory tubercle, amygdala, and substantia nigra reticulata. The cerebral cortex, caudate putamen, ventral pallidum, and nucleus accumbens showed lower labeling. The high affinity and selectivity of [(3)H]R214127 for mGlu1 receptors renders this compound the ligand of choice to study the native mGlu1 receptor in brain.     

Pharmacological characterization of the 5-HT1A, 5-HT2 and 5-HT3 receptors in the bovine ciliary muscle

We aimed to investigate the effects of serotonin (5-hydroxytryptamine, 5-HT) on the bovine ciliary muscle and subsequently to characterize and identify the subtypes of 5-HT receptors involved in the serotonin-evoked contractility muscle. The binding of [3H]ketanserin, [3H]granisetron and [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) was analyzed. All labelled compounds bound with high affinity to a single site in the membrane preparations studied. The affinity (K(d)) of the binding site was 7.5+/-1.2 nM for [3H]ketanserin, 6.9+/-0.8 nM for [3H]granisetron and 4.4+/-0.31 nM for [3H]8-OH-DPAT. The density of receptors (B(max)) was 1062+/-43.0 fmol/mg protein for [3H]ketanserin, 566+/-2.32 fmol/mg protein for [3H]granisetron and 205+/-4.63 fmol/mg protein for [3H]8-OH-DPAT. The serotonin-induced contraction appeared to be competitively antagonized by ketanserin (0.1, 1 and 10 microM) and ondansetron (0.1, 10 and 100 microM) which produced a pA(2) value of 8.5+/-0.12 and 8.0+/-0.19, respectively. 8-OH-DPAT and 5-carboxamidotryptamine (5-CT) proved to be completely ineffective. We conclude that serotonin induces bovine ciliary muscle contraction via 5-HT(2) and 5-HT(3) receptors while the 5-HT(1A) receptors, although present, do not mediate the contractile response.     

Syntheses and binding studies of new [(aryl)(aryloxy)methyl]piperidine derivatives and related compounds as potential antidepressant drugs with high affinity for serotonin (5-HT) and norepinephrine (NE) transporters

In a wide search program toward new, efficient, and fast-acting antidepressant drugs, we have prepared series of new compounds having an (aryl)(aryloxy)methyl moiety linked directly or through a methylene chain to different substituted and unsubstituted cycles (isoquinoline, piperazine, piperidine, tetrahydropyran, or cyclopentane). These compounds have been evaluated for their affinities for serotonin (5-HT) transporter (SERT) and 5-HT(1A) and 5-HT(2A) receptors. Racemic mixtures of 4-[(aryl)(aryloxy)methyl]piperidine derivatives showed much higher affinity values for SERT than fluoxetine and resulted in lack of affinity for 5-HT(1A) and 5-HT(2A) receptors. Some of these racemic mixtures were resolved to their enantiomers and tested for binding to norepinephrine (NE) transporter (NET), dopamine (DA) transporter (DAT), and alpha(2) receptor. Several of these enantiomers [(-)-15b, (-)-15j, (-)-15t, (+)-15u] displayed a dual binding profile with affinities for SERT and NET with K(i) < 25 nM and a NET/SERT ratio <10. Compound (-)-15j (coded as F-98214-TA for development studies) showed a dual binding profile with very high affinity values for SERT and NET (K(i) = 1.9 and 13.5 nM, respectively), and further pharmacological characterization is in progress for its evaluation as a antidepressant.     

Enhancing effect of zinc on astroglial and cerebral endothelial histamine uptake.

We have studied the effect of zinc ion on the uptake of histamine (HA) into cultured astroglial and cerebral endothelial cells and established that Zn(2+) enhances the uptake of the amine dose-dependently and in remarkable extents by increasing the V(max) to about 3-fold (from 3.25 +/- 0.42 to 8.50 +/- 0.97 pmol/mg protein/min in astroglial cells) without altering the K(M) (0.20 +/- 0.03 microM) significantly. The stimulatory effect of zinc ion showed strong sensitivity for VUF 8407, an inhibitory compound of astroglial and cerebral endothelial uptake of HA. In the presence of 20 microM VUF 8407 the zinc-enhanced uptake was reduced by about 50% in both cell types. Binding measurements revealed increased capacities of the zinc-exposed HA binding (B(max)= 0.41 +/- 0.05 increased to 1.21 +/- 0.16 pmol/mg protein in astroglial membranes and B(max) = 0.25 +/- 0.03 enhanced to 1.05 +/- 0.12 pmol/mg protein in cerebral endothelial membranes) but statistically unchanged affinity of the ligand for HA carrier (K(D) values calculated as 35.2 +/- 3.4 nM and 45.1 +/- 3.8 nM for astroglial bindings; whereas 25 +/- 2.1 nM and 30 +/- 2.6 nM for cerebral endothelial bindings of the amine). The compound VUF 8407 reduced the B(max) of zinc-exposed HA binding of astroglial membranes but did not modify the K(D) of the zinc-exposed membrane significantly. The ex vivo experiments confirmed our in vitro findings; an i.c.v. dose of 0.4 micromol/kg ZnSO(4,) 24 hr after the injection, enhanced the uptake of [(3)H]HA into dissociated hypothalamic and cerebellar cells to about 2- and 3-fold, respectively. Present data clearly showed that zinc exposures enhance the astroglial and the cerebral endothelial uptake of HA in vitro and it might be considered that zinc produces similar effects in vivo. Free zinc may participate in the regulation of the extraneuronal HA concentration and this metal ion (endogenous or exogenous) might be favored in the removal of the amine from the interstitial space especially in conditions with relatively high HA.     

S 14506: novel receptor coupling at 5-HT(1A) receptors

S 14506 is chemically related to the inverse agonist at 5-HT(1A) receptors, spiperone, but S 14506 behaves as one of the most potent agonists known at these receptors, both in vitro and in vivo. In hippocampal membranes, the specific binding of [(3)H]-S 14506 (K(d)=0.79+/-0.2 nM; B(max)=400+/-32 fmol/mg protein) to 5-HT(1A) receptors resembled that of an antagonist in that it was increased by GppNHp, whereas GppNHp reduced the binding of the classic agonist [(3)H]-8-OH-DPAT (K(d)=1.5+/-0.5 nM; B(max)=303+/-20 fmol/mg protein). Manganese, magnesium and calcium reduced the binding of [(3)H]-S 14506 to 5-HT(1A) receptors whereas the binding of [(3)H]-8-OH-DPAT was increased. Further, sodium markedly reduced the binding of [(3)H]-8-OH-DPAT, without affecting the binding of [(3)H]-S 14506. [(3)H]-S 14506 also bound with high affinity to h 5-HT(1A) receptors stably expressed in membranes of CHO cells (K(d)=0.13+/-0.05 nM; B(max)=2.99+/-0.60 pmol/mg protein): the B(max) was double that of [(3)H]-8-OH-DPAT. GppNHp strongly decreased [(3)H]-8-OH-DPAT binding but scarcely changed [(3)H]-S 14506 binding; calcium, magnesium and manganese had little effect on [(3)H]-S 14506 binding in CHO cells. Antagonists (WAY 100635, WAY 100135) and inverse agonists (spiperone and metitepine) displaced [(3)H]-S 14506 binding with high affinity and Hill slopes close to unity, whereas agonists (5-HT and 5-CT) displayed low affinity with low Hill slopes: partial agonists (buspirone, ipsapirone) showed intermediate properties. In fusion proteins of h 5-HT(1A) receptors with G(ialpha1) the compound potently increased high-affinity GTPase, with a steeper Hill slope than for 5-HT, which may indicate positive cooperativity. The maximum response for S 14506 in these assays was equivalent to 5-HT, indicating it to be a full agonist.In molecular modelling studies, using a three-site model of the 5-HT(1A) receptor, S 14506 spanned between the 5-HT recognition site and the "arginine switch" (DRY microdomain) postulated to activate the interaction of the receptor with the G protein. Thus it is possible to synthesise ligands at G-protein-coupled receptors which are highly potent agonists, but which are structurally related to inverse agonists and show some features of antagonist/inverse agonist binding.     

Modulation of affinity and density of LTB4 receptors on guinea pig lung membranes by divalent cations and guanine nucleotides.

We investigated the mechanism by which guanine nucleotides and divalent cations modulate the affinity and apparent density of high-affinity receptors for Leukotriene B4 (LTB4) on guinea pig lung membranes (GPLM). Divalent cations (Mg2+ = Ca2+ greater than Mn2+) stimulated, whereas EDTA inhibited (IC50 = 0.31 +/- 0.08 mM) binding of [3H]LTB4. Saturation analysis demonstrated that omission of divalent cations caused a two-fold reduction in apparent site density, (B max = 297 +/- 24 fmol/mg protein vs. 149 +/- 21 fmol/mg protein, P less than 0.01, for control and EDTA-treated respectively), but no significant change in receptor affinity (KD = 0.67 +/- 0.16 nM and 1.01 +/- 0.19 nM, P greater than 0.05). Competition experiments with LTB4 and the low-affinity (Ki = 165 nM) competitive LTB4-antagonist U75302, also demonstrated that EDTA caused a significant reduction (1.7 and 3.6-fold, P less than 0.05 and P less than 0.01, respectively), in affinity to both ligands. In the same experiments, the the guanine nucleotide analog GppNHp also reduced the affinity for LTB4 and U75302, similar to that observed with EDTA, suggesting that removal (Mg2+), or addition (GppNHp), of allosteric modulators of G-protein(s), causes reduction in receptor affinity. Saturation experiments also demonstrated that GppNHp, or GTP(gamma S), caused a significant reduction (40-50%) in receptor density. A larger reduction in affinity for U75302 (3- to 3.6-fold) than for LTB4 (1.7-fold) was induced by EDTA as well as GTP analogs.(ABSTRACT TRUNCATED AT 250 WORDS)     

An effect of ischemia on myocardial dihydropyridine binding sites

The [3H]nitrendipine binding activity of sarcolemmal fragments isolated from aerobically perfused or ischemic rat hearts was studied. After 90 min aerobic perfusion, two populations of binding sites were detected--high affinity sites with KD of 0.24 +/- 0.04 nM and Bmax 313 +/- 110 fmol/mg protein, and low affinity sites with KD of 47.6 +/- 8.7 nM and Bmax 12.4 +/- 1.88 pmol/mg protein. Sixty minutes global ischemia significantly reduced the KD of the low (15.8 +/- 2.9 nM, P less than 0.03) but not of the high (0.22 +/- 0.05 nM) affinity sites. Under these same conditions the Bmax of both the high (82.4 +/- 14.5 fmol/mg protein, P less than 0.03) and low (6.1 +/- 1.7 pmol/mg protein, P less than 0.01) affinity binding sites was reduced but the sites retained their selectivity, with nifedipine displacing bound [3H]nitrendipine more potently than D600. Bay K 8644, when added upon reperfusion, promoted a dose-related increase in Ca2+ entry which was reduced by nifedipine, indicating that dihydropyridine binding sites can be activated after 60 min ischemia.     

Synthesis and monoamine transporter binding of 2-(diarylmethoxymethyl)-3 beta-aryltropane derivatives

3 beta-Aryltropane analogues wherein the 2-position was substituted with various diarylmethoxyalkyl groups were synthesized and evaluated for binding at the dopamine transporter (DAT), serotonin transporter (SERT), norepinephrine transporter (NET), and muscarinic (M(1)) receptors. The 2 beta-analogues 9a-i generally demonstrated high to moderate binding affinities (K(i) = 34-112 nM) at the DAT with good selectivity over SERT, NET, and M(1) receptors. Alternatively, the 2 alpha-isomers 10a-i were 10-fold less potent at the DAT with poor selectivity over SERT. These SAR studies provide further evidence for the varied binding requirements of structurally diverse tropane-based ligands and support future studies to elucidate DAT binding requirements in relation to cocaine-like behavioral endpoints.     

Cloning and pharmacological characterization of the cat urotensin-II receptor (UT)

Urotensin-II (U-II), acting through its G-protein-coupled receptor, UT, is a possible contributor to hypertension. Variable functional responses to U-II, both within and between species studied to date, complicate the characterization of UT antagonists. In the cat, however, U-II causes systemic hypertension and constricts arterial segments isolated from several vascular beds. The purpose of this study was to clone and pharmacologically characterize cat recombinant UT to determine whether this system represents a model for characterizing UT antagonists. Cloned cat UT displayed 74% identity to primate UT, and 77% identity to rodent UT. [(125)I] hU-II bound in a saturable manner to a single site on recombinant cat UT with high affinity (K(D) 288+/-13pM) and high density (B(max) 747+/-66fmol/mg protein). U-II isopeptides displayed equipotent, high affinity binding to cat UT (K(i) 1.8-5.3nM). Cat UT was coupled to intracellular [Ca(2+)] release (EC(50) 0.6+/-0.2nM) and total inositol phosphate (IP) formation (EC(50) 0.4+/-0.1nM). Protein kinase C activation desensitized cat, but not human, UT-mediated IP formation. UT mRNA expression was detected in cat blood vessels, trachea, lung, and kidney, where the medulla (K(D) 815+/-34) and cortex and (K(D) 316+/-39pM) displayed high affinity binding for human U-II (hU-II). The cat urotensin-II receptor represents a suitable in vitro model to examine the role of the U-II/UT system in the etiology of hypertension, assisting in the evaluation of the UT antagonists to help treat cardiovascular disease.