High- and low-affinity binding sites for [3H]-alpha, beta-methylene ATP in rat urinary bladder membranes.

1. The characteristics of [3H]-alpha, beta-methylene adenosine 5'triphosphate ([3H]-alpha, beta-MeATP) binding to membrane preparations of rat urinary bladder detrusor were studied. 2. The rat bladder membrane preparation was obtained by multiple centrifugation. [3H]-quinuclidinyl benzilate [( 3H]-QNB) binding to this preparation demonstrated that the muscarinic receptor density was 4.32 times higher than that in the homogenate. [3H]-alpha, beta-MeATP binding was increased 3.88 times. 3. Saturation analysis revealed that the rat bladder membrane contained a high density of [3H]-alpha, beta-MeATP binding sites, which could be divided into a high-affinity component (Kd = 8.1-8.9 nM) and a low-affinity component (Kd = 67.0-119.8 nM). 4. Magnesium ions inhibited the maximum binding in a concentration-dependent manner. The maximum high-affinity binding was reduced from 10.32 pmol mg-1 protein in magnesium-free buffer to 4.62 pmol mg-1 protein with 25 mM MgCl2, while the maximum low-affinity binding was reduced from 58.84 pmol mg-1 protein to 14.24 pmol mg-1 protein. Kd values were not greatly affected. 5. The binding was a rapid reversible process. The association rate constants were 7.64 x 10(7) M-1 min-1 for high-affinity binding, and 7.31 x 10(6) M-1 min-1 for low-affinity binding. The dissociation rate constants were 0.2896 min-1 for high-affinity binding, and 0.6348 min-1 for the low-affinity binding. 6. Displacement experiments with unlabelled purinoceptor ligands confirmed that [3H]-alpha, beta-MeATP mainly binds to P2X-purinoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that the atypical 5-HT3 receptor ligand, [3H]-BRL46470, labels additional 5-HT3 binding sites compared to [3H]-granisetron.

1. The radioligand binding characteristics of the 3H-derivative of the novel 5-HT3 receptor antagonist BRL46470 were investigated and directly compared to the well characterized 5-HT3 receptor radioligand [3H]-granisetron, in tissue homogenates prepared from rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen. 2. In rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, [3H]-BRL46470 bound with high affinity (Kd (nM): 1.57 +/- 0.18, 2.49 +/- 0.30, 1.84 +/- 0.27, 3.46 +/- 0.36, respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 102 +/- 16, 44 +/- 4, 968 +/- 32 and 2055 +/- 105, respectively; mean +/- s.e. mean, n = 3-4) but failed to display specific binding in human putamen homogenates. 3. In the same homogenates of rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen as used for the [3H]-BRL46470 studies, [3H]-granisetron also bound with high affinity (Kd (nM): 1.55 +/- 0.61, 2.31 +/- 0.44, 1.89 +/- 0.36, 2.03 +/- 0.42 and 6.46 +/- 2.58 respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 39 +/- 4, 20 +/- 2, 521 +/- 47, 870 +/- 69 and 18 +/- 2, respectively; mean +/- s.e. mean, n = 3-4).(ABSTRACT TRUNCATED AT 250 WORDS)     

[3H] GR67330, a very high affinity ligand for 5-HT3 receptors

Summary GR67330 potently inhibited 5-hydroxytryptamine (5-HT)-induced depolarizations of the rat isolated vagus nerve. At the higher concentrations used (0.3 nmol/l–1 nmol/l) this was accompanied by a marked reduction in the maximum response to 5-HT. The calculated pK_B value was 10.2.The binding of the tritiated derivative of GR67330 to homogenates of rat entorhinal cortex was examined. Kinetic analysis revealed that specific [³H] GR67330 (0.1 nmol/l) binding was rapid and reversible. Association and dissociation rate constants were 1.48 ± 0.36 × 10⁸ mol/l^–1 s^–1 and 7.85 ± 0.41 × 10^–3 s^–1 respectively. Equilibrium saturation analysis revealed specific binding was to a single site (Bmax 22.6±0.21 fmol/mg protein) of high affinity (Kd 0.038±0.003 nmol/l). At low ligand concentrations, specific binding was up to 90% of total binding. If unlabelled GR67330 was used to define non-specific binding two sites were evident (Kd₁ 0.066 ± 0.007 nmol/l, Kd₂ 20.1 ± 9.7 nmol/l; Bmax₂ 31.5 ± 3.2 fmol/mg protein, Bmax₂ 1110 ± 420 fmol/mg protein). [³H] GR67330 binding was inhibited potently by 5-HT₃ antagonists and agonists. Ligands for other 5-HT receptors and other neurotransmitter receptors were either only weakly active or inactive at inhibiting binding. Hill numbers for antagonist inhibition of binding were close to unity, except for quipazine which was significantly greater than one. In common with other 5-HT₃ binding studies, all 5-HT₃ agonist tested had Hill numbers greater than one (1.51–1.71). GR38032 and GR65630 inhibited a greater proportion of binding than other 5-HT₃ antagonists, this additional binding was interpreted as inhibition from a second saturable site unrelated to the 5-HT₃ receptor.Homogenates of five areas of rat brain were examined for specific [³H]-GR67330 binding (entorhinal cortex, cingulate cortex, parietal cortex, hippocampus and nucleus accumbens/olfactory tubercle). In each brain area a site of very high affinity was labelled. Drug inhibition profiles were also very similar in each brain area. It is concluded that, because of its high affinity, [³H] GR67330 will be a useful ligand to label 5-HT₃ receptors especially in tissues with low receptor densities and to map 5-HT₃ receptors autoradiographically.Abbreviations 5-HT 5-Hydroxytryptamine - 8-OH-DPAT 8-hydroxy-2-di-N-propylaminotetralin - 5-CT 5-carboxyamidotryptamine - GR38032 (±) 1,2,3,9-tetrahydro-9-methyl-3[(2-methyl-1H-imidazol-1-yl)methyl]-4H-carbazol-4-one - GR65630 3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl) -1-propanone - GR67330 (±)1,2,3,9 - tetrahydro-9-methyl-3-[(5-methyl-1H-imidazol-4-yl)methyl]-4H-carbazol-4-one - MDL72222 1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate - ICS 205–930 (3-tropanyl)-1H-indole-3-carboxylic acid ester - BRL24924 endo-4-amino-5-chloro-2-methoxy-N-(1-azabicyclo[3,3,1]non-4-yl)benzamide - BRL43694 endo-N-(9-methyl-9-azabicyclo[3,3,1]non-3-yl)-1-methyl-indazole-3-carboxamide - SDZ 206-830 (3-homotropanyl)-1-methyl-5-fluoro-indole-3-carboxylic acid ester - mCPP meta-chlorophenylpiperazine Send offprint requests to G. J. Kilpatrick at the above address     

[3H]-lifarizine,a high affinity probe for inactivated sodium channels.

1. [3H]-lifarizine bound saturably and reversibly to an apparently homogeneous class of high affinity sites in rat cerebrocortical membranes (Kd = 10.7 +/- 2.9 nM; Bmax = 5.10 +/- 1.43 pmol mg-1 protein). 2. The binding of [3H]-lifarizine was unaffected by sodium channel toxins binding to site 1 (tetrodotoxin), site 3 (alpha-scorpion venom) or site 5 (brevetoxin), Furthermore, lifarizine at concentrations up to 10 microM had no effect on [3H]-saxitoxin (STX) binding to toxin site 1. Lifarizine displaced [3H]-batrachotoxinin-A 20-alpha-benzoate (BTX) binding with moderate affinity (pIC50 7.31 +/- 0.24) indicating an interaction with toxin site 2. However, lifarizine accelerated the dissociation of [3H]-BTX and decreased both the affinity and density of sites labelled by [3H]-BTX, suggesting an allosteric interaction with toxin site 2. 3. The binding of [3H]-lifarizine was voltage-sensitive, binding to membranes with higher affinity than to synaptosomes (pIC50 for cold lifarizine = 7.99 +/- 0.09 in membranes and 6.68 +/- 0.14 in synaptosomes). Depolarization of synaptosomes with 130 mM KCl increased the affinity of lifarizine almost 10 fold (pIC50 = 7.86 +/- 0.25). This suggests that lifarizine binds selectively to inactivated sodium channels which predominate both in the membrane preparation and in the depolarized synaptosomal preparation. 4. There was negligible [3H]-lifarizine and [3H]-BTX binding to solubilized sodium channels, although [3H]-STX binding was retained under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that [3H]dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

The interactions of dopaminergic agonists and antagonists with 3H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of [3H]dopamine and [3H]apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed "D3" binding site. Antagonist/[3H]dopamine competition curves are of uniformly steep slope (nH = 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific [3H]dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and [3H]flupentixol-binding activities. The affinities of agonists to inhibit D3 specific [3H]dopamine binding are also correlated with estimates of these agonists' affinities for the high affinity binding component of agonist/[3H]flupentixol competition curves. Both D3 specific [3H] dopamine binding and the high affinity agonist-binding component of dopamine/[3H]flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor.     

3H]-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and [3H] ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to [3H]ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding the serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both [3H]DOB and [3H]ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate [Gpp(NH)p] to this system caused a rightward shift and steepening of agonist competition curves for [3H] ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity [3H]DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that [3H]DOB and [3H]ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein. These observations are consistent with the classical view of interconvertible agonist affinity states of GTP-binding protein-coupled receptors and strongly support the "two state" over the "two receptor" model for DOB binding to the 5-HT2 receptor.     

[H]ATPA: a high affinity ligand for GluR5 kainate receptors

The pharmacological properties of [³H]ATPA ((RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) are described. ATPA is a tert-butyl analogue of AMPA (-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) that has been shown to possess high affinity for the GluR5 subunit of kainate receptors. [³H]ATPA exhibits saturable, high affinity binding to membranes expressing human GluR5 (GluR5) kainate receptors (K_d13 nM). No specific binding was observed in membranes expressing GluR2 and GluR6 receptors. Several compounds known to interact with the GluR5 kainate receptor inhibited [³H]ATPA binding with potencies similar to those obtained for competition of [³H]kainate binding to GluR5. Saturable, high affinity [³H]ATPA binding (K_d4 nM) was also observed in DRG neuron (DRG) membranes isolated from neonatal rats. The rank order potency of compounds to inhibit [³H]ATPA binding in rat DRG and GluR5 membranes were in agreement. These finding demonstrate that [³H]ATPA can be used as a radioligand to examine the pharmacological properties of GluR5 containing kainate receptors.     

The (S)-isomer of [3H]zacopride labels 5-HT3 receptors with high affinity in rat brain

The distribution of 5-HT3 receptor sites was examined in rat brain by autoradiography using 3H-enantiomers of zacopride. The (S)-3H-isomer labelled high densities of binding sites in the hippocampus, amygdala and cortex. The (R)-3H-isomer labelled considerably fewer sites than the (S)-isomer in nuclei of the lower medulla and did not exhibit any specific binding in the forebrain. These differences confirm that the (S)-isomer is specific for 5-HT3 binding sites and that it has a higher affinity than the (R)-isomer at these sites. These results are not consistent with the notion that 5-HT3 antagonist activity explains the anxiolytic effects of zacopride.     

Human cerebellar cortex possesses high affinity binding sites for [3H]somatostatin

Somatostatin binding sites have been identified in the human brain using [4-3H-(Phe6)]-somatostatin-14. In contrast to that of the rat, the human cerebellar cortex possesses a high density of somatostatin binding sites, comparable to that found in either the rat or human cerebral cortex. Autoradiographic localisation of somatostatin binding sites in the human cerebellum reveals that the highest density is associated with the granule cell layer.     

[3H]-guanfacine: a radioligand that selectively labels high affinity alpha2-adrenoceptor sites in homogenates of rat brain.

[3H]-guanfacine (N-amidino-2-(2,6-dichloro 3[3H] phenyl) acetamide hydrochloride; 24.2 Ci/mmol) has been used as a radioligand in homogenates of rat cerebral cortex. Specific binding of [3H]-guanfacine was linear with respect to tissue concentration (2.5-15 mg/ml), saturable and not markedly affected in the pH range 6.5-8.0. Analysis of the saturation of [3H]-guanfacine binding using an iterative least squares fitting procedure gave best fits to a single site model. [3H]-guanfacine binding was of high affinity (Kd 1.77 +/- 0.24 nM; n = 8) to a population of non interacting sites (nH 0.99 +/- 0.02; n = 8) with a density of 118.2 +/- 8.4 fmol/mg protein (n = 8). Highest levels of binding were achieved in cerebral cortex followed by thalamus greater than hypothalamus greater than medulla/pons greater than spinal cord greater than striatum greater than cerebellum. Binding was stereoselective with regard to the (-)-isomer of noradrenaline and the order of potency for displacement of [3H]-guanfacine by agonists was naphazoline greater than clonidine greater than (-)-adrenaline greater than (-)-alpha methylnoradrenaline greater than (-)-noradrenaline greater than (+/-)-alpha-methylnoradrenaline greater than (+)-noradrenaline greater than methoxamine greater than (+)-adrenaline greater than phenylephrine and by antagonists was phentolamine greater than dihydroergocryptine greater than piperoxane greater than yohimbine greater than prazosin greater than labetalol greater than indoramin suggested binding to alpha 2-adrenoceptors. The monovalent cations Na+ and K+ and also guanosine 5'-triphosphate (GTP) produced concentration-dependent inhibition whereas the divalent cations Ca2+, Mg2+, and Mn2+ first enhanced, then inhibited [3H]-guanfacine binding. Na+ (150 mM) or GTP (100 microM) produced marked reductions and Mn2+ (5 mM) marked increases in the number of receptor sites labelled by [3H]-guanfacine.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]ATPA: a high affinity ligand for GluR5 kainate receptors

The pharmacological properties of [3H]ATPA ((RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) are described. ATPA is a tert-butyl analogue of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) that has been shown to possess high affinity for the GluR5 subunit of kainate receptors. [3H]ATPA exhibits saturable, high affinity binding to membranes expressing human GluR5 (GluR5) kainate receptors (Kd approximately 13 nM). No specific binding was observed in membranes expressing GluR2 and GluR6 receptors. Several compounds known to interact with the GluR5 kainate receptor inhibited [3H]ATPA binding with potencies similar to those obtained for competition of [3H]kainate binding to GluR5. Saturable, high affinity [3H]ATPA binding (Kd approximately 4 nM) was also observed in DRG neuron (DRG) membranes isolated from neonatal rats. The rank order potency of compounds to inhibit [3H]ATPA binding in rat DRG and GluR5 membranes were in agreement. These finding demonstrate that [3H]ATPA can be used as a radioligand to examine the pharmacological properties of GluR5 containing kainate receptors.     

Affinity modulation of [3H]imipramine, [3H]paroxetine and [3H]citalopram binding to the 5-HT transporter from brain and platelets.

The dissociations of [3H]imipramine, [3H]paroxetine and [3H]citalopram from the 5-HT (serotonin 5-hydroxytryptamine) transporter were found to be markedly influenced by several drugs, although concentrations in the microM range were needed. Most of these drugs attenuated the dissociation rate, i.e. increased the affinity between the ligand and the binding site. A few increased the dissociation rate however. The binding of drugs to the affinity-modulating site was specific, although of low affinity and probably changing the conformation of the high-affinity binding site, thereby changing the fit between the ligand and the interacting amino acid side-chains. Although the drugs usually affected the dissociation rates of the three ligands in the same manner, there were some which had different effects on [3H]imipramine, [3H]paroxetine and [3H]citalopram. For example, 5-HT markedly attenuated the dissociation of [3H]imipramine, had a moderate effect on [3H]paroxetine and very little effect on [3H]citalopram dissociation. This indicates that the three ligands are bound to different domains on the 5-HT transporter. [3H]Citalopram dissociation from human brain and rat brain were differently affected by several drugs. Indalpine augmented the dissociation rate of the [3H]citalopram 5-HT transport complex in human brain but attenuated it in rat brain, thus revealing a species difference of the 5-HT transporter.     

3H]clonazepam, like [3H]flunitrazepam, is a photoaffinity label for the central type of benzodiazepine receptors

[³H]Clonazepam, like [³H]flunitrazepam, is irreversibly bound to membrane proteins of brain tissue when exposed to UV light. In polyacrylamide gel electrophoresis followed by fluoragraphy, the same pattern of photolabelled proteins was obtained in cerebellum and in hippocampus when either [³H]clonazepam or [³H]flunitrazepam was used as photoaffinity label. Since [³H]clonazepam does not interact with the peripheral type of benzodiazepine binding site present in the brain, these results confirm previous evidence that the proteins photolabelled with [³H]flunitrazepam are associated with the central type of benzodiazepine receptor.     

Effects of amiloride and its analogues on [3H]batrachotoxinin-A 20-alpha benzoate binding, [3H]tetracaine binding and 22Na influx

The ability of amiloride and its analogues to inhibit [3H]batrachotoxinin-A 20-alpha benzoate [( 3H]BTX-B) and [3H]tetracaine binding to rat synaptosomes and to a rat heart membrane preparation was tested. Their ability to inhibit 22Na influx was determined with rat synaptosomes. 5-N-substituted analogues were generally more potent in inhibiting [3H]BTX-B and [3H]tetracaine binding than compounds substituted on the guanidine group. However, the inhibition was not competitive. Amiloride and some of its analogues were as active or more active in inhibiting [3H]tetracaine binding than they were in inhibiting [3H]BTX-B binding. 22Na influx was inhibited with the same relative potencies as [3H]BTX-B binding and a good correlation was found between the two inhibitions. These results show an effect of amiloride and its analogues on the voltage-sensitive Na+ channels, which could partly explain the inotropic effects of these drugs.     

[3H]-idazoxan binds with high affinity to two sites on hamster adipocytes: an alpha 2-adrenoceptor and a non-adrenoceptor site.

1. [3H]-idazoxan labels a single population of high affinity sites (Kd 2.26 +/- 0.02 nM; Bmax 372 +/- 25 fmol mg-1 protein) in hamster adipocyte membranes. In the presence of 1 microM yohimbine to preclude binding to alpha 2-adrenoceptors, the density of [3H]-idazoxan binding sites was reduced (287 +/- 18 fmol mg-1 protein) without an apparent decrease in the affinity (Kd 2.19 +/- 0.24 nM) of the radioligand. 2. Displacement studies indicate that alpha-adrenoceptor ligands with an imidazoline side chain completely inhibit [3H]-idazoxan binding to hamster adipocyte membranes; in contrast, the alpha 2-adrenoceptor antagonists yohimbine, rauwolscine, BDF 6143 and phentolamine inhibited only 20-30% of the specific binding with affinity values consistent with an interaction at alpha 2-adrenoceptors. 3. The low potency of noradrenaline and adrenaline in displacing [3H]-idazoxan binding to the second site on hamster adipocyte membranes indicates that it is unlikely that this site is a type of adrenoceptor. 4. These results suggest that [3H]-idazoxan binds with high affinity to two sites in hamster adipocytes: an alpha 2-adrenoceptor and a non-adrenoceptor imidazoline site.