Autoradiographic localization of the GABAA receptor agonist [3H]-muscimol within rat kidney

By the use of combined radioreceptor binding and autoradiographic techniques, the pharmacological characteristics of (3H)-muscimol binding and the localization of the label were studied. (3H)-Muscimol was bound by sections of rat kidney in a manner consistent with the existence of specific gamma-aminobutyric acid 'A' (GABAA) receptors with KD and Bmax values of 23.7 nmol/l and 1.15 pmol/mg tissue, respectively. (3H)-Muscimol was bound by convoluted tubules of the renal cortex and by the collecting tubules. Our findings demonstrating the existence of recognition sites for the GABAA receptor agonist muscimol in the kidney suggest that GABA has a role in renal function.     

Inhibition by arylsulphatase A of Na-independent [3H]-GABA and [3H]-muscimol binding to bovine cerebellar synaptic membranes

Over an identical concentration range, both [³H]-GABA and [³H]-muscimol were bound to bovine cerebellar synaptic membranes through a sodium-independent and high-affinity process. Preincubating the synaptic membrane suspensions with arylsulphatase A inhibited [³H]-GABA and [³H]-muscimol binding. Furthermore, by using 4-nitrocatechol sulphate as a substrate for arylsulphatase A, a direct correlation appeared to exist between the amounts of SO^2?in4 released and the degree of inhibition of [³H]-GABA binding to synaptic membranes. The inhibitory effect of arylsulphatase A was blocked by known inhibitors of arylsulphatase such as AgNO₃. Scatchard analysis of specific [³H]-GABA binding to synaptic membranes showed that arylsulphatase decreased the number of [³H]-GABA binding sites from 2.65 to 1.75 pmol mg proteins without changing appreciably the affinity of [³H]-GABA binding to cerebellar synaptic membranes. These observations are interpreted to indicate that some components of GABA recognition sites may also contain sulpholipid(s) in addition to phospholipid(s).     

A double inhibition kinetic analysis of [3H]-muscimol binding to the gamma-aminobutyric acid receptor on calf brain synaptic membranes. Further studies on the mechanism of homocysteine-induced seizures

The simultaneous action of pyridoxal 5'-phosphate (PLP) and L-homocysteine on specific [3H]muscimol binding to the gamma-aminobutyric acid receptor on freeze-thawed, Triton-treated calf-brain synaptic membranes was examined kinetically by double inhibition analysis. PLP was found to be a pure inhibitor and L-homocysteine, a partial inhibitor, with respect to [3H]muscimol. Diagnostic analysis of the experimental data showed that the interaction constant (alpha) of the two inhibitors for the free receptor is between 0 and 1, confirming that the two inhibitors act synergistically. Further double inhibition analysis showed that no quaternary receptor-[3H]muscimol-homocysteine-PLP complex is formed although the ternary receptor-homocysteine-PLP complex is present. The localization and relationship of binding groups for both inhibitors is discussed as in their association with the ligand binding site.     

Binding of [3H]ADTN to rat striatal membranes

The conformationally restricted dopamine analogue ADTN binds in a specific saturable manner to rat striatal membranes. Analysis of the data suggests a single binding site. Binding of [3H]ADTN is displaced by a wide range of dopamine agonists and antagonists (both typical and atypical). The potency of ADTN derivatives to displace [3H]ADTN correlates well with their activity as agonists in other tests both in vivo and in vitro.     

Binding of [3H]naloxonazine to rat brain membranes

[3H]Naloxonazine binds to opioid-binding sites in rat brain homogenates. Prior administration of either morphine or D-Ala2-D-Leu5-enkephalin to the homogenates inhibits in a concentration-dependent manner the specific binding of [3H]naloxonazine. Most important, all the binding competed by unlabeled naloxonazine at 1 microM is also competed by morphine and D-Ala2-D-Leu5-enkephalin. [3H]Naloxonazine binding is linear with tissue up to 10 mg/ml wet weight of tissue, is temperature dependent, and has a pH maximum of approximately 7.7. Maximal binding is reached within 90 min at 25 degrees. The affinity of [3H]naloxonazine for its binding sites is quite high with half-maximal binding obtained at a concentration of approximately 2 nM. Approximately 40% of the total specific binding of [3H]naloxonazine is resistant to multiple washes and to displacement by levallorphan (1 microM) added 60 min after the [3H]naloxonazine, suggesting that a portion of [3H]naloxonazine binding is not freely reversible. The percentage of total [3H] naloxonazine binding which is not freely reversible varies 3-fold between regions, with the hypothalamus (60%) being the highest and the brainstem (18%) the lowest.     

Binding properties of [3H]MS-377, a novel sigma receptor ligand, to rat brain membranes

MS-377 ((R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]++ +methy l-2-pyrrolidinone L-tartrate) is a novel selective sigma receptor ligand, currently being developed for the treatment of schizophrenia. MS-377 showed anti-phencyclidine (PCP), anti-dopaminergic and anti-serotonergic activities, and we anticipated that the anti-psychotic activities of MS-377 were associated with sigma(1) receptors. However, its pharmacological profile is partly distinct from those of selective sigma(1) receptor ligands. Thus, one of the possible speculations is that MS-377 has another site of action. In the present study, we examined the binding properties of radiolabeled MS-377 ([3H]MS-377) to rat brain membranes. [3H]MS-377 showed saturable and reversible binding to rat brain membranes. Scatchard plot and Hill plot from saturation studies were linear, with K(d) of 15.2+/-6.6 nM, B(max) of 599.4+/-58.6 fmol/mg protein and Hill coefficient of 1.01+/-0.01, indicating that [3H]MS-377 bound to a single high-affinity site in rat brain membranes. Displacement studies revealed that the other sigma reference compounds with different structures inhibited the specific binding of [3H]MS-377 in a competitive manner. Stereoselectivity was observed for the inhibition of [3H]MS-377 binding, (+)-isomers were more potent than (-)-isomers. Non-sigma receptor ligand PCP showed weak inhibition of [3H]MS-377 binding. The rank order of potency for the sigma reference compounds to displace [3H]MS-377 binding were as following: haloperidol>MS-377=(+)-pentazocine>DTG (1, 3-Ditolylguanidine)=(-)-pentazocine>BMY14802 (alpha-(4-fluorophenyl)-4-(5-fluoro-2-pyramidinyl)-1-piperazine butanol)>(+)-SKF-10,047>(-)-SKF-10,047=PCP. These results suggested that the MS-377 selectively binds to sigma binding site with high affinity in rat brain membranes. Therefore, the anti-psychotic activities of MS-377 are attributable to association with sigma(1) receptors.     

Binding of [3H]clonidine to I1-imidazoline sites in bovine adrenal medullary membranes

Summary Imidazolines bind with high affinity not only to -adrenoceptors but also to specific imidazoline binding sites (IBS) labelled by either [³H]clonidine or [³H]idazoxan and termed I₁- and I₂-IBS, respectively. Since bovine adrenal chromaffin cells lack ₂-adrenoceptors, we investigated the pharmacological characteristics of [³H]clonidine binding sites in the bovine adrenal medulla. The binding of [³H]clonidine was rapid, reversible, partly specific (as defined by naphazoline 0.1 mmol/l; 55% specific binding at [³H]clonidine 10 nmol/l), saturable and of high affinity. The specific binding of [³H]clonidine to bovine adrenal medullary membranes was concentration-dependently inhibited by various imidazolines, guanidines and an oxazoline derivative but not, or with negligible affinity, by rauwolscine and (–)-adrenaline. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases the single detectable site) was as follows: naphazoline >- BDF 7579 (4-chloro-2-isoindolinyl guanidine) >-clonidine>- cirazoline >_ BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)isoindoline hydrochloride) > BDF 7572 (4,7-chloro-2-(2-imidazolin-2-ylamino)-isoindoline) > moxonidine = rilmenidine > BDF 6100 (2-(2-imidazolin-2-ylamino)-isoindoline) = idazoxan > phentolamine > aganodine = guanabenz > amiloride > histamine. This rank order is compatible with the pharmacological properties of the I₁-IBS. The non-hydrolysable GTP-analogue Gpp(NH)p (5guanylylimidodiphosphate; 100 mol/l) inhibited specific [³H]clonidine binding by about 50%. Equilibrium [³H]clonidine binding was also significantly reduced by K⁺ and Mg²⁺ In conclusion, [³H]clonidine labels non-adrenergic high-affinity sites in plasma membranes of the bovine adrenal medulla; these sites exhibit the pharmacological properties of I₁-IBS, but not of I₂-IBS. Furthermore, the IBS in the adrenal medulla appear to be coupled to a G-protein.Correspondence to G. J. Molderings at the above address     

Differential effects of triethyllead on synaptosomal [3H]dopamine vs. [3H]acetylcholine and [3H]gamma-aminobutyric acid release.

In vitro exposure to tetraethyllead (Et4Pb, 10 microM) did not alter the release of [3H] dopamine (DA), [3H]acetylcholine (ACh), or [3H]gamma-aminobutyric acid (GABA) from superfused synaptosomes isolated from rat brain striatum, hippocampus, and cortex, respectively. On the other hand, a concentration-dependent increase in the spontaneous release of these transmitters was observed following exposure to triethyllead (Et3Pb, 0.1-10 microM). The magnitude of 1 microM Et3Pb-induced [3H]DA release was 5-fold greater than that observed for [3H]ACh or [3H]GABA release. Removal of [Ca2+]e did not alter the Et3Pb-induced increase in the release of these three transmitter substances, nor did Et3Pb alter synaptosomal 45Ca efflux. EtePb-induced [3H]ACh and [3H]GABA release, but not [3H]DA release, was blocked by lowering [Na+]e from 140 to 50 mM. Similarly, the release of [3H]ACh and [3H]GABA, but not [3H]DA, induced by either Na,K-ATPase inhibition or veratridine (a Na(+)-ionophore), was attenuated by lowering [Na+]e from 140 to 50 mM. However, Et3Pb did not inhibit isolated synaptic membrane Na,K-ATPase, nor did the magnitude or temporal patterns of Et3Pb-induced transmitter release resemble transmitter release induced by Na,K-ATPase inhibition. Et3Pb and veratridine, but not Na,K-ATPase inhibition, produced an increase in synaptosomal [3H] deoxyglucose phosphate (dGluP) efflux, suggesting that both compounds increase membrane permeability. A Et3Pb-induced increase in membrane permeability is further supported by electrophysiological studies using the frog neuromuscular junction in which Et3Pb was found to reduce both the input resistance and membrane potential of muscle cells. As with [3H]ACh and [3H]GABA release, the Et3Pb-induced increase in synaptosomal [3H]dGluP efflux was attenuated by lowering [Na+]e.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of [3H]SCH23390 to a non-dopaminergic site in bovine kidney.

Binding of the D1 dopamine receptor antagonist [3H]SCH23390 to bovine renal cortical membranes has been studied. Specific binding of [3H]SCH23390 was saturable and reversible and stereoisomers of SCH23390 competed stereoselectively. In contrast, competition with the isomers of butaclamol was not stereoselective and dopamine failed to compete for the [3H]SCH23390 binding site. The site is therefore not a D1 dopamine receptor. Competition studies with a very wide range of compounds failed to define the nature of the [3H]SCH23390 binding sites in renal cortex whereas in parallel studies the characteristics of [3H]SCH23390 binding in caudate nucleus were entirely consistent with those of D1 dopamine receptors. The nature of [3H]SCH23390 binding in preparations of tubular and glomerular membranes was found to be virtually identical to those of crude renal cortical membranes indicating lack of compartmentation of these sites. Autoradiographic studies of [3H]SCH23390 binding in bovine kidney showed significantly higher levels of binding sites in renal cortex compared with renal medulla and this was confirmed by direct ligand binding studies.     

Binding of the radioligand [3H]-SCH 58261, a new non-xanthine A2A adenosine receptor antagonist,to rat striatal membranes.

1. The present study describes the binding to rat striatal 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-triazol o [1,5-c] pyrimidine, [3H]-SCH 58261. 2. [3H]-SCH 58261 specific binding to rat striatal membranes ( > 90%) was saturable, reversible and dependent upon protein concentration. Saturation experiments revealed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (Kd = 0.70 nM) and limited capacity (apparent Bmax = 971 fmol mg-1 of protein). The presence of 100 microM GTP in the incubation mixture did not modify [3H]-SCH 58261 binding parameters. 3. Competition experiments showed that [3H]-SCH 58261 binding is consistent with the labelling of A2A striatal receptors. Adenosine receptor agonists competed with the binding of 0.2 nM [3H]-SCH 58261 with the following order of potency: 2-hexynyl-5''-N-ethyl carboxamidoadenosine (2HE-NECA) > 5''-N-ethylcarboxamidoadenosine (NECA) > 2-[4-(2-carboxyethyl)-phenethylamino]-5''-N-ethylcarboxamidoadenosi ne (CGS 21680) > 2-phenylaminoadenosine (CV 1808) > R-N6-phenylisopropyladenosine (R-PIA) > N6-cyclohexyladenosine (CHA) = 2-chloro-N6-cyclopentyladenosine (CCPA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > 5-amino-8-(4-fluorobenzyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine (8FB-PTP) = SCH 58261 > xanthine amine congener (XAC) = (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3-dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > or = 8-phenyltheophylline (8-PT). 5. The Ki values for adenosine antagonists were similar to those labelled with the A2A agonist [3H]-CGS 21680. Affinities of agonists were generally lower. The A1-selective agonist, R-PIA, was found to be about 9 fold more potent than its stereoisomer, S-PIA, thus showing the stereoselectivity of [3H]-SCH 58261 binding. Except for 8-PT, the adenosine agonists and antagonists examined inhibited [3H]-SCH 58261 binding with Hill coefficients not significantly different from unity. 6. The present results indicate that [3H]-SCH 58261 is the first non-xanthine adenosine antagonist radioligand which directly labels A2A striatal receptors. High receptor affinity, good selectivity and very low non-specific binding make [3H]-SCH 58261 an excellent probe for studying the A2A adenosine receptor subtype in mammalian brain.     

Dopamine D2 receptor binding properties of [3H]U-86170, a dopamine receptor agonist.

U-86170F, an imidazoguinolinone, is a potent dopamine D2 agonist, binding with high affinity to the dopamine D2 receptor. A Kd of 0.99 nM was determined in membranes from Chinese hamster ovarian (CHO) cells transfected with the D2 receptor and a Kd of 1.72 nM was obtained in rat striatal homogenates. GTP sensitivity was demonstrated when its addition (300 microM) reduced [3H]U-86170 binding by 60%. This agonist ligand is especially effective in identifying agonists and partial agonists, as well as antagonists, and affords a more precise evaluation of their affinity for the dopamine D2 receptor, without the use of multiple site analysis, than does an antagonist [3H]-ligand.     

In vitro binding of the very potent and selective D-2 dopamine agonist, [3H]N-0437 to calf caudate membranes

N-0437, a non-catecholic aminotetralin has recently been described as a very potent and selective dopamine D-2 agonist. In this study the in vitro binding of [3H]N-0437 (specific activity 80.6 Ci/mmol) to calf caudate membranes is described. It was found that [3H]N-0437 binds with a high affinity (KD = 0.17 nM) and a low proportion of non-specific binding. Moreover the binding was saturable with a high number of binding sites (Bmax = 703 +/- 28 fmol/mg protein) and reversible (dissociation half-time = 68 min). Pharmacological analysis of [3H]N-0437 binding showed that it was selective for dopamine receptors and that it was also stereoselective for D-2 receptors. Non-dopaminergic drugs were without exception very poor displacers. Taken together the results suggest that [3H]N-0437 labels dopamine D-2 receptors with a high selectivity in the calf brain, and thus, that it should be a useful tool in studies of central dopamine receptors.     

Binding characteristics of the dopamine uptake inhibitor [3H]nomifensine to striatal membranes

Binding of the radiolabeled antidepressant [3H]nomifensine to rat and rabbit striatal membranes has been characterized. The specific binding of [3H]nomifensine to striatal membranes was stable, reversible and saturable. Saturation experiments indicated that [3H]nomifensine labeled a single site with an affinity (Kd) of 80 nM and a total number of binding sites (Bmax) of 6.5 pmoles/mg protein both in rat and rabbit striatal membranes. The affinity constants obtained from kinetic analyses and competition experiments were in fairly good agreement with those obtained in saturation experiments. Compounds known to inhibit [3H]dopamine uptake in vitro, such as nomifensine, 4-hydroxy-nomifensine, mazindol, amfonelic acid and benztropine, were the most potent competitors of nomifensine binding. Additionally, the absolute potencies of various drugs in competing for [3H]nomifensine binding to rat and rabbit striatal membranes correlated closely with their potencies in inhibiting [3H]dopamine uptake into striatal synaptosomes. Specific [3H]nomifensine binding was dependent on the presence of NaCl which is also consistent with its association with the dopamine uptake pump. The number, but not the affinity, of striatal [3H]nomifensine binding sites was reduced significantly following in vivo lesions with 6-hydroxydopamine. The number of [3H]nomifensine binding sites was found to be highest in areas rich in dopamine nerve terminals such as the striatum and olfactory tubercle. These results suggest that [3H]nomifensine binds to a site on dopaminergic nerve terminals associated with the dopamine uptake pump.     

Binding of [3H]CB 34, a selective ligand for peripheral benzodiazepine receptors, to rat brain membranes.

The 2-phenyl-imidazo[1,2-a]pyridine derivative CB 34 is a ligand for peripheral benzodiazepine receptors. The binding of [3H]CB 34 to rat cerebrocortical membranes was characterized. Specific binding was rapid, reversible, saturable and of high affinity. Kinetic analysis yielded association and dissociation rate constants of 0.2x10(8) M(-1) min(-1) and 0.29 min(-1), respectively. Saturation binding experiments revealed a single class of binding sites with a total binding capacity of 188+/-8 fmol/mg protein and an apparent dissociation constant of 0.19+/-0.02 nM. Specific [3H]CB 34 binding was inhibited by ligands selective for peripheral benzodiazepine receptors, whereas, with the exception of flunitrazepam and diazepam, ligands for central benzodiazepine receptors were inactive. Of the brain regions examined, the density of the [3H]CB 34-binding sites was greatest in the hypothalamus and lowest in the cerebral cortex. [3H]CB 34 is thus a potent and selective ligand for peripheral benzodiazepine receptors and should be proven useful for studies of the roles of these receptors.     

Purine inhibition of [3H]-γ-aminobutyric acid receptor binding to rat brain membranes

Several purines, including inosine and hypoxanthine, inhibit the binding of [^3H]-γ-amino-butyric acid (GABA) and [^3H]diazepam to freeze-thawed and extensively washed rat brain membranes. While purines have been reported to inhibit diazepam binding competitively, their interactions with GABA receptors in both mitochondrial and mitochondrial plus microsomal fractions are noncompetitive. The possibility that purines may bind at one site and affect the GABA receptor-ionophore-benzodi-azepine complex is discussed.     

Binding studies with the 5-HT1B receptor agonist [3H]CP-96,501 in brain tissues

Binding studies in rat brain membranes have shown that [³H]CP-96,501 is a selective, high affinity radioligand for 5-HT_1B receptors. Additional studies with [³H]CP-96,501 in brain tissues of several other species were undertaken to investigate its utility in detecting this receptor subtype. The presence of 5-HT_1B receptors in brain membranes of the hamster and gerbil was demonstrated with [³H]CP-96,501. This finding was confirmed by [³H]5-HT binding experiments that also indicated the presence of other 5-HT₁ subtypes (possibly 5-HT_1D, 5-HT_1E) in these species as well as the rat. Negligible specific binding of [³H]CP-96,501 was found in membranes of guinea pig brain, dog hypothalamus, bovine caudate, pig choroid plexus, and several human brain tissues, consistent with the reported absence of the 5-HT_1B receptor subtype in these species. Autoradiographic examination of rat brain sections labeled by [³H]CP-96,501 showed a dense localization in areas known to be enriched in 5-HT_1B receptors (e.g., globus pallidus, substantia nigra, superior colliculus, subiculum). On the other hand, brain sections of dog and guinea pig appeared to show no specific binding of [³H]CP-96,501 by autoradiography, in agreement with the lack of brain 5-HT_1B receptors in these tissues. © 1992 Wiley-Liss, Inc.     

Partial GABA agonist activity of SR 95531 on the binding of [35S]TBPS, [3H]DMCM and [3H]lormetazepam to rat brain membranes

A recently developed series of pyridazinyl-GABA derivatives has been classified as GABA antagonists in electrophysiological, behavioral and biochemical experiments. These substances seemed superior to the classical GABA antagonist bicuculline because of their water-solubility, high potency and apparent selectivity for GABAA receptors. In the present study the most potent representative of this class, SR 95531 almost completely reversed the stimulatory or inhibitory effect of GABA on [3H]lormetazepam and [35S]TBPS binding, respectively. To a lesser extent, it antagonized the inhibition of [3H]DMCM binding by GABA. However, the interaction of SR 95531 with the GABA receptor seems to be of a complex nature since the compound enhanced the binding of [3H]lormetazepam by 28% at 37 degrees in the presence of 200 mM Cl-. Bicuculline inhibited [3H]lormetazepam binding under these conditions, presumably by antagonizing the effect of residual endogenous GABA. Similar to GABA and THIP, SR 95531 potently inhibited the binding of [3H]DMCM and [35S]TBPS, suggesting SR 95531 to be a partial agonist at the GABAA receptor.     

Effects of acromelic acid A on the binding of [3H]-kainic acid and [3H]-AMPA to rat brain synaptic plasma membranes.

1. The ability of acromelic acid A to inhibit [3H]-kainic acid and [3H]-(RS)-alpha-amino-3-hydroxy-5-methyloxazole-4-propionic acid ([3H]-AMPA) binding to rat brain synaptic plasma membranes was investigated by equilibrium radioligand binding assay. 2. Kinetic analysis of [3H]-kainic acid binding demonstrated the existence of two kainate binding sites in this tissue preparation and yielded equilibrium dissociation constants for [3H]-kainic acid of KD = 0.4 nM and KD = 20.8 nM. 3. Kainic acid and domoic acid both appeared to displace [3H]-kainic acid from a single binding site with equilibrium binding constants of KD = 19.4 nM and Ki = 14.5 nM respectively. Acromelic acid A exhibited a biphasic inhibition of [3H]-kainic acid binding to synaptic membranes with binding affinities of Ki = 15.1 nM and Ki = 1.49 microM. 4. In the absence of chaotropic ions, the order of potency of inhibition of [3H]-AMPA binding was acromelic acid A (Ki = 26 nM) greater than AMPA (KD = 184 nM) greater than domoic acid (Ki = 499 nM). 5. The inclusion of 100 mM thiocynanate ion in the [3H]-AMPA binding assay resulted in a change in the order of potency to: AMPA (KD = 160 nM) greater than acromelic acid A (Ki = 289 nM) greater than domoic acid (Ki = 9.02 microM). 6. These results show that acromelic acid A distinguishes two kainate binding sites in rat brain synaptic plasma membranes and in addition, that in the absence of chaotropic ions, acromelic acid A is the most potent displacer of [3H]-AMPA binding yet described.