Pharmacology of [3H]apomorphine binding to bovine brain tissue

Activity against high-affinity (nM), and over 80 per cent dopamine (DA)-agonist-displaceable binding of [³H]apomorphine (APO) to a “subsynaptosomal” membrane fraction of bovine caudate nucleus was evaluated using amino-6,7-dihydroxytetralin (ADTN) as a blank. Aporphines with 10,11-catechol and small N-alkyl groups were potent inhibitors ($\text{IC}{}_{50}\text{< 25}\text{nM}$) with parallel inhibition curves (n_h = 0.8 to 1.0). For phenethylamines, catechol and amine moieties were important structural requirements; α or β substitution markedly reduced potency. Thus, epinephrine and isoproterenol were weak while DA and its N-methylated congeners were potent; (? )norepinephrine (NE) and fluoro-DAs were intermediate in potency and ( + )NE, other catechols and hydroxylated phenethylamines were virtually inactive; m-tyramine, while weak, was more active than p-tyramine. Other DA agonists [ADTN > lergotrile >N-n-propyl-3-(3-hydroxyphenyl)-piperidine (3-PPP) > bromocriptine] were active. Neuroleptics competed relatively weakly, with imperfect correspondence to in vivo activities. Stereoselectivity was found with several aporphines, phenethylamines, and antipsychotic drugs. Many other neuropharmacologically active agents, including inhibitors of amine uptake and adrenergic receptors, were inactive. These characteristics strongly suggest that APO interacts with a DA agonist binding site in mammalian brain tissue.     

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 characteristics of [3H]ucb 30889 to levetiracetam binding sites in rat brain

Levetiracetam (2S-(2-oxo-1-pyrrolidinyl)butanamide, KEPPRA, a novel antiepileptic drug, has been shown to bind to a specific binding site located in brain (levetiracetam binding site [Eur. J. Pharmacol. 286 (1995) 137]). However, [3H]levetiracetam displayed only micromolar affinity for these sites making it an unsuitable probe for further characterization. The present study describes the binding properties of an analogue of levetiracetam: [3H]ucb 30889, (2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide. [3H]ucb 30889 binds reversibly to specific binding sites in rat brain. Kinetics at 4 degrees C were biphasic with half-times of association and dissociation of, respectively, 3 and 4 min for the fast component and 47 and 61 min for the slow component. [3H]ucb 30889 saturation binding curves were compatible with the labelling of a homogenous population of binding sites having a B(max) of 4496+/-790 fmol/mg protein (mean+/-S.D., n=5) and a K(d) of 62+/-20 nM (mean+/-S.D., n=5), a 20-fold increase in affinity compared to [3H]levetiracetam. Competition binding curves with ligands known to interact with levetiracetam binding sites and tissue distribution restricted to the brain indicated that [3H]ucb 30889 and [3H]levetiracetam bind to the same site. Although levetiracetam binding sites and GABA(A) (gamma-aminobutyric acid) receptors share some ligands such as pentobarbital and pentylenetetrazol, experiments performed with [35S]TBPS (tert-butyl-bicyclo[2.2.2]phosporothionate), a probe for the GABA(A) Cl(-) channel do not support the hypothesis that levetiracetam binding sites are part of the GABA(A) receptor complex. Preliminary autoradiography studies in rat brain revealed that [3H]ucb 30889 labels specific sites in all brain regions and that this binding is concentration-dependently displaced by levetiracetam.     

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     

Effects of cations and temperature on the binding of [3H]spiperone to sheep caudate nucleus

The specific [3H]spiperone binding by sheep caudate nucleus homogenate is increased by divalent cations. The effect of Ca2+ or Mn2+ (5 mM) is temperature-dependent, and it is optimal at about 37 degrees, but is relatively low below 15 degrees and above 50 degrees. In the absence of added Ca2+ or Mn2+, the maximal specific [3H]spiperone binding is observed at about 25 degrees, and the cations shift the optimum to about 37 degrees. Under the experimental conditions used, the KD is about 0.6 nM and is not influenced by Ca2+ or Mn2+, or by temperature (25 and 37 degrees). In addition to Ca2+ and Mn2+, Mg2+ and Zn2+ also increase the specific [3H]spiperone binding, but to a smaller extent. At the concentrations of Ca2+, Mn2+, Mg2+ and Zn2+ which produce a maximal increase in the [3H]spiperone binding, the membranes are nearly saturated with the cations which bind about 100 nmoles of Ca2+ or Mg2+/mg of protein, 170 nmoles Zn2+/mg of protein and at least 300 nmoles Mn2+/mg of protein. It is suggested that the cations increase the [3H]spiperone binding by either exposing more binding sites, by preventing denaturation or by increasing the solubility of [3H]spiperone in the membrane phase, or by a combination of these processes.     

Characterization of adenosine binding sites in bovine testicular tissue using 8-cyclopentyl-1,3-[3H]dipropylxanthine

The existence of specific adenosine binding sites in bovine testicular tissue was evaluated using the novel antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX). Saturation analysis revealed specific binding that was saturable at approximately 1 nM. Scatchard analysis indicated a single class of binding sites with a KD = 0.26 nM and a Bmax = 0.37 pmol/mg protein. Affinity profiles suggest an A1 subtype recognition site that is different from the classical A1 adenosine receptor. The results presented should prove useful in subsequent studies concerning heterogeneity among adenosine receptors and also aid in discerning the role of adenosine in reproduction.     

Binding of [3H]cimetidine to rat brain tissue

Binding of [3H]cimetidine to rat brain tissue was investigated, and a saturable binding with dissociation constant 0.22 +/- 0.05 microM found. This binding is inhibited by a range of imidazole-derived histamine H2-receptor antagonists, but not by a number of non-imidazole H2-receptor antagonists. It is concluded that the [3H]cimetidine binding site in rat brain tissue that is labelled in these experiments is not the histamine H2-receptor.     

Cocaine and dopamine differentially protect [3H]mazindol binding sites from alkylation by N-ethylmaleimide.

The binding of cocaine, d-amphetamine and dopamine to the site on the dopamine transporter labeled by [3H]mazindol was investigated in rat striatal membranes. N-Ethylmaleimide inhibited about 95% of the specific binding of 5 nM [3H]mazindol in a concentration-dependent manner. The effect of 10 mM N-ethylmaleimide was completely prevented by cocaine (EC50 of 3 microM), but neither 300 microM dopamine nor d-amphetamine afforded any significant protection. On the other hand, high concentrations of cocaine, d-amphetamine and dopamine provided similar protection against inhibition by 0.1 mM N-ethylmaleimide. Taken together these data support the hypothesis that a significant portion of the cocaine binding domain on the transporter is distinct from that of either dopamine or amphetamine. This distinction may be sufficient to allow properly designed drugs to prevent cocaine binding without inhibiting DA transport.     

The effect of melanotropin release inhibiting factor, its metabolites and analogs on [3H]spiroperidol and [3H]apomorphine binding sites

The effects of melanotrophin release inhibiting factor (Pro-Leu-Gly-NH2, MIF), its possible metabolites, Pro-Leu-OH, Leu-Gly-NH2, Leu-Gly-OH and an analogue, cyclo(Leu-Gly), on [3H]spiroperidol binding sites in the striatum and on [3H]apomorphine binding sites in the striatum and hypothalamus of male Sprague-Dawley rats were determined. [3H]Spiroperidol binding to dopamine receptors in striatal membranes was unaffected by any of the above peptides in concentration up to 0.1 mM. The binding of [3H]apomorphine was enhanced by MIF, Pro-Leu-OH and cyclo(Leu-Gly) in both striatal and hypothalamic membranes in submicromolar concentrations. Leu-Gly-NH2 and Leu-Gly-OH did not affect [3H]apomorphine binding to dopamine receptors in striatum of hypothalamus. The enhancement in binding of [3H]apomorphine by MIF and cyclo(Leu-Gly) was not related to the changes in the number of binding sites but to an increase in the affinity to the receptors. The results indicate that MIF and some of its related peptides do not affect dopamine receptor binding sites labeled by the neuroleptic [3H]spiroperidol but facilitate the transmission in those sites labeled by [3H]apomorphine. Since [3H]apomorphine and [3H]spiroperidol predominantly label pre- and post-synaptic dopamine receptors, it is concluded that MIF and its active analogs interact with presynaptic dopamine receptors.     

Binding of [3H]Ro 11-2465. Possible identification of a subclass of [3H]imipramine binding sites

Ro 11-2465 is a cyanide derivative of imipramine. In cerebral cortex homogenates, [3H] Ro 11-2465 displays a binding profile similar to that of [3H]imipramine. Agents compete with binding of [3H]Ro 11-2465 in an order of potency similar to their ability to block serotonin uptake, and raphe lesions greatly decrease the binding of [3H]Ro 11-2465. These observations suggest that the sites labeled by [3H]Ro 11-2465 are presynaptic. The binding of [3H]Ro 11-2465 is sodium ion-dependent, as are both the binding of [3H] imipramine and the serotonin uptake mechanism. In the presence of sodium ions, binding of [3H]Ro 11-2465 to brain tissue or platelets at 4 degrees is apparently irreversible. Binding is not displaced by high concentrations of the displacing agent desipramine or by repeated washing. However, by either removing sodium or increasing the assay temperature to 23 degrees, the ligand dissociates from the tissue. In tissue where [3H]Ro 11-2465 is irreversibly bound to receptor at 4 degrees, subsequent [3H]imipramine binding is decreased by about 50%. At temperatures greater than 23 degrees, [3H]Ro 11-2465 binding displays a temperature dependency similar to that of [3H]imipramine; that is, when temperatures are raised from 23 degrees to 30 degrees or 37 degrees there is no change in the Bmax, but the affinity of the ligand for the receptor is decreased. These data suggest that [3H]Ro 11-2465 binds to a discrete population of [3H]imipramine binding sites, comprising about one-half of the total [3H] imipramine binding sites.     

Pharmacologic characterization of [3H]dopamine and [3H]spiperone binding in mouse cerebellum

• 1.1. [³H]Dopamine and [³H]spiperone binding to cerebellar homogenates was characterized utilizing dopaminergic agonists, antagonists and non-dopaminergic drugs.
• 2.2. The [³H]DA binding to low affinity binding sites reveals a heterogenous population consisting of dopaminergic as well as serotonergic and noradrenergic sites. However, the high affinity binding of [³H]DA reflects dopaminergic sites, although a small contribution of serotonergic and noradrenergic binding sites cannot be excluded.
• 3.3. [³H]Spiperone also labels a heterogenous population of binding sites which, however, are mainly dopaminergic.

     

Characterization of L-[3H]glutamate binding sites in bovine brain coated vesicles.

Clathrin-coated vesicles isolated from bovine brain exhibit an L-[3H]glutamate-specific binding. Coated vesicles were purified from bovine brain by differential centrifugation and gel filtration. High purity of coated vesicles was established previously by several enzyme markers and electron microscopy. The binding activity was performed in the absence of Na+, Ca2+, and Cl- ions to avoid binding and/or uptake to uptake sites. Coated vesicles were frozen, thawed, treated with 0.04% Triton X-100 and washed before incubation with L-[3H]glutamate. Saturation binding experiments revealed a single binding site with a Kd = 439 +/- 87 nM and a Bmax = 11.74 +/- 3.4 pmol/mg protein, consistent with kinetics characteristic for glutamate receptors. The glutamate-specific binding was stereospecific for glutamate and aspartate, showing higher affinity for L-forms than D-forms. Pharmacological characterization indicated that specific binding was sensitive to quisqualate and almost insensitive to kainate and N-methyl-D-aspartate. 200 microM guanosine triphosphate (GTP) produced a decrease of 50% in L-[3H]glutamate binding activity and competition experiments produced an affinity shift to the right of the glutamate dose-response curve. These results support the evidence that glutamate receptors are present in bovine brain coated vesicles and, at least in part, are associated to a G-protein.     

Characterization of binding sites for [3H]spiroperidol

Experiments were designed to investigate the biochemical properties of binding sites for [3H]spiroperidol ([3H]SPD) solubilized from canine caudate and to define the effect of detergent on the binding of the radioligand. Extraction of canine caudate with 0.75-1.0% digitonin was found to generate the maximum yield of binding sites for [3H]SPD while minimizing extraction of membrane proteins. Although binding sites were solubilized with 1.0% digitonin, a 10-fold reduction in detergent concentration was necessary to achieve maximal binding of [3H]SPD. The rank order of affinity for agonists and antagonists was consistent with the pharmacologic properties of the D2 subtype of the dopamine receptor. However, the binding of antagonists was found to be complex. Studies with some preparations of pooled canine caudate resulted in competition curves for the D2-selective antagonists domperidone and sulpiride that best fit a single-site model. Other preparations exhibited biphasic inhibition curves with these antagonists. The class of binding sites for [3H]SPD with low affinity for D2-selective antagonists constituted as much as 30-40% of the binding sites. Enrichment of solubilized binding sites for [3H]SPD was achieved by size exclusion HPLC followed by adsorption to DEAE-Sephadex and elution with buffer of increasing ionic strength. Enrichment of binding sites was accompanied by a decrease in the affinity of solubilized sites for [3H]SPD.     

Characterization of [3H]tryptamine binding sites in brain

[3H]Tryptamine binds with high affinity to sites on rat brain membranes. The sites have the characteristics of tryptamine receptor recognition sites. These sites are widely distributed among rat brain regions with the highest density occurring in the cerebral cortex, striatum and hippocampus. The site is also found in human cerebral cortex. The binding site is localized mainly to the synaptosomal fraction. Drug competition studies indicate that the [3H]tryptamine binding site is distinct from serotonin receptors. Drugs that are potent inhibitors of [3H]tryptamine binding include tetrahydro-beta-carboline, quipazine, phenylethylamine, amphetamine, p-chloroamphetamine and methamphetamine.     

Characterization of the [3H]-desipramine binding site of the bovine adrenomedullary plasma membrane

Summary The specific (i.e. nisoxetine-sensitive) binding of [³H]desipramine was studied in membranes prepared from bovine adrenal medullae. (1) [³H]desipramine bound reversibly and with high affinity (K _D = 2.8 nmol/l) to a single class of non-interacting binding sites (Hill coefficient = 0.96); the maximal number of binding sites (B_max) was 2.1 pmol/mg protein. (2) Binding of [³H]desipramine was dependent on [Na⁺] and [Cl^–]. Increasing the concentrations of these ions increased binding. (3) Substrates and inhibitors of the neuronal noradrenaline transport system (uptake,) inhibited binding of [³H]desipramine with a rank order of potency typical for an interaction with the uptake, carrier.The characteristics of [³H]desipramine binding remained essentially unchanged after solubilization of adrenomedullary membranes with the non-ionic detergent digitonin.The results indicate that the plasma membrane of bovine adreno-medulary cells is endowed with the neuronal uptake₁ transporter. Correspondence to: H. Bönisch     

3H]cocaine binding and inhibition of [3H]dopamine uptake is similar in both the rat striatum and nucleus accumbens

Cocaine binds with high affinity to the dopamine transporter in both the striatum and the nucleus accumbens. We examined Na(+)-dependent [3H]cocaine binding, mazindol inhibition of [3H]cocaine binding and cocaine inhibition of [3H]dopamine uptake in both rat brain areas. The striatum and nucleus accumbens demonstrated Na(+)-dependent [3H]cocaine binding with similar densities. Mazindol inhibited [3H]cocaine binding with a similar IC50 in both the striatum and nucleus accumbens. Likewise, cocaine inhibited [3H]dopamine uptake in both brain regions with equivalent efficacy. From these data we conclude that the dopamine transporter is similar in both the striatum and nucleus accumbens.