Ionic Requirements for the Specific Binding of [3H]GBR 12783 to a Site Associated with the Dopamine Uptake Carrier

At 0°C, when Na⁺ was the only cation present in the incubation medium, increasing the Na⁺ concentration from 3 to 10 mM enhanced the affinity of [³H]l-[2-(di-phenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([³H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the 5_max. For higher Na⁺ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na⁺. In a 10 mM Na⁺ medium, the K_D and the B_max were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [³H]GBR 12783, the half-maximal specific binding occurred at 5 mM Na⁺. A similar Na⁺ dependence was observed at 20°C. Scatchard plots indicated that K⁺, Ca²⁺, Mg²⁺, and Tris⁺ acted like competitive inhibitors of the specific binding of [³H]GBR 12783. The inhibitory potency of various cations (K⁺, Ca²⁺, Mg²⁺, Tris⁺, Li⁺ and choline) was enhanced when the Na⁺ concentration was decreased from 130 to 10 mM. In a 10 mM Na⁺ medium, the rank order of inhibitory potency was Ca²⁺ (0.13 mM) > Mg²⁺ > Tris⁺ > K⁺ (15 mM). The requirement for Na⁺ was rather specific, because none of the other cations acted as a substitute for Na⁺. No anionic requirement was found: Cl^-, Br^-, and F^- were equipotent. These results suggest that low Na⁺ concentrations are required for maximal binding; higher Na⁺ concentrations protect the specific binding site against the inhibitory effect of other cations.     

Kinetic Analysis of the Chloride Dependence of the Neuronal Uptake of Dopamine and Effect of Anions on the Ability of Substrates to Compete with the Binding of the Dopamine Uptake Inhibitor GBR 12783

The specific binding of [3H]1-[2-(diphenyl-methoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([3H]GBR 12783) to the dopamine (DA) neuronal carrier present in membranes prepared from rat striatum was not affected when Cl- was replaced by either Br- or NO3-. In media containing Cl-, Br-, or NO3-, d-amphetamine and DA competed with the radioligand in a monophasic manner with Hill coefficients of close to 1 (0.94-1.12). Replacement of Cl- by Br- impaired the ability of some substrates (d-amphetamine, DA, p-hydroxyamphetamine, and m-tyramine) to compete with [3H]GBR 12783. The potency of Br- to decrease the affinity of substrates for the specific binding site was significantly correlated (t = 7.07, p less than 0.001) with their affinity for this binding site. These results suggest that the various substrates tested could bind to recognition sites in which Cl- is differently involved; as a consequence, substrates could bind to the neuronal carrier by means of partly different links. In experiments dealing with the specific uptake of [3H]DA, F-, NO3-, isethionate-, or acetate- was unable to substitute for Cl-, whereas Br- was quite a total substitute. Replacement of Cl- by equimolar concentrations of either NO3- or isethionate- resulted in inhibition curves of DA specific uptake with Hill coefficients of close to 1 (0.77 and 1.04 respectively); this indicates that both NO3- and isethionate- are devoid of inhibitory effects on neuronal uptake and are quite ineffective substitutes for Cl-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic mechanism of dopamine transporter interaction with 1-(2-(bis-(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909)

Interaction of piperazine-based dopamine transporter inhibitor GBR12909 with rat dopamine transporters has been studied by means of competition kinetics analysis, employing [(3)H]PE2I as the reporter ligand. It has been found that GBR12909 is capable of inducing so-called "slow isomerization step" upon binding to DAT, probably consisting of a conformational change in the transporter protein. The mechanism exhibited by GBR12909 appears to be similar to the mechanism of PE2I that has been reported earlier and also confirms previous observations for GBR12783 made by Do-Rego and co-workers using dopamine uptake data. It appears that the isomerization phenomenon previously described for PE2I is not limited to tropane-based DAT inhibitors, but is, in fact, a general property of dopamine transporter protein, similar to "isomerization" process reported previously for G-protein coupled receptors. The rapid first step of association of the GBR 12909 is characterized by the equilibrium constant K(L)=34+/-11nM and the second slow step by k(i)=0.033+/-0.005s(-1).     

Effects of Several Cations on the Neuronal Uptake of Dopamine and the Specific Binding of [3H]GBR 12783: Attempts to Characterize the Na+ Dependence of the Neuronal Transport of Dopamine

We have studied the effects of several cations on (1) the neuronal uptake of [3H]dopamine ([3H]DA) and (2) the specific binding of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-[1-3H]propenyl)piperazi ne ([3H]GBR 12783) to a site associated with the neuronal carrier of DA, in preparations obtained from rat striatum. When studied under the same experimental conditions, both the uptake of [3H]DA and the binding of [3H]GBR 12783 were similarly impaired by the gradual replacement of NaCl by sucrose. In both processes, no convenient substitute for Na+ was found. Furthermore, potential substitutes of Na+ acted as inhibitors of the uptake with a rank order of potency as follows: K+ = Li+ > or = Cs+ > or = Rb+ > choline+ > Tris+ > sucrose, which was somewhat different from that observed in binding studies, i.e., Cs+ > Rb+ > choline+ > or = K+ > Li+ > Tris+ > sucrose. In the presence of either 36 mM or 136 mM Na+, [3H]DA uptake was optimal with 2 mM Mg2+, 1 mM K+, or 1 mM Ca2+. In contrast, higher concentrations of divalent cations competitively blocked the uptake process. K+ concentrations > 50 mM impaired the specific binding, whereas in the millimolar range of concentrations, K+ noncompetitively inhibited the uptake. Decreasing the Na+ concentration increased the inhibitory effect of K+, Ca2+, and Mg2+ on the specific uptake. An increase in NaCl concentration from 0 to 120 mM elicited a significant decline in the affinity of some substrates for the [3H]GBR 12783 binding site. An uptake study performed using optimal experimental conditions defined in the present study revealed that decreasing Na+ concentration reduces the affinity of DA for the neuronal transport. We propose a hypothetical model for the neuronal transport of DA in which both Na+ and K+ membrane gradients are involved.     

Modeling of the Interaction of Na+ and K+ with the Binding of the Cocaine Analogue 3β-(4-[125I]Iodophenyl)tropane-2β-Carboxylic Acid Isopropyl Ester to the Dopamine Transporter

Abstract: The present study examines the interaction of Na⁺ and K⁺ with the binding of the cocaine analogue 3β-(4-[¹²⁵I]iodophenyl)tropane-2β-carboxylic acid isopropyl ester to dopamine transporters (DATs) in rat striatal synaptosomal membranes at 37°C. The binding increases with [Na⁺] from 10 to 100 mM and decreases with higher [Na⁺]. The presence of K⁺ reduces the maximal stimulatory effect of Na⁺ and causes a nonlinear EC₅₀ shift for Na⁺. K⁺ strongly inhibits the binding at low [Na⁺]. Increasing [Na⁺] produces a linear IC₅₀ shift for K⁺. Saturation analysis indicates a single binding site changing its affinity for the radioligand depending on [K⁺]/[Na⁺] ratio in the assay buffer. A reduced B_max was observed in the presence of 10 mM Na⁺ and 30 mM K⁺. Both high [Na⁺] and high [K⁺] accelerate the dissociation of the binding, and K⁺-induced acceleration was abolished by increasing [Na⁺]. Least squares model fitting of equilibrium data and kinetic analysis of dissociation rates reveal competitive interactions between Na⁺ and K⁺ at two sites allosterically linked on the DAT: One site mediates the stimulatory effect of Na⁺, and the other site involves the radioligand binding and the inhibitory effect of cations on the binding. Various uptake blockers and substrates, dopamine in particular, display reduced potency in inhibiting the binding at a higher [K⁺]/[Na⁺] ratio.     

Further exploration of 1-[2-[Bis-(4-fluorophenyl)methoxy]ethyl]piperazine (GBR 12909): role of N-aromatic,N-heteroaromatic,and 3-oxygenated N-phenylpropyl substituents on affinity for the dopamine and serotonin transporter

A series of N-aromatic, N-heteroaromatic, and oxygenated N-phenylpropyl derivatives of 1-(2-benzhydryloxyethyl)-piperazine and 1-[2-[bis-(4-fluorophenyl)methoxy]ethyl]piperazine, analogues of GBR 12909 (1a) and 12935 (1b), was synthesized and examined for their dopamine (DAT) and serotonin (SERT) transporter binding properties. One of these compounds, racemic 3-[4-(2-benzhydryloxyethyl)piperazin-1-yl]-1-(3-fluorophenyl)-propan-1-ol (33), had DAT affinity as good as, or better than, GBR 12909 and 12935, and was more selective for DAT over SERT than the GBR compounds. Both trans- (43) and cis- (47) (+/-)-2-(4-[2-[bis-(4-fluorophenyl)-methoxy]ethyl]piperazin-1-ylmethyl)-6-methoxy-1,2,3,4-tetrahydronaphthalen-1-ol had relatively good SERT selectivity and, as well, showed high affinity for SERT.     

Cationic and Anionic Requirements for the Binding of 2β-Carbomethoxy-3β-(4-Fluorophenyl)[3H]tropane to the Dopamine Uptake Carrier

Abstract: The present study reports the ion dependency of 2β-carbomethoxy-3β-(4-fluorophenyl)[³H]tropane ([³H]- CFT) binding to the dopamine transporter in the rat striaturn. The results indicate that [³H]CFT binding to synaptosomal P₂ membranes requires low concentrations of Na⁺ (peak binding between 20 and 50 m M Na⁺), is stimulated by phosphate anion or l^-, but is unaffected or only slightly affected by F^-, Cl^-, Br^-, NO₃^-, or SO₄^2-, Concentrations of Na⁺ of >50 m M become inhibitory except in the presence of l^-, which shifts peak binding levels toward higher Na⁺ concentrations and also elevates the peak binding level. K⁺ strongly decreased [³H]CFT binding with a shallow inhibition curve, and Na⁺ could not overcome this effect. Saturation analysis of [³H]CFT binding revealed a single binding site changing its affinity for CFT depending on the concentration of sodium phosphate buffer (6, 10, 30, 50, 130, or 200 m M ; 1 mM plus 49 mM NaCIversus 10 m M plus 40 m M NaCI; or 1 mM plus 129 m M Nal versus 10 m M plus 120 m M Nal). No differences were observed in the density of CFT binding sites between any of the conditions examined.     

Tissue distribution, autoradiography, and metabolism of 4-(2'-methoxyphenyl)-1-[2' -[N-2"-pyridinyl)-p-[(18)F]fluorobenzamido]ethyl]piperazine (p-[(18)F]MPPF), a new serotonin 5-HT(1A) antagonist for positron emission tomography: An In vivo study in rats

The in vivo behavior of 4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-p-[(18)F]fluorobenzamido ]ethyl]-piperazine (p-[(18)F]MPPF), a new serotonin 5-HT(1A) antagonist, was studied in awake, freely moving rats. Biodistribution studies showed that the carbon-fluorine bond was stable in vivo, that this compound was able to cross the blood-brain barrier, and that a general diffusion equilibrium could account for the availability of the tracer. The great quantity of highly polar metabolites found in plasma did not contribute to the small amounts of metabolites found in hippocampus, frontal cortex, and cerebellum. Exvivo p-[(18)F]MPPF and in vitro 8-hydroxy-2-(di-n-[(3)H]propylamino)tetralin autoradiography were compared both qualitatively and quantitatively. Qualitative evaluation proved that the same brain regions were labeled and that the p-[(18)F]MPPF labeling is (a) in total agreement with the known distribution of 5-HT(1A) receptors in rats and (b) characterized by very low nonspecific binding. Quantitative comparison demonstrated that the in vivo labeling pattern obtained with p-[(18)F]MPPF cannot be explained by differences in regional blood flow, capillary density, or permeability. The 5-HT(1A) specificity of p-[(18)F]MPPF and binding reversibility were confirmed in vivo with displacement experiments. Thus, this compound can be used to evaluate parameters characterizing 5-HT(1A) binding sites in the brain.     

Syntheses and pharmacokinetic evaluations of four metabolites of 2-(4-(2-((1H-benzo[d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis-(methylthio)pyridin-3-yl)acetamide hydrochloride [K-604], an acyl-CoA:cholesterol O-acyltransferase-1 inhibitor

We synthesized and identified four metabolites of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT)-1 inhibitor, K-604 (1). Two of the metabolites M1 and M2, were prepared from 1 using a combination reagent of hydrogen peroxide and sodium tungstate with either phosphoric acid or trifluoroethanol as the solvent to control the regioselectivity. Upon exposure of 4b to tert-butyl hypochlorite at −78 °C, the monosulfoxidation afforded synthetic intermediate of M3 in excellent yield. The efficient synthesis of M4 was established. The in vitro metabolic study exhibited a high clearance value (720 μL/min/mg protein) of 1 using human liver microsomes. We orally administered a single dose of 10 mg/kg of 1 to monkeys because the in vitro metabolic patterns are quite similar. Fortunately, the drug concentration of 1 was much higher than those of M1, M2, M3 and M4.     

Methyl 3β-(4-[125I]Iodophenyl)Tropane-2β-Carboxylate In Vitro Binding to Dopamine and Serotonin Transporters Under "Physiological" Conditions

Abstract: Methyl 3β-(4-[¹²⁵I]iodophenyl)tropane-2β-carboxylate ([¹²³I]β-CIT) is a single photon emission computed tomographic radiotracer for in vivo labeling of dopamine (DA) and serotonin (5-HT) transporters. Single photon emission computed tomographic experiments in nonhuman primates showed that [¹²³I]β-CIT in vivo binding to DA transporters had a much slower washout than binding to 5-HT transporters. This observation was not predicted from previously published in vitro studies. These studies, performed at 22°C in nonphysiological buffer, reported similar affinity of [¹²⁵I]β-CIT for DA and 5-HT transporters. We now report [¹²⁵I]β-CIT binding parameters to fresh rat membranes at 22°C and 37°C, in a buffer mimicking the composition of cerebrospinal fluid. At both temperatures, binding to DA transporters was best fit by a twosite model, whereas binding to 5-HT transporters was compatible with one population of sites. At 22°C, [¹²⁵I]β-CIT showed similar affinity to high-affinity DA (0.39 n M ) and 5-HT transporter sites (0.47 n M ). Increasing the incubation temperature from 22°C to 37°C reduced binding to DA transporters by 60%, whereas binding to 5-HT transporters was only marginally affected. In vitro kinetic experiments failed to detect significant differences in on or off rates that could explain the observed in vivo kinetics. These experiments thus failed to explain [¹²³ I]β-CIT in vivo uptake kinetics, suggesting the existence of specific factors affecting the in vivo situation.     

Inhibitory effects in vitro of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-glutathione,a proposed metabolite of l-histidine,on γ-glutamyltransferase activity

A transfer of the γ-glutamyl moiety of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (I), an adduct of glutathione and l-histidine metabolite urocanic acid, has been investigated by using γ-glutamyltransferase preparation from bovine kidney. When an equimolar mixture of two diastereomers of compound I in a phosphate buffer was allowed to react with glycylglycine in the presence of the transferase, two diastereomers of N-{S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteinyl}glycine (II) were formed in the same yield with each other and this was accompanied by a formation of γ-glutamylglycylglycine. Kinetics of compound I with the transferase indicated high affinity between the two materials, while the maximal reaction velocity of the γ-glutamyl transfer was low. Effects of compound I in vitro on the transfer of γ-glutamyl moiety of γ-glutamyl-p-nitroanilide to glycylglycine with the transferase were also studied, and the results indicated that the transfer was suppressed by compound I based on its competitive and non-competitive inhibitions. These results suggest that little variation in reactivities of two diastereomers of compound I as the substrate is given by the difference in stereomerism of their asymmetric carbon atoms and that inhibitory effects of compound I on the catalytic action of the transferase is of sufficient physiological importance to decrease the degradation of natural γ-glutamyl compounds, such as glutathione and its analogs.     

Functionalised dithiocarbamate complexes: Complexes based on indoline, indole and substituted piperazine backbones - X-ray crystal structure of [Ni(S2CNC3H6C6H4)2]

A range of new nickel, copper and zinc bis(dithiocarbamate) complexes has been prepared from secondary amines with functionalised backbones. These include complexes derived from iso-indoline, tetrahydro-isoindoline, 1,2,3,4-tetrahydroisoquinoline and a number of functionalised piperazines. The crystal structure of [Ni(S₂CNC₃H₆C₆H₄)₂] derived from 1,2,3,4-tetrahydroisoquinoline is reported.