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.     

Anticholinergic activity of the dopamine receptor agonist, TL-68 (N,N-dipropyl-2-aminotetralin)

The anticholinergic properties of a dopamine receptor agonist, a non-hydroxylated derivative of N,N-dipropylaminotetralin (TL-68), were evaluated using the guinea-pig isolated tracheal strip and rat phrenic nerve-diaphragm preparations. TL-68 competitively antagonized carbachol-induced contractions in guinea-pig trachea with a pA2 value of 5.88 +/- 0.05. In the rat phrenic nerve-diaphragm preparation, TL-68 was found to be inactive in blocking nicotinic cholinergic receptors.     

Characterization of [3H]zetidoline binding to rat striatal membranes

The binding of [3H]zetidoline, a novel neuroleptic agent, to rat brain striatal membranes was investigated in-vitro. The optimal binding conditions for [3H]zetidoline differed from those for [3H]spiperone in pH, temperature and time. [3H]Zetidoline has high affinity for striatal dopamine receptors. Its binding is saturable, stereo-specific, has a low non-specific component and is reversible and tissue specific. The Scatchard analysis gave a biphasic curve, indicating that [3H]zetidoline interacts with more than one population of receptor sites (B'max = 67 fmol mg-1 protein, K'd = 0.11 nM; B"max = 500 fmol mg-1 protein, K'd = 2.49 nM). Kinetic analysis of rates of association and dissociation yielded a Kd value in agreement with that measured at equilibrium. Inhibition studies indicated that only dopamine and dopaminergic agents are able to displace [3H]zetidoline from its binding sites, and in a different rank order from that for displacement of [3H]spiperone. (-)-Sulpiride was especially effective in inhibiting [3H]zetidoline specific binding. Furthermore, like that of [3H]benzamides, [3H]zetidoline binding appears to be highly Na+-dependent and Li+ only partially substitutes Na+.     

Further in vitro and in vivo studies with the putative presynaptic dopamine agonist N,N-dipropyl-7-hydroxy-2-aminotetralin

Summary The in vitro binding of the putative dopamine autoreceptor agonist [³H]DP-7-ATN to rat striatal membrane homogenates was investigated. The maximum number of binding sites B _max was 497.5 ± 50.2 fmol/mg protein and the affinity constant K _D was 8.3 ± 1.5 nM using 10 M (+) butaclamol to define non-specific binding. Lesion of the left medium forebrain bundle by 6-hydroxydopamine resulted in an almost complete loss of dopamine in the striatum but did not affect the binding of [³H]DP-7-ATN. The binding of [³H]DP-7-ATN to the homogenates of the dopaminergic cell bodies in the substantia nigra revealed a B _max of 542.4 ± 40.1 fmol/mg protein and a K _D of 11.1 ± 1.3 nM. The pharmacological profile of the binding was characterized as being to D-2 receptors. No direct in vitro evidence could be found for a selective binding to DA autoreceptors. The dopamine uptake inhibitor GBR 12909 interacted in a noncompetitive manner with the in vitro binding of [³H]DP-7-ATN and the latter compounds uptake into isolated synaptosomes was not through the specific dopamine uptake system but rather through diffusion. GBR 12909 failed to reveal any agonistic or antagonistic activity in the GBL model but was able to antagonize the hypomotility in rats induced by 0.25 mg/kg DP-7-ATN. The inhibitory effect of DP-7-ATN on DA release was also demonstrated using in vivo brain dialysis in conscious rats. Based on the above results, the possibility is discussed that the release regulating DA autoreceptors, which might be coupled to the reuptake complex, and the DA biosynthesis regulating autoreceptors, are different entities. Send offprint requests to A. S. Horn     

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.     

6,7-Dihydroxy-3-chromanamine: synthesis and pharmacological activity of an oxygen isostere of the dopamine agonist 6,7-dihydroxy-2-aminotetralin

6,7-Dihydroxy-3-chromanamine, the oxygen isostere of 6,7-dihydroxy-2-aminotetralin (6,7-ADTN), has been synthesized and its dopaminergic activity in various test systems determined. Following bilateral injection into the rat nucleus accumbens, a pattern of locomotor activity similar to that produced by 6,7-ADTN was observed. Its ability to displace N-n-propyl[3H]norapomorphine binding to homogenates of rat brain corpus striatum was found to be about 15 times weaker than 6,7-ADTN and apomorphine. Like 6,7-ADTN it failed to influence dopamine metabolism following an intraperitoneal injection. It is suggested that in addition to the 2-aminotetralins, the 3-chromanamines may be a potential source of new dopamine receptor agonists.     

Synaptic concentration of dopamine in rat striatal slices in relationship to [3H]raclopride binding to the dopamine D2 receptor

The in vivo binding of dopamine (DA) radioligands to D2 receptors can be affected by competition with endogenous dopamine. In the present study, we used a brain slice preparation that provides more controlled conditions than in vivo preparations in order to examine the relationship between synaptic DA and the binding of [3H]raclopride to D2 receptors. We also estimated the synaptic DA concentration in rat striatal slices by determining the changes in [3H]raclopride binding. To correlate the changes in [3H]raclopride binding with the concentration of synaptic DA, the kinetic parameters were determined. [3H]Raclopride reached equilibrium binding conditions within two hours. The Ki value for DA in inhibiting [3H]raclopride binding was about 2.2 nM. The increase in synaptic DA evoked by electrical stimulation decreased the striatal binding of [3H]raclopride in a frequency-dependent manner. Increases in the DA concentration evoked by amphetamine (AMPH) or cocaine decreased [3H]raclopride binding by 74% or 20%, respectively, corresponding to increases in the synaptic DA concentrations of 1.6 nM or 0.6 nM, respectively. Pargyline also decreased [3H]raclopride binding by 36% corresponding at a concentration of 1.2 nM. In contrast, the depletion of synaptic DA by alpha-methyl-p-tyrosine (alpha-MpT) increased the specific binding of [3H]raclopride by 43% when the DA concentration was decreased to 0.7 nM. The changes in the DA concentration at the synapse were responsible for the changes in the striatal binding of [3H]raclopride. The values calculated in this study may therefore approximate the changes in the synaptic DA concentration in rat striatal slices following manipulation.     

Paraxanthine displaces the binding of [3H]SCH 23390 from rat striatal membranes

We present evidence showing that paraxanthine (1,7-dimethylxanthine), the main metabolite of caffeine in man, displaces the binding of [3H]SCH 23390, a radioligand which selectively labels dopamine D-1 receptors when used at low concentrations, from striatal membranes of the rat. The displacement was competitive and indicated the existence of two affinity states (Hill coefficient = 0.49; K(high) = 0.15 microM; K(low) = 95.9 microM, %R(high) = 32.4). When the stable GTP analog Gpp(NH)p was included, the displacement curve indicated the presence of only the low-affinity state (Hill coefficient = 1.16; Ki = 72.1 microM). However, paraxanthine did not displace the specific binding of [3H]spiperone. After injection of 30 mg/kg s.c. of caffeine, a maximum of 10 microM of paraxanthine was found in striatal homogenates, which could be sufficient to occupy dopamine D-1 receptors. Our results suggest that a dopaminergic action of paraxanthine could be involved in the behavioural stimulation produced by caffeine.     

The binding of [3H]N-(chloroethyl)-norapomorphine to rat striatal membranes

On the stretch-induced discharge activity of the isolated crayfish sensory neuron flurazepam (less than or equal to 3 X 10(-4) M) and Ro 15-1788 (less than or equal to 10(-3) M) produced reversible concentration-dependent excitation, but oxazepam only produced depression (less than or equal to 5 X 10(-4) M). Similar divergent effects on the membrane properties were observed. Oxazepam increased the threshold to firing without changing resting potential, membrane resistance or the GABA-mediated IPSP. In contrast flurazepam and Ro 15-1788 produced a concentration-dependent decrease in threshold. Flurazepam did not alter membrane resistance or resting potential but facilitated GABA transmission. Ro 15-1788 had the opposite effect on the GABA synapse, and also depolarized the resting potential but did not alter membrane resistance. The change in spike threshold appeared to be an important component in producing discharge excitation or depression. These results not only demonstrate the capability of the sensory neuron to discriminate between structures of benzodiazepines, but also that these agents can produce divergent effects on synaptic and non-synaptic properties of a single neuron.     

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.     

[3H] sertraline binding to rat brain membranes

Tritiated sertraline, a radiolabeled form of a potent and selective inhibitor of serotonin uptake, was found to bind with high affinity to rat whole brain membranes. Characterization studies showed that [³H] sertraline binding occurred at a single site with the following parameters:K _d 0.57 nM,B _max 821 fmol/mg protein,n _h 1.06. This binding was reversible; the dissociation constant calculated from kinetic measurements (K _d 0.81 nM) agreed with that determined by saturation binding experiments. [³H] Sertraline binding in the presence of serotonin, paroxetine, fluoxetine or imipramine suggested competitive inhibition of binding (large increase inK _d with little change inB _max). The rank order of potency of inhibition of [³H] sertraline binding was similar to that of inhibition of serotonin uptake for known uptake inhibitors and the 1-amino-4-phenyltetralin uptake blockers. A marked decrease in ex vivo [³H] sertraline binding in the brain of rats 7 days after treatment withp-chloroamphetamine was consistent with the loss of serotonin uptake sites induced by this agent. The results of our study indicated that [³H] sertraline labels serotonin uptake sites in rat brain.     

Interaction of dihydroxy-2-aminotetralin derivatives at sites labelled with [3H]clonidine, [3H]prazosin and [3H]spiperone in rat brain membranes

The interactions of 5,6- and 6,7-dihydroxy derivatives of 2-aminotetralin with [³H]clonidine and [³H]clonidine and [³H]prazosin as well as with [³H]spiperone binding sites in rat cerebral cortex membrane preparations were investigated. The hydroxy derivatives of 2-aminotetralin tested showed significant interaction with [³H]clonidine as well as with [³H]spiperone binding sites while for [³H]prazosin binding site these agents appeared virtually inactive. For interaction with [³H]clonidine binding site 6,7-dihydroxy substitutions impart greater potency that 5,6-dihydroxy substitutions and N-alkyl substitutions either make no difference or reduce the affinity of these compounds. N-alkyl substitutions, however, markedly enhance the affinity of 5,6-dihydroxy derivatives for interactions with [³H]spiperone binding site. The results suggest that some hydroxy derivatives of aminotetralin have significant interaction with both central α₂-adrenoceptor and D₂-dopamine receptor systems.     

The effects of kainic acid and 6-hydroxydopamine lesions, metal ions and GTP on in vitro binding of the D-2 dopamine agonist, [3H]N-0437, to striatal membranes

The kinetic and pharmacological profiles of the potent and selective D-2 dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin ([3H]N-0437) have recently been described. This report concerns the effects of chemical lesions and metal ions on the radioreceptor binding of [3H]N-0437. Kainic acid lesions reduced the maximum number of binding sites (Bmax) in the rat striatum by 50%. The affinity of [3H]N-0437 for dopamine receptors was reduced by half. 6-Hydroxydopamine lesions had no measurable effect on the Bmax or on the KD. Of the physiological metal ions tested only Na+ had a significant effect on the binding. Sodium ions reduced the affinity of [3H]N-0437 for striatal receptors from 5.0 +/- 1.1 nM to 8.4 +/- 0.3 nM. In addition GTP lowered the Bmax from 1121 +/- 44 to 868 +/- 84 fmol/mg protein. The trace ions Li+ and Mn2+ had no effect at a concentration of 3.0 mM, while the exogenous ion Hg2+ at the same concentration prevented the specific binding of [3H]N-0437. Together, the results suggest that [3H]N-0437 labels both pre- and postsynaptic receptors, although postsynaptic receptors are labelled preferentially. Moreover, there is an indication that GTP shifts the affinity state of the D-2 receptor from high to low, while Na+ seems to be an allosteric inhibitor.     

Effect of transition or heavy metals on [3H]haloperidol binding in rat striatal membranes in vitro

Our previous experiments have shown that several metal cations affect dopaminergic uptake and release processes in synaptosomes in vitro. It is thus possible that other membrane-related steps of neurotransmission, such as receptor binding, are affected as well. We studied the effect of Mn2+, Cu2+, Cd2+, Zn2+, Hg2+, Pb2+ and of two organometals, methyl mercury and triethyl lead, on [3H]haloperidol binding in the striatal P2 fraction assuming that such a study would reveal direct effects of the ions on dopaminergic D2 receptor binding. According to non-linear curve fitting and Scatchard analysis, [3H]haloperidol bound to two sites in striatal tissue. The Kd of the higher affinity site was 0.14 +/- 0.05 nM and the Bmax 226.3 +/- 50.3 fmol/mg protein. The respective values for the lower affinity site were 2.49 +/- 0.56 nM and 678.3 +/- 111.4 fmol/mg protein. Among the divalent cations, Hg2+ (IC50 0.7 microM) and Cu2+ (IC50 2.9 microM) inhibited the high affinity [3H]haloperidol binding most potently. The inhibition by Cu2+ was due to a decrease in the binding affinity (increase in the Kd) while the number of binding sites remained unchanged. Zn2+ inhibited the binding by 41.8% and Cd2+ by 38.7% at 10 microM concentration while Pb2+ and Mn2+ did not affect binding significantly at this or lower concentrations. Methyl mercury (IC50 0.9 microM) and triethyl lead (IC50 2.6 microM) inhibited binding as well. Both these organometallic cations decreased the binding affinity but did not change significantly the number of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological profile of a chromanamine analogue (DP-6OH-3CA) of the selective presynaptic dopamine agonist N,N-dipropyl-7-hydroxy-2-aminotetralin

The pharmacological profile of an oxygen isostere of the selective presynaptic dopamine agonist DP-7OH-AT, i.e. dipropyl-6-hydroxy-3-chromanamine (DP-6OH-3CA) has been evaluated in various receptor binding, neurobiochemical and behavioural experiments. The chromanamine displaced the 3H-labelled dopamine ligands, 5,6-DPAT and N-0437, with Ki values of 106 and 143 nM, respectively. In in-vivo biochemical models for presynaptic activity the chromanamine induced a half-maximal effect in the gamma-butyrolactone reversal test at 6.8 mumol kg-1 and had an ED70 value of 40 mumol kg-1 for HVA decrease in the striatum. In behavioural models for postsynaptic dopaminergic activity a half-maximal effect for the induction of stereotypy was reached at 100 mumol kg-1 and reversal of the effects of reserpine to a level of 200 counts was induced at 11 mumol kg-1. On comparison of these results with the results obtained with the carbon analogue of DP-6OH-3CA, i.e. DP-7OH-AT, it is apparent that the chromanamine has a reduced potency for dopamine D2 receptors in in-vitro and in-vivo models. The selectivity for presynaptic dopamine receptors was lower than with DP-7OH-AT and the isomeric chromanamine, DP-8OH-3CA, indicating that the optimal position of the hydroxyl group for presynaptic selectivity is in the 8 and not in the 6 position for the chromanamines.     

The purported dopamine agonist DPI inhibits [3H]noradrenaline release from rat cortical slices but not [3H]dopamine and [14C]acetylcholine release from rat striatal slices in-vitro

The effects of the purported dopamine (DA) receptor agonist (3,4-dihydroxyphenylimino)-2-imidazolidine (DPI) upon the in-vitro K+-induced release of [3H]DA and [14C]acetylcholine from rat neostriatal slices, and of [3H]noradrenaline from rat neocortical slices have been investigated and compared with those of the DA receptor agonist TL-99 and the alpha-adrenoceptor agonist clonidine, respectively. The rapid decomposition of the catechol compounds DPI and TL-99 in the Krebs-Ringer bicarbonate superfusion medium was shown to be inhibited by both the chelating agent EDTA and the reducing agent ascorbic acid. The results suggest that in-vitro DPI is unable to stimulate striatal DA receptors, whereas it is effective in stimulating cortical alpha 2-adrenoceptors (EC50 = 61 nM). It is concluded that DPI should be considered as a mixed alpha 1/alpha 2-adrenoceptor agonist and that the designation of DPI as a DA receptor agonist should be abandoned.     

Acetylcholinesterase potentiates [3H]fluorowillardiine and [3H]AMPA binding to rat cortical membranes

In addition to its action at cholinergic synapses acetylcholinesterase (AChE) has been proposed to modulate neuronal activity by mechanisms unrelated to the hydrolysis of acetylcholine. We have investigated the effects of AChE on the binding of the specific AMPA receptor agonists (S)-[3H]5-fluorowillardiine ([3H]FW) and [3H]AMPA to rat cortical membranes. Pretreatment of membranes with AChE causes a dose-dependent increase in the binding of both radiolabelled agonists with a maximal increase to approximately 60% above control. This increase is completely blocked by the specific AChE inhibitors propidium, physostigmine, DFP and BW 284C51. AChE pretreatment had no effect on [3H]kainate binding. [3H]FW binding to membranes from young (15-day-old) rats is four orders of magnitude more sensitive to AChE modulation than membranes from adult rats (EC50 values of 4x10(-5) and 0.1 unit/ml, respectively) although the total percentage increase in binding is similar. Furthermore, the AChE-induced potentiation of [3H]FW binding is Ca2+ - and temperature-dependent suggesting an enzymatic action for AChE in this system. Saturation binding experiments with [3H]FW to adult membranes reveal high and low affinity binding sites and demonstrate that the main action of AChE is to increase the Bmax of both sites. These findings suggest that modulation of AMPA receptors could provide a molecular mechanism of action for the previously reported effects of AChE in synapse formation, synaptic plasticity and neurodegeneration.     

Activation of presynaptic alpha-noradrenaline receptors in rat brain by the potent dopamine-mimetic N,N-dipropyl-5,6-ADTN

Rat cerebral cortex slices labeled with 3H-noradrenaline (NA) were superfused and 3H-NA release was induced with 20 mM K+. The release of 3H-NA was not affected by DA (1 micro M), but was inhibited by the 2-aminotetralins, 5,6-ADTN and N,N-dipropyl-5,6-ADTN (at a concentration of 1 micro M) by about 27% and 65%, respectively. The inhibitory effect of N,N-dipropyl-5,6-ADTN was concentration-dependent (3 x 10(-8) to 3 x 10(-7) M) and was antagonized by phentolamine but not by fluphenazine. The data indicate that, in addition to activating dopamine receptors, N,N-dipropyl-5,6-ADTN stimulates presynaptic alpha-NA receptors.