Peter H. Andersen

The dopamine uptake inhibitor GBR 12909: selectivity and molecular mechanism of action

Abstract The neurochemical profile of GBR 12909 (1-(2-(bis(4-fluorphenyl)-methoxy)-ethyl)-4-(3-phenyl-propyl)piperazine) was investigated. GBR 12909 was a potent and selective inhibitor of synaptosomal dopamine uptake (KI = 1 nM), with a 20-fold lower affinity for the histamine H1-receptor and a more than 100-fold lower affinity for the noradrenaline and 5-HT uptake carriers, the dopamine D-1, D-2, 5-HT1A and α1-receptors and voltage-dependent sodium channels. GBR 12909 (3 μM) was without effect on muscarinic, α2, β1+2, γ-aminobutyric acid (GABA) and benzodiazepine receptors, and on choline and GABA uptake carriers. The selective dopamine uptake inhibitory profile of GBR 12909 was confirmed by ex vivo uptake experiments. GBR 12909 inhibited dopamine uptake in vitro in a competitive manner as did cocaine and methylphenidate. [3H]GBR 12935 binding was competitively inhibited by GBR 12909 as well as by dopamine, cocaine and methylphenidate. Off-rate analysis of the [3H]GBR 12935 binding excluded the presence of allosteric binding sites on the dopamine carrier complex. Instead, the data favored the notion that GBR 12909 inhibits dopamine uptake by binding to the dopamine binding site on the carrier protein itself, thereby blocking the carrier process. In conclusion, GBR 12909 is a highly selective inhibitor of dopamine uptake, both in vivo and in vitro. At the moment GBR 12909 is the only compound with this neurochemical profile. The selective effect of GBR 12909 on this neuronal system makes it an interesting experimental tool and a potential antidepressant agent.

Dopamine receptor subtypes: beyond the D1/D2 classification

The D1/D2 dopamine receptor classification is widely accepted. However, intense investigative efforts over the last several years using pharmacological, biochemical and behavioral approaches have produced results that are increasingly difficult to reconcile with the existence of only two dopamine receptor subtypes. Recent developments, including cloning of the cDNAs and/or genes for several members of the large family of G-protein-coupled receptors, have revealed that heterogeneity in the pharmacological or biochemical characteristics of individual receptors often indicates the presence of previously unsuspected molecular subtypes. In this article, Marc Caron and colleagues have assembled the main lines of evidence that suggest the presence of several novel subtypes for both D1 and D2 dopamine receptors and predict that molecular cloning will, in the near future, confirm their existence.

Biochemical and pharmacological characterization of [^3H] GBR 12935 binding in vitro to rat striatal membranes : labeling of the dopamine uptake complex

Abstract: Binding of the selective dopamine (DA) uptake inhibitor [3H]GBR 12935 to rat striatal membranes was characterized biochemically and pharmacologically. [3H]‐GBR 12935 binding at 0°C was reversible and saturable and Scatchard analysis indicated a single binding site with a KD of 5.5 nM and a Bmax of 760 pmol/mg tissue. [3H]GBR 12935 labeled two binding sites. One binding site was identified as the classic DA uptake site, since methylphenidate, cocaine, diclofensine, and Lu 19–005 potently inhibited [3H]GBR 12935 binding to it. Binding to the second site was inhibited by high concentrations of the above compounds. IC50 values for inhibition of [3H]GBR 12935 binding to the DA uptake site were proportional to IC50 values for inhibition of DA uptake. However, substrates of DA uptake, e.g., DA and 1‐methyl‐4‐phenylpyridine, and DA releasers, e.g., the amphetamines, inhibited [3H]GBR 12935 binding less than DA uptake. Rate experiments excluded the possibility that these “weak” inhibitors affected the binding by alloste‐ric coupled binding sites. The second binding site was not a noradrenergic, serotonergic, or GABAergic uptake site. Neither was it a dopaminergic, acetylcholinergic, histaminic, serotonergic, or adrenergic receptor. However, [3H]GBR 12935 was potently displaced from it by disubstituted piper‐azine derivatives, i.e., flupentixol and piflutixol. DA uptake and the DA uptake binding site of [3H]GBR 12935 were located primarily in the striatum, but the piperazine acceptor site was distributed uniformly throughout the brain. Also only the DA uptake binding site was destroyed by 6‐OH‐DA. Thus, [3H]GBR 12935 labels the classic DA uptake site in rat striatum and also a piperazine acceptor site. Substrates for DA uptake and releasers of DA inhibited [3H]GBR 12935 binding with low potency, but did not alter the rate constants for [3H]GBR 12935 binding. Therefore inhibitors of DA uptake label the carrier site and prevent the carrier process.

Dopamine receptor agonists: selectivity and dopamine D1 receptor efficacy

Dopamine receptor selectivity was investigated for a number of dopamine receptor agonists. In vitro, the benzazepine derivatives, e.g., SKF 38393 and SKF 75670 as well as the isoquinoline derivatives, SKF 89626 and SKF 89615, were D1 receptor-selective. All other compounds like apomorphine, CY 208-243, 6,7-ADTN and 3-PPP were either D2-selective or did not discriminate between subtypes. In general, the same receptor profile seen in vitro was observed in vivo. The exceptions to this pattern were: compounds which did not cross the blood-brain barrier, like 6,7-ADTN and SKF 89626, and compounds which appeared nonselective in vitro but demonstrated D2 selectivity in vivo like apomorphine, CI 201-678 and CY 208-243. A number of compounds were characterized in detail with respect to a GTP-induced affinity shift in inhibition of [3H]SCH 23390 binding, and potency and efficacy in stimulating adenylate cyclase from rat striatum. Inhibition of specific [3H]SCH 23390 binding by these agonists in the absence of GTP occurred with Hill slopes below unity and could best be explained by a two-site model with a high (KH)- and low-affinity (KL) component. Inhibition of [3H]SCH 23390 binding in the presence of 15 microM GTP occurred with Hill slopes of unity. The KI values obtained in the presence of 15 microM GTP were similar to the KL values, the low-affinity component observed in the absence of GTP. The capability of the agonists to stimulate the adenylate cyclase was analyzed in relation to dopamine (efficacy = 100%). The efficacy of the benzazepine derivatives varied from 24 (SKF 75670) to 100% (SKF 83189), dependent on the substituents on the benzazepine core. The isoquinolines, SKF 89626 and SKF 89615 had full efficacy, whereas most other agonists tested appeared to have only partial efficacy. In summary, the present paper presents data on dopamine receptor selectivity and efficacy in stimulating adenylate cyclase for a number of dopaminergic agonists. These data may create a basis for selection of agonists in future characterizations of dopaminergic-mediated events.

(R)-N-[4,4-Bis(3-Methyl-2-Thienyl)but-3-en-1-yl]Nipecotic Acid Binds with High Affinity to the Brain γ-Aminobutyric Acid Uptake Carrier

(R)‐N‐[4,4‐Bis(3‐methyl‐2‐thienyl)but‐3‐en‐l‐yl]nipecotic acid (NO 328) has previously been shown to be a potent anticonvulsant in both mice and rats. Here, we report that NO 328 is a potent inhibitor of γ‐[3H]aminobutyric acid ([3H]GABA) uptake in a rat forebrain synaptosomal preparation (IC50= 67 nM) and in primary cultures of neurons and astrocytes. Inhibition of [3H]GABA uptake by NO 328 is apparently of a mixed type when NO 328 is preincubated before [3H]GABA uptake; the inhibition is apparently competitive without preincubation. NO 328 itself is not a substrate for the GABA uptake carrier, but NO 328 is a selective inhibitor of [3H]GABA uptake. Binding to benzodiazepine receptors, histamine H1 receptors, and 5‐hydroxytryptaminelA receptors was inhibited by NO 328 at 5—30 μM, whereas several other receptors and uptake sites were unaffected. [3H]NO 328 showed saturable and reversible binding to rat brain membranes in the presence of NaCI. The specific binding of [3H]NO 328 was inhibited by known inhibitors of [3H]GABA uptake; GABA and the cyclic amino acid GABA uptake inhibitors were, however, less potent than expected. This indicates that the binding site is not identical to, but rather overlapping with, the GABA recognition site of the uptake carrier. The affinity constant for binding of [3H]NO 328 is 18 nM, and the Bmax is 669 pmol/g of original rat forebrain tissue. The regional distribution of NaCl‐dependent [3H]NO 328 binding followed that of synaptosomal [3H]GABA uptake. It is concluded that NO 328 is a potent and selective inhibitor of neuronal and glial GABA uptake and that [3H]NO 328 is a useful radioligand for labeling the GABA uptake carrier in brain membranes. In the mouse brain in vivo, [3H]NO 328 likewise showed saturable and reversible binding that could be displaced by analogues of NO 328. Further studies are needed to demonstrate whether the uptake carrier is indeed labeled by [3H]NO 328 in vivo.

A comparison between dopamine-stimulated adenylate cyclase and 3H-SCH 23390 binding in rat striatum

Methods for measuring 3H-SCH 23390 binding and dopamine (DA) stimulated adenylate cyclase (AC) were established in identical tissue preparations and under similar experimental conditions. Pharmacological characterization revealed that both assays involved interaction with the D1 receptor or closely associated sites. In order to investigate whether the binding sites for 3H-SCH 23390 and DA in fact are identical, the antagonistic effects of a variety of pharmacologically active compounds were examined. Surprisingly, the Ki-values obtained from Schild-plot analysis of the antagonism of DA-stimulated AC, were 80-240 times higher than the Ki-values obtained from competition curves of 3H-SCH 23390 binding. Since both assays were performed under identical conditions, the differences in Ki-values indicate the possibility of different binding sites for DA and 3H-SCH 23390 or, that DA and 3H-SCH 23390 label different states of the same receptor.

Comparison of the pharmacological characteristics of [3H]raclopride and [3H]SCH 23390 binding to dopamine receptors in vivo in mouse brain

In vivo binding of the benzamide derivative [3H]raclopride was studied in mouse brain. The binding was saturable, reversible and stereospecific. Non-specific binding was 5-15% of the total binding. Pharmacological characterization of the binding indicated labelling of dopamine D2 receptors since the binding was potently inhibited by compounds with high affinity for this receptor in vitro. On the other hand, compounds with low affinity in vitro i.e., dopamine D1-selective compounds were weak or inactive as inhibitors of [3H]raclopride binding. A comparison of the pharmacological characteristics of [3H]raclopride and [3H]SCH 23390 binding in vivo indicated that compounds with selectivity in vitro retained this selectivity in vivo. Thus, spiroperidol, haloperidol, 1-sulpiride, clebopride, LY 171555 and (-)-NPA ((-)-N-propyl-norapomorphine) were D2 selective while SCH 23390, SKF 38393 and SKF 75670 were D1 selective. Clozapine, tilozepine, cis-flupentixol, chlorpromazine and butaclamol were non-selective both in vitro and in vivo. However, a few compounds changed profile in vivo compared to in vitro. Thus, fluperlapine and fluphenazine had a dual D1-D2 receptor profile in vitro but were D1- or D2-selective in vivo, respectively. Pergolide and molindone which were D2-selective in vitro both had a dual D1-D2 receptor profile in vivo. In conclusion, [3H]raclopride, in vivo, selectively labels the dopamine D2 receptor. Comparison of the pharmacological characteristics of [3H]raclopride and [3H]SCH 23390 binding in vivo supported the that the dopamine D1 receptor is an important target for a variety of neuroleptics, especially of the clozapine type. This may indicate that blockade of the dopamine D1 receptor conveys antipsychotic action.

Evidence for Different States of the Dopamine Dl Receptor: Clozapine and Fluperlapine May Preferentially Label an Adenylate Cyclase‐Coupled State of the Dl Receptor

Abstract: It has been shown previously that typical neuroleptics have higher affinities for 3,4‐dihydroxyphenyl‐ethylamine (dopamine) Dl receptors as labeled by(R)‐ (+)‐ 8‐chloro‐2,3,4,5‐tetrahydro‐3‐methyl‐5‐phenyl‐1 ‐N‐3‐benzazepine‐7‐ol ([3H]SCH 23390) than for inhibiting dopamine‐stimulated adenylate cyclase. We now report that the atypical neuroleptics, clozapine and fluperlapine, exhibit characteristics opposite to typical neuroleptics, i.e., they have higher affinity for inhibiting dopamine‐stimulated adenylate cyclase than [3H]SCH 23390 binding. A variety of compounds, i.e., clozapine, fluperlapine, and dopamine, were tested for their capacity to affect the rate constants of [3H]SCH 23390 binding; these experiments revealed no effect of any tested compound on on‐rate or off‐rate of [3H]SCH 23390 binding. Treatment of striatal membranes with phospholipase A2 (PLA2) caused a rapid decrease in the Bmax value of the [3H]SCH 23390 binding with no effect on the Kd value. The adenylate cyclase, both the unstimulated, the dopamine‐, fluoride‐, and forskolin‐stimulated activity, was far less sensitive than [3H]SCH 23390 binding to PLA2. Treatment of striatal membranes with filipine and (NH4SO4 produced, as did PLA2 treatment, a rapid decline in [3H]SCH 23390 binding. However, opposite to PLA2 treatment, these agents stimulated the adenylate cyclase. In conclusion, a comparison of the pharmacological characteristics of [3H]SCH 23390 binding and dopamine‐stimulated adenylate cyclase suggests the existence of two different Dl binding sites. The rate experiments exclude the possibility of allosterically coupled sites. Instead our results favor that the Dl receptor exists in different states/conformations, i.e., both adenylate cyclase‐coupled and uncoupled, and further, that the atypical neuroleptics clozapine and fluperlapine may have adenylate cyclase‐coupled dopamine Dl receptors as target.

NNC-112, NNC-687 and NNC-756, new selective and highly potent dopamine D1 receptor antagonists

The neurochemical properties of three novel benzazepine derivatives NNC-112, NNC-687 and NNC-756 were assessed. These compounds inhibited dopamine D1 receptor binding in vitro with low nanomolar to picomolar dissociation constants whereas those for the D2 receptor were in the micromolar range. Contrary to classical neuroleptics, but similar to the atypical neuroleptics, clozapine and fluperlapine, NNC-112, NNC-687 and NNC-756 were relatively more potent in inhibiting dopamine-stimulated adenylyl cyclase than [3H]SCH 23390 binding. Both NNC-112 and NNC-756 had high affinity for the 5-HT2 receptor whereas NNC-687 had low affinity for this receptor. The affinity for other receptors or neurotransmitter transporters was very low. In vivo, the dopamine D1 receptor selective profile of NNC-112, NNC-687 and NNC-756 was evident from the potent inhibition of D1 receptor binding whereas no effect on D2 receptor binding was apparent. In addition, the compounds blocked D1 receptor-mediated rotation in unilaterally 6-hydroxydopamine-lesioned rats, but had no effect on D2-induced rotation. Thus, NNC-112, NNC-687 and NNC-756 are potent and selective dopamine D1 receptor antagonists that may be useful in the treatment of schizophrenia.

Surface Expression of the AMPA Receptor Subunits GluR1, GluR2, and GluR4 in Stably Transfected Baby Hamster Kidney Cells

Abstract: The surface expression of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)‐type glutamate receptor (GluR) subunits GluR1, GluR2, and GluR4 was studied in cultures of stably transfected baby hamster kidney (BHK)‐570 cells. Two methods were used to quantify surface expression: cross‐linking with the membrane‐impermeant reagent bis(sulfosuccinimidyl)suberate (BS3) and labeling of surface receptors with the membrane‐impermeant biotinylating reagent sulfosuccinimidyl 2‐(biotinamido)ethyl‐1,3‐dithiopropionate (NHS‐ss‐biotin) followed by precipitation with neutravidin beads. Western blot analyses of control versus treated cultures revealed that, for all three GluR subunits examined, 25–40% of the total GluR population is located in the plasma membrane of the BHK‐570 cells. This finding was corroborated by analyses of the surface expression of [3H]AMPA binding sites in the GluR‐expressing BHK‐570 cells performed via the biotinylation/precipitation method; these studies revealed that 30–40% of the total binding site population is found in the plasma membrane. Analyses of combinations of the subunits, both GluR1 + GluR2 and GluR2 + GluR4, revealed that heteromeric combinations of the subunits are not trafficked to the surface more efficiently than homomeric receptors. For each of the three subunits, western blots revealed two distinct bands; removal of surface receptors reduced immunoreactivity for the upper band of each subunit by >90%, whereas immunoreactivity for the lower band was reduced by only 10–20%. Treatment of extracts from the various cell lines with glycopeptidase F resulted in the collapse of the two bands into a single band of lower molecular weight, suggesting that the two original bands represent differentially glycosylated forms of the same polypeptides. These data indicate that the majority of the stably expressed GluR subunits in these cell lines are incompletely glycosylated and that complete glycosylation is associated with trafficking of the GluR subunits to the cell surface.