Nora S. Kula

Lack of increase in dopamine transporter binding or function in rat brain tissue after treatment with blockers of neuronal uptake of dopamine

Rats were pretreated daily for 10 days with a dopamine (DA) uptake blocker ([+]amphetamine, benztropine, cocaine, GBR-12909, mazindol, or nomifensine) or control vehicle and, after 1-4 days of no treatment, striatal tissue was fractionated to provide synaptosomes and membranes for assays of transport of 3H-DA or binding of 3H-GBR-12935. There were no significant increases of apparent maxima for uptake (Vmax) or binding (Bmax) or consistent changes in ligand affinity. Pharmacologic characterization of 3H-GBR-12935 binding extended the impression that this ligand has high affinity and selectivity for many agents which block neuronal uptake of DA uptake and much less for those which interact with DA receptors or other amine transporters. The results suggest that dopamine transporters are not regulated in the same way as receptors, nor influenced similarly toward upregulation and supersensitization by repeated treatment with antagonists.

Regional distribution of dopamine D4 receptors in rat forebrain

BINDING of the D2-like (D2/D3/D4) radioligand [3H]nemonapride under selective conditions (with 300 nM S[−]-raclopride and other masking agents to occlude D2/D3 receptors and non-specific binding sites) revealed a subset of raclopride-insensitive binding sites considered D4-like receptors. These sites were stereoselective to R(−)− N-n-propylnorapomorphine (NPA) over its S(+)-NPA in a similar fashion to cloned D4 receptors expressed in cell lines. In addition, the highly D4-selective agent L-745,870 displaced 74–83% of these sites in rat brain regions, suggesting that most were D4 receptors. These apparent D4 receptors represented a relatively high proportion of D2-like receptors in hippocampus, dorsolateral frontal, medial prefrontal and entorhinal cortex, but fewer in caudate-putamen and nucleus accumbens.

Compounds selective for dopamine receptor subtypes

Novel dopamine (DA) receptor proteins of relatively low natural abundance and uncertain physiology can be expressed selectively in genetically transfected cultured cells to facilitate screening of novel DA receptor ligands. Selective agonists or antagonists for most of the five major DA receptor types are emerging, but better D 4 and D 5 agonists, D 3 and D 5 antagonists, and more selective D 2 antagonists are needed. Clinical development of such compounds as diagnostic neuroradiopharmaceuticals or neuropsychiatric drugs remains empirical and somewhat unpredictable. The search for novel receptor-selective agents can be enhanced by better understanding of the physiology and pharmacology of DA neuroreceptors.

Interactions of fluoxetine with metabolism of dopamine and serotonin in rat brain regions.

Given evidence of inhibitory effects of serotonin on dopaminergic neurotransmission, a series of experiments sought neurochemical evidence of interactions between the selective serotonin transport inhibitor fluoxetine and the metabolism of dopamine (DA) or serotonin (5-HT) in regions of rat brain that might account for extrapyramidal side-effects associated with clinical use of fluoxetine. There were significant inhibitory effects of acute or repeated fluoxetine treatment on the turnover of 5-HT (accumulation of 5-hydroxytryptophan, or ratio of [5-hydroxyindoleacetic acid]/[5-HT]) in striatum, nucleus accumbens and frontal cerebral cortex, but only minor effects on metabolism of DA (accumulation of dihydroxyphenylalanine, or [homovanillic acid]/[DA] ratio), even at high doses or with repeated treatment, and no significant inhibition of the DA metabolism-increasing actions of haloperidol.

Pharmacology of binding of 3H-SCH-23390 to D-1 dopaminergic receptor sites in rat striatal tissue

3H-SCH-23390, a selective antagonist of D-1 dopamine (DA) receptors, was used in a radioreceptor assay with rat brain striatal tissue, optimized biochemically, and extensively characterized pharmacologically with striatal membranes. Nonspecific binding, defined with excess cis(Z)-flupenthixol (300 nM), averaged 20-25% of total counts bound. Specific binding was linearly dependent on the amount of original striatal tissue (0-4 mg) or protein (0-250 micrograms), temperature dependent, saturable and reversible, and appeared to involve a single site at ligand concentrations limited to less than 10 nM. Binding in rat brain regions ranked as: striatum greater than accumbens greater than prefrontal cortex greater than posterior cerebral cortex greater than cerebellum. Association was virtually complete within 30 min at 30 degrees, and the rate of dissociation at 30 degrees was 0.0377 min-1 (half-time = 18.4 min). Affinity (Ka or Kd) determined from association and dissociation rate constants and from concentration isotherms averaged 0.349 and 0.340 nM respectively. Including Na+ at 150 mM increased apparent maximum specific binding (Bmax) by 19%, with a 29% increase in affinity; other monovalent cations alone had small effects on specific binding; Ca2+ and Mg2+ reduced binding by 42%. Agents (N = 85) were tested for potency (Ki or IC50) in competition with the ligand (at 0.30 nM). Those known to have selective effects at D-1 receptors, generally, were most potent and stereoselective. Na+ (150 mM) had little effect on the affinity of cis-thioxanthenes but decreased that of most other agents tested with high D-1 affinity. For antipsychotic agents, the correlation of typical clinical daily doses versus Ki at D-1 sites (r = 0.06) was much lower than at D-2 sites (r = 0.94). (-)Thioridazine was discovered to be D-1 selective, whereas the (+) enantiomer was selective for D-2 sites labeled with 3H-spiperone. Relatively sedating antidepressants had greater D-1 affinity than their less-sedating, secondary amine congeners.

S-(+)-aporphines are not selective for human D3 dopamine receptors

Summary1. Our aim was to test the hypothesis that selectivity for D3 dopamine (DA) receptors may contribute to limbic anti-DA selectivity ofS-(+)-aporphine DA partial agonists.2. Affinity was tested with3H-emonapride, using human D3 receptors in mouse fibroblasts and D2 receptors in rat striatal tissue.3. D3 receptors showed a picomolar affinity for3H-emonapride, Na+ dependence, and reversible saturability, as well as stereoselectivity. Confirmatory or novel D3/D2 pharmacologic selectivity was found with several benzamides, thioxanthenes, buspirone, GBR-12909, and DA agonists including hydroxyaminotetralins [ADTN, (+)-7-OH-DPAT, (−)-PPHT and its fluorescein derivative], (−)-N-propylnorapomorphine, (−)-3-PPP, (−)-quinpirole, and SDZ-205-502, but neither aminoergoline nor (+)-aporphine partial agonists.4. The results extend pharmacologic characterization of D3-transfected cell membranes but fail to account for the high limbic anti-DA selectivity ofS-(+)-aporphines.

[3H]β-CIT: a radioligand for dopamine transporters in rat brain tissue

Abstract [ 3 H]2-β-carbomethoxy-3-β-[4′-iodophenyl]tropane (β-CIT) was prepared and evaluated. With rat forebrain tissue, [ 3 H]β-CIT showed high affinity for dopamine transporters (DAT), with selectivity for DAT over norepinephrine transporters, but not serotonin transporters, as well as DAT-stereoselectivity with β-CIT, amphetamine and methylphenidate. Affinity and selectivity for 53 compounds assayed with [ 3 H]β-CIT and standard DAT radioligand [ 3 H]GBR-12935 were highly correlated ( r >0.95). [ 3 H]β-CIT is proposed as a useful, high-affinity DAT radioprobe.

RBI-257: A highly potent dopamine D4 receptor-selective ligand

RBI-257 (1-[4-iodobenzyl]-4-[N-(3-isopropoxy-2-pyridinyl)-N-methyl]-aminopiperid ine), the p-iodobenzyl analog of U-101,958 (1-benzyl-4-[N-(3-isopropoxy-2-pyridinyl)-N-methyl]-aminopiperidine) had a lower dissociation constant (Ki = 0.3 vs. 2.7 nM) and higher selectivity than U-101,958 at dopamine D4 receptors, over dopamine D2 and D3 receptors in transfected cell membranes and D2-like sites in rat forebrain. Dopamine D4 receptor affinity of iodo-isomers of RBI-257 ranked: para > meta > ortho. RBI-257 had much lower affinity at D1 and D5 dopamine receptors in transfected cells, as well as dopamine D1-like receptors, alpha1, alpha2 or beta(1,2) adrenoceptors, sigma(1,2) receptors and 5-HT1A or 5-HT2A receptors, and transporters for dopamine, norepinephrine or serotonin in rat forebrain tissue. RBI-257 may be a useful probe or radioligand for brain dopamine D4 receptors.

Prolonged antidopaminergic actions of single doses of butyrophenones in the rat

Rats were treated once with doses of haloperidol or of droperidol below and above the acute ID50 vs the dopamine agonist apomorphine; they were later challenged with an acute dose of apomorphine (0.3mg/kg, SC) and rated for stereotyped behavioral responses. The two neuroleptics were similar in acute anti-apomorphine potency (ID50=0.12 and 0.18mg/kg for haloperidol and droperidol, respectively). The antidopaminergic effects of droperidol persisted for nearly 1 week and those of haloperidol lasted for 20–40 days, depending on the dose given. The computed half-time of disappearance of their antidopaminergic effects was 7.6±1.0 days and 0.59±0.17 days for haloperidol and droperidol, respectively, following a dose of 0.3 mg/kg, and these indices of duration of action did not vary significantly at doses between 0.1 and 1.0mg/kg. Haloperidol reduced the acute entry of 3H-apomorphine into brain by 21.5% 1 week later. Treatment with apomorphine alone just prior to haloperidol (both at 0.3 mg/kg) prevented the prolonged antidopaminergic effects of the neuroleptic evaluated 1 week later. These results indicate that some neuroleptics may have very prolonged activity or retention in tissue at sites of action, even after moderate, single doses. Caution is recommended in the interpretation of studies which assume “neuroleptic-free” conditions of subjects previously exposed to a neuroleptic agent.

Isomeric selectivity at dopamine D3 receptors.

Racemic 7-hydroxy-N,N-dipropylaminotetralin (7-OH-DPAT) shows greater affinity for limbic-selective dopamine D3 receptors than for more ubiquitous dopamine D2 receptors. R(+)-7-OH-DPAT was prepared and evaluated in radioreceptor assays using membranes of fibroblasts expressing the human dopamine D3 receptor as well as rat striatal membranes containing dopamine D2 receptors. This enantiomer had 2-fold greater D3 affinity than the racemate and similarly greater D3 vs. D2 selectivity (64-fold). The results may facilitate development of D3 selective agents and evaluation of functions of these receptors.