The relationships between the density of dopamine D 4.4 receptors and the agonist efficacies of L‐745,870 (3‐(4‐[4‐chlorophhenyl]piperazin‐1‐yl)‐methyl‐1H‐pyrrolo [2,3‐b]pyridine) and U‐101958 ((1‐benzyl‐piperidin‐4‐yl)‐(3‐isopropoxy‐pyridin‐2‐yl)‐methyl‐amine) were investigated in Chinese hamster ovary (CHO) cells, after treatment with the gene expression enhancer, sodium butyrate. In CHO cells expressing D 4.4 receptors (CHO/D 4 cells), dopamine inhibited forskolin‐stimulated cyclic AMP accumulation (E max 56±1% inhibition, pEC 50 7.4±0.1, n =10). U‐101958 behaved as a partial agonist (39±7% the efficacy of dopamine, pEC 50 8.1±0.3, n =4), whereas L‐745,870 had no detectable agonist effect. Receptor density, as estimated by [ 3 H]‐spiperone saturation binding was 240±30 fmol mg −1 protein ( n =8) in CHO/D 4 cell homogenates. It reached 560±150 ( n =6), 1000±190 ( n =4) and 840±120 ( n =4) fmol mg −1 protein after treatment with sodium butyrate (5 m M ) for 6, 18 and 48 h, respectively. The increase in receptor density was associated with a gradual enhancement of the agonist effects (increased E max and pEC 50 values) of dopamine. The efficacy of U‐101958 (relative to dopamine) doubled and L‐745,870 was turned into a partial agonist (efficacy 49% relative to dopamine, pEC 50 8.6±0.2, n =6, after 48 h treatment with sodium butyrate). These agonist effects of U‐101958 and L‐745,870 could be antagonized by spiperone (0.1 μ M ) but not by raclopride (10 μ M ). The results show that U‐101958 and L‐745,870 are partial agonists at human dopamine D 4.4 receptors expressed in CHO cells. Their efficacy is governed by receptor density. Agonist effects of these two compounds in vivo cannot be excluded under circumstances of increased receptor levels. British Journal of Pharmacology (1999) 128 , 613–620; doi: 10.1038/sj.bjp.0702849
1. Dopamine D4 receptor antagonists are being developed by several pharmaceutical companies as putative novel antipsychotics, possibly with low propensity to side-effects. Two such compounds, L-745,870 and U-101958 have been recently introduced. 2. The radioligand binding and functional activities of L-745,870 and U-101958 were investigated in human embryonic kidney (HEK)293 cells expressing the human recombinant dopamine D4.4 receptor (HEK293/D4 cells). [3H]-spiperone binding experiments were performed and inhibition of forskolin-stimulated cyclic AMP accumulation was used as the functional response. 3. [3H]-spiperone was found to label a homogeneous and saturable population of specific binding sites in HEK293/D4 cell homogenates (Bmax 505+/-90 fmol mg(-1) protein, pK(D) 9.5+/-0.1, n=3). Inhibition of specific [3H]-spiperone binding was observed with spiperone (pKi 9.6+/-0.1, n=3), clozapine (pKi 7.4+/-0.1, n=4), L-745,870 (pKi 8.5+/-0.1, n=3) and U-101958 (pKi 8.9+/-0.1, n=3). By contrast, raclopride was very weak (pKi < 5, n=3). 4. Dopamine inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D4 cells in a concentration-dependent fashion (Emax 71+/-2% inhibition of forskolin-stimulated levels, pEC50 8.7+/-0.1, n=10). This effect was mimicked by the dopamine D2-like receptor agonists, quinpirole and 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT). 5. L-745,870 and U-101958 also inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D4 cells in a concentration-dependent way. L-745,870 was less efficacious than dopamine (71% the efficacy of dopamine), whereas U-101958 behaved as a full agonist compared to dopamine. Potencies (pEC50) values of L-745,870 and U-101958 were 9.0+/-0.2 (n=4) and 8.7+/-0.3 (n=3), consistent with pKi values determined in radioligand binding studies. 6. Dopamine, L-745,870 and U-101958 (up to 1 microM) were devoid of effect on forskolin-stimulated cyclic AMP accumulation in control, non-transfected HEK293 cells. 7. The agonist effects of dopamine, L-745,870 and U-101958 in HEK293/D4 cells could be antagonized by spiperone (pK(B) 8.2-8.8) and clozapine (pK(B) 7.1), but not by raclopride (pK(B) < 5). None of these antagonists had any significant agonist activity at concentrations up to 10 microM. 8. These results show that the putative dopamine D4 receptor antagonists, L-745,870 and U-101958 are not devoid of intrinsic activity at human recombinant dopamine D4.4 receptors. Therefore, they may not represent the most appropriate drugs for testing the benefit of D4 receptor antagonism in schizophrenic patients, if agonism should translate in vivo.
