1 Dopamine D-4 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 D-4.4 receptor (HEK293/D-4 cells). [H-3]-spiperone binding experiments were performed and inhibition of forskolin-stimulated cyclic AMP accumulation was used as the functional response. 3 [H-3]-spiperone was found to label a homogeneous and saturable population of specific binding sites in HEK293/D-4 cell homogenates (B-max 505 +/- 90 fmol mg(-1) protein, pK(D) 9.5 +/- 0.1, n=3). Inhibition of specific [H-3]-spiperone binding was observed with spiperone (pK(i) 9.6 +/- 0.1, n=3), clozapine (pK(i) 7.4 +/- 0.1, n=4), L-745,870 (pK(i) 8.5 +/- 0.1, n=3) and U-101958 (pK(i) 8.9 +/- 0.1, n=3). By contrast, raclopride was very weak (pK(i) < 5, n = 3). 4 Dopamine inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D-4 cells in a concentration-dependent fashion (E-max 71 +/- 2% inhibition of forskolin-stimulated levels, pEC(50) 8.7 +/- 0.1, n=10). This effect was mimicked by the dopamine D-2-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/D-4 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 (pEC(50)) values of L-745,870 and U-101958 were 9.0 +/- 0.2 (n = 4) and 8.7 +/- 0.3 (n = 3), consistent with pK(i) values determined in radioligand binding studies. 6 Dopamine, L-745,870 and U-101958 (up to 1 mu M) 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/D-4 cells could be antagonized by spiperone (pK(B) 8.2-8.8) and clozapine (pKB 7.1), but not by raclopride (pK(B)<5). None of these antagonists had any significant agonist activity at concentrations up to 10 mu M. 8 These results show that the putative dopamine D-4 receptor antagonists, L-745,870 and U-101958 are not devoid of intrinsic activity at human recombinant dopamine D-4.4 receptors. Therefore, they may not represent the most appropriate drugs for testing the benefit of D-4 receptor antagonism in schizophrenic patients, if agonism should translate in vivo.
