Receptor density as a factor governing the efficacy of the dopamine D4 receptor ligands, L-745,870 and U-101958 at human recombinant D4.4 receptors expressed in CHO cells.

1. The relationships between the density of dopamine D4.4 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-a min e) were investigated in Chinese hamster ovary (CHO) cells, after treatment with the gene expression enhancer, sodium butyrate. 2. In CHO cells expressing D4.4 receptors (CHO/D4 cells), dopamine inhibited forskolin-stimulated cyclic AMP accumulation (Emax 56+/-1% inhibition, pEC50 7.4+/-0.1, n=10). U-101958 behaved as a partial agonist (39+/-7% the efficacy of dopamine, pEC50 8.1+/-0.3, n=4), whereas L-745,870 had no detectable agonist effect. 3. Receptor density, as estimated by [3H]-spiperone saturation binding was 240+/-30 fmol mg-1 protein (n=8) in CHO/D4 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 mM) for 6, 18 and 48 h, respectively. 4. The increase in receptor density was associated with a gradual enhancement of the agonist effects (increased Emax and pEC50 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, pEC50 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 microM) but not by raclopride (10 microM). 5. The results show that U-101958 and L-745,870 are partial agonists at human dopamine D4.4 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.

The agonist activities of the putative antipsychotic agents, L-745,870 and U-101958 in HEK293 cells expressing the human dopamine D4.4 receptor.

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.

Pharmacological characterisation of human cerebral cortex somatostatin SRIF1 and SRIF2 receptors.

Radioligand binding studies were performed in membranes of human cerebral cortex using [125I]Tyr3-octreotide in the presence of 5 mM MgCl2, [125I]SRIF-14 ([125I]Tyr11-SRIF-14) and [125I]CGP 23996 ([125I]c[Asu- Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) both in the presence of 120 mM NaCl, to characterise the nature of the somatostatin (SRIF) receptors. The pharmacological profile of human brain SRIF recognition sites was compared with that of recombinant human SRIF1 (sst2-sst3-sst5) or SRIF2 receptors (sst1-sst4) and with that of native rat sst1, sst2, and sst4 receptors. [125I]Tyr3-octreotide labelled binding sites in human cerebral cortex: Bmax = 238 +/- 36 fmol/mg protein and pKd = 9.73 +/- 0.08. The pharmacological profile of [125I]Tyr3-octreotide labelled sites correlated very significantly with that of recombinant human sst2 receptors (r = 0.98) and much less with those of recombinant human sst3 (r = 0.65) or sst5 receptors (r = 0.72). The correlation between [125I]Tyr3-octreotide binding to native sst2 receptors in human and rat cerebral cortex was also highly significant (r = 0.97). [125I]SRIF-14 and [125I]CGP 23996 binding (performed in the presence of 120 mM NaCl) in the human cerebral cortex identified very similar populations of sites Bmax = 44 +/- 7 and 36 +/- 5 fmol/mg protein and pKd = 9.44 +/- 0.08 and 9.48 +/- 0.10, respectively. The pharmacological profiles of the sites labelled with [125I]SRIF-14 and [125I]CGP 23996 correlated highly significantly with those of recombinant human sst1 (r = 0.97-0.99) or sst4 receptors (r = 0.91-0.94). Similarly, the correlations between [125I]SRIF-14 or [125I]CGP 23996 binding in human cortex and [125I]SRIF-14 binding to native sst1 sites in rat cerebral cortex were also highly significant (r = 0.97 and 0.94, respectively). Finally, the pharmacological profile of native rat lung sst4 sites determined with [125I]LTT-SRIF-28 ([Leu8,D-Trp22, 125I-Tyr25]SRIF-28) correlated with [125I]SRIF-14 and [125I]CGP 23996 binding in human cortex; r = 0.91 and 0.87, respectively. The present data show that in human cerebral cortex, [125I]Tyr3-octreotide labels SRIF1 receptor sites which are best characterised as of the sst2 type, whereas [125I]SRIF-14 and [125I]CGP 23996 (both in the presence of 120 mM NaCl), label sites which fit almost equally well with sst1 or sst4 receptors and therefore are best described as of the SRIF2 type. Under the conditions used, there was no evidence that either of these ligands would label sst3 or sst5 receptors in human cerebral cortex.

Functional effects of D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Trp-NH2 and differential changes in somatostatin receptor messenger RNAs, binding sites and somatostatin release in kainic acid-treated rats.

In situ hybridization histochemistry for somatostatin receptors-1, -2, -3 and -4 section and receptor autoradiography using [125I]CGP 23996, [125I]somatostatin-28, [125I]seglitide and [125I]Tyr3 octreotide were carried out to determine the expression of somatostatin receptor messenger RNAs and binding sites in the hippocampus and cerebral cortex of rats 21 days following generalized limbic seizures induced by subcutaneous injection of 12mg/kg kainic acid. In control rats, somatostatin-1 to somatostatin-4 receptor messenger RNAs were found in the pyramidal layer and granule cell layer of the dentate gyrus. After kainate treatment, the CA1 subfield displayed a selective decrease in somatostatin-3 and somatostatin-4 receptor hybridization signals of 35 and 41%, respectively, whereas no changes were observed in the remaining hippocampal areas. Somatostatin-1 and somatostatin-2 receptor messenger RNA expression in the hippocampus remained unaffected by kainate treatment. No effect of kainate was observed in the expression of somatostatin receptor messenger RNAs in the cerebral cortex. In control rats, the selective somatostatin-2 receptor ligands, [125I]seglitide and [125I]Tyr3 octreotide and the non-selective somatostatin receptor ligands [125I]CGP 23996 and [125I]somatostatin-28, labelled preferentially the stratum oriens and radiatum CA1, the granule and molecular layers of the dentate gyrus and the deep layers of the cerebral cortex. [125I]somatostatin-28 and [125I]CGP 23996 labelled sites were selectively decreased by 32 and 39%, respectively, in the stratum radiatum CA1 after kainate treatment. [125I]CGP 23996 binding was also decreased by 35% in the stratum oriens CA1 and by 36% on average in the stratum oriens and radiatum CA3. [125I]seglitide and [125I]Tyr3 octreotide binding was not affected by kainate in any hippocampal region. The granule and molecular layers of the hippocampus and the layers IV-VI of the cerebral cortex did not show changes in binding sites for any of the radioligands analysed. A 18 and 35% decrease in the spontaneous and 50 mM KCl-induced somatostatin release from hippocampal slices was found two days after kainate, a likely reflection of neuronal cell loss. No differences in somatostatin release were observed 21 days after kainate treatment. At this latter time, the rats had an enhanced susceptibility to tonic-clonic seizures induced by intraperitoneal injection of 30 mg/kg pentylenetetrazol, a subconvulsant dose in naive rats. Bilateral infusion of 6 micrograms RC 160, a selective somatostatin-2 receptor agonist, in the dentate gyrus 21 days after kainate, significantly reduced (P < 0.05) the number of animals with tonic-clonic seizures induced by pentylenetetrazol.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacological identity between somatostatin SS-2 binding sites and SSTR-1 receptors.

Somatostatin (SRIF) SS-2 binding sites were originally defined in rat brain cerebral cortex membranes using [125I]Tyr11-SRIF-14 in the presence of 120 mM NaCl. These sites were characterized by their high affinity for SRIF-14 and SRIF-28, but very low affinity for cyclic peptides such as octreotide (SMS 201-995) and seglitide (MK 678). The characteristics of SS-2 sites are reminiscent of 125I]CGP 23996-labelled sites in rat brain which have been termed SRIF-2 sites. In the present study, the pharmacological profile of SS-2 sites was determined in radioligand binding studies performed in rat cortex membranes using [125I]SRIF-14 in the presence of 120 mM NaCl and compared to that of human SSTR-1 receptors expressed in human embryonic kidney (HEK 293) cells, using [125I]SRIF-14. The rank orders of affinity of a variety of SRIF analogues and synthetic peptides for SS-2 binding sites and recombinant human SSTR-1 receptors were very similar and correlated highly significantly (r = 0.99). However, SS-2 binding correlated also with binding to recombinant SSTR-4 receptors (r = 0.91). Autoradiographic studies were performed using the radioligand [125I]CGP 23996 which has been claimed to label selectively SRIF-2 binding sites and compared with the distribution of SSTR-1 receptor mRNA determined using in situ hybridization in rat brain. Although some overlap was observed between the distribution of SSTR-1 mRNA and [125I]CGP 23996 binding sites, the latter were clearly more widespread, suggesting this ligand to label SSTR-1 and other sites. In addition, inhibition of forskolin-stimulated adenylate cyclase was investigated in HEK 293 cells transfected with human SSTR-1 receptors; a variety of SRIF analogues and short synthetic peptides behaved as agonists at adenylate cyclase and displayed a rank order of potency highly similar to that observed for these compounds at SS-2 binding sites. Seglitide acted as an antagonist at SSTR-1 receptor mediated inhibition of adenylate cyclase activity with a pKB of 4.42. It is concluded that the pharmacological profile of SS-2 binding sites resembles most closely that of SSTR-1 receptors (although similarities with SSTR-4 receptors were observed), that [125I]CGP 23996 labels presumably several SRIF receptors in rat brain, and that SSTR-1 receptors are negatively and efficiently coupled to adenylate cyclase activity.

Characterization and distribution of somatostatin SS-1 and SRIF-1 binding sites in rat brain: identity with SSTR-2 receptors.

Somatostatin (SRIF) SS-1 binding sites were initially defined in radioligand binding studies performed in rat brain cerebral cortex membranes using [125I]204-090 (a radiolabelled Tyr3 analogue of SMS 201-995, octreotide). SRIF-1 recognition sites were defined in binding studies performed with [125I]MK 678 (seglitide). Both SS-1 and SRIF-1 sites were characterized by their high affinity for SRIF-14, SRIF-28 and for cyclic peptides such as octreotide and seglitide, in marked contrast to SS-2 and SRIF-2 sites which have very low affinity for these synthetic SRIF analogues. In the present study, SS-1 and SRIF-1 radioligand binding studies were performed in rat cortex membranes and compared to results obtained in cloned Chinese hamster ovary cells expressing human SSTR-2 receptors using [125I]204-090 and/or [125I]MK-678. The rank orders of affinity of a variety of SRIF analogues and synthetic peptides for SS-1/SRIF-1 binding sites and recombinant SSTR-2 receptors were very similar and correlated highly significantly (r = 0.94-0.99); by contrast, correlation between SS-1 and SSTR-5 (r = 0.44) or SSTR-3 binding (r = 0.07) was not significant. Autoradiographic studies were performed in rat brain using both radioligands [125I]204-090 and [125I]MK-678 and compared with the distribution of SSTR-2 receptor mRNA determined using in situ hybridization. A clear overlap was observed between the distribution of SSTR-2 mRNA and binding sites labelled with both radioligands. SSTR-2 receptor-mediated inhibition of forskolin-stimulated adenylate cyclase in Chinese hamster ovary cells by a variety of SRIF analogues and short synthetic peptides displayed a rank order of potency highly similar to their rank order of affinity at SS-1/SRIF-1 binding sites. It is concluded that SS-1 and SRIF-1 binding sites respectively labelled with [125I]204-090 and [125I]MK 678, both display the pharmacological profile of SSTR-2 receptors, that the distribution of [125I]204-090 and [125I]MK-678 binding sites in rat brain is superimposable and largely comparable to that of SSTR-2 mRNA expression. It is also shown that neither [125I]204-090 nor [125I]MK-678 label SSTR-3 or SSTR-5 receptors in rat brain. Finally, it is demonstrated that SSTR-2 receptors can very efficiently couple to adenylate cyclase activity in an inhibitory manner.