The effect of interleukin‐8 (IL‐8) and growth‐related oncogene α (GROα) on [ 35 S]‐guanosine 5′‐ O ‐(3‐thiotriphosphate) ([ 35 S]GTPγS) binding, forskolin‐stimulated cyclic AMP accumulation and cytosolic calcium concentration were determined in recombinant CHO cells expressing HA‐tagged CXC‐chemokine receptors 1 and 2 (CXCR1 and CXCR2). Radioligand binding assays confirmed that the binding profiles of the recombinant receptors were similar to those of the native proteins. IL‐8 displaced [ 125 I]‐IL‐8 binding to CXCR1 and CXCR2 with pK i values of 8.89±0.05 and 9.27±0.03, respectively. GROα, a selective CXCR2 ligand, had a pK i value of 9.66±0.39 at CXCR2 but a pK i >8 at CXCR1. Calcium mobilization experiments were also consistent with previous reports on native receptors. Activation of both receptors resulted in stimulation of [ 35 S]GTPγS binding and inhibition of adenylyl cyclase. A comparison of the functional data at CXCR1 showed that a similar potency order (IL‐8>>GROα) was obtained in all three assays. However, at CXCR2 whilst the potency orders for calcium mobilization and inhibition of adenylyl cyclase were similar (IL‐8GROα), the order was reversed for stimulation of [ 35 S]GTPγS binding (GROα>IL‐8). All of the functional responses at both receptors were inhibited by pertussis toxin (PTX), suggesting coupling to a Gi/Go protein. However, the calcium mobilization induced by IL‐8 at CXCR1 was not fully inhibited by PTX, suggesting an interaction with a G‐protein of the Gq family. Our results with pertussis toxin also suggested that, in the [ 35 S]GTPγS binding assay, CXCR1 displays some constitutive activity. Thus, we have characterized the binding and several functional responses at HA‐tagged CXCRs 1 and 2 and have shown that their pharmacology agrees well with that of the native receptors. We also have preliminary evidence that CXCR1 displays constitutive activity in our cell line and that CXCR2 may traffic between different PTX sensitive G‐proteins. British Journal of Pharmacology (1999) 126 , 810–818; doi: 10.1038/sj.bjp.0702329

N-[2-[2,3,7,8-tetrahydro-1H-furo(2,3-g)indol-1-yl]ethyl]acetamide (GR196429) is a novel, nonindolic melatonin receptor agonist. GR196429 had high affinity for human mt1 (pKi 9.9) and MT2 (pKi 9.8) receptors expressed in Chinese hamster ovary cells and for 2-[125I]-iodomelatonin binding sites in human cerebellum, guinea pig superior colliculus and hypothalamus and chicken retina and tectum (pKi 8.8-9.5). GR196429 was inactive at a wide range of other hormone and neurotransmitter receptors. In Chinese hamster ovary cells expressing human mt1 or MT2 receptors, both melatonin and GR196429 dose-dependently inhibited forskolin-stimulated cAMP accumulation. In rabbit isolated retina, GR196429 inhibited calcium-dependent [3H]-dopamine release with potency (IC50 30 pM) and maximum effect (76 +/- 5% at 1 nM) similar to those of melatonin. The response was antagonized by the melatonin receptor antagonist luzindole (1 microM). In slices of rat brain suprachiasmatic nucleus, perfusion (1 h) with GR196429 at zeitgeber time 10 phase advanced the circadian peak in neuronal activity measured on the following day, with a maximum phase advance of 2.7 +/- 0.3 h at 10 pM and an EC50 of 0.6 pM, results that indicated a melatonin-like action on the phase of the circadian clock. CNS penetration and duration of receptor occupancy was determined in an ex vivo radioligand binding assay. In membranes of guinea pig superior colliculus prepared 30 min after administration of GR196429 (s.c.), 2-[125I]-iodomelatonin binding was inhibited with an ED50 of 0.04 mg/kg. After a dose of 1 mg/kg, binding was significantly inhibited for at least 3 h. Thus GR196429 is a potent and selective agonist at high-affinity melatonin receptors, which modulates circadian rhythms in an in vitro model of the circadian clock and which readily penetrates the CNS.