Agonist activity at G protein-coupled receptors (GPCRs) that regulate heterotrimeric G proteins of the Galpha(i/o) or Galpha(q) families has been shown to result in activation of the mitogen-activated protein (MAP) kinase cascade. To facilitate compound screening for these classes of GPCR, we have developed a reporter gene that detects the activation of the ternary complex transcription factor Sap1a following MAP kinase activation. In contrast to other reporter gene assays for Galpha(i/o)-coupled GPCRs, the MAP kinase reporter generates an increase in signal in the presence of agonist. The reporter gene has been transfected into Chinese hamster ovary cells to generate a "host" reporter gene-containing cell line. The Galpha(i)-coupled human CXCR1 chemokine receptor was subsequently transfected into this cell line in order to develop a 384-well format screen for both agonists and antagonists of this receptor. Agonists activated the reporter gene with the expected rank order of potency and with similar concentration dependence as seen with the regulation of other signal transduction cascades in mammalian cells: interleukin-8 (IL-8) (pEC(50) = 7.0 +/- 0.1) > GCP-2 (pEC(50) = 6.3 +/- 0.1) > NAP-2 (pEC(50) < 6). CXCR1-mediated activation of MAP kinase was inhibited by pertussis toxin and the MEK inhibitor PD98059, demonstrating that receptor activation of MAP kinase is due to pertussis toxin-sensitive Galpha(i/o)-family G proteins to cause the activation of MEK kinase. Using the 384-well format, assay performance was unaffected by solvent concentrations of 0.5% ethanol, 0.15% glycerol, or 1% DMSO. Signal crosstalk between adjacent wells was less than 1%. The assay exhibited a Z factor of 0.53 and a coefficient of variation of response to repeated application of IL-8 (100 nM) of 15.9%.

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

In this study we have examined the ability of melatonin and four synthetic melatonin receptor agonists to entrain endogenous melatonin secretion in rats, free running in constant darkness. The circadian melatonin profile was measured by trans-pineal microdialysis, which not only reveals the time of onset and end of production (phase), but also the amplitude of the rhythm. Exogenous melatonin given at the onset of subjective darkness (clock time 12 h) was effective to entrain endogenous melatonin production. Only one agonist, 2-chloroacetamido-8-methoxytetralin (AH-017), mimicked this action. Two other agonists, 4-methoxy-2-(methylene propylamide)indan (GG-012) and N-[2-[2,3,7,8-tetrahydro-1H-furo(2,3-g)indol-1-yl]ethyl]acetamide (GR196429), induced a phase-delay under free running conditions, possibly by increasing tau (τ) period. One agonist, 2-acetamido-8-methoxytetralin (AH-001) did not show any phase effect on the free running rhythm. Unexpectedly, all melatonin receptor agonists increased the amplitude of melatonin secretion. The amount of the increase varied from just below the level of significance (AH-001) to an approximately 2-fold increase (GG-012 and GR196429). This is in clear contrast to entrainment with melatonin, which significantly decreased the amplitude. It is hypothesized that entrainment and effects on amplitude of melatonin secretion are mediated by different mechanisms which can be differentially modulated using specific ligands.

The activation of G-proteins by melatonin mt(1) receptors was studied by measuring [S-35]-guanosine-5'-(3-thiotriphosphate) ([S-35]-GTP gamma S) binding to membranes prepared from Chinese hamster ovary (CHO) cells stably expressing human mt(1) receptors. Melatonin stimulated [S-35]-GTP gamma S binding in a concentration-dependent manner (pEC(50), 8.77 +/- 0.02). The optimal (212 +/- 4%) increase over basal levels of binding (basal = 100%) was observed following incubation of membranes (12.5 mu g protein/well) for 120 min at 30 degrees with [S-35]-GTP gamma S (0.1 nM), in the presence of GDP (10 mu M), NaCl (100 mM), and MgCl2 (10 mM). Melatonin analogues stimulated [S-35]-GTP gamma S binding with a rank order (2-iodomelatonin > melatonin = S20098 > GR196429 > 6-chloromelatonin = 6-hydroxymelatonin much greater than N-acetylserotonin greater than or equal to GR135531 = mt(1) luzindole = 5-HT = 0), which was identical to their affinities for the high affinity state of the receptor (correlation coefficient 0.94). All agonists evoked similar maximum increases in [S-35]-GTP gamma S binding. EC50 values were 14- to 63-fold lower than binding affinities. The melatonin receptor antagonist luzindole (0.1-10 mu M) evoked a parallel rightward shift in the melatonin concentration-response curve, with a pK(B) Of 7.19 +/- 0.13, which is similar to its affinity in radioligand binding studies for human mt(1) receptors. Stimulation of [S-35]-GTP gamma S binding was abolished by pretreatment of cells with pertussis toxin (18 hr, 100 ng/mL) prior to preparation of membranes. Melatonin was without effect in CHO cells which lacked the mt(1) receptor. Thus, melatonin and melatonin analogues stimulate [S-35]-GTP gamma S binding with a profile which is consistent with binding to mt(1) receptors causing activation of G(i)/G(o) G-proteins. (C) 1998 Elsevier Science Inc.

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.