Melatonin (5-methoxy-N-acetyltryptamine), dubbed the hormone of darkness, is released following a circadian rhythm with high levels at night. It provides circadian and seasonal timing cues through activation of G protein-coupled receptors (GPCRs) in target tissues (1). The discovery of selective melatonin receptor ligands and the creation of mice with targeted disruption of melatonin receptor genes are valuable tools to investigate the localization and functional roles of the receptors in native systems. Here we describe the pharmacological characteristics of melatonin receptor ligands and their various efficacies (agonist, antagonist, or inverse agonist), which can vary depending on tissue and cellular milieu. We also review melatonin-mediated responses through activation of melatonin receptors (MT1, MT2, and MT3) highlighting their involvement in modulation of CNS, hypothalamic-hypophyseal-gonadal axis, cardiovascular, and immune functions. For example, activation of the MT1 melatonin receptor inhibits neuronal firing rate in the suprachiasmatic nucleus (SCN) and prolactin secretion from the pars tuberalis and induces vasoconstriction. Activation of the MT2 melatonin receptor phase shifts circadian rhythms generated within the SCN, inhibits dopamine release in the retina, induces vasodilation, enhances splenocyte proliferation and inhibits leukocyte rolling in the microvasculature. Activation of the MT3 melatonin receptor reduces intraocular pressure and inhibits leukotriene B4-induced leukocyte adhesion. We conclude that an accurate characterization of melatonin receptors mediating specific functions in native tissues can only be made using receptor specific ligands, with the understanding that receptor ligands may change efficacy in both native tissues and heterologous expression systems.

The neurotrophic peptide PACAP (pituitary adenylate cyclaseactivating polypeptide) elevates cAMP in PC12 cells. Forskolin and dibutyryl cAMP mimic PACAP's neuritogenic and cell morphological effects, suggesting that they are driven by cAMP. Comparison of microarray expression profiles after exposure of PC12 cells to either forskolin, dibutyryl cAMP, or PACAP revealed a small group of cAMP-dependent target genes. Neuritogenesis induced by all three agents is protein kinase A (PKA)-independent [not blocked by N- [2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89)] and extracellular signal-regulated kinase (ERK)-dependent [blocked by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio) butadiene (U0126)], and therefore cAMP-dependent target genes potentially mediating neuritogenesis were selected for further analysis based on the pharmacological profile of their induction by PACAP (i.e., mimicking that of neuritogenesis). Small interfering RNA (siRNA) targeting one of these genes, Egr1, blocked PACAPinduced neuritogenesis, and siRNA targeting another, Vil2, blocked a component of the cell size increase elicited by PACAP. Neither siRNA blocked PACAP's PKA-dependent antiproliferative effects. PACAP signaling to neuritogenesis was also impaired by dominant-negative Rap1 expression but was not affected by inhibition of protein kinase C (PKC), indicating a G-protein-coupled receptor-mediated differentiation pathway distinct from the one activated by receptor tyrosine kinase ligands such as nerve growth factor (NGF), that involves both Rap1 and PKC. We have thus identified a cAMP-dependent, PKA-independent pathway proceeding through ERK that functions to up-regulate the transcription of two genes, Egr1 and Vil2, required for PACAP-dependent neuritogenesis and increased cell size, respectively. Dominant-negative Rap1 expression impairs both PACAP-induced neuritogenesis and Egr1 activation by PACAP, suggesting that cAMP elevation and ERK activation by PACAP are linked through Rap1.Neurotrophic factors activating receptor tyrosine kinases, such as nerve growth factor (NGF), promote neurite extension through a cAMP-independent signaling pathway involving Ras, PKC, and ERK (Ginty et al., 1991;Vaudry et al., 2002b), although other effects of NGF, such as induction of sodium channel expression, do require cAMP (Ginty et al., 1992;Yao et al., 1998). A significant literature also implicates cAMP in a broad range of neuronal differentiation responses, including neuritogenesis, survival, regeneration, repair, and expression of genes encoding neuron-specific proteins, such as neurotransmitter biosynthetic enzymes, neuropeptides, receptors, and ion channels (Qiu et al., 2002),

The hormone melatonin phase shifts circadian rhythms generated by the mammalian biological clock, the suprachiasmatic nucleus (SCN) of the hypothalamus, through activation of G protein-coupled MT2 melatonin receptors. This study demonstrated that pretreatment with physiological concentrations of melatonin (30-300 pM or 7-70 pg/mL) decreased the number of hMT2 melatonin receptors heterologously expressed in mammalian cells in a time and concentration-dependent manner. Furthermore, hMT2-GFP melatonin receptors heterologously expressed in immortalized SCN2.2 cells or in non-neuronal mammalian cells were internalized upon pretreatment with both physiological (300 pM or 70 pg/mL) and supraphysiological (10 nM or 2.3 ng/mL) concentrations of melatonin. The decrease in MT2 melatonin receptor number induced by melatonin (300 pM for 1 h) was reversible and reached almost full recovery after 8 h; however, after treatment with 10 nM melatonin full recovery was not attained even after 24 h. This recovery process was partially protein synthesis dependent. Furthermore, exposure to physiological concentrations of melatonin (300 pM) for a time mimicking the nocturnal surge (8 h) desensitized functional responses mediated through melatonin activation of endogenous MT2 receptors, i.e., stimulation of protein kinase C (PKC) in immortalized SCN2.2 cells and phase shifts of circadian rhythms of neuronal firing in the rat SCN brain slice. We conclude that in vivo the nightly secretion of melatonin desensitizes endogenous MT2 melatonin receptors in the mammalian SCN thereby providing a temporally integrated profile of sensitivity of the mammalian biological clock to a melatonin signal.

Pituitary adenylate cyclase–activating polypeptide (PACAP) is a neuropeptide that elevates adenosine 3′,5′-monophosphate (cyclic AMP, also abbreviated cAMP) to elicit neuritogenesis in PC12 cells. This effect appears to be independent of cAMP-dependent protein kinase (PKA) yet dependent on cAMP, leading to the conclusion that another cAMP-binding protein and subsequent signaling pathway must exist to mediate this PKA-independent signaling mechanism. Such a protein was identified as exchange protein directly activated by cAMP (EPAC). Although EPAC may play an indirect role in PACAP-mediated neuritogenesis, it does not serve as the only PKA-independent link from cAMP that leads to neuritogenesis. Thus, the challenge remains to construct a signaling network that incorporates the known mediators, working independently of PKA, that are ultimately responsible for PACAP-mediated neuritogenesis.

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The hormone melatonin mediates a variety of physiological functions in mammals through activation of pharmacologically distinct MT(1) and MT(2) G protein-coupled melatonin receptors. We therefore sought to investigate how the receptors were regulated in response to short melatonin exposure. Using 2-[(125)I]iodomelatonin binding, cAMP functional assays, and confocal microscopy, we demonstrated robust differences in specific 2-[(125)I]iodomelatonin binding, receptor desensitization, and cellular trafficking of hMT(1) and hMT(2) melatonin receptors expressed in Chinese hamster ovary (CHO) cells after short (10-min) exposure to melatonin. Exposure to melatonin decreased specific 2-[(125)I]iodomelatonin binding to CHO-MT(2) cells (70.3 +/- 7.6%, n = 3) compared with vehicle controls. The robust decreases in specific binding to the hMT(2) melatonin receptors correlated both with the observed functional desensitization of melatonin to inhibit forskolin-stimulated cAMP formation in CHO-MT(2) cells pretreated with 10 nM melatonin (EC(50) of 159.8 +/- 17.8 nM, n = 3, p < 0.05) versus vehicle (EC(50) of 6.0 +/- 1.2 nM, n = 3), and with the arrestin-dependent internalization of the receptor. In contrast, short exposure of CHO-MT(1) cells to melatonin induced a small decrease in specific 2-[(125)I]iodomelatonin binding (34.2 +/- 13.0%, n = 5) without either desensitization or receptor internalization. We conclude that differential regulation of the hMT(1) and hMT(2) melatonin receptors by the hormone melatonin could underlie temporally regulated signal transduction events mediated by the hormone in vivo.

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Pituitary adenylate cyclase-activating polypeptide (PACAP) is an evolutionarily well conserved neuropeptide with multiple functions in the nervous, endocrine, and immune systems. PACAP provides neuroprotection from ischemia and toxin exposure, is anti-inflammatory in gastric inflammatory disease and sepsis, controls proliferative signaling pathways involved in neural cell transformation, and modulates glucohomeostasis. PACAP-based, disease-targeted therapeutics might thus be both effective and benign, enhancing homeostatic responses to behavioral, metabolic, oncogenic, and inflammatory stressors. PACAP signal transduction employs synergistic regulation of calcium and cyclic adenosine monophosphate (cAMP), and noncanonical activation of both calcium- and cAMP-dependent processes. Pharmacological activation of PACAP signaling should consequently have highly specific effects even in vivo. Here, a combined cellular biochemical, pharmacologic, transcriptomic, and bioinformatic approach to understanding PACAP signal transduction by identifying PACAP target genes with oligonucleotide- and cDNA-based microarray is described. Calcium- and cAMP-dependent PACAP signaling pathways for regulation of genes encoding proteins required for neuritogenesis, changes in cell morphology, and cell survival have been traced in PC12 cells. Pharmacological experiments have linked gene expression to cell physiological responses in this system, in which gene silencing can also be employed to confirm the functional significance of induction of specific transcripts. Differential transcriptional responses to metabolic, ischemic, and other stressors in wild type compared to PACAP-deficient mice establish in principle which PACAP-responsive transcripts in culture are PACAP-dependent in vivo. Bioinformatic approaches aid in creating a pipeline for identifying neuropeptide-regulated genes, validating their cellular functions, and defining their expression in the context of neuropeptide signaling physiology, required for discovery of new targets for drug action.