Monica I. Masana

Molecular pharmacology, regulation and function of mammalian melatonin receptors.

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.

Selective MT2 melatonin receptor antagonists block melatonin-mediated phase advances of circadian rhythms

This study demonstrates the involvement of the MT2 (Mel1b) melatonin receptor in mediating phase advances of circadian activity rhythms by melatonin. In situ hybridization histochemistry with digoxigenin‐labeled oligonucleotide probes revealed for the first time the expression of mt1 and MT2 melatonin receptor mRNA within the suprachiasmatic nucleus of the C3H/HeN mouse. Melatonin (0.9 to 30 μg/mouse, s.c.) administration during 3 days at the end of the subjective day (CT 10) to C3H/HeN mice kept in constant dark phase advanced circadian rhythms of wheel running activity in a dose‐dependent manner [EC50 = 0.72 μg/mouse; 0.98 ± 0.08 h (n = 15) maximal advance at 9 μg/mouse]. Neither the selective MT2 melatonin receptor antagonists 4P‐ADOT and 4P‐PDOT (90 μ/mouse, s.c.) nor luzindole (300 μg/mouse, s.c.), which shows 25‐fold higher affinity for the MT2 than the mt1 subtype, affected the phase of circadian activity rhythms when given alone at CT 10. All three antagonists, however, shifted to the right the dose‐response curve to melatonin, as they significantly reduced the phase shifting effects of 0.9 and 3 mg melatonin. This is the first study to demonstrate that melatonin phase advances circadian rhythms by activation of a membrane‐bound melatonin receptor and strongly suggests that this effect is mediated through the MT2 melatonin receptor subtype within the circadian timing system. We conclude that the MT2 melatonin receptor subtype is a novel therapeutic target for the development of subtype‐selective analogs for the treatment of circadian sleep and mood‐related disorders.—Dubocovich, M. L., Yun, K., Al‐Ghoul, W. M., Benloucif, S., Masana, M. I. Selective MT2 melatonin receptor antagonists block melatonin‐mediated phase advances of circadian rhythms. FASEB J. 12, 1211–1220 (1998)

Melatonin receptor antagonists that differentiate between the human Mel1a and Mel1b recombinant subtypes are used to assess the pharmacological profile of the rabbit retina ML1 presynaptic heteroreceptor

Abstract We have identified subtype selective agonists, partial agonists and antagonists, which distinguish the human recombinant Mel1a and Mel1b melatonin receptors expressed in COS-7 cells. Melatonin receptor agonists showed higher affinity for competition of 2-[125I]-iodomelatonin binding for the Mel1b than the Mel1a melatonin receptor. The dissociation constants (Ki) of 16 agonists determined on the recombinant human Mel1a and Mel1b melatonin receptor subtypes showed a significant correlation (r2 = 0.85, slope = 0.97, P < 0.0001, n = 16). However, six agonists showed 10 to 60 fold higher affinity for the Mel1b melatonin receptor as indicated by the affinity selectivity ratios (Mel1a/Mel1b) [8-methoxy-2-acetamidotetraline (11); S20098 (14); 8-methoxy-2-propionamidotetraline (20); 6, 7 di-chloro-2-methylmelatonin (21); 6-chloromelatonin (57); 6-methoxymelatonin (59)]. Dissociation constants for competition of 11 partial agonists and antagonist for 2-[125I]-iodomelatonin binding were between 15.5 (luzindole, pKi: 7.7) to 362 (4-phenyl-2-chloroacetamidotetraline, pKi: 9.1) fold higher for the Mel1b than for the Mel1a melatonin receptor. The lack of correlation between the pKi values (r2 = 0.23, P > 0.1, n = 11) strongly suggest that the two human melatonin receptor subtypes can be distinguished pharmacologically. The partial agonist: 5-methoxyluzindole (pKi: 9.6) and the competitive melatonin receptor antagonists: GR128107 (pKi: 9.6), 4-phenyl-2-chloroacetamidotetraline (pKi: 9.1), 4-phenyl-2-acetamidotetraline (pKi: 8.9) and 4-phenyl-2-propionamidotetraline (pKi: 8.8) are selective Mel1b melatonin receptor analogues as their affinity selectivity ratios (Mel1a/Mel1b) are bigger than 100. We conclude that the 40% overall amino acid difference in the sequence of the human recombinant Mel1a and Mel1b melatonin receptors is reflected in distinct pharmacological profiles for the subtypes.We compared the pharmacological profile of the presynaptic ML1 melatonin heteroreceptor of rabbit retina mediating inhibition of the calcium-dependent release of dopamine to that of the recombinant Mel1a and Mel1b melatonin receptors. Melatonin inhibited [3H]dopamine release by 50% (IC50) at 20 pM with a maximal inhibitory effect (80%) at 1 nM. The partial agonists, i.e., N-acetyltryptamine (IC50: 5.6, maximal inhibition 55%) and 5-methoxyluzindole (IC50: 1.3, maximal inhibition 40%) showed various degrees of efficacy while none of the competitive melatonin receptor antagonists did inhibit [3H]dopamine release on their own. The potency (IC50) of full melatonin receptor agonists significantly correlated with their affinity to compete for 2-[125I]-iodomelatonin binding to either the Mel1a (r2 = 0.76, slope = 0.77, P<0.0001, n = 17) or Mel1b (r2 = 0.63, slope = 0.75, P<0.001, n = 17) human melatonin receptors. By contrast, the apparent dissociation constants (KB) for partial agonists and antagonists to antagonize the inhibition of [3H]dopamine release mediated by activation of the ML1 heteroreceptor by melatonin, significantly correlated with the affinity constants (Ki) for 2-[125I]-iodomelatonin binding determined on the Mel1b (r2 = 0.77, slope = 0.55, P<0.001; n = 11) but not the Mel1a (r2 = 0.27, P<0.1, n = 11) subtype. Together these results demonstrate that the pharmacological profile of the human recombinant Mel1b melatonin receptor is similar to that of the functional presynaptic melatonin heteroreceptor of rabbit retina, which we referred as an ML1B subtype. We conclude that the selective Mel1b melatonin partial agonists and antagonists described here can be used to identify melatonin receptor subtypes in native tissues and to search for subtype selective analogues with therapeutic potential.

Melatonin desensitizes endogenous MT2 melatonin receptors in the rat suprachiasmatic nucleus: relevance for defining the periods of sensitivity of the mammalian circadian clock to melatonin

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.—Gerdin, M. J., Masana, M. I., Rivera‐Bermúdez, M. A., Hudson, R. L., Earnest, D. J., Gillette, M. Ú., Dubocovich, M. L. Melatonin desensitizes endogenous MT2 melatonin receptors in the rat suprachiasmatic nucleus: relevance for defining the periods of sensitivity of the mammalian circadian clock to melatonin. FASEB J. 18, 1646–1656 (2004)

Circadian rhythm of mt1 melatonin receptor expression in the suprachiasmatic nucleus of the C3H/HeN mouse

This report studied the diurnal and circadian rhythms of mt1 melatonin receptor expression in the SCN of C3H/HeN mice maintained in either a light:dark (LD) cycle or in constant dark for a minimum of 6 wk. Diurnal times (ZT) were assessed with reference to the onset of the light period (ZT0) and circadian times (CT) were established by determining the phase of wheel running activity of each mouse before sacrifice. 2‐[125I]‐Iodomelatonin binding in the SCN revealed low amplitude diurnal and circadian rhythms with highest levels of binding 2 hr after lights on (41.3±1.7 fmol/mg protein, n=5, at ZT2) or at the beginning of the subjective day (48.6±2.1 fmol/mg protein, n=6, CT2), respectively. The expression of mt1 mRNA, determined by in situ hybridization with a 35 S‐labeled mouse mt1 riboprobe, showed robust diurnal and circadian rhythms. In animals housed under a LD cycle, low levels of expression were observed during the day, with a rapid rise in mt1 melatonin receptor expression at the beginning of the dark period (ZT14), coincident with an abrupt increase in levels of circulating melatonin measured by radioimmunoassay. In animals housed under constant dark conditions, a robust peak of mt1 mRNA expression occurred in the middle of the subjective night (CT18), 8 hr before the peak of protein expression, while the lowest levels of mt1 mRNA expression were observed during the day (CT10). Results suggest that mt1 melatonin receptor rhythm in the C3H/HeN mouse SCN is regulated both by light and by the biological clock as distinct rhythms of both mRNA and protein are differentially expressed under a LD cycle and constant dark conditions.

Effect of MT1 melatonin receptor deletion on melatonin‐mediated phase shift of circadian rhythms in the C57BL/6 mouse

Abstract:  In the mouse suprachiasmatic nucleus (SCN), melatonin activates MT1 and MT2 G‐protein coupled receptors, which are involved primarily in inhibition of neuronal firing and phase shift of circadian rhythms. This study investigated the ability of melatonin to phase shift circadian rhythms in wild type (WT) and MT1 melatonin receptor knockout (KO) C57BL/6 mice. In WT mice, melatonin (90 μg/mouse, s.c.) administered at circadian time 10 (CT10; CT12 onset of activity) significantly phase advanced the onset of the circadian activity rhythm (0.60 ± 0.09 hr, n = 41) when compared with vehicle treated controls (−0.02 ± 0.07 hr, n = 28) (P < 0.001). In contrast, C57 MT1KO mice treated with melatonin did not phase shift circadian activity rhythms (−0.10 ± 0.12 hr, n = 42) when compared with vehicle treated mice (−0.12 ± 0.07 hr, n = 43). Similarly, in the C57 MT1KO mouse melatonin did not accelerate re‐entrainment to a new dark onset after an abrupt advance of the dark cycle. In contrast, melatonin (3 and 10 pm) significantly phase advanced circadian rhythm of neuronal firing in SCN brain slices independent of genotype with an identical maximal shift at 10 pm (C57 WT: 3.61 ± 0.38 hr, n = 3; C57 MT1KO: 3.45 ± 0.11 hr, n = 4). Taken together, these results suggest that melatonin‐mediated phase advances of circadian rhythms of neuronal firing in the SCN in vitro may involve activation of the MT2 receptor while in vivo activation of the MT1 and possibly the MT2 receptor may be necessary for the expression of melatonin‐mediated phase shifts of overt circadian activity rhythms.

Melatonin receptors in the mammalian suprachiasmatic nucleus

The SCN of the hypothalamus, the site of the circadian pacemaker in mammals, is endowed with melatonin receptors of the ML-1 subtype. Here, we present evidence suggesting that activation of melatonin receptors in the SCN regulates circadian rhythms of behavior in the mouse. In a paradigm simulating a eastbound transmeridian flight, timed administration of melatonin may either accelerate or decrease the rate of reentrainment. Moreover, under constant environmental conditions, exogenous melatonin phase shifts circadian rhythms only during times when the production of the hormone is inhibited. Similarly, light shows periods of circadian sensitivity only at times when light is not present in a natural photoperiod. The maximal phase shifts elicited by melatonin and light coincide with the subjective light-dark (dusk) and subjective dark-light (dawn) transitions. The periods of sensitivity for melatonin, occur at the same circadian times in mouse and in man. Under a short photoperiod the duration of the nocturnal melatonin production may overlap with periods of sensitivity for the hormone, and therefore, melatonin, may be important in synchronizing circadian rhythms to changes in the natural photoperiod. It follows that the identification of periods of circadian sensitivity to melatonin in mammals is important for the development of effective treatments with melatonin and related analogues for sleep disorders characterized by alterations of circadian rhythmicity.

In vivo evidence that lithium inactivates Gi modulation of adenylate cyclase in brain.

In vivo microdialysis of cyclic AMP from prefron‐tal cortex complemented by ex vivo measures was used to investigate the possibility that lithium produces functional changes in G proteins that could account for its effects on adenylate cyclase activity. Four weeks of lithium administration (serum lithium concentration of 0.85 ±0.05 mM; n= 11) significantly increased the basal cyclic AMP content in dialysate from prefrontal cortex of anesthetized rats. Forskolin infused through the probe increased dialysate cyclic AMP, but the magnitude of this increase was unaffected by chronic lithium administration. Inactivation of the inhibitory guanine nucleotide binding protein Gi with pertussis toxin increased dialysate cyclic AMP in control rats, as did stimulation with cholera toxin (which activates the stimulatory guanine nucleotide binding protein Gs). The effect of pertussis toxin was abolished following chronic lithium, whereas the increase in cyclic AMP after cholera toxin was enhanced. In vitro pertussis toxin‐catalyzed ADP ribosyla‐tion of αi (and αo) was increased by 20% in prefrontal cortex from lithium‐treated rats, but the αi and αs contents (as determined by immunoblot) as well as the cholera toxin‐catalyzed ADP ribosylation of αs were unchanged. Taken together, these results suggest that chronic lithium administration may interfere with the dissociation of Gi into its active components and thereby remove a tonic inhibitory influence on adenylate cyclase, with resultant enhanced basal and cholera toxin‐stimulated adenylate cyclase activity.

Molecular Pharmacology and Function Ofmelatonin Receptor Subtypes

The secretion of melatonin primarily from the vertebrate retina and pineal gland with high levels at night is controlled by circadian clocks locally within the retina and the hypothalamic suprachiasmatic nucleus and is synchronized by environmental light (1,2). The first biological activity of melatonin can be traced back to 1917 when McCord and Allan (3) discovered that extracts of bovine pineal gland caused blanching of Rana pipiens tadpole skin. This bioassay was used to isolate melatonin from pineal extracts which led to the elucidation of its chemical structure (4). The property of melatonin to aggregate pigment granules (melanosomes) of amphibian dermal melanophores was used 1) to postulate the presence of melatonin receptors and to propose that Nacetyltryptamine is a melatonin receptor antagonist (5), 2) to establish the first structure-activity relationships of melatonin analogues (5), and 3) to demonstrate, in cultured Xenopus laevis melanophores, that activation of melatonin receptors inhibits cAMP formation through coupling to a pertussis toxin-sensitive G-protein (6). Subsequently, expression cloning led to the isolation of the first cDNA encoding a melatonin receptor from the Xenopus laevis melanophore (7). This landmark discovery facilitated the cloning and characterization of mammalian melatonin receptor subtypes, belonging to a novel subfamily of seven transmembrane domain G-protein coupled receptors

Light-induced phase shifts of circadian activity rhythms and immediate early gene expression in the suprachiasmatic nucleus are attenuated in old C3H/HeN mice.

Alterations in the mechanisms of entrainment and/or response of the circadian pacemaker to zeitgebers may contribute to age related changes in sleep/wake rhythms. This study examined the effect of age on light-induced phase shifts of circadian activity rhythms and on the expression of the immediate early genes c-fos and jun-B in the suprachiasmatic nucleus (SCN) of young and old C3H/HeN mice. Mice (4 months or 16 months at the beginning of the experiment) were housed in constant darkness with circadian rhythms assessed by running wheel activity. Mice were exposed to light pulses of 30, 100, 300 or 1000 lux and steady state phase shifts of circadian activity rhythms determined. In young mice exposed to light at circadian time (CT) 14, light pulses of 30, 100, 300 or 1000 lux induced phase delays of circadian activity rhythms of similar magnitude (averaging 2.8 h). Phase delays following photic stimulation were reduced in the old mice at all light levels (averaging 1.1 h, P < 0.001). Following behavioral testing, mice were exposed to light (1000 lux) at CT 14 for determination of the light-induced expression of c-fos and jun-B mRNA in the SCN by in situ hybridization histochemistry. Immediate early gene expression following light exposure was reduced by 42% (c-fos) and 48% (jun-B) in the SCN of old mice compared to young controls (P < 0.001). Together, these results suggest an age related reduction in responsiveness to light by the circadian pacemaker.