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M L Dubocovich

Publications and source records attributed to M L Dubocovich.

At least 19 recordsLinked to original sources
PubMed2005

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

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 microg/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 MT(1)KO: 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.

Animals↗
PubMed2005

The antidepressant-like effect of the melatonin receptor ligand luzindole in mice during forced swimming requires expression of MT2 but not MT1 melatonin receptors.

We previously reported an antidepressant-like effect in C3H/HeN mice during the forced swimming test (FST) following treatment with the MT1/MT2 melatonin receptor ligand, luzindole. This study investigated the role melatonin receptors (MT1 and/or MT2) may play in the effect of luzindole in the FST using C3H/HeN mice with a genetic deletion of either MT1 (MT1KO) or MT2 (MT2KO) melatonin receptors. In the light phase (ZT 9-11), luzindole (30 mg/kg, i.p.) significantly decreased immobility during swimming in both wild type (WT) (135.6 +/- 25.3 s, n = 7) and MT(1)KO (132.6 +/- 13.3 s, n = 8) as compared with vehicle-treated mice (WT: 207.1 +/- 6.0 s, n = 7; MT1KO: 209.5 +/- 6.2 s, n = 8) (P < 0.001). In the dark phase (ZT 20-22), luzindole also decreased time of immobility in both WT (89.5 +/- 13.9 s, n = 8) and MT1KO (66.5 +/- 6.4 s, n = 8) mice as compared with the vehicle treated (WT: 193.8 +/- 3.5, n = 6; MT1KO: 176.6 +/- 6.2 s, n = 8) (P < 0.001). Genetic disruption of the MT1 gene did not alter the diurnal rhythm of serum melatonin in MT1KO mice (ZT 9-11: 1.3 +/- 0.6 pg/mL, n = 7; ZT 20-22: 10.3 +/- 1.1 pg/mL, n = 8) as compared with WT (ZT 9-11: 1.4 +/- 0.7 pg/mL; ZT 20-22: 10.6 pg/mL). Swimming did not alter the serum melatonin diurnal rhythm in WT and MT1KO mice. Decreases in immobility of WT and MT1KO mice by luzindole treatment were not affected by gender or age (3 months versus 8 months). In contrast, luzindole did not decrease immobility during the FST in MT2KO mice. We conclude that the antidepressant-like effect of luzindole may be mediated through blockade of MT2 rather than MT1 melatonin receptors.

Animals↗
PubMed2003

Short-term exposure to melatonin differentially affects the functional sensitivity and trafficking of the hMT1 and hMT2 melatonin receptors.

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.

Animals↗
PubMed2001

Melatonin receptor signaling: finding the path through the dark.

Melatonin, dubbed "the hormone of darkness," is involved in relaying photoperiodic information to the organism. Not only is melatonin involved in the regulation of circadian rhythms and sleep, but it also has roles in visual, cerebrovascular, reproductive, neuroendocrine, and neuroimmunological functions. Melatonin mediates its effects through G protein-coupled receptors: MT(1), MT(2), and, possibly, MT(3). Pharmacological agents have been instrumental in identifying these receptor types. Masana and Dubocovich discuss how the level of receptor expression may alter their efficacy, so that caution is necessary when extrapolating the pharmacological properties of ligands defined on recombinant systems to the receptors in the organism. With these cautions in mind, they describe the various signaling pathways and physiological roles ascribed to the three melatonin receptor types.

Animals↗
PubMed2001

Melatonin and N-acetylserotonin inhibit leukocyte rolling and adhesion to rat microcirculation.

The hormone melatonin produced by the pineal gland during the daily dark phase regulates a variety of biological processes in mammals. The aim of this study was to determine the effect of melatonin and its precursor N-acetylserotonin on the microcirculation during acute inflammation. Arteriolar diameter, blood flow rate, leukocyte rolling and adhesion were measured in the rat microcirculation in situ by intravital microscopy. Melatonin alone or together with noradrenaline did not affect the arteriolar diameter or blood flow rate. Melatonin inhibited both leukocyte rolling and leukotriene B(4) induced adhesion while its precursor N-acetylserotonin inhibits only leukocyte adhesion. The rank order of potency of agonists and antagonist receptor selective ligands suggested that the activation of MT(2) and MT(3) melatonin binding sites receptors modulate leukocyte rolling and adhesion, respectively. The effect of melatonin and N-acetylserotonin herein described were observed with concentrations in the range of the nocturnal surge, providing the first evidence for a possible physiological role of these hormones in acute inflammation.

Animals↗
PubMed2001

Familial advanced sleep phase syndrome.

BACKGROUND: The circadian rhythms of sleep propensity and melatonin secretion are regulated by a central circadian clock, the suprachiasmatic nucleus of the hypothalamus. The most common types of sleep disorders attributed to an alteration of the circadian clock system are the sleep/wake cycle phase disorders, such as delayed sleep phase syndrome and advanced sleep phase syndrome (ASPS). Advanced sleep phase syndrome is characterized by the complaint of persistent early evening sleep onset and early morning awakening. Although the complaint of awakening earlier than desired is relatively common, particularly in older adults, extreme advance of sleep phase is rare. OBJECTIVE: To phenotypically characterize a familial case of ASPS. METHODS: We identified a large family with ASPS; 32 members of this family gave informed consent to participate in this study. Measures of sleep onset and offset, dim light melatonin onset, the Horne-Ostberg morningness-eveningness questionnaire, and clinical interviews were used to characterize family members as affected or unaffected with ASPS. RESULTS: Affected members rated themselves as "morning types" and had a significant advance in the phase of sleep onset (P<.001) and offset (P =.006) times. The mean sleep onset was 2121 hours for the affected family members and 0025 hours for the unaffected family members. The mean sleep offset was 0507 hours for the affected members and 0828 hours for the unaffected members. (Times are given in military form.) In addition, the phase of the circadian rhythm of melatonin onset for the affected family members was on average 3-1/2 hours earlier than for the unaffected members. CONCLUSIONS: The ASPS trait segregates with an autosomal dominant mode of inheritance. The occurrence of familial ASPS indicates that human circadian rhythms, similar to those in animals, are under genetic regulation. Genetic analysis of familial sleep and circadian rhythm disorders is important for identifying a specific gene(s) responsible for the regulation of sleep and circadian rhythms in humans.

Adult↗
PubMed2001

Activation of MT(2) melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian clock.

The aim of this study was to identify the melatonin receptor type(s) (MT(1) or MT(2)) mediating circadian clock resetting by melatonin in the mammalian suprachiasmatic nucleus (SCN). Quantitative receptor autoradiography with 2-[(125)I]iodomelatonin and in situ hybridization histochemistry, with either (33)P- or digoxigenin-labeled antisense MT(1) and MT(2) melatonin receptor mRNA oligonucleotide probes, revealed specific expression of both melatonin receptor types in the SCN of inbred Long-Evans rats. The melatonin receptor type mediating phase advances of the circadian rhythm of neuronal firing rate in the SCN slice was assessed using competitive melatonin receptor antagonists, the MT(1)/MT(2) nonselective luzindole and the MT(2)-selective 4-phenyl-2-propionamidotetraline (4P-PDOT). Luzindole and 4P-PDOT (1 nM-1 microM) did not affect circadian phase on their own; however, they blocked both the phase advances (approximately 4 h) in the neuronal firing rate induced by melatonin (3 pM) at temporally distinct times of day [i.e., subjective dusk, circadian time (CT) 10; and dawn, CT 23], as well as the associated increases in protein kinase C activity. We conclude that melatonin mediates phase advances of the SCN circadian clock at both dusk and dawn via activation of MT(2) melatonin receptor signaling.

Action Potentials↗
PubMed2000

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 35S-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 (CTI10). 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.

Animals↗
PubMed2000

Siberian hamsters that fail to reentrain to the photocycle have suppressed melatonin levels.

Siberian hamsters readily reentrain to a 3-h phase delay of the photocycle (16 h light/day) but fail to reentrain to a 5-h phase delay. This study tested whether melatonin production was suppressed in animals that failed to reentrain. Melatonin was measured on the day before, day of, or several days after each phase shift. Melatonin levels measured 4 h after dark onset were approximately 83 microg/ml on the day before each phase delay and undetectable (<6 microg/ml) during the light phase on the day of the phase shift. Activity onsets regained their prior phase relationship to the photocycle 4 (3 h) or 5 (5 h) days after the phase shift; on that day, melatonin levels were measured 4 h after dark onset. Melatonin levels were unaffected by the 3-h phase delay (>57.6 microg/ml) but were undetectable after a 5-h phase delay (<8 microg/ml). Thus melatonin remained suppressed only after the phase delay to which hamsters also fail to reentrain. This relationship suggests that the propensity for reentrainment may be influenced by changes in melatonin production following a phase shift of the photocycle.

Animals↗
PubMed1999

Nimodipine potentiates the light-induced suppression of melatonin.

In mammals the phase shifting response of the circadian clock to light can be enhanced by administration of the calcium channel antagonist nimodipine. In the present study we assessed the potential for nimodipine to affect the responsiveness of the human circadian clock to light by measuring the light-induced suppression of melatonin levels in plasma. Seven healthy young subjects (3M, 4F, 27.3 +/- 1.8 years old) were admitted on four occasions to the Clinical Research Center at Northwestern University Medical School. Blood was collected during the night to assess the effect of nimodipine (30 mg, orally, 01:30 h) on plasma melatonin levels in the presence or absence of light (500 lux, 2-3 am). Melatonin levels in plasma were measured by radioimmunoassay. Exposure to light for 1 h suppressed melatonin levels in plasma by nearly 38% relative to samples obtained at the same time in the absence of light (P = 0.013). Nimodipine administration did not modify plasma melatonin levels. However, combined treatment with nimodipine and light suppressed melatonin levels in plasma by 59%. Levels of plasma melatonin were significantly lower following treatment with nimodipine and light than following treatment with placebo/light (P = 0.014). Thus, the calcium channel antagonist nimodipine potentiated the suppressive effect of light on melatonin levels in plasma. These results suggest that the calcium channel antagonist nimodipine may also potentiate the response of the human circadian clock to light, and might thus be useful in combination with phototherapy for the treatment of sleep and circadian rhythm disorders.

Adult↗
PubMed1999

Interactions between light and melatonin on the circadian clock of mice.

Melatonin and light synchronize the biological clock and are used to treat sleep/wake disturbances in humans. However, the two treatments affect circadian rhythms differently when they are combined than when they are administered individually. To elucidate the nature of the interaction between melatonin and light, the present study assessed the effect of melatonin on circadian timing and immediate-early gene expression in the suprachiasmatic nucleus (SCN) when administered in the presence of light. Male C3H/HeN mice, housed in constant dark in cages equipped with running wheels, were treated with either melatonin (90 microg, s.c.) or vehicle (3% ethanol-saline) 5 min prior to exposure to light (15 min, 300 lux) at various times in the circadian cycle. Combined treatment resulted in lower magnitude phase delays of circadian activity rhythms than those obtained with light alone during the early subjective night and advances in phase when melatonin and light were administered during the subjective day (p < .001). The reduction in phase delays with combined treatment at Circadian Time (CT) 14 was significant when light exposure measured 300 lux but not at lower light levels (p < .05). When light preceded melatonin administration, the inhibition of phase delays attained significance only when the light exposure reached 1000 lux (p < .05). Neither basal nor light-induced expression of c-fos mRNA in the SCN was modified by melatonin administration at CT 14 or CT 22. Together, these results suggest that combined administration of melatonin and light affect circadian timing in a manner not predicted by summing the two treatments given individually. Furthermore, the interaction is not likely to be due to inhibition of photic input to the clock by melatonin but might arise from a photically induced enhancement of melatonin's actions on circadian timing.

Animals↗
PubMed1998

Melatonin receptor subtype expression in human cerebellum.

We report the distinct cellular distribution in the human cerebellar cortex of the mammalian mt1 (Mel1a) and MT2 (Mel1b) (1) melatonin receptor subtypes. Specific binding of the non-selective radioligand 2-[125I]iodomelatonin to the outer molecular layer was significantly higher than to the granule cell layer. Melatonin receptor subtype expression was assessed by in situ hybridization using selective and specific digoxigenin-labeled antisense oligonucleotide probes. This is the first demonstration of MT2 melatonin receptor mRNA expression in human cerebellar Bergmann glia and astrocytes. On the other hand, the mt1 melatonin receptor mRNA was expressed in both basket-stellate cells and granule cells. We conclude that mt1 and MT2 melatonin receptors are heterogeneously expressed in human cerebellar cortex.

Astrocytes↗
PubMed1998

Melatonin mediates two distinct responses in vascular smooth muscle.

The pineal hormone melatonin was found to produce two distinct contractile responses in vascular smooth muscle. In isolated rat caudal artery segments, denuded of endothelium, melatonin (10(-10)-10(-7) M) potentiated phenylephrine-induced contractions in a concentration-dependent manner. At higher melatonin concentrations (10(-7)-10(-5) M), however, the potentiating effect was attenuated. In the presence of the melatonin MT2 receptor antagonist, 4-phenyl-2-acetamidotetraline (4P-ADOT), the attenuated constrictor responses were selectively enhanced. These results are consistent with the hypothesis that melatonin activates two receptor subtypes in vascular smooth muscle; MT2 receptors may induce relaxation, while a second receptor subtype mediates vasoconstriction.

Adrenergic alpha-Agonists↗
PubMed1998

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 microg/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 microg/mouse; 0.98+/-0.08 h (n=15) maximal advance at 9 microg/mouse]. Neither the selective MT2 melatonin receptor antagonists 4P-ADOT and 4P-PDOT (90 microg/mouse, s.c.) nor luzindole (300 microg/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 microg 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.

Animals↗
PubMed1998

Physiological exposure to melatonin supersensitizes the cyclic adenosine 3',5'-monophosphate-dependent signal transduction cascade in Chinese hamster ovary cells expressing the human mt1 melatonin receptor.

Here, we report the effects of short exposure to melatonin on the human mt1 (h mt1) melatonin receptor-mediated signaling in Chinese hamster ovary (CHO) cells, and the consequences of an exposure that resembles the physiological pattern of melatonin release on cAMP-mediated signal transduction. Short exposure (10 min) of h mt1 melatonin receptors to melatonin (400 pM) inhibited forskolin-stimulated cAMP formation, cAMP-dependent protein kinase activity, and phosphorylation of the cAMP response element-binding protein. However, treatment of mt1-CHO cells with melatonin in a manner that closely mimics the in vivo activation of melatonin receptors (i.e. 400 pM melatonin for 8 h to mimic darkness) resulted in a supersensitization of the cAMP-dependent signal transduction cascade during the period of withdrawal (i.e. 16 h without melatonin to mimic the light cycle of a diurnal photoperiod). During the period of withdrawal, forskolin induced a time-dependent (1-16 h) increase in cAMP formation (approximately 200% of control cells). This effect of melatonin was dependent on the presence of the h mt1 melatonin receptor, as no potentiation of forskolin-induced cAMP formation was observed in CHO cells transfected only with the neomycin resistance plasmid. The time-dependent increase in forskolin-stimulated cAMP levels resulted in a potentiation of cAMP-dependent protein kinase activity 1 h after withdrawal (approximately 130% of control cells; P < 0.05) and in the number of cells containing the phosphorylated form of cAMP response element-binding protein (approximately 75% of cells at 1 and 16 h compared with 30% in control cells; P < 0.05). An increase in the undissociated state (G alphabetagamma) of Gi proteins may underlie this phenomenon as demonstrated by the increase in pertussis toxin-catalyzed ADP-ribosylation of G proteins (217 +/- 48% of control; P < 0.05) after melatonin withdrawal. This increase in the ribosylation was not due to an up-regulation of Galpha(i) protein, as no significant change in Galpha(i) protein levels occurred at this time. We demonstrated that activation of the h mt1 melatonin receptor in a manner that resembles the physiological pattern of melatonin exposure alters signaling, as potentiation of cAMP-mediated signal transduction events is observed after hormone withdrawal. The CHO cells expressing the human melatonin receptor may provide an in vitro cellular model in which to investigate the putative signaling mechanisms leading to gene regulation by melatonin.

Animals↗
PubMed1998

GR196429: a nonindolic agonist at high-affinity melatonin receptors.

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.

Animals↗