RESUMEN
MAP kinases (MAPKs) play a key role in photic entrainment signaling in the suprachiasmatic nuclei (SCN), the mammalian circadian clock. The control of MAPKs is a fine balance between specific kinases (MEKs) and phosphatases (MKPs), whose orchestration in the SCN is still unresolved. We have found MKP1/2 and MKP3 immunoreactive-cells in the hamster SCN, whose levels are rapidly increased in response to transient light stimulation in the subjective night (CT 18), when light is able to entrain the clock. Moreover, the expression level of MKP3 varies under light-dark cycles and constant darkness, peaking at noon, when MAPKs are in their activated state and begin their inactivation. These results show a different perspective on MAPKs in the SCN, which includes its regulation by a complex net of phosphatases.
Asunto(s)
Ritmo Circadiano/fisiología , Fosfatasa 1 de Especificidad Dual/metabolismo , Fosfatasa 6 de Especificidad Dual/metabolismo , Fotoperiodo , Núcleo Supraquiasmático/enzimología , Animales , Relojes Biológicos/fisiología , Cricetinae , Luz , Masculino , MesocricetusRESUMEN
In mammals, the mechanism for the generation of circadian rhythms and entrainment by light-dark (LD) cycles resides in the hypothalamic suprachiasmatic nuclei (SCN), and the principal signal that adjusts this biological clock with environmental timing is the light:dark cycle. Within the SCN, rhythms are generated by a complex of molecular feedback loops that regulate the transcription of clock genes, including per and cry. Posttranslational modification plays an essential role in the regulation of biological rhythms; in particular, clock gene phosphorylation by casein kinase I , both epsilon (CKIepsilon) and delta (CKIdelta), regulates key molecular mechanisms in the circadian clock. In this paper, we report for the first time that CKI activity undergoes a significant circadian rhythm in the SCN (peaking at circadian time 12, the start of the subjective night), and its pharmacological inhibition alters photic entrainment of the clock, indicating that CKI may be a key element in this pathway.
Asunto(s)
Quinasa de la Caseína I/antagonistas & inhibidores , Ritmo Circadiano/fisiología , Isoquinolinas/farmacología , Núcleo Supraquiasmático/efectos de los fármacos , Núcleo Supraquiasmático/enzimología , Animales , Quinasa de la Caseína I/metabolismo , Cricetinae , Inhibidores Enzimáticos/farmacología , Luz , Fototransducción/efectos de los fármacos , Fototransducción/fisiología , Masculino , Mesocricetus , Ratones , Núcleo Supraquiasmático/metabolismoRESUMEN
Circadian entrainment involves photic stimulation of the suprachiasmatic molecular oscillator, including activation of the ERK/MAP kinase, which is phosphorylated endogenously during the day and in response to light during the night. We aimed to disrupt the diurnal cycle of ERK phosphorylation by in vivo transfection of a constitutively active form of MEK, a MAPK kinase. This procedure did not affect normal circadian parameters, but completely inhibited light-induced phase advances. Therefore, circadian regulation of the ERK pathway is not essential for the normal mechanism of the biological clock, but it is fundamental as an interface with environmental entrainment by light.
Asunto(s)
Ritmo Circadiano/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Mesocricetus/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Núcleo Supraquiasmático/enzimología , Núcleo Supraquiasmático/fisiología , Animales , Cricetinae , Activación Enzimática , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Actividad Motora/fisiología , TransfecciónRESUMEN
Mammalian circadian rhythms are entrained by light pulses that induce phosphorylation events in the suprachiasmatic nuclei (SCN). Ca(2+)-dependent enzymes are known to be involved in circadian phase shifting. In this paper, we show that calcium/calmodulin-dependent kinase II (CaMKII) is rhythmically phosphorylated in the SCN both under entrained and free-running (constant dark) conditions while neuronal nitric oxide synthase (nNOS) is rhythmically phosphorylated in the SCN only under entrained conditions. Both p-CaMKII and p-NOS (specifically phosphorylated by CaMKII) levels peak during the day or subjective day. Light pulses administered during the subjective night, but not during the day, induced rapid phosphorylation of both enzymes. Moreover, we found an inhibitory effect of KN-62 and KN-93, both CaMKII inhibitors, on light-induced nNOS activity and nNOS phosphorylation respectively, suggesting a direct pathway between both enzymes which is at least partially responsible of photic circadian entrainment.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Ritmo Circadiano/fisiología , Óxido Nítrico Sintasa/biosíntesis , Fotoperiodo , Núcleo Supraquiasmático/enzimología , Núcleo Supraquiasmático/fisiología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Bencilaminas/farmacología , Western Blotting , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Cricetinae , Inhibidores Enzimáticos/farmacología , Inyecciones Intraventriculares , Luz , Mesocricetus , Óxido Nítrico Sintasa de Tipo I , Fosforilación , Sulfonamidas/farmacología , Núcleo Supraquiasmático/efectos de los fármacosRESUMEN
Entrainment of mammalian circadian rhythms requires the activation of specific signal transduction pathways in the hypothalamic suprachiasmatic nuclei (SCN). We have tested the participation of heme oxygenase (HO) in the SCN, by assessing HO specific activity at different time points and photic conditions. HO activity was determined by the conversion of hemin to bilirubin. HO enzymatic activity in the SCN was significantly higher during the night than during the day; this difference persisted when animals were placed under constant darkness, suggesting an endogenous circadian control. HO inhibition by Zn-protoporphyrin did not affect light-induced phase shifts in vivo, suggesting that the enzyme is not necessary for light input to the clock.
Asunto(s)
Ritmo Circadiano/fisiología , Hemo Oxigenasa (Desciclizante)/metabolismo , Núcleo Supraquiasmático/enzimología , Animales , Conducta Animal , Bilirrubina/metabolismo , Western Blotting , Ritmo Circadiano/efectos de los fármacos , Cricetinae , Oscuridad , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Hígado/efectos de los fármacos , Hígado/enzimología , Actividad Motora/efectos de los fármacos , Estimulación Luminosa , Protoporfirinas/farmacología , Núcleo Supraquiasmático/efectos de los fármacos , Factores de TiempoRESUMEN
The levels of cyclic AMP and protein kinase A, as well as the activity of this enzyme, were measured in the hamster suprachiasmatic nuclei at different time points throughout the daily or circadian cycle. Significant diurnal variations for levels of AMPc and the catalytic subunit of protein kinase A and the activity of this enzyme were found. All of these parameters tended to increase throughout the nocturnal phase, reaching higher values at the end of the night and the beginning of the day and minimal values around the time of lights off. This rhythmicity appears to be under exogenous control, since constant darkness abolished fluctuations throughout the circadian cycle. In vitro incubation in the presence of melatonin during the day significantly decreased cyclic AMP levels and basal protein kinase A activity in the SCN, while neither neuropeptide Y nor light pulses affected these parameters. These results suggest a significant diurnal regulation of the cyclic AMP-dependent system in the hamster circadian clock.
Asunto(s)
Ritmo Circadiano/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Periodicidad , Núcleo Supraquiasmático/enzimología , Animales , Relojes Biológicos/efectos de los fármacos , Relojes Biológicos/efectos de la radiación , Western Blotting , Cricetinae , Oscuridad , Técnicas In Vitro , Luz , Masculino , Melatonina/metabolismo , Melatonina/farmacología , Mesocricetus , Neuropéptido Y/metabolismo , Neuropéptido Y/farmacología , Estimulación LuminosaRESUMEN
Circadian rhythms are controlled by an endogenous clock, which in mammals is located in the hypothalamic suprachiasmatic nuclei (SCN). A role for nitric oxide in circadian responses to light has been indicated. To test the role of nitric oxide synthase (NOS) in the SCN and in circadian responses to light, we examined NOS specific activity at different time points and photic conditions. NOS activity was determined by the conversion of 3H-arginine to 3H-citrulline. NOS enzymatic activity in the SCN was significantly higher during the dark phase than during the day, without any changes in the levels of the NOS protein. However, this difference disappeared when animals were placed under constant darkness, and NOS activity was similar at CT 8 and CT 18 (with CT 12 defined as the onset of the subjective night). When 5-min light pulses were administered at these time points (when light would induce no phase shift or a phase advance, respectively), NOS activity was significantly increased almost equally. A spectrophotometric assay was used to determine NO content in the SCN, showing relatively high constitutive levels enhanced by 100 microM glutamate. These results suggest that NOS activity is not controlled by the circadian clock, although it might mediate some of the effects of light on biological rhythms.
Asunto(s)
Ritmo Circadiano/fisiología , Óxido Nítrico Sintasa/metabolismo , Núcleo Supraquiasmático/enzimología , Animales , Western Blotting , Cricetinae , Oscuridad , Ácido Glutámico/fisiología , Masculino , Mesocricetus , Neurotransmisores/fisiología , Óxido Nítrico/análisis , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/análisis , Estimulación Luminosa , Núcleo Supraquiasmático/químicaRESUMEN
Activities of the antioxidant enzymes superoxide dismutase (E.C.1.15.1.1) and glutathione peroxidase (E.C.1.11.1.9) were measured in the pineal gland of adult rats. For comparative purpose superoxide dismutase was also measured in the anterior hypothalamic area (suprachiasmatic region) and the liver and glutathione peroxidase in the liver. The mean activities obtained for superoxide dismutase were: pineal (103.13 +/- 4.70 N.U.), anterior hypothalamus (132.88 +/- 4.73 N.U.), liver (253.91 +/- 0.10 N.U.) and, for glutathione peroxidase were: pineal (0.027 +/- 0.005 I.U.) and liver (0.103 +/- 0.031 I.U.). It was shown for the first time that superoxide dismutase and glutathione peroxidase are present in pineal gland being part of its antioxidant defense system.