Signaling in the mammalian circadian clock: the NO/cGMP pathway.

Mammalian circadian rhythms are generated by a hypothalamic suprachiasmatic nuclei (SCN) clock. Light pulses synchronize body rhythms by inducing phase delays during the early night and phase advances during the late night. Phosphorylation events are known to be involved in circadian phase shifting, both for delays and advances. Pharmacological inhibition of the cGMP-dependent kinase (cGK) or Ca2+/calmodulin-dependent kinase (CaMK), or of neuronal nitric oxide synthase (nNOS) blocks the circadian responses to light in vivo. Light pulses administered during the subjective night, but not during the day, induce rapid phosphorylation of both p-CAMKII and p-nNOS (specifically phosphorylated by CaMKII). CaMKII inhibitors block light-induced nNOS activity and phosphorylation, suggesting a direct pathway between both enzymes. Furthermore, SCN cGMP exhibits diurnal and circadian rhythms with maximal values during the day or subjective day. This variation of cGMP levels appears to be related to temporal changes in phosphodiesterase (PDE) activity and not to guanylyl cyclase (GC) activity. Light pulses increase SCN cGMP levels at circadian time (CT) 18 (when light causes phase advances of rhythms) but not at CT 14 (the time for light-induced phase delays). cGK II is expressed in the hamster SCN and also exhibits circadian changes in its levels, peaking during the day. Light pulses increase cGK activity at CT 18 but not at CT 14. In addition, cGK and GC inhibition by KT-5823 and ODQ significantly attenuated light-induced phase shifts at CT 18. This inhibition did not change c-Fos expression SCN but affected the expression of the clock gene per in the SCN. These results suggest a signal transduction pathway responsible for light-induced phase advances of the circadian clock which could be summarized as follows: Glu-Ca2+-CaMKII-nNOS-GC-cGMP-cGK-->-->clock genes. This pathway offers a signaling window that allows peering into the circadian clock machinery in order to decipher its temporal cogs and wheels.

Participation of transcription factors from the Rel/NF-kappa B family in the circadian system in hamsters.

We have studied the presence and activity of components of the nuclear factor-kappaB (NF-kappaB) transcription factor in the hamster circadian system analyzing wheel-running activity, protein expression and DNA binding activity by electrophoresis mobility shift assays (EMSA). Non-rhythmic specific immunoreactive bands corresponding to a NF-kappaB subunit (p65) were found in hamster suprachiasmatic nuclei (SCN) homogenates. The active form of NF-kappaB evidenced by EMSA was clear and specific in SCN nuclear extracts. The administration of the NF-kappaB inhibitor pyrrolidine-dithiocharbamate (PDTC) blocked the light-induced phase advance at circadian time 18 (vehicle+light pulse: 2.08+/-0.46 h, PDTC+light: 0.36+/-0.35 h). These results demonstrate the presence and activity of Rel/NF-kappaB family proteins in the hamster SCN and suggest that these proteins may be related to the entrainment and regulation of circadian rhythms.

Diurnal, circadian and photic regulation of calcium/calmodulin-dependent kinase II and neuronal nitric oxide synthase in the hamster suprachiasmatic nuclei.

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.

Circadian heme oxygenase activity in the hamster suprachiasmatic nuclei.

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.

Circadian and photic regulation of ERK, JNK and p38 in the hamster SCN.

Circadian rhythms are entrained by light-activated signal transduction pathways in the biological clock. Among these, circadian and photic control of mouse suprachiasmatic ERK MAP kinase activation has been reported. In this paper we extend these results to hamsters and to the two other major members of the MAPK family: JNK and p38. The three kinases are rhythmically phosphorylated under light-dark and constant conditions, with maximal values during the day or subjective day. Light pulses during the subjective night induce rapid activation of the three enzymes, suggesting that the three MAP kinases might be implicated in mammalian photic entrainment.

From light to genes: moving the hands of the circadian clock.

Mammalian circadian rhythms are generated by the hypothalamic suprachiasmatic nuclei and finely tuned to environmental periodicities by neurochemical responses to the light-dark cycle. Light reaches the clock through a direct retinohypothalamic tract, primarily through glutamatergic innervation, and its action is probably regulated by a variety of other neurotransmitters. A key second messenger in circadian photic entrainment is calcium, mobilized through membrane channels or intracellular reservoirs, which triggers the activation of several enzymes, including a calcium/calmodulin-dependent protein kinase and nitric oxide synthase. Other enzymes activated by light are mitogen-activated- and cGMP-dependent protein kinase; all of the above have been reported to be involved in the circadian responses to nocturnal light pulses. These mechanisms lead to expression of specific clock genes which eventually set the phase of the clock and of clock-controlled circadian rhythms.