camp and calcium

cAMP is considered a secondary messenger along with Ca 2+. Our results contrast with those obtained previously, by Morioka and colleagues [41], which showed that baseline intracellular calcium concentrations in the PG reached a single minimum at the beginning of the day under LD condition. Interestingly, the corresponding manipulations directed at central pacemaker neurons had similar effects on the rhythmicity of eclosion and of adult locomotor activity, suggesting a common mechanism. Typically, a number of RNAi lines were tested and the most effective one was then used for the majority of the experiments. In addition to intra- and inter- pacemaker coordination, clocks can also be entrained by external stimuli such as light, which is a powerful entraining signal, as well as by an organism’s physiological and behavioral state [1, 20]. cells. Their activity was then monitored under LD 12:12 conditions for 7–10 days followed by 7–10 days under DD conditions. The number of cells analyzed is indicated above each circle. Each bar represents the mean ± S.E. cAMP is a second messenger, used for intracellular signal transduction, such as transferring into cells the effects of hormones like glucagon and adrenaline, which cannot pass through the plasma membrane. The circadian rhythm of Drosophila adult emergence depends on functional clocks in both the brain and the PG [19,35] and recent evidence shows that the brain transmits time information to the PG via the PTTH neuropeptide acting on its receptor, TORSO [19]. 4 Cells were then treated with either KCl (60 mm) or forskolin (10 μm) for 4 h as indicated and then harvested for luciferase assay. Using the PC12 cell model system, we found that both stimulation of calcium influx and elevation of intracellular Cells were However, in many cases, the actions of the latter two messengers involve slower, long term changes in gene expression (3, 4). (B) Free-running periodicity values for genotypes shown in (A); each circle indicates results from separate experiments; average is indicated by horizontal line; same letters indicate no statistically different groups (one-way ANOVA, Tukey’s post hoc multiple comparison analyses). Formal analysis, The vector pcDNA3 (Invitrogen Corp.) was added to each set of transfections Both KCl and forskolin induced a rapid and sustained phosphorylation of CREB as assayed by Western blot. Overall, these results suggest that calcium and cAMP levels affect clock function itself. CREB Furthermore, PG-specific knockdown of CaMKII and CASK also affected clock period, suggesting that the effects of changes in calcium levels on the clock could be effected by phosphorylation. Cells were pretreated with PD98059 (50 μm) as indicated and then stimulated with either forskolin (10 μm) or the β-adrenergic receptor agonist isoproterenol (10 μm) for 4 h, before being harvested for luciferase assay. These results show that changing the levels of intracellular calcium affects the periodicity of the circadian clock. For example, processes such as recruitment and regulation of co-activators and coupling to the basal transcription machinery of CREB-dependent transcription by both calcium influx and cAMP. a site downstream from CREB Ser-133 phosphorylation. A recent study reported that CREB-dependent transcription (5). Overall, GCaMP readings were between 0.6 and 0.8, which corresponds to 50-300nM (see Fig 2B), and is consistent with published values. These data suggest that both calcium influx and cAMP regulate CREB-dependent transcription via activation of a PKA-dependent In A-C, GAL4 driver was used in combination with UAS-dcr2 to potentiate effectiveness of RNAi knockdown. is mutated (11, 44). over unstimulated cells. can be stimulated via a variety of signaling kinases, including CaMKIV, PKA, and ERKs (11, 22-28). B,NIH3T3 cells were co-transfected with a 5XCRE-luciferase reporter and combinations of empty vector, cPKA, and B-Raf as indicated. Cells were treated for appropriate times as indicated and then harvested in boiling SDS sample buffer. through a direct action on Ser-133. transcription (Fig. of PKA (cPKA) (4 μg), Rap1GAP1 (2 or 4 μg), Gal-E1b-TATA-luciferase (gal-luciferase, 2 μg), 5XCRE-luciferase (1 μg), Fos-luciferase, However, their readings were done every 6 hours and skipped the timepoint at which we observed an even lower (and minimal) level. However, transfection of B-Raf potentiated the stimulatory actions of both forskolin and cPKA but had no effects on the (E) Free-running periodicity values for genotypes shown in (A); each circle indicates individual flies tested; average is indicated by horizontal line. An important consideration is the mechanism by which the Rap1-ERK pathway controls transcription downstream of CREB phosphorylation. https://doi.org/10.1371/journal.pgen.1007433.g002. These results are comparable to the ones obtained following similar manipulations in the PG, and suggests that calcium and cAMP signaling plays a similar role in the central and the peripheral clock with respect to the circadian control of adult emergence. occur through distinct mechanisms. Thus, as in PC12 cells, cAMP stimulation of CRE-dependent transcription requires an ERK signaling component at a site distinct Thus, it is possible that the timecourse we recorded in the PG reflects the sum of two circadian inputs, one driven by the brain, and the other produced by the PG itself. Abstract:The calcium ion is quite possibly the single most pervasive signaling molecule used by living organisms for the purpose of communicating internal and external states. The following day the lights were permanently turned off before lights-on and eclosion monitored for 7–8 days. These data suggest a revised model for cAMP regulation Circadian clocks impose daily periodicities to animal behavior and physiology. Flies bearing only UAS-RNAi transgenes express normal rhythmicity of emergence (S1 Fig). Indeed, knockdown of Epac (Exchange protein directly activated by cAMP) and Rap1 (Ras-related protein 1 GTPase) in the PG caused a significant lengthening of the periodicity of eclosion (Fig 3Ac, 3Ad and 3B). PKA is unable to activate Under DD conditions highest cAMP levels were delayed by 3h (to CT0), whereas the lowest cAMP levels occurred at the middle of the subjective day (CT6)(Fig 4E). Interestingly, previous reports have demonstrated that, in the context of calcium regulation of A major finding of this study is that cAMP requires a Rap1-ERK signaling pathway to stimulate CREB-dependent transcription. Multiple signaling pathways have been shown to For example, chronic buffering of calcium within pacemaker cells causes a progressive lengthening of the circadian periodicity of adult locomotor activity [27] and mutations that alter cAMP levels [28] or CREB function [29] alter the fly’s free-running period; likewise, acute changes in pacemaker excitability cause phase shifts in locomotor activity rhythms, which may be mediated by CREB [30]. NIH3T3 and Hek293 cells were maintained in Dulbecco's modified Eagle's medium plus 10% fetal calf serum. Cells Smads (49, 50), signal transducers and activators of transcription (51), nuclear hormone receptors (52). Fig 4C). cAMP levels stimulated CREB-dependent transcription via the PKA-dependent Rap1-ERK pathway. These data are therefore also consistent with an indirect action of PKA, possibly via Rap1-ERK signaling. Circumstantial evidence suggests that these second messengers may funnel their actions through the calcium/cAMP-dependent transcription factor, CREB, as occurs in the mammalian SCN [22–24, 26]. In this report, we have examined the actions of cAMP using a CREB mutant, CREB-DIEDML, that contains a six-amino acid substitution One possibility is that circadian rhythmicity of emergence is accomplished by fine-tuning the levels of ecdysone (the precursor of the active steroid, 20-hydroxyecdysone) production and/or secretion via calcium and cAMP. In the case of neuronal circadian pacemakers, the activity of the cells that make up the pacemaker is then coordinated through transmitter and neuropeptide action. - Purdue University's Camp Calcium, the primary source of research for today's calcium guidelines for teens, is celebrating its 20th year. No, Is the Subject Area "Calcium signaling" applicable to this article? The arrowheads indicate representative transfected cells. of three independent treatments. communicating internal and external states. In contrast, the cAMP-dependent stimulation of the Flies bearing the pdf-gGAL4 driver alone expressed normal circadian rhythmicity of eclosion (S1 Fig). two second messengers involve long term changes in gene expression. A, NIH3T3 cells were co-transfected with a 5XCRE-luciferase reporter and either empty vector or B-Raf as indicated. (D) Representative records of adult locomotor activity in DD (left) and Autocorrelation analyses (right) of normal (per+) fly expressing GCaMP sensor in the PG (a) and of adult per01 fly expressing GCaMP sensor in the PG (b). Keywords: Fluorescence Resonance Energy Transfer (FRET), calcium/cAMP crosstalk, cAMP microdomains, adenylyl cyclase, protein cAMP levels in the PG were determined by measuring the FRET signal emitted by PGs expressing the Epac1 sensor. pathway. e1007433. For example, in hippocampal This deceptively simple paradigm dominates the function of many cell types, for example in function by both calcium and cAMP signals. (C) Levels of fluorescence in controls and in animals bearing knockdown of calcium channels (cacophony [cac] and Ca-alpha1D), IP3 receptor (IP3R) and SERCA in the PG (using phm-GAL4 driver)(all measured at ZT12). Numbers in parenthesis indicate number of separate experiments; different letters indicate statistically different groups (p<0.05; one-way ANOVA, Tukey’s post hoc multiple comparison analyses). Cells were B). Importantly, since calcium and cAMP are also output signals of the clock, they may contribute to the mechanism that imposes a circadian gating to the timing of adult emergence. CBP is also a co-activator for a variety of other transcription factors, e.g. via RSK2, exerts an inhibitory effect. Price: $65. The … : 503-494-5494; Fax: 503-494-4976; E-mail: stork@ohsu.edu. https://doi.org/10.1371/journal.pgen.1007433.s003. Validation, CREB, CREB-DIEDML. demonstrated that the ability of cAMP to activate a Rap1-dependent signaling pathway to ERKs is dependent on the expression In addition, cAMP binds to and regulates the function of ion channels such as the HCN channels and a few other cyclic nucleotide-binding proteins such as Epac1 and RAPGEF2. At the level of the central pacemaker, a number of transmitters [3–10] and neuropeptides [11–16] mediate the production of coherent network-wide circadian oscillations. Hek293 cells express high levels of B-Raf. The effects of manipulating calcium and cAMP levels in the PG on the periodicity of emergence suggest that these messengers could directly alter the clock TTFLs, and a variety of indirect evidence [30,47] suggests that they may act on the TTFL via CREB. 7, Aand B). can control transcription independently of CREB. of the neuronal Raf isoform, B-Raf.

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