Differential regulation of melatonin synthesis genes and phototransduction genes in embryonic chicken retina and cultured retinal precursor cells. (9/63)

PURPOSE: Photoreceptor differentiation involves the activation of two specific sets of genes; those encoding the proteins of the phototransduction cascade and those encoding the enzymes of the melatonin synthesis pathway, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT). The purpose of the present study was to examine the conditions of AANAT and HIOMT gene activation, relative to that of selected phototransduction markers (alpha-transducin and opsins), in both in vivo and in vitro differentiating photoreceptors of the chicken retina. METHODS: Neural retina RNA was obtained between embryonic day 7 (E7) and posthatch day 8 (P8) and analyzed on northern blots with cDNA probes to AANAT, HIOMT, visinin, alpha-transducin, rhodopsin, and the four cone opsins. Cell cultures were prepared from E7 chicken neural retina and incubated for two to four days in vitro, either in basal medium or in serum-supplemented medium or in medium containing an insulin-based supplement. RNA from the cultured cells was analyzed on northern blots as above. Real time RT-PCR was used to confirm in vitro changes in HIOMT and red opsin mRNA levels. The cultured cells were transfected with promoter-reporter plasmids for direct analysis of HIOMT promoter regulation by the dual luciferase method. RESULTS: The different mRNAs composing the photoreceptor phenotype appeared at E7 (visinin), E10 (alpha-transducin), E14 (HIOMT), E15 (rhodopsin, red opsin, and green opsin), E16 (AANAT), E17 (blue opsin), and E18 (violet opsin). In the early differentiating cones of the central retina, HIOMT mRNA appeared two days earlier than red opsin and green opsin mRNAs (E12 rather than E14). In cultured embryonic neural retina cells, basal medium was sufficient to activate alpha-transducin gene transcription, an insulin-based supplement was sufficient to activate HIOMT gene transcription, whereas serum was required for red opsin gene transcription after two days in vitro. All serum batches were able to activate red opsin gene transcription, whereas some of them failed to activate HIOMT gene transcription. Activation of the HIOMT gene promoter by an insulin-based supplement and by serum was confirmed after transfection of chicken embryonic neural retina cells with promoter-reporter plasmids. CONCLUSIONS: Activation of the melatonin synthesis genes in vivo takes place in a time window very close to that of early opsins. However, a 24-48 h lead of HIOMT gene expression over early opsins was clearly observed. Our in vitro experiments indicate that different exogenous signals are required to activate the different genes encoding photoreceptor specific functions. Significantly, marker genes for light sensitivity (red opsin) and for melatonin synthesis (HIOMT) appear to be activated in response to different signals.  (+info)

Rhythmic expression of adenylyl cyclase VI contributes to the differential regulation of serotonin N-acetyltransferase by bradykinin in rat pineal glands. (10/63)

The rhythmic nocturnal production of melatonin in pineal glands is controlled by the periodic release of norepinephrine from the superior cervical ganglion. Norepinephrine binds to the beta-adrenergic receptor and stimulates an increase in intracellular cAMP levels, leading to the transcriptional activation of serotonin N-acetyltransferase, which in turn promotes melatonin production. In the present study, we report that bradykinin inhibits basal- and forskolin-stimulated adenylyl cyclase activity, norepinephrine-induced cAMP generation, and N-acetyltransferase expression in a calcium-dependent manner. These effects were blocked by pretreatment with U73122 (a selective phospholipase C inhibitor), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (a Ca(2+) chelator), but not pertussis toxin. The calcium ionophore, ionomycin, inhibited isoproterenol-mediated cAMP generation, similar to bradykinin. Interestingly, the inhibitory effect of bradykinin was evident only during the daytime. At midday, bradykinin inhibited the cAMP level by approximately 50% but markedly stimulated cAMP production (by approximately 50%) at night. Northern blotting and immunoblotting data disclosed circadian expression of calcium-inhibitable adenylyl cyclase type 6. Expression of adenylyl cyclase type 6 was maximal at Zeitgeber Time (ZT) 01 and very low at ZT 13. Our results suggest that bradykinin-induced calcium inhibits melatonin synthesis through the mediation of adenylyl cyclase type 6 expression.  (+info)

Protein kinase structure and function analysis with chemical tools. (11/63)

Protein kinases are the largest enzyme superfamily involved in cell signal transduction and represent therapeutic targets for a range of diseases. There have been intensive efforts from many labs to understand their catalytic mechanisms, discover inhibitors and discern their cellular functions. In this review, we will describe two approaches developed to analyze protein kinases: bisubstrate analog inhibition and phosphonate analog utilization. Both of these methods have been used in combination with the protein semisynthesis method expressed protein ligation to advance our understanding of kinase-substrate interactions and functional elucidation of phosphorylation. Previous work on the nature of the protein kinase mechanism suggests it follows a dissociative transition state. A bisubstrate analog was designed against the insulin receptor kinase to mimic the geometry of a dissociative transition state reaction coordinate distance. This bisubstrate compound proved to be a potent inhibitor against the insulin receptor kinase and occupied both peptide and nucleotide binding sites. Bisubstrate compounds with altered hydrogen bonding potential as well as varying spacers between the adenine and the peptide demonstrate the importance of the original design features. We have also shown that related bisubstrate analogs can be used to potently block serine/threonine kinases including protein kinase A. Since many protein kinases recognize folded protein substrates for efficient phosphorylation, it was advantageous to incorporate the peptide-ATP conjugates into protein structures. Using expressed protein ligation, a Src-ATP conjugate was produced and shown to be a high affinity ligand for the Csk tyrosine kinase. Nonhydrolyzable mimics of phosphoSer/phosphoTyr can be useful in examining the functionality of phosphorylation events. Using expressed protein ligation, we have employed phosphonomethylene phenylalanine and phosphonomethylene alanine to probe the phosphorylation of Tyr and Ser, respectively. These tools have permitted an analysis of the SH2-phosphatases (SHP1 and SHP2), revealing a novel intramolecular stimulation of catalytic activity mediated by the corresponding phosphorylation events. They have also been used to characterize the cellular regulation of the melatonin rhythm enzyme by phosphorylation.  (+info)

Norepinephrine activates store-operated Ca2+ entry coupled to large-conductance Ca2+-activated K+ channels in rat pinealocytes. (12/63)

Norepinephrine (NE) is one of the major neurotransmitters that determine melatonin production in the pineal gland. Although a substantial amount of Ca(2+) influx is triggered by NE, the Ca(2+) entry pathway and its physiological relevance have not been elucidated adequately. Herein we report that the Ca(2+) influx triggered by NE significantly regulates the protein level of serotonin N-acetyltransferase, or arylalkylamine N-acetyltransferase (AANAT), a critical enzyme in melatonin production, and is responsible for maintaining the Ca(2+) response after repetitive stimulation. Ca(2+) entry evoked by NE was dependent on PLC activation. NE evoked a substantial amount of Ca(2+) entry even after cells were treated with 1-oleoyl-2-acetyl-sn-glycerol (OAG), an analog of diacylglycerol. To the contrary, further OAG treatment after cells had been exposed to OAG did not evoke additional Ca(2+) entry. Moreover, NE failed to induce further Ca(2+) entry after the development of Ca(2+) entry induced by thapsigargin (Tg), suggesting that the pathway of Ca(2+) entry induced by NE might be identical to that of Tg. Interestingly, Ca(2+) entry evoked by NE or Tg induced membrane hyperpolarization that was reversed by iberiotoxin (IBTX), a specific inhibitor of large-conductance Ca(2+)-activated K(+) (BK) channels. Moreover, IBTX-sensitive BK current was observed during application of NE, suggesting that activation of the BK channels was responsible for the hyperpolarization. Furthermore, the activation of BK channels triggered by NE contributed to regulation of the protein level of AANAT. Collectively, these results suggest that NE triggers Ca(2+) entry coupled to BK channels and that NE-induced Ca(2+) entry is important in the regulation of AANAT.  (+info)

The candidate gene, Clock, localizes to a strong spawning time quantitative trait locus region in rainbow trout. (13/63)

We applied a candidate gene mapping approach to an existing quantitative trait loci (QTL) data set for spawning date in rainbow trout (Oncorynchus mykiss) to ascertain whether these genes could potentially account for any observed QTL effects. Several genes were chosen for their known or suspected roles in reproduction, circadian, or circannual timing, including salmon-type gonadotropin-releasing hormone 3A and 3B (GnRH3A and GnRH3B), Clock, Period1, and arylalkylamine N-acetlytransferase-1 and -2 (AANAT-1 and AANAT-2). Genes were sequenced, and polymorphisms were identified in parents of two rainbow trout mapping families, one of which was used previously to detect spawn timing QTL. Interval mapping was used to identify associations between genetic markers and spawning date effects. Using a genetic map that was updated with 574 genetic markers (775 total), we found evidence for 11 significant or suggestive QTL regions. Most QTL were only localized within one of the parents; however, a strong QTL region was identified in both female and male parents on linkage group RT-8 that explained 20% and 50% of trait variance, respectively. The Clock gene mapped to this region. Period1 mapped to a region in the female parent associated with a marginal effect (P = .056) on spawn timing. Other candidate genes were not associated with significant QTL effects.  (+info)

Intraocular injection of kainic acid does not abolish the circadian rhythm of arylalkylamine N-acetyltransferase mRNA in rat photoreceptors. (14/63)

PURPOSE: Melatonin synthesis in mammalian retinal photoreceptors is under photic and circadian control and regulated by changes in the activity of arylalkylamine N-acetyltransferase (AANAT). Recent studies have suggested that retinal dopaminergic neurons contain a circadian pacemaker, and dopamine is the neurotransmitter that drives circadian rhythmicity in the mammalian retina. METHODS: To investigate the role of inner retinal neurons, including dopamine neurons, in generating the rhythm of melatonin synthesis, rat retinas were lesioned with kainic acid (KA), which was shown previously to induce degeneration of neurons in the inner nuclear layer and to eliminate rhythmicity in the dopaminergic system. Aanat, rhodopsin, medium wavelength (mwl) opsin, short wavelength (swl) opsin, and period1 (Per1), and period2 (Per2) mRNA levels were measured using real-time quantitative RT-PCR in KA injected and control eyes. RESULTS: Our data show that intraocular injections of KA did not abolish the daily and circadian rhythms of Aanat mRNA in the photoreceptors, but it did shift the phase of the Aanat transcript rhythm in constant darkness. Surprisingly, KA injections reduced the levels and eliminated daily rhythms of mwl and swl opsin transcripts, but not of rhodopsin mRNA. Per1 and Per2 mRNA levels were rhythmic in saline injected and in KA-treated retinas, and Per2 mRNA levels were significantly reduced (20-50%) in KA-treated retinas. CONCLUSIONS: These findings demonstrate that the circadian clock generating melatonin rhythmicity is largely KA insensitive and likely to be located in the rod photoreceptors, although KA-sensitive neurons do influence its timing. More important, our data demonstrate that dopamine rhythmicity is not necessary for generating the circadian rhythm of Aanat mRNA in the photoreceptors. Our data also indicate that Per1 and Per2 are rhythmically transcribed in the rat retina and KA treatment has a dramatic effect on the overall levels of Per2 mRNA.  (+info)

Circadian time-keeping during early stages of development. (15/63)

The zebrafish pineal gland is a photoreceptive organ containing an intrinsic central circadian oscillator, which drives daily rhythms of gene expression and the melatonin hormonal signal. Here we investigated the effect of light, given at early developmental stages before pineal gland formation, on the pineal circadian oscillator. Embryos that were exposed to light at 0-6, 10-13, or 10-16 h after fertilization exhibited clock-controlled rhythms of arylalkylamine-N-acetyltransferase (zfaanat2) mRNA in the pineal gland during the third and fourth day of development. This rhythm was absent in embryos that were placed in continuous dark within 2 h after fertilization (before blastula stage). Differences in the phases of these rhythms indicate that they are determined by the time of illumination. Light treatments at these stages also caused a transient increase in period2 mRNA levels, and the development of zfaanat2 mRNA rhythm was abolished by PERIOD2 knock-down. These results indicate that light exposure at early developmental stages, and light-induced expression of period2, are both required for setting the phase of the circadian clock. The 24-h rhythm is then maintained throughout rapid proliferation and, remarkably, differentiation.  (+info)

Zebrafish arylalkylamine-N-acetyltransferase genes - targets for regulation of the circadian clock. (16/63)

Daily rhythms of melatonin production are controlled by changes in the activity of arylalkylamine-N-acetyltransferase (AANAT). Zebrafish possess two aanats, aanat1 and aanat2; the former is expressed only in the retina and the latter is expressed in both the retina and the pineal gland. Here, their differential expression and regulation were studied using transcript quantification and transient and stable in vivo and in vitro transfection assays. In the pineal gland, the aanat2 promoter exhibited circadian clock-controlled activity, as indicated by circadian rhythms of Enhanced green fluorescent protein (EGFP) mRNA in AANAT2:EGFP transgenic fish. In vivo transient expression analyses of the aanat2 promoter indicated that E-box and photoreceptor conserved elements (PCE) are required for expression in the pineal gland. In the retina, the expression of both genes was characterized by a robust circadian rhythm of their transcript levels. In constant darkness, the rhythmic expression of retinal aanat2 persisted while the aanat1 rhythm disappeared; indicating that the former is controlled by a circadian clock and the latter is also light driven. In the light-entrainable clock-containing PAC-2 zebrafish cell line, both stably transfected aanat1 and aanat2 promoters exhibited a clock-controlled circadian rhythm, characteristic for an E-box-driven expression. Transient co-transfection experiments in NIH-3T3 cells revealed that the two, E-box- and PCE-containing, promoters are driven by the synergistic action of BMAL/CLOCK and orthehodenticle homeobox 5. This study has revealed a shared mechanism for the regulation of two related genes, yet describes their differential phases and photic responses which may be driven by other gene-specific regulatory mechanisms and tissue-specific transcription factor profiles.  (+info)