Retinal tyrosine hydroxylase: comparison of short-term and long-term stimulation by light

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Retinal tyrosine hydroxylase: comparison of short-term and long-term stimulation by light

Dopamine (DA) is a putative neurotransmitter within some retinal amacrine neurons. Exposure of rats to light for either 15 min or 96 hr increases retinal tyrosine hydroxylase activity. Concomitant with the increase of enzyme activity is a 4-fold increase of DA formation. The molecular mechanism for the increased enzyme activity for the two exposures to light is apparently different. Short-term exposure to light decreases the K(m) but not the V(max) of the enzyme for the pteridine cofactor, while 96 hr of exposure to light increases the V(max) for tyrosine but has no significant effect on the K(m) for tyrosine or cofactor. Enzyme activity of rats exposed to 15 min of light decreases to the level found in 96 hr dark-adapted rats within 30 min of darkness, while more than 6 hr of darkness are required after 96 hr of light exposure. These studies are consistent with the hypothesis that retinal DA formation is modulated by different molecular mechanisms depending on the duration of exposure of light. Short-term exposure to light activates tyrosine hydroxylase while long-term exposure to light results in the formation of more active molecules of enzyme

Expression of cMOP4, a putative clock component in retina, pineal gland and peripheral tissues

Purpose: The avian circadian system is regulated by a multi-oscillatory system consisting of clocks in the retina, the pineal gland and the hypothalamus. In chickens, retinal and pineal circadian clocks govern the daily rhythmic secretion of melatonin via clock- dependent expression of arylalkylamine N-acetyltransferase (AANAT). In order to further characterize the avian circadian pacemaking system, we have examined the temporal expression patterns of the putative clock gene, cMOP4, in the retina, pineal gland and peripheral tissues. Methods: One-day old chicks were housed for two weeks on a 12 h light/12 h dark (LD) cycle, with lights on at zeitgeber time (ZT) 0. Following this, the animals were transferred to constant (24 h/day) light (LL) or darkness (DD). The animals were sacrificed at the times indicated in the figures. A partial fragment of cMOP4 (approx. 1.2 Kb) was amplified using degenerate-PCR and was used for northern hybridization. Total RNA was reverse transcribed using M-MLV reverse transcriptase. cDNA from each sample was amplified with specific primers for cMOP4 using quantitative real time RT-PCR. Results: Real time RT-PCR and northern blot analyses indicate that cMOP4 is widely expressed in neuronal and peripheral tissues. Retina expresses two transcripts of cMOP4, with the sizes of 4.7 Kb and 7.0 Kb. Retina and pineal gland express robust rhythms of cMOP4 mRNA with peak levels at ZT 12 in LD, LL and DD. cMOP4 mRNA is also rhythmically expressed in liver and heart. However, the peak of cMOP4 mRNA is 4 h delayed in liver and 4 h advanced in heart as compared to the retina and pineal gland. Conclusions: Our results indicate that cMOP4 expression is controlled as a circadian rhythm. cMOP4 may serve as a component of the central oscillator in retinal and pineal clocks and/or as an output mechanism, coupling the circadian clocks to rhythmic physiology

Circadian expression of Bmal1 in chicken retina, pineal gland, and peripheral tissues

Purpose: Circadian rhythms are generated in pacemaker cells and are entrained by environmental cues such as the light:dark (LD) cycle. The chicken retina and pineal gland contain endogenous clocks that govern the rhythmic synthesis of melatonin. The dynamic circadian control of melatonin production occurs in part through clock-controlled regulation of arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the melatonin biosynthetic pathway. The transcription of the chicken AANAT gene is regulated directly by the interaction of circadian transcription factors with the AANAT promoter. The aim of the present study was to examine the temporal expression pattern of Bmal1, one of the clock genes implicated in AANAT induction, in chick retina, pineal gland and peripheral tissues. Methods: Newborn chickens were housed for two weeks on a 12 hr light/12 hr dark (LD) cycle with light on at zeitgeber time (ZT) 0. A fragment of cBmal1 cDNA (1.9 kb) was used as a probe for northern hybridization. Northern blot and quantitative real-time RT-PCR analyses were performed to investigate the temporal expression patterns of Bmal1 in LD, in constant light (LL), and in constant darkness (DD). Tissue distribution of chicken Bmal1 splice variants was studied by RT-PCR and Southern blot analysis. Results: Northern blot and RT-PCR analyses showed that cBmal1 mRNA is expressed in a circadian manner in the retina, with high levels during early subjective night (ZT12) in both LL and DD. The phase of the circadian rhythm of cBmal1 mRNA in DD was reversed by reversing the prior LD cycle, indicative of photoentrainment. The expression of the Bmal1 is also rhythmic in the pineal gland, with a similar phase relationship to that observed in retina. Rhythmic expression of Bmal1 was also observed in heart and liver, but the peaks of Bmal1 mRNA are 8 h delayed in the liver and 4 h advanced in the heart relative to the Bmal1 peaks in retina and pineal gland. Alternatively spliced variants of cBmal1, with Bmal1b' the primary splice variant, are expressed in retina and other tissues. Conclusions:Our results indicate that cBMAL1 is widely expressed in the chick and may function as a dimeric partner of MOP4 and CLOCK for control of clock-related physiology in central and peripheral tissues

Retinal melatonin biosynthesis: interactions of A/T–rich regions and CRE–like sequences contribute to cAMP–dependent regulation of the chicken AANAT promoter

Purpose:Arylalkylamine N–acetyltransferase (AANAT) is the key regulatory enzyme in the biosynthesis of melatonin. Previous studies have shown that AANAT activity and the abundance of AANAT mRNA in the chicken photoreceptor cells are regulated by cAMP–dependent mechanisms. The purpose of this study was to identify regulatory elements within the AANAT gene promoter responsible for cAMP–dependent induction. Methods: Photoreceptor–enriched retinal cell cultures were prepared from 6–day–old embryos and were incubated for 5 days. Cells were transfected with pGL3 plasmid containing the wild type and mutated AANAT promoter constructs fused to luciferase. To examine the influence of cAMP, cells were treated with 10µM forskolin and incubated for 6 h before harvesting for measurement of luciferase activity. For DNA–protein binding experiments, electrophoretic mobility shift assay (EMSA) of nuclear proteins was performed with wild type and mutated oligonucleotides. Results:Forskolin–treatment stimulated luciferase activity driven by a 4 kb reporter construct and all 5’–deletion constructs except the smallest, AANAT (–217 to +120)luc. Both the basal and forskolin–stimulated expression levels were maximal with AANAT (–484 to +120)luc, which contains an 8 x TTATT repeat (TTATT8 ) and three CRE–like sequences, designated CLS1–3. Basal expression of AANAT (–217 to +120)luc, which does not contain the TTATT8 sequence, was minimal and unresponsive to forskolin. EMSA demonstrated a number of nuclear protein complexes that bind to the TTATT8 sequence and CLS1–3. Proteins that bound to the TTATT8 sequence were displaced by unlabeled TTATT oligonucleotides, and also by an oligonucleotide containing CLS1, suggesting an interaction between these sites. Mutations of TTATT8 or CLS1 reduced protein binding and eliminated forskolin–stimulated promoter activity. Supershift EMSA using c–Fos and delta CREB antibodies identified these factors as probable components of the protein complexes that bind to the TTATT8 and CLS sites. Conclusions:Transcription driven by this promoter is enhanced by activation of the cAMP/PKA signaling cascade. This appears to be mediated by CLS elements in the promoter acting in concert with the TTATT8 motif, and that combination mediates the full cAMP–induced transcriptional response of a 484 bp segment of AANAT proximal promoter, in the absence of a consensus CRE

Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons

Retinal dopamine-containing amacrine neurons are rapidly activated by light, as shown by an increase in the rate of dopamine formation in vivo and a concomitant increase in the activity of tyrosine hydroxylase, measured in vitro with a subsaturating concentration of pteridine cofactor. Activation of tyrosine hydroxylase also occurs when isolated eyes from rats killed in the dark are exposed to a strobe light. Studies of amacrine neurons should provide basic data about the biochemical processing of visual information, as well as the physiological presynaptic regulatory mechanisms of dopamine-containing neurons

Circadian expression of tryptophan hydroxylase mRNA in the chicken retina

Many aspects of retinal physiology are controlled by a circadian clock located within the eye. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate-limiting enzyme in melatonin biosynthesis in retina appears to be tryptophan hydroxylase (TPH)[G.M. Cahill and J.C. Besharse, Circadian regulation of melatonin in the retina of Xenopus laevis: Limitation by serotonin availability, J. Neurochem. 54 (1990) 716-719]. In this report, we found that TPH mRNA is strongly expressed in the photoreceptor layer and the vitread portion of the inner nuclear layer; the message is also expressed, but to a lesser extent, in the ganglion cell layer. The abundance of retinal TPH mRNA exhibits a circadian rhythm which persists in constant light or constant darkness. The phase of the rhythm can be reversed by reversing the light:dark cycle. In parallel experiments we found a similar pattern of expression in the chicken pineal gland. However, whereas a pulse of light at midnight suppressed retinal TPH mRNA by 25%, it did not alter pineal TPH mRNA, suggesting that there are tissue-specific differences in photic regulation of TPH mRNA. In retinas treated with kainic acid to destroy serotonin-containing amacrine and bipolar cells, a high amplitude rhythm of TPH mRNA was observed indicating that melatonin-synthesizing photoreceptors are the primary source of the rhythmic message. These observations provide the first evidence that chick retinal TPH mRNA is under control of a circadian clock