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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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Madame Curie Bioscience Database [Internet].

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Diurnal 5-HT Production and Melatonin Formation

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We have provided evidence that pineal 5-hydroxytryptomine (5-HT or serotonin) production is up regulated at night, and is controlled by beta-adrenergic signaling.1 In this paper, we demonstrate that the increased 5-HT synthesis is due to increased protein expression of tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT synthesis.

Melatonin is synthesized from dietary tryptophan through actions of four enzymes (see Fig. 3 and ref. 2). Tryptophan hydroxylase (TPH) controls the first step of the pathway in which tryptophan is converted to 5-hydroxytryptophan (5-HTP). Aromatic amino acid decarboxylase (AADC) catalyzes the conversion of 5-HTP to 5-hydroxytryptamine (5-HT, serotonin). Melatonin formation from 5-HT requires two pineal/retina specific enzymes: serotonin N-acetyltransferase (NAT), which forms N-acetylserotonin (NAS) from serotonin, and hydroxyindole-O-methyltransferase (HIOMT), which produces melatonin from NAS. Of the four enzymes involved, NAT has long been viewed as the “rate-limiting” enzyme for melatonin production, due to its diurnal pattern of activity, and has been extensively analyzed.2 In comparison, the regulation of TPH and its contribution in melatonin production is less well understood. A few decades ago, TPH, the rate-limiting enzyme for 5-HT production, was found to be diurnally regulated in the rat pineal with a two-fold increase in activity at night.3,4 Recently, we have shown that 5-HT synthesis is increased early at night, and that the increase in 5-HT production is abolished when beta-adrenergic signaling is blocked.1

Figure 3. Scheme of signal transduction pathways for pineal 5-HT and melatonin synthesis and release.

Figure 3

Scheme of signal transduction pathways for pineal 5-HT and melatonin synthesis and release.

In this study, we analyze TPH mRNA and protein expression in the rat pineal gland and compare it with the expression of two rhythmically expressed messages patched 15 and PL22 (manuscript submitted), normalized to GAPDH control. We find no detectable difference in TPH mRNA levels throughout a diurnal cycle (Fig. 1). In contrast, TPH protein levels vary diurnally as shown in (Fig. 2). The fact that TPH protein levels increase immediately after the lights are turned off (Fig. 2) and that 5-HT production increases within 20 min of lights-off1 supports the idea that post-transcriptional mechanisms are responsible for the increased 5-HT production at night in the rat pineal gland.

Figure 1. TPH mRNA expression in the rat pineal assayed by Northern blot analysis.

Figure 1

TPH mRNA expression in the rat pineal assayed by Northern blot analysis. Pineal glands of adult (8 wks) male Sprague Dawley rats, housed in a temperaturecontrolled room under 14:10 h light/dark cycle, were harvested at the indicated times. Total RNA from (more...)

Figure 2. TPH protein expression in the rat pineal assayed by Western analysis.

Figure 2

TPH protein expression in the rat pineal assayed by Western analysis. Pineals at indicated times were harvested as in (Fig. 1). Electroblotted protein extracts from 1/5 of a single pineal per lane were probed with anti-TPH antibody (Calbiochem, Cat#OP71L). (more...)

A number of studies have demonstrated an increase in TPH activity in rat pineals at night.3,4,6 It is also well established that beta-adrenergic signaling activates the increase in TPH activity,4,7 and 5-HT synthesis.1 Furthermore, purified rat brain TPH is phosphorylated in vitro by cAMP-dependent protein kinase (PKA),6,8 and beta-adrenergic signaling leads to an increase in intracellular cAMP levels in night pineal gland.2 On the other hand, alpha-adrenergic signaling has been shown to increase 5-HT secretion in vitro9,10 and in vivo.1 These data support the model of intracellular control of 5-HT synthesis and release shown in (Fig. 3). Nighttime release of norepinephrine activates both alpha- and beta-adrenergic receptors. Beta-adrenergic receptor, upon stimulation induces an increase in intracellular cAMP level that serves to stimulate NAT transcription and increase NAT protein stability, which leads to increased metabolism of 5-HT. In addition to the beta-receptor mediated increase in 5-HT consumption, activated alpha-adrenoceptor increases 5-HT secretion.1 Both types of adrenergic signaling effectively lower the intracellular concentration of 5-HT. It is no wonder, then that the pineal has developed a simultaneous mechanism to increase 5-HT production required for melatonin biosynthesis. Studies mentioned above and presented in this paper suggest that cAMP signaling increases 5-HT synthesis via either stimulation of TPH protein synthesis or stabilization of TPH protein level.

References

1.
Sun X, Deng J, Liu T. et al. Circadian 5-HT production regulated by adrenergic signaling. Proc Natl Acad Sci USA. 2002;99:4686–4691. [PMC free article: PMC123708] [PubMed: 11917109]
2.
Borjigin J, Li X, Snyder SH. The pineal gland and melatonin: Molecular and pharmacologic regulation. Annu Rev Pharmacol Toxicol. 1999;39:53–65. [PubMed: 10331076]
3.
Sitaram BR, Lees GJ. Diurnal rhythm and turnover of tryptophan hydroxylase in the pineal gland of the rat. J Neurochem. 1978;31:1021–1026. [PubMed: 151732]
4.
Shibuya H, Toru M, Watanabe S. A circadian rhythm of tryptophan hydroxylase in rat pineals. Brain Res. 1978;138:364–368. [PubMed: 589481]
5.
Borjigin J, Deng J, Wang MM. et al. Circadian rhythm of patched1 transcription in the pineal regulated by adrenergic stimulation and cAMP. J Biol Chem. 1999;274:35012–35015. [PubMed: 10574978]
6.
Ehret M, Pevet P, Maitre M. Tryptophan hydroxylase synthesis is induced by 3',5'-cyclic adenosine monophosphate during circadian rhythm in the rat pineal gland. J Neurochem. 1991;57:1516–1521. [PubMed: 1655976]
7.
Toru M, Watanabe S, Nishikawa T. et al. Physiological and pharmacological properties of circadian rhythm of tryptophan hydroxylase in rat pinealsIn: Passouant P, Oswald I, eds.Advances in Biosciences, Vol. 21: Pharmacology of the States of AlertnessOxford: Pergamon Press,1979253–255. [PubMed: 755723]
8.
Johnansen PA, Jennings I, Cotton RGH. et al. Tryptophan hydroxylase is phosphorylated by protein kinase A. J Neurochem. 1995;65:882–888. [PubMed: 7616249]
9.
Aloyo VJ, Walker RF. Alpha-adrenergic control of serotonin release from rat pineal glands. Neuroendocrinology. 1988;48:61–66. [PubMed: 2845293]
10.
Miguez JM, Simonneaux V, Pevet P. The role of the intracellular and extracellular serotonin in the regulation of melatonin production in rat pinealocytes. J Pineal Res. 1997;23:63–71. [PubMed: 9392444]
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