Diurnal 5-HT Production and Melatonin Formation

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

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.

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 vitro^9,10 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.

Figures

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 single pineals was loaded in each lane, electrophoresed, blotted, and probed as indicated.

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). Nighttime levels of TPH were normalized against the daytime average. The data is representative of five independent experiments.

Figure 3

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