Serotonin modulates melatonin synthesis as an autocrine neurotransmitter in the pineal gland

The pineal gland secretes melatonin principally at night. Regulated by norepinephrine released from sympathetic nerve terminals, adrenergic receptors on pinealocytes activate aralkylamine N-acetyltransferase that converts 5-hydroxytryptamine (5-HT, serotonin) to N-acetylserotonin, the precursor of melatonin. Previous studies from our group and others reveal significant constitutive secretion of 5-HT from pinealocytes. Here, using mass spectrometry, we demonstrated that the 5-HT is secreted primarily via a decynium-22–sensitive equilibrative plasma membrane monoamine transporter instead of by typical exocytotic quantal secretion. Activation of the endogenous 5-HT receptors on pinealocytes evoked an intracellular Ca²⁺ rise that was blocked by RS-102221, an antagonist of 5-HT_2C receptors. Applied 5-HT did not evoke melatonin secretion by itself, but it did potentiate melatonin secretion evoked by submaximal norepinephrine. In addition, RS-102221 reduced the norepinephrine-induced melatonin secretion in strips of pineal gland, even when no exogenous 5-HT was added, suggesting that the 5-HT that is constitutively released from pinealocytes accumulates enough in the tissue to act as an autocrine feedback signal sensitizing melatonin release.

The principal role of pinealocytes of the pineal gland (PG) is to synthesize and secrete the hormone melatonin, driven by the neurotransmitter norepinephrine (NE) released at night from sympathetic nerve terminals. Acting through α₁- and β₁-adrenergic receptors on pinealocytes, the adrenergic signal generates Ca²⁺ and cyclic AMP (cAMP) to activate up to 150-fold (in rat) a key synthetic enzyme aralkylamine N-acetyltransferase (AANAT) (1). This signaling pathway is shown in Fig. 1. AANAT in turn converts serotonin (5-HT) from a constitutive pool to generate N-acetylserotonin (NAS), from which hydroxyindole-O-methyltransferase generates melatonin (1). The secretion of melatonin and NAS can be increased 100-fold at night (2). In addition to 5-HT, many other neurotransmitters are present elsewhere in the PG, including acetylcholine (ACh), glutamate, GABA, and neuropeptides (3). Their roles in modulating the activity of pinealocytes and melatonin synthesis remain to be established. Here we focus on modulatory effects of 5-HT.Open in a separate windowFig. 1.Synthesis and release of 5-HT and melatonin in rat pinealocytes. The enzymatic pathway from L-tryptophan to 5-HT and melatonin uses four enzymes. AANAT is regulated by adrenergic signaling. Two possibilities are shown (red dotted arrows) for the mechanism of release of 5-HT from pinealocytes: through vesicular exocytosis or through monoamine transporters running in reverse (orange). Released serotonin activates serotonin receptors (blue) on the plasma membrane of pinealocytes. We are suggesting that the receptors up-regulate AANAT acting as an auto- or paracrine mechanism to promote melatonin synthesis. AADC, aromatic amino acid decarboxylase; HIOMT, hydroxyindole-O-methyltransferase; TPH, tryptophan hydroxylase.Serotonergic signaling plays diverse roles in the CNS and peripheral tissues. It is critically involved in mood control, the sleep–wake cycle, breathing, locomotion, and more (4, 5) and acts through 14 distinct, mostly G protein–coupled 5-HT receptors. In the mammalian PG, 5-HT content shows a circadian rhythm, higher during daytime and somewhat lower at night while melatonin is being produced (6, 7). Compared to melatonin release, the 5-HT release from the gland shows only modest (±twofold) circadian variation, continuing in appreciable amounts throughout (8–10). The large pineal pool of 5-HT is continually turning over through constitutive synthesis and secretion (11). Exogenous NE increases 5-HT production and secretion in PGs partly by elevating tryptophan hydroxylase activity (10, 12–17).Secretion of 5-HT from the PG is larger than the elevated nocturnal release of melatonin (9, 10), but the physiological significance of this secretion has not been clear. Released 5-HT is suggested to prime the cAMP-dependent activation of AANAT for melatonin synthesis (17–20). We tested the hypothesis that 5-HT secretion acts as an autocrine signal to promote melatonin synthesis and secretion (Fig. 1) by answering the following questions: What is 1) the secretory mechanism, 2) the type of 5-HT receptor used, and 3) the effect of 5-HT on melatonin secretion? Using optical, electrical, and mass spectrometric (MS) measurements, we found that 5-HT is released from pinealocytes via a nonconventional mechanism and sensitizes NE-induced melatonin synthesis and secretion by activating 5-HT_2C receptors present in the PG. We show that 5-HT qualifies as an autocrine transmitter in the PG.