The activity of N-acetyltransferase in the rat pineal gland is more than 15 times higher at night than during the day. This circadian rhythm persists in complete darkness, or in blinded animals, and is suppressed in constant lighting. The N-acetyltransferase rhythm is 180 degrees out of phase with the serotonin rhythm and is similar to the norepinephrine and melatonin rhythms. Experiments in vitro indicate that norepinephrine, not serotonin, regulates the activity of N-acetyl-transferase through a highly specific receptor.

The pineal gland plays an essential role in vertebrate chronobiology by converting time into a hormonal signal, melatonin, which is always elevated at night. Here we have analyzed the rodent pineal transcriptome using Affymetrix GeneChip technology to obtain a more complete description of pineal cell biology. The effort revealed that 604 genes (1,268 probe sets) with Entrez Gene identifiers are differentially expressed greater than 2-fold between midnight and mid-day (false discovery rate <0.20). Expression is greater at night in ϳ70%. These findings were supported by the results of radiochemical in situ hybridization histology and quantitative real time-PCR studies. We also found that the regulatory mechanism controlling the night/ day changes in the expression of most genes involves norepinephrine-cyclic AMP signaling. Comparison of the pineal gene expression profile with that in other tissues identified 334 genes (496 probe sets) that are expressed greater than 8-fold higher in the pineal gland relative to other tissues. Of these genes, 17% are expressed at similar levels in the retina, consistent with a common evolutionary origin of these tissues. Functional categorization of the highly expressed and/or night/day differentially expressed genes identified clusters that are markers of specialized functions, including the immune/inflammation response, melatonin synthesis, photodetection, thyroid hormone signaling, and diverse aspects of cellular signaling and cell biology. These studies produce a paradigm shift in our understanding of the 24-h dynamics of the pineal gland from one focused on melatonin synthesis to one including many cellular processes.A defining feature of the pineal gland is a 24-h rhythm in melatonin synthesis. Melatonin provides vertebrates with a circulating signal of time and is essential for optimal integration of physiological functions with environmental lighting on a daily and seasonal basis (1-4).The melatonin rhythm in mammals is driven by a circadian clock located in the suprachiasmatic nucleus (SCN), 13 which is hard-wired to the pineal gland by a polysynaptic pathway that courses through central and peripheral neuronal structures. The pineal gland is innervated by projections from the superior cervical ganglia (SCG) in the form of a dense network of catecholamine-containing sympathetic fibers. Activation of the SCN 3 pineal pathway occurs at night and results in the release

Pineal serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, or AA-NAT) generates the large circadian rhythm in melatonin, the hormone that coordinates daily and seasonal physiology in some mammals. Complementary DNA encoding ovine AA-NAT was cloned. The abundance of AA-NAT messenger RNA (mRNA) during the day was high in the ovine pineal gland and somewhat lower in retina. AA-NAT mRNA was found unexpectedly in the pituitary gland and in some brain regions. The night-to-day ratio of ovine pineal AA-NAT mRNA is less than 2. In contrast, the ratio exceeds 150 in rats. AA-NAT represents a family within a large superfamily of acetyltransferases.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N -acetyltransferase (AANAT; serotonin N -acetyltransferase, EC 2.3.1.87 ). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN → RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the K m for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

The nocturnal increase in circulating melatonin in vertebrates is regulated by 10- to 100-fold increases in pineal serotonin N-acetyltransferase (AA-NAT) activity. Changes in the amount of AA-NAT protein were shown to parallel changes in AA-NAT activity. When neural stimulation was switched off by either light exposure or L-propranolol-induced beta-adrenergic blockade, both AA-NAT activity and protein decreased rapidly. Effects of L-propranolol were blocked in vitro by dibutyryl adenosine 3',5'-monophosphate (cAMP) or inhibitors of proteasomal proteolysis. This result indicates that adrenergic-cAMP regulation of AA-NAT is mediated by rapid reversible control of selective proteasomal proteolysis. Similar proteasome-based mechanisms may function widely as selective molecular switches in vertebrate neural systems.

The nocturnal increase in circulating melatonin in vertebrates is regulated by the activity of arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the melatonin pathway (serotonin 3 N-acetylserotonin 3 melatonin). Large changes in activity are linked to cyclic AMP-dependent protein kinase-mediated phosphorylation of AANAT T31. Phosphorylation of T31 promotes binding of AANAT to the dimeric 14-3-3 protein, which activates AANAT by increasing arylalkylamine affinity. In the current study, a putative second AANAT cyclic AMP-dependent protein kinase phosphorylation site, S205, was found to be Ϸ55% phosphorylated at night, when T31 is Ϸ40% phosphorylated. These findings indicate that ovine AANAT is dual-phosphorylated. Moreover, light exposure at night decreases T31 and S205 phosphorylation, consistent with a regulatory role of both sites. AANAT peptides containing either T31 or S205 associate with 14-3-3 in a phosphorylation-dependent manner; binding through phosphorylated (p)T31 is stronger than that through pS205, consistent with the location of only pT31 in a mode I binding motif, one of two recognized high-affinity 14-3-3-binding motifs AANAT protein binds to 14-3-3 through pT31 or pS205. Two-site binding lowers the K m for arylalkylamine substrate to Ϸ30 M. In contrast, single-site pS205 binding increases the K m to Ϸ1,200 M. Accordingly, the switch from dual to single pS205 binding of AANAT to 14-3-3 changes the K m for substrates by Ϸ40-fold. pS205 seems to be part of a previously unrecognized 14-3-3-binding motif-pS͞pT (X 1-2)-COOH, referred to here as mode III.pineal ͉ circadian ͉ cAMP ͉ kinase T he daily rhythm in circulating melatonin is a highly conserved feature of vertebrate physiology; in all cases, high levels occur at night. Day͞night differences in circulating melatonin levels provide a hormonal analog signal of environmental lighting, which is used in a variety of ways to optimize circadian and circannual rhythms in physiology (1). The melatonin rhythm is controlled by large changes in the rate of melatonin production in the pineal gland; these changes reflect changes in the penultimate enzyme in melatonin synthesis, arylalkylamine Nacetyltransferase (AANAT) (2, 3). Changes in the activity of this enzyme are strongly influenced by cAMP-dependent binding to the bowl-shaped, dimeric 14-3-3 protein (4, 5). Binding is thought to protect the enzyme against proteasomal proteolysis (6, 7); moreover, binding to 14-3-3 increases the affinity of AANAT by Ϸ10-fold for arylalkylamine substrates, e.g., serotonin and tryptamine (4, 5).Pinealocyte cAMP is controlled in mammals through a photoneural system, which includes the eyes and the suprachiasmatic nucleus, the site of the master circadian clock (1). In lower vertebrates, cAMP is regulated by light acting directly on pinealocytes (8). It is likely that cAMP-dependent binding to 14-3-3 occurs in all vertebrates, based on the ubiquitous presence of 14-3-3 proteins and the presence of two putative cyclic AMP-dependent protein kinase (PKA) sit...

The adrenergic control of cAMP and 3',5'-cyclic GMP (cGMP) in dispersed adult rat pinealocytes was investigated. Norepinephrine treatment increased cAMP and cGMP content 60- and 400-fold, respectively; both alpha- and beta-adrenoceptors had to be activated for these responses to occur. Beta-Adrenergic stimulation alone produced only about 6- and 2-fold increase in cAMP and cGMP content, respectively. Alpha-Adrenergic stimulation, which alone had no effect on either cyclic nucleotide concentration, markedly amplified the beta-adrenergic stimulation of both cAMP and cGMP. The relative potency of alpha-adrenergic agonists and antagonists indicates the alpha 1-subclass of adrenoceptors is involved. A role of alpha 1-adrenoceptors in the control of pineal cAMP is consistent with published evidence of the presence of alpha 1-adrenoceptors on pinealocytes and their role in the regulation of N-acetyltransferase activity and melatonin production.

The role played by postsynaptic et-adrenergic receptors in the stimulation of pineal N-acetyltransferase (EC 2.3.1.5) and [3H]melatonin production was investigated in the rat. In vivo studies indicated that phenylephrine, an a-adrenergic agonist, potentiated and prolonged the effects of isoproterenol, a P adrenergic agonist. Similar observations were made in organ culture with glands devoid of functional nerve endings. In addition, a combination of 1 FAM prazosin, an a1-adrenergic blocking agent, and 1 FM propranolol, a -adrenergic blocking agent, was many times more potent then either agent alone in blocking the stimulatory effects of norepinephrine on N-acetyltransferase activity and [3Hlmelatonin production. These findings establish that norepinephrine acting through ar-and (3-adrenergic receptors stimulates rat pineal N-acetyltransferase activity and, as a result, the production of melatonin. Apparently, (-adrenergic activation is an absolute requirement, and an et-adrenergic receptor mechanism potentiates -adrenergic activation. These findings are significant because they demonstrate a-adrenergic potentiation of (-adrenergic effects. In addition, they indicate that the widely held belief that melatonin production is regulated exclusively by a postsynaptic -adrenergic mechanism must be revised.A daily rhythm in circulating melatonin occurs in all mammals (1). In the rat this is generated by a large rhythm in the activity of pineal arylamine N-acetyltransferase (EC 2.3.1.5) (1-4), the enzyme that converts serotonin to the immediate precursor of melatonin, N-acetylserotonin.The activity of N-acetyltransferase is regulated by a neural circuit that stimulates the nocturnal release of norepinephrine from sympathetic nerves in the pineal gland (5)(6)(7)(8). Norepinephrine acts through an adrenergic mechanism to increase intracellular cyclic AMP (9, 10) which induces and activates Nacetyltransferase (3, 4), leading to an increase in melatonin production (11,12).It is now generally thought, based on a number of studies (11)(12)(13)(14)(15)(16)(17), that norepinephrine acts exclusively through postsynaptic /3-adrenergic receptors to increase cyclic AMP, N-acetyltransferase-activity, and melatonin production. We were surprised, therefore, by recent observations (unpublished data) which seem to indicate that norepinephrine increases pineal cyclic AMP through an action involving both a-and (3-adrenergic receptors. We found that a combination of isoproterenol (10 ,tM) and phenylephrine (10 ,uM) produced a stimulation at 10 min, of cyclic AMP and cyclic GMP equivalent to that produced by norepinephrine (10 ,uM) and that the effect of phenylephrine (10 ,uM) or isoproterenol (10 ,uM) alone was <20% of that produced by the combination of isoproterenol (10 UM) and phenylephrine (10 MM).This raised the obvious possibility that N-acetyltransferase activity and melatonin production are also regulated by both aand (3adrenergic receptors located on postsynaptic structures. We have studied this question and our results, pr...