Spatial and temporal expression patterns of Bmal delineate a circadian clock in the nervous system of Branchiostoma lanceolatum.

We cloned the homologue of the clock gene Bmal from a cephalochordate, Branchiostoma lanceolatum (syn. amphioxus). Amphioxus possesses a single copy of this gene (amphiBmal) that encodes for a protein of 649 amino acids, which is quite similar to BMALs of other chordates. The gene is expressed by a restricted cell group in the anterior vesicle of the neural tube, and its expression site coincides with that of another clock gene, namely, amphiPer. The expression of amphiBmal shows a rhythmic fluctuation that persists under constant darkness and is, thus, circadian. Similar to the situation in craniates, the peak phases of the amphiBmal and amphiPer expression are offset by 12 hours. Based on these observations and the putative homology between the diencephalon of vertebrates and the anterior vesicle of lancelets, we suggest a homology between the suprachiasmatic nucleus of craniates and the amphiBmal/amphiPer-expressing cell group of amphioxus.

Nocturnal behavior and rhythmic period gene expression in a lancelet, Branchiostoma lanceolatum.

The authors here present the first anatomical, molecular biological, and ethological data on the organization of the circadian system of a lancelet, Branchiostoma lanceolatum, a close invertebrate relative of vertebrates. B. lanceolatum was found to be a nocturnal animal and, since its rhythmic activity persisted under constant darkness, it also appears to possess an endogenous, circadian oscillator. The authors cloned a homolog of the clock gene Period (Per), which plays a central (inhibitory) role in the biochemical machinery of the circadian oscillators of both vertebrates and protostomians. This gene from B. lanceolatum was designated as amphiPer. Both the sequence of its cDNA and that of the predicted protein are more similar to those of the Per paralogs of vertebrates than to those of the single protostomian Per gene. A strong expression of amphiPer was found in a small cell group in the anterior neural tube. The amphiPer mRNA levels fluctuated in a rhythmic manner, being high early in the day and low late at night. The authors' data suggest a homology of the amphiPer expessing cells to the suprachiasmatic nucleus of vertebrates.

Norepinephrine-dependent phosphorylation of the transcription factor cyclic adenosine monophosphate responsive element-binding protein in bovine pinealocytes.

Norepinephrine (NE)-dependent activation of transcription factors is of central importance for the rhythmic production of melatonin in the rodent pineal gland. At variance with rodents, NE regulates melatonin biosynthesis through post-translational mechanisms in ungulates, and it is not yet known whether transcription factors play any role in ungulate pineal functions. Here, we investigated in isolated bovine pinealocytes the NE-dependent phosphorylation of the transcription factor cyclic adenosine monophosphate (cAMP) responsive element-binding protein (CREB) and compared the effects of NE with those of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Treatment with 10(-7) m NE for 30 min induced a strong nuclear phosphorylated CREB (pCREB) immunoreaction in cells that were identified as pinealocytes by immunocytochemical demonstration of serotonin, a pinealocyte-specific marker. Immunoblots showed that the NE-induced immunoreaction was due to phosphorylation of the transcription factor CREB and another protein, presumably the activating transcription factor 1 (ATF-1). 10(-7) m isoproterenol (ISO) or 10(-5) m forskolin mimicked the response to NE indicating that NE acts through the beta-adrenergic/cAMP pathway. Also 10(-7) m PACAP, but not 10(-7) m VIP-enhanced CREB phosphorylation; however, only a subpopulation of cells was responsive to PACAP. Our results suggest that, irrespective of whether or not melatonin production is controlled via transcriptional mechanisms, NE-induced CREB phosphorylation represents a very conserved element in pineal physiology of mammals because NE increases pCREB levels in all mammalian species investigated so far. However, the genes targeted by pCREB may vary from one mammalian species to the other. Our results also suggest that transcription factors other than pCREB, like ATF-1, may play a role in pineal functions of mammals.

Signal transduction and regulation of melatonin synthesis in bovine pinealocytes: impact of adrenergic, peptidergic and cholinergic stimuli.

Limited studies of the regulation of pineal melatonin biosynthesis in ungulates indicate that it differs considerably from that in rodents. Here we have investigated several signal transduction cascades and their impact on melatonin synthesis in bovine pinealocytes. Norepinephrine increased the intracellular calcium ion concentration ([Ca2+]i) via alpha(1)-adrenergic receptors. Activation of beta-adrenergic receptors enhanced cAMP accumulation and rapidly elevated arylalkylamine N-acetyltransferase (AANAT) activity and melatonin secretion. The beta-adrenergically evoked increases in AANAT activity were potentiated by alpha(1)-adrenergic stimulation, but this was not seen with cAMP or melatonin production. PACAP treatment caused small increases in cAMP, AANAT activity and melatonin biosynthesis, apparently in a subpopulation of cells. VIP and glutamate did not influence any of these parameters. Activation of nicotinic and muscarinic acetylcholine receptors increased [Ca2+]i, but did not alter cAMP levels, AANAT activity or melatonin production. Our study reveals that discrete differences in pineal signal transduction exist between the cow and rodent, and emphasizes the potential importance that the analysis of ungulate pinealocytes may play in understanding regulation of pineal melatonin biosynthesis in primates and man, whose melatonin-generating system appears to be more similar to that in ungulates than to that in rodents.