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Cannabinoids modulate neuronal and neuroendocrine circuits by binding to cannabinoid receptors acting upon cAMP/Ca(2+)-mediated intracellular signaling cascades. The rat pineal represents an established model to investigate intracellular signaling processes because a well defined input, the neurotransmitter norepinephrine, is transformed via cAMP/Ca(2+)-dependent mechanisms into an easily detectable output signal, the biosynthesis of melatonin. Here we investigated the impact of cannabinoids on norepinephrine-regulated melatonin biosynthesis in the rat pineal. We demonstrated that treatment of cultured rat pineals with 9-carboxy-11-nor-delta-9-tetrahydrocannabinol (THC), cannabidiol or cannabinol significantly reduced norepinephrine-induced arylalkylamine N-acetyltransferase (AANAT) activity and melatonin biosynthesis. These effects were not mimicked by the cannabinoid receptor agonist WIN55,212-2 and were not blocked by cannabinoid 1 and 2 receptor antagonists. The cannabinoids used did not affect norepinephrine-induced increases in cAMP/Ca(2+) levels. Notably, cannabinoids were found to directly inhibit AANAT activity in lysates of the pineal gland. This effect was specific in so far as cannabinoids did not influence the activity of hydroxyindole-O-methyltransferase (HIOMT), the last enzyme in melatonin biosynthesis. Taken together, our data strongly suggest that cannabinoids inhibit AANAT activity and attenuate melatonin biosynthesis through intracellular actions without involvement of classical cannabinoid receptor-dependent signaling cascades.

We have previously shown that the extracellular nucleoside triphosphate-hydrolyzing enzyme NTPDase2 is highly expressed in situ by stem/progenitor cells of the two neurogenic regions of the adult murine brain:the subventricular zone (type B cells) and the dentate gyrus of the hippocampus (residual radial glia). We explored the possibility that adult multipotent neural stem cells express nucleotide receptors and investigated their functional properties in vitro. Neurospheres cultured from the adult mouse SVZ in the presence of epidermal growth factor and fibroblast growth factor 2 expressed the ecto-nucleotidases NTPDase2 and the tissue non-specific isoform of alkaline phosphatase, hydrolyzing extracellular ATP to adenosine. ATP, ADP and, to a lesser extent, UTP evoked rapid Ca2+ transients in neurospheres that were exclusively mediated by the metabotropic P2Y1 and P2Y2 nucleotide receptors. In addition,agonists of these receptors and low concentrations of adenosine augmented cell proliferation in the presence of growth factors. Neurosphere cell proliferation was attenuated after application of the P2Y1-receptor antagonist MRS2179 and in neurospheres from P2Y1-receptor knockout mice. In situ hybridization identified P2Y1-receptor mRNA in clusters of SVZ cells. Our results infer nucleotide receptor-mediated synergism that augments growth factor-mediated cell proliferation. Together with the in situ data, this supports the notion that extracellular nucleotides contribute to the control of adult neurogenesis.

The rat pineal gland is a suitable model to investigate neurotransmitter-controlled gene expression, because it is well established that the stimulation of melatonin biosynthesis by norepinephrine (NE) depends on the activation of the gene that encodes arylalkylamine N-acetyltransferase (AANAT), the melatonin rhythm enzyme. The mechanisms responsible for downregulation of Aanat transcription are less clear. In this in vitro study we investigated the role of pCREB dephosphorylation for termination of Aanat gene transcription. Immunosignals for pCREB, strongly induced after NE stimulation, rapidly decreased after withdrawal of NE. The immunoreactivity of the inhibitory transcription factor ICER increased twofold after NE treatment for 6 h, but did not change within 30 min after removal of the stimulus. Application of protein serine/threonine phosphatase (PSP) inhibitors prevented pCREB dephosphorylation and blocked the decreases in Aanat mRNA levels, AANAT protein amount and melatonin biosynthesis all of which occurred rapidly after NE withdrawal. PSPs in the rat pineal gland were characterized by immunocytochemistry and immunoblotting. NE-stimulation for 8 h induced accumulation of PSP1-catalytic subunit (CSU) in pinealocyte nuclei, but did not affect the distribution of PSP2A-CSU. The results identify dephosphorylation of pCREB by PSPs as an essential mechanism for downregulation of Aanat transcription in the rat pineal gland.

The molecular processes underlying neural transmission are central issues in neurobiology. Here we describe a novel mechanism through which noradrenaline (NA) activates its target cells, using the mammalian pineal organ as a model. In this neuroendocrine transducer, NA stimulates arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87), the key enzyme regulating the nocturnal melatonin production. In rodents, AANAT protein accumulates as a result of enhanced transcription, but in primates and ungulates, the AANAT mRNA level fluctuates only marginally, indicating that other mechanisms regulate AANAT protein and activity. These were investigated in cultured bovine pinealocytes. AANAT mRNA was readily detectable in unstimulated pinealocytes, and levels did not change following NA treatment. In contrast, NA increased AANAT protein levels in parallel with AANAT activity, apparently through a cyclic AMP-mediated mechanism. Immunocytochemistry revealed that the changes in AANAT protein levels occurred in virtually all pinealocytes. Inhibition of AANAT degradation by proteasomal proteolysis alone was found to switch-on enzyme activity by increasing AANAT protein levels five- to 10-fold. Accordingly, under unstimulated conditions AANAT protein is continually synthesized and immediately destroyed by proteasomal proteolysis. NA appears to act via cyclic AMP to protect AANAT from proteolytic destruction, resulting in accumulation of the protein. These findings show that tightly regulated control of proteasomal proteolysis of a specific protein alone can play a pivotal role in neural regulation.

The subcommissural organ (SCO), an ependymal (glial) circumventricular organ, releases glycoproteins into the cerebrospinal fluid; however, the regulation of its secretory activity is largely unknown. To identify neuroactive substances that may regulate SCO activity, we investigated immunocytochemically identified bovine SCO cells by means of calcium imaging. This analysis was focused on: (1) serotonin (5HT) and substance P (SP), immunocytochemically shown to be present in axons innervating the bovine SCO; and (2) ATP, known to activate glial cells. 5HT had no effect on the intracellular calcium concentration ([Ca(2+)](i)), and its precise role remains to be clarified. SP elicited rises in [Ca(2+)](i) in approx. 30% and ATP in even 85% of the analyzed SCO cells. These effects were dose-dependent, involved NK(3) and P2Y(2) receptors linked to G protein and phospholipase C (PLC) activation, and could not be mimicked by forskolin or 8-bromo-cAMP. In 50% of the SP-sensitive cells, the increases in [Ca(2+)](i) comprised calcium release from thapsigargin-sensitive intracellular stores and an influx of extracellular calcium via protein kinase C (PKC)-induced opening of L-type voltage-gated calcium channels (VGCCs). In the remaining SP-sensitive cells, the increase in [Ca(2+)](i) was caused exclusively by influx of extracellular calcium via VGCCs of the L-type. In all ATP-sensitive cells the increase in [Ca(2+)](i) involved calcium release from thapsigargin-sensitive intracellular stores and a PKC-mediated influx of extracellular calcium via L-type VGCCs. Our data suggest that SP and ATP are involved in regulation of the activity of SCO cells.

Pineal organ of the lamprey, Lampetra japonica, is essential to keep the circadian locomotor activity rhythm as previously reported. In this paper, we tried to show that an endogenous oscillator is located and is working in the pineal organ. When the pineal organs were excised and cultured in a plastic tube with M199 medium at 20 degrees C, melatonin secretion rhythms were clearly observed under both light-dark and continuous dark conditions. The circadian secretion of melatonin continued for more than five cycles under the continuous dark condition. This indicates that the pineal organ has an endogenous oscillator and that the melatonin secretion rhythm is controlled by this oscillator. These findings suggest the possibility that the locomotor activity rhythm of the lamprey is under the control of the oscillator in the pineal organ.

Together with cAMP, calcium ions play an important role in the regulation of melatonin synthesis in the pineal organ of all vertebrate species, irrespective of the conspicuous phylogenetic transformation of the melatonin-producing cell, the pinealocyte. Here we address the question how the intracellular concentration of free calcium ions [Ca2+]i is regulated in directly light-sensitive trout pinealocytes and in rat pinealocytes which have lost the direct light sensitivity and respond to norepinephrine. Isolated pinealocytes identified by the S-antigen immunoreaction were investigated by means of the fura-2 technique, image analysis and patch clamp recordings. Approximately 30% of the trout pinealocytes exhibited spontaneous [Ca2+]i oscillations that were not affected by light or dark adaptation of the cells. Removal of extracellular Ca2+ or application of 10 microM nifedipine caused a reversible breakdown of the [Ca2+]i oscillations. Treatments with 60 mM KCl and nifedipine suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca2+]i in both oscillating and nonoscillating trout pinealocytes. Experiments with thapsigargin (2 microM) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role in the regulation of [Ca2+]i remains elusive. Norepinephrine had no apparent effect on [Ca2+]i in any trout pinealocyte. In rat pinealocytes, [Ca2+]i did not show spontaneous oscillations. Norepinephrine evoked a dramatic biphasic rise in [Ca2+]i in more than 95% of the cells via stimulation of alpha1-adrenergic receptors. The response reflects a combination of calcium mobilization from intracellular, thapsigargin-sensitive calcium stores and an increased calcium influx. Voltage-gated calcium channels of the L-type are present in the rat pinealocyte membrane, but they are not involved in the norepinephrine-induced calcium response. These channels, however, mediate the increase in calcium influx which is observed in virtually all rat pinealocytes upon stimulation with acetylcholine or nicotine. The results show that the mechanisms which regulate [Ca2+]i in pinealocytes are complex and differ considerably between poikilothermic and mammalian species.

The addition of ammonium sulfate to starved yeast cells leads to a 3- to 4-fold rapid increase of the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), the products of phosphoinositide-specific phospholipase C (PI-PLC). This response is reduced by dissecting the RAS-activating Cdc25 protein, and is completely abolished by the cdc25-1 mutation even at permissive temperature. Starved cdc25-1 mutant cells have a strongly reduced IP3 content, but an at least 10-fold increased DAG level compared to the isogenic wild-type strain. NH4 does not stimulate cAMP synthesis, and glucose does not induce IP3 and DAG. Our data suggest that the Cdc25 protein controls a nitrogen-specific signalling pathway involving the effector PI-PLC, in addition to the glucose-induced activation of adenylyl cyclase (AC).

In adult rat pinealocytes, acetylcholine activates nicotinic receptors whose stimulation causes a depolarization of the cells, opening of voltage-gated cation channels of the L-type and sub- sequent increase in the intracellular calcium ion concentration. These events trigger a release of glu- tamate that, by its action on metabotropic glutamate type 3 receptors, activates an inhibitory cyclic AMP cascade and suppresses norepinephrine-induced melatonin biosynthesis. The nicotinic res- ponse is fully developed in the third postnatal week. Prior to this timepoint, rat pinealocytes possess functional muscarinic receptors whose activation causes a rise in the intracellular calcium ion concen- tration through a calcium release from thapsigargin-sensitive intracellular calcium stores and an opening of store-operated calcium channels. This cascade may influence tissue differentiation and matu- ration of the melatonin pathway. The demonstration of functional cholinoreceptors and the ontoge- netic switch from muscarinic to nicotinic signalling in rat pinealocytes supports the concept that pineal functions in mammals are influenced by neuronal inputs other than the sympathetic innerva- tion which serves as the major regulatory system. © Inra/Elsevier, Paris

1. Membrane voltage (Vm) recordings were obtained from isolated rat pinealocytes using the patch-clamp technique. In parallel to the electrophysiological experiments, intracellular Ca2+ measurements were performed using fura-2. 2. The resting Vm averaged -43 mV and replacement of extracellular NaCl by KCl completely depolarized the cells. This indicates that the resting Vm is dominated by a K+ conductance. Single-channel recordings revealed the presence of a large conductance Ca(2+)-activated charybdotoxin-sensitive K+ channel. 3. Application of ACh (100 microM) depolarized the pinealocytes on average by 16 mV. The depolarizing effect of ACh was mimicked by nicotine (50 microM) and was prevented by tubocurarine (100 microM). 4. The ACh-induced depolarization was largely abolished in the absence of extracellular Na+, but was not significantly affected by extracellular Ca2+ removal. 5. Application of ACh (100 microM) caused an increase in [Ca2+]i. This increase was completely dependent on the presence of extracellular Ca2+ and was largely reduced after extracellular Na+ removal. Nifedipine (1 microM) reduced the ACh-induced increase in [Ca2+]i by about 50%. 6. Our findings indicate that in rat pinealocytes stimulation of a nicotinic ACh receptor (nAChR) induces depolarization mainly by Na+ influx via the nAChR. The depolarization then activates L-type Ca2+ channels, which are responsible for the nifedipine-sensitive portion of the intracellular Ca2+ increase. Ca2+ influx via the nAChR probably also contributes to the observed rise in [Ca2+]i.