The role of PACAP in the control of circadian expression of clock genes in the chicken pineal gland.

Several features of the molecular circadian oscillator of the chicken pineal gland show homology with those in the mammalian SCN. Studies have shown the effects of PACAP on the mammalian SCN, but its effects on the expression of clock genes in the avian pineal gland have not yet been demonstrated. Clock and Cry1 expression was analyzed in pineal glands of chicken embryos after exposure to PACAP-38 in vitro. PACAP reduced expression of both clock genes within 2h. Ten hours after exposure, mRNA contents exceeded that of the controls. Our results support the hypothesis that the molecular clock machinery in the chicken pineal gland is also sensitive to PACAP.

The avian pineal gland.

The pineal gland plays a key role in the control of the daily and seasonal rhythms in most vertebrate species. In mammals, rhythmic melatonin (MT) release from the pineal gland is controlled by the suprachiasmatic nucleus via the sympathetic nervous system. In most non-mammalian species, including birds, the pineal gland contains a self-sustained circadian oscillator and several input channels to synchronize the clock, including direct light sensitivity. Avian pineal glands maintain rhythmic activity for days under in vitro conditions. Several physical (light, temperature, and magnetic field) and biochemical (Vasoactive intestinal polypeptide (VIP), norepinephrine, PACAP, etc.) input channels, influencing release of melatonin are also functional in vitro, rendering the explanted avian pineal an excellent model to study the circadian biological clock. Using a perifusion system, we here report that the phase of the circadian melatonin rhythm of the explanted chicken pineal gland can be entrained easily to photoperiods whose length approximates 24 h, even if the light period is extremely short, i.e., 3L:21D. When the length of the photoperiod significantly differs from 24 h, the endogenous MT rhythm becomes distorted and does not follow the light-dark cycle. When explanted chicken pineal fragments were exposed to various drugs targeting specific components of intracellular signal transduction cascades, only those affecting the cAMP-protein kinase-A system modified the MT release temporarily without phase-shifting the rhythm in MT release. The potential role of cGMP remains to be investigated.

Circadian expression of clock genes clock and Cry1 in the embryonic chicken pineal gland.

Clock and Cry1 expression were examined in the pineal gland of chicken embryos incubated under constant darkness from embryonic day (ED) 0. From ED13, Clock and Cry1 mRNA levels showed episodic alterations. After ED17, circadian pattern of clock gene expression was seen both in vivo and in vitro. Our results support the idea that rhythmic environmental factors are not necessary for the generation of circadian patterns of clock gene expression during development.

Expression of Cry2 in the chicken pineal gland: effects of changes in light-dark conditions.

Pineal expression of Cry2 mRNA has been examined in chickens under normal (LD) and reversed (DL) light-dark conditions. In vivo the peak of Cry2 mRNA content at late subjective day under LD diminished after switching to a DL schedule. In vitro, Cry2 mRNA levels showed a steady decrease during light exposure at subjective night. Our data show that light-sensitive clock components in the pinealocytes may be involved in the repression of Cry2 transcription at night, which may contribute to resetting the phase of the clock within 24 h.

Cry1 expression in the chicken pineal gland: effects of changes in the light/dark conditions.

Cryptochromes (Cry) are core components in the gene regulation of circadian rhythmic processes. It was shown earlier, that Cry1 mRNA content of the avian pineal gland was increased after a 4h exposure to light during subjective night; however, a 30min exposure was ineffective. In this study, changes in pineal Cry1 expression were detected in chickens during and after being placed into reversed light/dark environment. Cry1 mRNA content was higher if light was on during the night; however, in the first 2h of light exposure at night, Cry1 mRNA contents were decreased. Following the first overnight light exposure, the peak of the mRNA expression was delayed for 12h compared to controls. Our results suggest that environmental illumination activates a complex regulatory cascade that includes both up- and down-regulation of the Cry1 gene which inverses the 24h pattern of Cry1 mRNA expression within one period.

Development of the rhythmic melatonin secretion in the embryonic chicken pineal gland.

In order to elucidate details on the development of the circadian clock, the effects of light on the in vitro melatonin (MT) release and the presence of mRNAs of several clock genes in the embryonic chicken pineal gland were investigated. Chicken embryos of various developmental stages were exposed to stimuli of light in vitro in dynamic, four day long bioassay (perifusion). MT secretion and mRNA levels of Cry1, Cry2, Clock and Bmal2 clock genes were determined. Our conclusions: (1) environmental illumination modified MT secretion from explanted embryonic pineal glands as early as on the 13th embryonic day, (2) daily rhythm of MT release develops between embryonic days 16 and 18 under periodic environmental illumination. (3) Chicken Cry1, Cry2, Clock and Bmal2 clock gene mRNAs were also detected in glands of animals of 15th embryonic day. Although both MT secretion and clock genes have been developed by then, the circadian MT rhythm appears first on the 17th embryonic day. Either the mechanisms coupling the clock with the melatonin output or the synchronization of the individual pinealocytes develop around this age. Rhythmic MT release in the embryonic chicken pineal gland evolves only if the egg is exposed to rhythmic environmental stimuli.

Receptor (MT(1)) mediated influence of melatonin on cAMP concentration and insulin secretion of rat insulinoma cells INS-1.

Recent functional, autoradiographic, and molecular investigations have shown that the pineal secretory product melatonin reduces the forskolin-stimulated insulin secretion from isolated pancreatic islets of neonate rats. Autoradiographic and binding studies as well as reverse transcriptase-polymerase chain reaction (RT-PCR) experiments proved that these effects are mediated through specific, high-affinity pertussis-toxin-sensitive Gi-protein-coupled MT(1) receptors and subsequent inhibition of the adenylyl cyclase/cyclic adenosine monophosphate (cAMP) system. This hypothesis was proved by blocking the intracellular signal transduction pathway using the non-hydrolyzable guanosine triphosphate analog guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) or the competitive melatonin receptor antagonist luzindole. Both GTPgammaS and luzindole diminished the melatonin effect. We have published these prior results elsewhere. So far, however, no information is available on both whether the MT1 receptors are located on the beta-cells and whether the consecutive functional reactions are based on a direct influence of melatonin on the insulin producing beta-cells. In order to examine this question, we used a glucose responsive insulin producing insulinoma cell line INS-1 isolated from rats. Comparable with the results of islets the competitive receptor antagonist luzindole diminished the insulin-decreasing effect of melatonin. In addition, our RT-PCR experiments, using specific primers for the rat melatonin receptor MT(1) showed that this melatonin receptor mRNA is also expressed in the INS-1 cells. Furthermore we radioimmunologically analyzed the forskolin-stimulated cAMP concentration in the superfusate. Similar to insulin secretion, the cAMP concentration was significantly reduced by melatonin. Following the hypothesis that cAMP is actively secreted from INS-1 cells by an energy-dependent mechanism based on either a OAT1/ROAT1 like anion exchanger or MDR-like transport systems, we used probenecid (p-[dipropylsulfamoyl] benzoic acid), a known inhibitor of cAMP extrusion. Probenecid blocks the export of cAMP by acting on transport mechanisms which are as yet not completely understood. Consistently, insulin secretion was increased and cAMP concentration diminished. The application of the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) caused a marked rise of insulin secretion as well as cAMP concentration in the perifusate. From these data we conclude that the MT1 receptor is located on the INS-1 cell and therefore in general on pancreatic beta-cells.