Daily injections of melatonin entrain the circadian activity rhythms of nocturnal rats but not diurnal chipmunks.

It is well known that daily injections of melatonin entrain the free-running rhythms of nocturnal rodents (rats and hamster) and diurnal sauropside (birds and lizard). Here, we asked whether daily injections of melatonin entrain the free-running rhythm of the chipmunk, a diurnal rodent, and, if they do, is the phase relationship between the time of injection and onset of the activity interval similar to that in sauropside rather than that of nocturnal rodents? Contrary to our expectations, daily injections of melatonin did not entrain the free-running rhythm in 9 of 10 chipmunks, even when a high dose of melatonin (1 mg/kg, b.wt.) was used. These results indicate that the entraining effect of daily injections of melatonin on free-running rhythm varies among mammalian species.

Effects of daily injections of melatonin on locomotor activity rhythms in rats maintained under constant bright or dim light.

It has been demonstrated that daily melatonin injections entrain free-running locomotor activity rhythms in rats kept in constant darkness, and synchronize disrupted circadian patterns of wheel-running activity under constant light. Contrary to these previous observations, our result did not show that daily injections of melatonin synchronize disrupted locomotor activity in rats maintained under constant bright light. On the other hand, daily treatment with melatonin entrained the intact free-running rhythm in rats kept in constant dim light. This entrainment took place only when the time of injection corresponded to the activity onset time, and similar results were obtained in blinded rats. Pinealectomy had no influences on either the free-running rhythm or melatonin-induced entrainment. To examine whether a behavioral feedback mechanism is involved in melatonin-induced entrainment, rats were immobilized for 3 h after each daily melatonin injections. This did not interfere with melatonin-induced entrainment. These results suggest that the mechanism underlying melatonin-induced entrainment of activity rhythms may be different from those in photic and behavioral entrainment.

Melatonin accelerates reentrainment of circadian locomotor activity rhythms to new light-dark cycles in the rat.

We examined whether continuous melatonin administration through subcutaneously implanted silastic tubing accelerates reentrainment of circadian rhythms of locomotor activity to shifted illumination cycles. We found that rats required less time to reentrain to an advanced or delayed phase shift of a light-dark cycle while carrying a silastic implant filled with melatonin than while carrying an empty implant. These results suggest that continuous administration of melatonin accelerates the reentrainment of the circadian locomotor activity rhythm to a new light-dark cycle.

Intrapituitary distribution and effects of annexin 5 on prolactin release.

Annexin 5 is expressed by rat anterior pituitary cells and a depolarizing stimulus results in increased extracellular display and, depending on local calcium concentrations, potential release into the extracellular environment. In order to further investigate the role of annexin 5 in anterior pituitary function, we have examined the intracellular distribution by immunocytochemistry and the effects of annexin 5 on the release of a major secretory product, prolactin. Prolactin was chosen because we could easily monitor effects on basal release and effects on the immediate and sustained phases of thyroid stimulating hormone releasing hormone (TRH)-stimulated release. Immunocytochemical localization of annexin 5 showed staining of the majority of anterior pituitary cells. Labeling was predominantly on the nuclear envelope and plasma membrane. For the chosen secretory product, prolactin, annexin 5 was found in most, but not all prolactin positive cells. When recombinant annexin 5 (50 ng/mL) was added to a 3 h static culture incubation of rat anterior pituitary cells, prolactin release was inhibited by about 30% (p<0.05). A lower dose had a reduced effect and higher doses had no further inhibitory effect, indicating that the effect was specific to annexin 5 and not a nonspecific toxic effect of some contaminant in the preparation. This interpretation was further strengthened in a time-course experiment demonstrating that when TRH and annexin 5 were added together, there was no effect of annexin 5 on the amount of prolactin released. After a 3 h preincubation in annexin 5, however, prolactin release, in response to TRH, was suppressed by about 30% in both the acute and sustained phases. These data suggest that annexin 5 may be a local regulator of release in the anterior pituitary, but a slow onset effect on both phases of TRH-stimulated release suggests that this is not an effect at the plasma membrane such as local extracellular calcium depletion by plasma membrane-bound annexin 5.

Comparison of circadian oscillation of melatonin release in pineal cells of house sparrow, pigeon and Japanese quail, using cell perfusion systems.

We compared the circadian oscillation of melatonin release from cultured pineal cells in Japanese quail, pigeon and house sparrow, to determine whether the pineal gland of these species retains the circadian oscillator function in vitro. After dissociated pineal cells have been cultured for 4 days under 12 h: 12 h light-dark (LD) cycle, they were perfused at a flow rate of 0.25 ml/h for 6-7 days under LD or constant darkness (DD). Melatonin release increased during the dark period and low during the light period in all pineal cell cultures. Under DD conditions, the circadian rhythm of melatonin release persisted for up to 3-4 cycles in pigeon and house sparrow pineal cells, but the amplitude of the rhythm decreased gradually. However, in Japanese quail pineal cell culture the circadian oscillation of melatonin release was weak or abolished under DD conditions. These results strengthen the argument that the avian pineal gland's role in circadian organization differs between species. The direct demonstration of species-specific differences in the mechanism of the circadian oscillation of melatonin release from pineal cells should provide a useful model for the analysis of the pineal's circadian system at the cellular level.

Secretion of recombinant rat annexin 5 by insect cells in a baculovirus expression system.

Rat annexin 5 was expressed in insect cells using a baculovirus vector, Autographa californica nuclear polyhedrosis virus. The rat annexin 5 cDNA was prepared by a polymerase chain reaction using mRNA from rat pituitary glands and placed under the control of the polyhedrin promoter. The gene product was 36 k dalton and was recognized by anti-rat annexin 5 serum. The calcium dependent binding of the recombinant annexin 5 to membranes was confirmed. The recombinant protein appeared in the medium by 21 hours post-inoculation in high amount and this was specific to this recombinant virus. High potassium milieu (20 mM KCl) for two hours increased the release of the recombinant protein but not for the recombinant beta-galactosidase prepared for a control. These results reveal that the product of the annexin 5 gene, which lacks a signal sequence, follows a secretory pathway in insect cells.

Circadian oscillation of 64-kDa polypeptide in the rat suprachiasmatic nucleus.

In cell suspensions of suprachiasmatic nucleus harvested every 3 hr from rats kept under 12 hr: 12 hr light-dark cycle and constant darkness, we have detected a M(r) 64-kDa protein whose synthesis exhibits two distinct daily peaks in SDS-PAGE. Analysis of densitometer tracings revealed that the synthesis of other proteins was independent of the time of day or not reproducible. Maximum synthesis of the 64-kDa polypeptide occurred at around CT6 and CT21, which are almost coincident with the phase advance regions of circadian activity rhythm induced by anisomycin and light pulses [15], respectively. These results suggest that the 64-kDa protein in SDS-PAGE may be a part of the circadian clock mechanism.