Daily and circadian variation in survival from ultraviolet radiation in Chlamydomonas reinhardtii.

The survival of organisms depends on their ability to adapt to their environment, one important aspect of which is the daily cycle of day and night. During the day, organisms use a variety of strategies to protect themselves from deleterious ultraviolet (UV) wavelengths of sunlight. Among those strategies could be timing of UV-sensitive cellular processes to occur at night to avoid UV-induced damage. We tested whether the unicellular alga Chlamydomonas reinhardtii uses this strategy by measuring the survival of cells following exposure to UV radiation at different phases of the day. Chlamydomonas cells displayed a rhythm of survival from UV radiation where the most sensitive phases occurred during the end of the day and at the beginning of the night. This phase of sensitivity corresponds to the time of nuclear division. The rhythm continues in constant light indicating control by a circadian clock. The results presented here suggest a hypothesis of how circadian clocks may have evolved; a temporal program whereby light-sensitive processes are timed to avoid sunlight-induced damage would be advantageous and therefore selected.

Day/night differences in the stimulation of adenylate cyclase activity by calcium/calmodulin in chick pineal cell cultures: evidence for circadian regulation of cyclic AMP.

In chick pineal cell culture, stimulation of adenylate cyclase with the diterpene forskolin was greater during the subjective night than during the subjective day. This rhythm of cyclic AMP (cAMP) stimulation mimicked the rhythm of unstimulated cAMP measured previously during LD cycles from flow-through culture. Direct measurement of adenylate cyclase activity in permeabilized cells revealed an adenylate cyclase activity activated by Ca2+/calmodulin during the night but not during the day. However, this difference in adenylate cyclase activity at two times of the circadian cycle is apparent only when permeabilized cells were prewashed with buffer containing GTE When cAMP was measured from flow-through cultures maintained in continuous darkness to determine whether a circadian clock may regulate cAMP, a low-amplitude rhythm was measured. The circadian rhythm of cAMP was similar to the cAMP rhythm previously measured on LD cycles except that the rhythm in darkness had a lower amplitude. Similar to the suppression of melatonin, cAMP was suppressed by light presented during the middle of the night. LD differences in nocturnal cAMP levels were abolished with dipyridamole, an inhibitor of cyclic GMP (cGMP) phosphodiesterase. These results suggest that the rhythm of cAMP in chick pineal cells involves the stimulation of adenylate cyclase by Ca2+/calmodulin during the night and a GTP-dependent suppression of adenylate cyclase activity during the day. The photic suppression of cAMP at night involves the activation of a dipyridamole-sensitive, cGMP phosphodiesterase.

Calcium modulates circadian variation in cAMP-stimulated melatonin in chick pineal cells.

In chick pineal cells, melatonin synthesis is regulated by both calcium and cAMP. Calcium-dependent regulation of melatonin is suggested by the monotonic decrease in melatonin observed with decreasing extracellular calcium ion concentrations ([Ca2+]0), the stimulation of melatonin with Bay K8644, and the inhibition of nocturnal melatonin by several calmodulin antagonists. At submicromolar [Ca2+]0, a stimulation of melatonin was observed in the presence of 8-Br cAMP, but not with Bay K8644, suggesting that this amount of stimulation of melatonin by 8-Br cAMP is independent of Ca2+ influx through dihydropyridine-sensitive Ca2+ channels. At micromolar [Ca2+]0, there was a further increase in the stimulation of melatonin by 8-Br cAMP that was not blocked by nifedipine, a dihydropyridine-sensitive Ca2+ channel antagonist. Micromolar [Ca2+]0 is required for the greater stimulation of melatonin by 8-Br cAMP during the night than during the day. Melatonin was stimulated by 8-Br cAMP to higher levels during the night than during the subjective day under normal [Ca2+]0 (1.3 mM). This difference in the amount of melatonin stimulated by 8-Br cAMP during the subjective night versus the subjective day was blocked by lowering [Ca2+]0 to a submicromolar concentration (0.2 microM). Both nifedipine and calmidazolium partially blocked nocturnal increases in melatonin, but were ineffective during the day. These results suggest that Ca2+ plays an important role in the differential ability of cAMP to stimulate melatonin during the night versus the day.

Automated sampling and RNA isolation at room temperature for measurements of circadian rhythms in Chlamydomonas reinhardtii.

The development of techniques allowing the unattended collection of RNA from cell samples at room temperature makes practical accurate and facile monitoring of circadian rhythms in Chlamydomonas reinhardtii. The utility of these methods was demonstrated by collecting RNA samples for three days from cells maintained in continuous darkness. Every hour, cells were automatically collected and lysed with buffer containing SDS and proteinase K. Samples were maintained at room temperature with little or no evidence of degradation of RNA. Strong, non-damping circadian rhythms of cab mRNA abundance were measured. Free-running rhythms of about 24 h were measured from cultures maintained at 16, 20, 25 and 30 degrees C, thus demonstrating temperature compensation of circadian period. Simultaneous collections from cultures previously entrained to 12 h light/12 h dark cycles of opposite phase displayed circadian rhythms of cab mRNA abundance that were in phase with their previous entraining light cycles. Thus, this result suggests that the measured circadian rhythms of cab mRNA abundance was not an artifact of the collection procedure.

Twenty-four hour oscillation of cAMP in chick pineal cells: role of cAMP in the acute and circadian regulation of melatonin production.

Chick pineal cells contain circadian oscillators that regulate a rhythm of melatonin biosynthesis. We explored the role of cAMP in regulating this melatonin rhythm. Chick pineal cells expressed a 24 hr oscillation of cAMP efflux with a waveform similar to that of melatonin. Elevation of cAMP in chick pineal cells stimulated melatonin. These results suggest that an oscillation of cAMP regulates the rhythm of melatonin. We investigated whether cAMP was a component of the circadian oscillator by determining the effects of 8-Br cAMP pulses on the phase of the circadian melatonin rhythm. Six hour pulses of 8-Br cAMP did not cause steady-state phase shifts of the rhythm. The acute regulation of melatonin by cAMP, the 24 hr oscillation of cAMP, and the inability of cAMP to phase-shift the melatonin rhythm strongly suggest that cAMP acts as an output signal of the circadian oscillator.