Light Effects on Cyclic Nucleotide Levels and Phase Shifting of the Circadian Clock in Neurospora crassa

Abstract— Mycelia of Neurospora crassa (band bd] mutant) were exposed to white light (blue light intensity 3.5 μ.mol s^?1 m^?2) of different durations during constant darkness. The concentrations of different second messenger molecules and the phase shifting of the circadian rhythm were determined during light exposures at circadian time (ct) 12 and thereafter. These light exposures elicited 8-12 ct units delay phase shifts but did not change the amount of inositol 1,4,5-triphosphate (InsP₃). In contrast, significant effects of light were observed on cyclic adenosine 3′,5′-monophosphate (cAMP) levels, which increased transiently about 30-90 s after the onset of light. The same kinetics was observed under continued exposure to light as well as after 10 s and 2 s of light followed by darkness. The relative amount of the cAMP-dependent protein kinase A (PKA) in the form of its catalytic sub-units was determined in isolated nuclei of the bd strain in relation to total nuclear proteins by means of western blot analysis, using a heterologous antibody. The nuclear PKA content changed parallel to the cAMP changes. The transient increase of cAMP did not occur in the “blind”white collar mutant (wc-2). Long-term kinetics of cAMP changes after different light pulses in bd showed the initial increase and a return to control levels about 10 min after the onset of the light pulse and a subsequent longer lasting decrease. Light-induced cAMP changes and light-induced phase shifts showed different duration dependencies, thus indicating that cAMP may not play a role in the signal transduction pathway to the clock. Light exposures, furthermore, led to a significant decrease of the cyclic guanosine 3′,5′-monophosphate (cGMP) level. Long-term kinetics of the cGMP content again showed the immediate decrease after 2 min and a slow recovery to (or above) control values after several hours. Various calcium channel blockers (nickel, cobalt, nifedipine, dantrolene, lanthanum) and the calcium calmodulin inhibitor chlorpromazine did not affect the phase shifting by light nor did they significantly phase shift the circadian rhythm in the dark themselves (again determined at ct 12). The data showed that InsP₃ did not change after exposure to light, whereas cAMP increased and cGMP decreased. There was no evidence, however, that these changes play a role in light signal transmission to the clock.