LIGHT EFFECTS ON CYCLIC-NUCLEOTIDE LEVELS AND PHASE-SHIFTING OF THE CIRCADIAN CLOCK IN NEUROSPORA-CRASSA

Mycelia of Neurospora crassa (band [bd] mutant) were exposed to white light (blue light intensity 3.5 mu mol s(-1) m(-2)) of different durat ions during constant darkness. The concentrations of different second messenger molecules and the phase shifting of the circadian rhythm wer e determined during light exposures at circadian time (ct) 12 and ther eafter, These light exposures elicited 8-12 ct units delay phase shift s but did not change the amount of inositol 1,4,5-triphosphate (InsP(3 )). In contrast, significant effects of light were observed on cyclic adenosine 3',5'-monophosphate (cAMP) levels, which increased transient ly about 30-90 s after the onset of light. The same kinetics was obser ved 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 subunits was dete rmined in isolated nuclei of the bd strain in relation to total nuclea r proteins by means of western blot analysis, using a heterologous ant ibody, The nuclear PKA content changed parallel to the cAMP changes, T he transient increase of cAMP did not occur in the ''blind'' white col lar 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 subseque nt longer lasting decrease, Light-induced cAMP changes and light-induc ed phase shifts showed different duration dependencies, thus indicatin g 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 k inetics of the cGMP content again showed the immediate decrease after 2 min and a slow recovery to (or above) control values after several h ours, Various calcium channel blockers (nickel, cobalt, nifedipine, da ntrolene, lanthanum) and the calcium calmodulin inhibitor chlorpromazi ne did not affect the phase shifting by light nor did they significant ly phase shift the circadian rhythm in the dark themselves (again dete rmined at ct 12), The data showed that InsP, did not change after expo sure to light, whereas cAMP increased and cGMP decreased, There was no evidence, however, that these changes play a role in light signal tra nsmission to the clock. (C) 1996 American Society for Photobiology