Effects of Respiratory Inhibitors on Respiration, ATP Contents, and the Circadian Conidiation Rhythm of Neurospora crassa

Abstract

Effects of respiratory inhibitors on the circadian clock, respiratory activity, and ATP content were examined in Neurospora crassa. AU inhibitors, potassium cyanide, sodium azide, antimycin A, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), shifted the phase of the conidiation rhythm. All the phase response curves were similar and resembled that for cycloheximide, but were different from the phase response curve for light. Phase shifting by azide and CCCP was proportional to the lowering of respiratory activity and ATP content, but such a correlation was not observed for cyanide and antimycin A. In particular, cyanide at a concentration of 0.5 millimolar completely depleted ATP of the cultures but did not significantly shift their phase. Their results suggest that large shifts caused by these inhibitors are not due to a decrease in energy from respiratory activity.Circadian rhythms are observed in many organisms from lower eukaryotic plants to higher animals. However, the molecular mechanism ofthe clock which regulates these rhythms is still not known. Bunning (2) proposed that the clock is composed of two different phases: a physiological night phase which requires energy and a physiological day phase in which energy is not required. In fact, deprivation of oxygen for short times (1, 18) or treatment with respiratory inhibitors (3, 6, 9-11) resulted in phase shifting at specific phases in several organisms. These results suggest that certain phases of the clock, in particular the subjective night, may be energy dependent. Koyama and Feldman treated Neurospora with several respiratory inhibitors to examine the energy dependence of clock function (cited in Feldman and Dunlap [7]). They obtained phase-shifting data but did not measure inhibition ofrespiration or ATP levels, nor have such measurements been made in other organisms. The correlations such measurements might provide are needed before it can be concluded that energy depletion caused by these inhibitors results in phase shifting of the clock. In this report, both phase shifting by respiratory inhibitors and levels of the respiratory activity and ATP were measured. MATERIALS AND METHODSCulture Conditions and Analysis of the Conidiation Rhythm. The bd (band) strain of Neurospora crassa was used. Procedures for maintaining stock cultures and for liquid culture were the same as reported previously (12). Conidia (13 x 1O') suspended in distilled H20 were added to 25 ml ofliquid medium containing Fries' salts (8), 0.3% glucose, and 0.5% arginine. Conidial con-'Supported in part by the Japan-United States Cooperative Program. centration were determined by A at 480 nm. After culturing for 36 h in continuous light at 26C, discs were cut from the mycelial mats with a cork borer 11 mm in diameter. Six discs from each mat were transferred to a 125-ml Erlenmeyer flask with 25 ml of liquid medium containing Fries' salts, 0.03% glucose and 0.05% arginine and cultured in continuous darkness with reciprocal shaking (100 cycles per min) at 26C. The mediu...

Phase Shifting of the Circadian Conidiation Rhythm in Neurospora crassa by Calmodulin Antagonists

“…On the phase response curve for trifluoperazine, the crossover point from phase delay to phase advance was CT 8, which is very close to that (CT 4) of the phase response curves for cycloheximide , diethylstilbestrol (Nakashima, 1982), and respiratory inhibitors (Nakashima, 1984b). It has been suggested that phase shifting by diethylstilbestrol is due to inhibition of some mitochondrial functions, but not of activity of plasma membrane ATPase (Nakashima, 1982).…”

ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

“…2). Although cyanide is known as an inhibitor of the respiratory electron-transport chain that depletes ATP (34), it also can directly damage cell membranes by altering the membrane's resistance (increasing its permeability) (35). We tested this possibility by monitoring the potential changes of the latent ATPhydrolyzing activity of the right-side-out plasma membrane vesicles after KCN treatment.…”