ATP DEPLETION INHIBITS GLUCOCORTICOID-INDUCED THYMOCYTE APOPTOSIS

In quiescent thymocytes, mitochondrial de-energization was not correla ted to apoptotic death. In fact, thymocytes treated with oligomycin, a highly specific inhibitor of ATP synthase, alone or with atractylosid e to block ATP translocation from the cytoplasm, were alive, even if t heir mitochondria were depolarized, as revealed by flow cytometry afte r Rhodamine 123 staining, Furthermore, oligomycin was a powerful inhib itor of apoptosis induced in rat thymocytes by dexamethasone and, to a lesser extent, by the calcium ionophore A23187 and etoposide, but was without effect when apoptosis was induced by staurosporine, and incre ased cell death in mitogen-treated thymocytes, The inhibition of apopt osis was confirmed by morphological criteria, inhibition of inter-nucl eosomal DNA fragmentation and inhibition of the loss of membrane integ rity. The anti-apoptotic effect of oligomycin in cells treated with A2 3187 or etoposide was correlated to the inhibition of protein synthesi s, while inhibition of apoptosis induced by dexamethasone, already evi dent at an oligomycin concentration of 10 ng/ml, was instead strictly correlated to the effect exerted on the cellular ATP level. Thymocyte apoptosis triggered by dexamethasone was blocked or delayed by inhibit ors of respiratory-chain uncouplers, inhibitors of ATP synthase and an tioxidants: a lasting protection from dexamethasone-induced apoptosis was always correlated to a drastic and rapid reduction in ATP level (3 1-35 % of control), while a delay in the death process was characteriz ed by a moderate decrease in ATP (73-82 % of control). Oligomycin inhi bited the specific binding of radioactive corticosteroid to thymocyte nuclei, confirming the inhibitory effect of ATP depletion on glucocort icoid binding and suggesting that ATP depletion is a common mediator o f the anti-apoptotic action of different effecters in glucocorticoid-i nduced apoptosis. In conclusion, the reported data indicate that ATP m ay act as a cellular modulator of some forms of apoptosis, depending o n the death trigger, and that in quiescent cells the de-energization o f mitochondria is not necessarily linked to apoptosis.