Intracellular water motion decreases in apoptotic macrophages after caspase activation

Triggering of the macrophage cell line RAW 264.7 with lipopolysaccharide an d interferon-gamma promoted apoptosis that was prevented by inhibitors of t ype 2 nitric oxide synthase or caspase. Using H-1 NMR analysis, we have inv estigated the changes of the intracellular transverse relaxation time (T-2) and apparent diffusion coefficient (ADC) as parameters reflecting the rota tional and translational motions of water in apoptotic macrophages. T-2 val ues decreased significantly from 287 to 182 ms in cells treated for 18 h wi th NO-donors. These changes of T-2 were prevented by caspase inhibitors and were not due to mitochondrial depolarization or microtubule depolymerizati on. The decrease of the intracellular values of T-2 and ADC in apoptotic ma crophages was observed after caspase activation, but preceded phosphatidyls erine exposure and nucleosomal DNA cleavage. The changes of water motion we re accompanied by an enhancement of the hydrophobic properties of the intra cellular milieu, as detected by fluorescent probes. These results indicate the occurrence of an alteration in the physicochemical properties of intrac ellular water during the course of apoptosis.