Intracellular water motion decreases in apoptotic macrophages after caspase activation.
Triggering of the macrophage cell line RAW 264.7 with lipopolysaccharide and interferon-gamma promoted apoptosis that was prevented by inhibitors of type 2 nitric oxide synthase or caspase. Using (1)H NMR analysis, we have investigated the changes of the intracellular transverse relaxation time (T(2)) and apparent diffusion coefficient (ADC) as parameters reflecting the rotational and translational motions of water in apoptotic macrophages. T(2) values decreased significantly from 287 to 182 ms in cells treated for 18 h with NO-donors. These changes of T(2) were prevented by caspase inhibitors and were not due to mitochondrial depolarization or microtubule depolymerization. The decrease of the intracellular values of T(2) and ADC in apoptotic macrophages was observed after caspase activation, but preceded phosphatidylserine exposure and nucleosomal DNA cleavage. The changes of water motion were accompanied by an enhancement of the hydrophobic properties of the intracellular milieu, as detected by fluorescent probes. These results indicate the occurrence of an alteration in the physicochemical properties of intracellular water during the course of apoptosis.
Hortelano
S
S
Instituto de Bioquímica, Centro Mixto CSIC-UCM, Facultad de Farmacia, Universidad Complutense. 28040 Madrid, Spain.
García-Martín
M L
ML
Cerdán
S
S
Castrillo
A
A
Alvarez
A M
AM
Boscá
L
L
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D001834:Body Water / Q000737:chemistry*; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003593:Cytoplasm / Q000737:chemistry; D004058:Diffusion; D004789:Enzyme Activation; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008264:Macrophages / Q000166:cytology*; D009682:Magnetic Resonance Spectroscopy; D009068:Movement; D009569:Nitric Oxide / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052247:Nitric Oxide Synthase Type II
D000590:Amino Acid Chloromethyl Ketones; D015853:Cysteine Proteinase Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D020169:Caspases