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Intracellular water motion decreases in apoptotic macrophages after caspase activation
S Hortelano1, M L García-Martín, S Cerdán
1Instituto de Bioquímica, Centro Mixto CSIC-UCM, Facultad de Farmacia, Universidad Complutense. 28040 Madrid, Spain.
Abstract:
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.
Insights
Apoptosis in macrophages alters intracellular water motion, evidenced by decreased water diffusion and relaxation times. These changes, linked to caspase activation, precede key apoptotic events and suggest altered intracellular physicochemical properties.
Area of Science:
- Cell Biology
- Biophysics
- Biochemistry
Background:
- Macrophages play a crucial role in immune responses.
- Apoptosis is a critical process in cellular homeostasis and disease.
- Nitric oxide synthase (NOS) and caspases are key regulators of apoptosis.
Purpose of the Study:
- To investigate the changes in intracellular water motion during macrophage apoptosis.
- To correlate these water dynamics with specific apoptotic events and molecular pathways.
Main Methods:
- Utilized RAW 264.7 macrophage cell line.
- Induced apoptosis using lipopolysaccharide and interferon-gamma.
- Employed (1)H NMR spectroscopy to measure intracellular transverse relaxation time (T(2)) and apparent diffusion coefficient (ADC).
- Assessed mitochondrial depolarization, microtubule depolymerization, and hydrophobic properties using fluorescent probes.
Main Results:
- Lipopolysaccharide and interferon-gamma induced apoptosis, which was inhibited by type 2 nitric oxide synthase or caspase inhibitors.
- A significant decrease in T(2) values (from 287 to 182 ms) was observed in cells treated with NO-donors.
- These T(2) changes were prevented by caspase inhibitors and were independent of mitochondrial or microtubule status.
- Decreased intracellular T(2) and ADC values occurred after caspase activation but before phosphatidylserine exposure and DNA fragmentation.
- Apoptosis-associated water motion changes correlated with enhanced intracellular hydrophobicity.
Conclusions:
- Macrophage apoptosis involves significant alterations in intracellular water physicochemical properties.
- Changes in water diffusion and relaxation dynamics are early indicators of apoptosis, linked to caspase activation.
- These findings provide novel biophysical insights into the cellular events during apoptosis.