NMR studies of intracellular water at 300 MHz: T₂‐specific relaxation mechanisms in synchronized or EGF‐stimulated cells

Abstract

Responses specific to the spin-spin relaxation time (T2) have been observed in two time-dependent studies of the intracellular water in normal and transformed Syrian hamster fetal fibroblasts. At 300-MHz (7.0 T), the spin-lattice relaxation time (T1) was insensitive to several aspects of cellular physiology that produced changes in the T2 and the apparent self-diffusion coefficient (Dapp) of intracellular water. In normal cells stimulated with epidermal growth factor (EGF), T1 was insensitive to time-dependent changes detected by T2 and Dapp. In synchronized tumor cells, T1 was insensitive to cell-cycle-dependent changes detected by T2. The strongly coupled behavior of T2 and Dapp that was observed as a function of time in EGF-stimulated cells indicates that the diffusion of intracellular water through inhomogeneous local magnetic field gradients produced effects observable in T2. Conformational changes in large intracellular macromolecular assemblies such as chromatin or the cytoskeleton may alter the magnitude and inhomogeneity of local field gradients, producing responses in T2 and Dapp only.

Influence of cell cycle phase on apparent diffusion coefficient in synchronized cells detected using temporal diffusion spectroscopy

“…Dynamic variations of major intracellular structures may reflect the physiological states of cells, and some of these, such as nuclear size, may be characteristic of specific diseases. For example, there is massive synthesis and rearrangement of intracellular macromolecules and organelles when cells undergo division, as previously reported for relaxation times (7, 8). Moreover, proliferating tumors usually contain a much higher fraction of cells that are in active cell division phases, such as the S (synthesis) and M (mitosis) phases, than normal tissues, so for a full understanding of the diffusion properties of tumors, it is necessary to understand how the changes that occur inside cells in different phases affect the ADC (8, 9).…”

Intracellular water motion decreases in apoptotic macrophages after caspase activation

“…Parallel changes were observed in ADC, and these results have been interpreted as reflecting alterations in the diffusion of intracellular water through non-homogeneous local magnetic field gradients, these effects also influencing T 2 values. 35,36 In addition to this, decreases in ADCs of water and small metabolites were measured under hypoxia in brain slices, 37 and in models of intact/relaxed and skinned/rigor muscle fibers of frog skeletal muscle, where the intracellular water located in the overlap region of actin and myosin filaments was less structured in the rigor state than in the relaxed situation. 38 Data on ADC and T 2 values have previously been studied in apoptotic cells.…”

In-cell NMR: Why and how?

“…These measurements neglected the effects of magnetic susceptibility inhomogeneities in cell samples, which generate intrinsic field gradients and thus additional signal decay during PFG NMR experiments (These can themselves be used to measure intracellular diffusion coefficients of water in live and fixed cells [83,84]); the effects of such inhomogeneities on NMR signals are rather complicated and are treated in Section 3.2. They were also ignored in the later PFG NMR studies on cells published since the 1990's, which aimed at measuring transmembrane water exchange in erythrocytes with varying cholesterol contents [85] and in the Gram positive Corynebacterium glutamicum [86], or self-diffusion coefficients of water in cultured mammalian cells through the cell cycle [87] and in camel erythrocytes [88]. PFG NMR experiments have also been used to measure simultaneously both water self-diffusion coefficients and transmembrane exchange in various systems: in yeast at different growth phases [89], in unicellular algae Chlorella [90], in erythrocytes [91][92][93][94], in cultured mammalian cells [95][96][97], or in plant systems like banana tissues [98], lupin roots upon exposure to lead [99], in maize roots of different lengths, or upon oxidative stress or at varying CO 2 concentrations [100][101][102].…”

“…Rubidium has for long been established as a good surrogate for cellular potassium uptake [285], and used in quantitative studies primarily in its radioactive form 86 Rb [286]. Its stable isotope 87 Rb was considered in the late 1980's for achieving NMR studies of potassium uptake in intact rat kidney and heart [287][288][289], in mammalian cultured cells [290], in erythrocytes [291,292], in rat salivary glands [293] and in skeletal muscles of living rats [294]. Sensitivity assessment in erythrocytes indeed showed that direct 87 Rb NMR detection was likely to provide about 19 times more signal than 39 K [291].…”

“…Its stable isotope 87 Rb was considered in the late 1980's for achieving NMR studies of potassium uptake in intact rat kidney and heart [287][288][289], in mammalian cultured cells [290], in erythrocytes [291,292], in rat salivary glands [293] and in skeletal muscles of living rats [294]. Sensitivity assessment in erythrocytes indeed showed that direct 87 Rb NMR detection was likely to provide about 19 times more signal than 39 K [291]. However, as with 23 Na or 39 K, the quadrupolar nature of 87 Rb causes fast, bi-exponential relaxation in cells [295,296].…”