Effects of osmotic manipulation of intracellular hydration of HeLa S-3 cells on their proton NMR relaxation times

Abstract

Pellets of HeLa from suspension cultured cells in isotonic medium (300 mosmolar) were introduced into a Bruker CXP100 NMR spectrophotometer at 80 mHz within 5 min of the start of centrifugation. T1 and T2 times were measured within a total elapsed time of 20-25 min at 80 mHz and 37 degrees C, and averaged 1430 msec and 120 msec, respectively. Extrapolation to zero extracellular space gave a corrected T1 of 1370 msec. For cells collected after 10 min in hypotonic medium (down to 30 mosmolar) increased proton density correlated well with increased cell water content, but relaxation times did not rise in proportion to that predicted for the entry of "bulk" water (T1 of 4700 msec), except when swelling approached lysis point. Cells partially dehydrated by 10 min in hypertonic medium of up to 1500 mosmolar have also been analyzed, but once again the shortening of T1 was not proportional to the loss of "free" (bulk phase) water. At the upper limit of hypertonic treatment, lacunae or vacuoles of a watery nature separated within the cytomatrix, preventing maximum dehydration. The relationship of cell water to T1 is complex over the whole range of tonicity that HeLa S-3 cells tolerate. The data indicate, however, that hypotonically induced water probably has an average T1 time considerably lower than bulk phase water. In contrast, raising the total extracellular volume with medium had precisely the predicted effect on T1 time, further strengthening the case that water taken up by cell acquires a shorter T1 time. Cells adapting to hypotonic conditions oscillated in size and water content over 2-3 hr before returning to near their initial volume. Under these circumstances, T1 oscillated in the same way but with a reduced amplitude, consistent with the above findings.