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Gruwel ML, Culic O, Schrader J: A 133Cs nuclear magnetic resonance study of endothelial Na (+)-K (+)-ATPase activity: can actin regulate its activity?. Biophys J. 1997 Jun;72(6):2775-82.
Using (133) Cs+ NMR, we developed a technique to repetitively measure, in vivo, Na (+)-K (+)-ATPase activity in endothelial cells. The measurements were made without the use of an exogenous shift reagent, because of the large chemical shift of 1.36 +/- 0.13 ppm between intra- and extracellular Cs+. Intracellularly we obtained a spin lattice relaxation time (T1) of 2.0 +/- 0.3 s, and extracellular T1 was 7.9 +/- 0.4 s. Na (+)-K+ pump activity in endothelial cells was determined at 12 +/- 3 nmol Cs+ x min (-1) x (mg Prot)[-1] under control conditions. When intracellular ATP was depleted by the addition of 5 mM 2-deoxy-D-glucose (DOG) and NaCN to about 5% of control, the pump rate decreased by 33%. After 80 min of perfusion with 5 mM DOG and NaCN, reperfusion with control medium rapidly reestablished the endothelial membrane Cs+ gradient. Using (133) Cs+ NMR as a convenient tool, we further addressed the proposed role of actin as a regulator of Na (+)-K+ pump activity in intact cells. Two models of actin rearrangement were tested. DOG caused a rearrangement of F-actin and an increase in G-actin, with a simultaneous decrease in ATP concentration. Cytochalasin D, however, caused an F-actin rearrangement different from that observed for DOG and an increase in G-actin, and cellular ATP levels remained unchanged. In both models, the Na (+)-K (+)-pump activity remained unchanged, as measured with (133) Cs NMR. Our results demonstrate that (133) Cs NMR can be used to repetitively measure Na (+)-K (+)-ATPase activity in endothelial cells. No evidence for a regulatory role of actin on Na (+)-K (+)-ATPase was found. |
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