A CS-133 NUCLEAR-MAGNETIC-RESONANCE STUDY OF ENDOTHELIAL NA-K+-ATPASEACTIVITY - CAN ACTIN REGULATE ITS ACTIVITY()

Using Cs-133(+) NMR, we developed a technique to repetitively measure, in vivo, Na+-K+-ATPase activity in endothelial cells. The measurement s 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 extra cellular Cs+. Intracellularly we obtained a spin lattice relaxation ti me (T-1) of 2.0 +/- 0.3 s, and extracellular T-1 was 7.9 +/- 0.4 s, Na +-K+ pump activity in endothelial cells was determined at 12 +/- 3 nmo l Cs+. min(-1) . (mg Prot)(-1) under control conditions. When intracel lular 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%. Afte r 80 min of perfusion with 5 mM DOG and NaCN, reperfusion with control medium rapidly reestablished the endothelial membrane Cs+ gradient. U sing Cs-133(+) NMR as a convenient tool, we further addressed the prop osed role of actin as a regulator of Na+-K+ pump activity in intact ce lls. Two models of actin rearrangement were tested. DOG caused a rearr angement of F-actin and an increase in G-actin, with a simultaneous de crease in ATP concentration. Cytochalasin D, however, caused an F-acti n rearrangement different from that observed for DOG and an increase i n G-actin, and cellular ATP levels remained unchanged. In both models, the Na+-K+-pump activity remained unchanged, as measured with Cs-133 NMR. Our results demonstrate that Cs-133 NMR can be used to repetitive ly measure Na+-K+-ATPase activity in endothelial cells. No evidence fo r a regulatory role of actin on Na+-K+-ATPase was found.