A 133Cs nuclear magnetic resonance study of endothelial Na(+)-K(+)-ATPase activity: can actin regulate its activity?

• Published in: Biophysical journal 1997-06, Vol.72 (6), p.2775-2782
• Main Authors: Gruwel, M.L., Culíc, O., Schrader, J.
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Adenosine Triphosphate - metabolism; Animals; Biophysical Phenomena; Biophysics; Cesium; Cytochalasin D - pharmacology; Deoxyglucose - pharmacology; Endothelium, Vascular - drug effects; Endothelium, Vascular - enzymology; In Vitro Techniques; Kinetics; Magnetic Resonance Spectroscopy; Models, Biological; Sodium Cyanide - pharmacology; Sodium-Potassium-Exchanging ATPase - chemistry; Sodium-Potassium-Exchanging ATPase - metabolism; Swine
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(97)78920-3

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.