Stimulation Of Human And Mouse Erythrocyte Na+-K+-2cl-Cotransport By Osmotic Shrinkage Does Not Involve Amp-Activated Protein Kinase, But Is Associated With Ste20/Sps1-Related Proline/Alanine-Rich Kinase Activation

This study was undertaken to investigate whether the mechanism of increased Na+-K+-2Cl- (NKCC1) cotransporter activity by osmotic shrinkage involved AMP-activated protein kinase (AMPK) activation. AMPK was found to phosphorylate a recombinant GST-dogfish (1-260) NKCC1 fragment at Ser38 and Ser214, corresponding to Ser77 and Ser242 in human NKCC1, respectively. Incubation of human erythrocytes with 20 mu m A769662 AMPK activator increased Ser242 NKCC1 phosphorylation but did not stimulate 86Rb+ uptake. Under hypertonic conditions in human red blood cells (RBCs) incubated with 0.3 m sucrose, NKCC1 activity increased as measured by bumetanide-sensitive 86Rb+ uptake and AMPK was activated. However, there was no effect of AMPK alpha 1 deletion in mouse RBCs on the increased rate of 86Rb+ uptake induced by hyperosmolarity. AMPK activation by osmotic shrinkage of mouse RBCs was abrogated by 10 mu m STO-609 CaMKK beta inhibitor, but incubation with STO-609 did not affect the increase in 86Rb+ uptake induced by hyperosmolarity. Osmotic shrinkage of human and mouse RBCs led to activation loop phosphorylation of the STE20/SPS1-related proline/alanine-rich kinase (SPAK) at Thr233, which was accompanied by phosphorylation of NKCC1 at Thr203/207/212, one of which (Thr207) is responsible for cotransporter activation. Therefore, phosphorylation-induced activation of NKCC1 by osmotic shrinkage does not involve AMPK and is likely to be due to SPAK activation.