Growth factors stimulate the Na-K-2Cl cotransporter NKCC1 through a novel Cl--dependent mechanism

The Na-K-2Cl cotransporter NKCC1 is an important volume-regulatory transporter that is regulated by cell volume and intracellular Cl-. This regulation appears to be mediated by phosphorylation of NKCC1, although there is evidence for additional, cytoskeletal regulation via myosin light chain (MLC) kinase. NKCC1 is also activated by growth factors and may contribute to cell hypertrophy, but the mechanism is unknown. In aortic endothelial cells, NKCC1 (measured as bumetanide-sensitive Rb-86(+) influx) was rapidly stimulated by serum, lysophosphatidic acid, and fibroblast growth factor, with the greatest stimulation by serum. Serum increased bumetanide-sensitive influx significantly more than bumetanide-sensitive efflux (131% vs. 44%), indicating asymmetric stimulation of NKCC1, and produced a 17% increase in cell volume and a 25% increase in Cl- content over 15 min. Stimulation by serum and hypertonic shrinkage were additive, and serum did not increase phosphorylation of NKCC1 or MLC, and did not decrease cellular Cl- content. When cellular Cl- was replaced with methanesulfonate, influx via NKCC1 increased and was no longer stimulated by serum, whereas stimulation by hypertonic shrinkage still occurred. Based on these results, we propose a novel mechanism whereby serum activates NKCC1 by reducing its sensitivity to inhibition by intracellular Cl-. This resetting of the Cl- set point of the transporter enables the cotransporter to produce a hypertrophic volume increase.