Lithium and protein kinase C modulators regulate swelling-activated K-Cl cotransport and reveal a complete phosphatidylinositol cycle in low K sheep erythrocytes

K-Cl cotransport (COT), a ouabain insensitive, Cl-dependent bidirectional Ii flux, is ubiquitously present in all cells, plays a major role in ion and volume homeostasis. and is activated by cell swelling and a variety of chemical interventions. Lithium modulates several cation transport pathways and inhibits phospholipid turnover in red blood cells (RBCs). Lithium also inhibits K-Cl COT by an unknown mechanism. To test the hypothesis whereby Li inhibits swelling-activated K-Cl COT by altering either its osmotic response, its regulation, or by competing with K for binding sites, low K (LK) sheep (S) RBCs were loaded with Li by Na/Li exchange or the cation ionophore nystatin. K-Cl COT was measured as the Cl-dependent, ouabain-insensitive K efflux or Rb influx. The results show that Li altered the cell morphology, and increased both cell volume and diameter. Internal (Li-i) but not external (Li-o) Li inhibited swelling-activated K-Cl COT by 85% with an apparent K-i of similar to 7 mM. In Cl, Li-i decreased K efflux at relative cell volumes between 0.9 and 1.2, and at external pHs between 7.2 and 7.4. Li-i reduced the V-max and increased the K-m for K efflux in Cl. Furthermore, Li-i increased the production of diacylglycerol in a bimodal fashion, without significant effects on the phosphatidylinositol concentration, and revealed the presence of a complete PI cycle in LK SRBCs. Finally, phorbol ester treatment and PD89059, an inhibitor of mitogen-activated protein kinase (ERK2) kinase, caused a time-dependent inhibition of K-CI GOT. Hence, Li-i appears to inhibit K-Cl COT by acting at an allosteric site on the transporter or its putative regulators, and by modulation of the cellular phospholipid metabolism and a PKC-dependent regulatory pathway, causes an altered response of K-Cl COT to pH and volume.