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 K 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 approximately 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-Cl COT. 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.
Ferrell
C M
CM
Department of Pharmacology and Toxicology, Wright State University, Dayton, Ohio 45435, USA.
Lauf
P K
PK
Wilson
B A
BA
Adragna
N C
NC
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D048429:Cell Size; D004912:Erythrocytes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008094:Lithium / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D012413:Rubidium / Q000378:metabolism; D012756:Sheep; D015398:Signal Transduction; D027981:Symporters; D000096922:K Cl- Cotransporters
D002352:Carrier Proteins; D008094:Lithium; D010716:Phosphatidylinositols; D010743:Phospholipids; D011188:Potassium; D011493:Protein Kinase C; D012413:Rubidium; D027981:Symporters; D000096922:K Cl- Cotransporters