Lithium and Protein Kinase C Modulators Regulate Swelling-Activated K-Cl Cotransport and Reveal a Complete Phosphatidylinositol Cycle in Low K Sheep Erythrocytes C.M. Ferrell 1 , P.K. Lauf 2 , B.A. Wilson 3 , N.C. Adragna 1 Departments of Pharmacology and Toxicology 1 , Physiology and Biophysics 2 , and Biochemistry and Molecular Biology 3 , Wright State University, Dayton, Ohio 45435, USA Received: 1 November 1999/Revised: 6 June 2000 Abstract. K-Cl cotransport (COT), a ouabain- insensitive, Cl-dependent bidirectional K flux, is ubiqui- tously 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 modu- lates several cation transport pathways and inhibits phos- pholipid 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 re- sponse, its regulation, or by competing with K for bind- ing 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 ∼7mM. 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. Further- more, Li i increased the production of diacylglycerol in a bimodal fashion, without significant effects on the phos- phatidylinositol 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 re- sponse of K-Cl COT to pH and volume. Key words: Lithium — K-Cl Cotransport — Phospha- tidylinositol — Erythrocytes — Protein kinase C — Cell Swelling Introduction Red blood cells (RBCs) from various species, including sheep, toadfish, dog, rabbit and man, have been used as models for the study of K-Cl cotransport (COT) and cell volume regulation [13, 21, 23]. K-Cl COT is defined as a Na-independent, Cl-dependent, ouabain-insensitive K flux. In sheep (S) RBCs, this system is stimulated by acid pH, cell swelling, thiol modification, oxidative stress, nitrite (a nitric oxide derivative), Mg depletion and vasodilators [2, 3, 4, 24, 29, 36]). K-Cl COT is inhibited by quinine and quinidine, stilbene derivatives, bumetanide and furosemide [1, 25, 29] and has a low affinity for both of its carried ions. These features dis- tinguish it from other K transport pathways [29]. K-Cl COT is activated by staurosporine and is in- hibited by genistein, both kinase inhibitors [8, 15]. In addition, the protein phosphatase 1 (PP1) inhibitors, oka- daic acid and calyculin, are inhibitors of the system [19, 22]. The effect of these compounds on K-Cl COT ac- tivity suggests control by phosphorylation/dephosphoryl- ation reactions [19]. K-Cl COT is mediated by a ∼150 kDa protein with 12 putative transmembrane domains and cytoplasmic N- and C-termini [16]. Of the four iso- forms identified, KCC1 is ubiquitously distributed and possesses kinase consensus sites (i.e., phosphorylation by casein kinase and PKC) located on the C-terminal domain [16]. Aside from cytosolic regulation, LK Correspondence to: N.C. Adragna J. Membrane Biol. 177, 81–93 (2000) DOI: 10.1007/s002320001101 The Journal of Membrane Biology © Springer-Verlag New York Inc. 2000