Functional and molecular characterization of a newly identified human granulocyte H+/K+-ATPase

Polymorphonuclear leucocytes (PMNs) migrate to sites of infection upon activation by various chemotactic stimuli. During this stimulated migration as well as during phagocytosis, PMNs are subjected to morphological changes like cell swelling as we1l as metabolic changes like the generation of free oxygen radicals and intracellular protons. Therefore, this so called respiratory burst leads to an enormous intracellular acid load, and accurate extrusion of protons is of critical importance for the survival and normal function of PMNs. One H + ion extruding mechanism has been identified as the Na+ /H+ exchanger. Its activation tends to restore the initial intracellular pH and leads to cell swelling. We have shown that this cell swelling is a prerequisite for stimulated migration of PMNs. Inhibition of the Na+ /H+ exchanger deteriorates the intracellular acidosis, prevents cell swelling and inhibits chemotaxis stimulated by the peptide fMLP. However, several pathophysiological conditions like extracellular acidosis or altered ambient osmolarity are unfavorable for the Na+ /H+ exchange activity. Recently, we have succeeded in showing for the first time the existence of a K+ -dependent proton extruding mechanism in human PMNs that is involved in intracellular pH regulation and functionally linked to chemotaxis via a cell volume-dependent mechanism. Stimulation of cell migration with fMLP was accompanied by rapid intracellular acidosis and cell swelling. Proton extrusion, cell swelling and chemotaxis could be inhibited by omeprazole and SCH 28080, both being specific inhibitors of the gastric H+ /K+ -ATPase. By using a monoclonal antibody specific for the beta- subunit of gastric H+ /K+ -ATPase for immunoblotting experiments, we detected a highly glycosylated 35kD core protein in lysates of human PMNs. These results strongly indicate the existence of a new isoform of an H + /K+ - ATPase in human PMNs that is involved in the regulation of intracellular pH and chemotaxis via a cell volume- dependent mechanism [1]. Since the H+ /K+ -ATPase is a primary active ion transporter, such a mechanism would support cell migration even under pathological conditions like those in the highly acidic milieu of abscesses or in the urinary tract. The aim of this project is to characterize this H+ /K+ -ATPase on a functional and molecular level. 1 Ritter M. et al.: Effect of inhibitors of Na+ /H+ -exchange and gastric H+ /K+ -ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes. British Journal of Pharmacology, 1998:124:627-638

Polymorphonuclear leucocytes (PMNs) are white blood cells that play a key role in the immune defense against pathogens. They are attracted to sites of infection by various stimuli, a process called chemotaxis. At the site of the infection they ingest and destroy the pathogens by a mechanism termed phagocytosis. These processes are accompanied by an enormous acid load in the cells interior that is harmful to the PMNs. Hence, accurate extrusion of acid (in the form of protons, H+ ) is of critical importance for the normal function and survival of the cells. Some of the molecular mechanisms fulfilling this task are known and well characterized. Recently we have shown for the first time the existence of a H+ -extruding mechanism in human PMNs that is involved in intracellular pH regulation and functionally linked to the regulation chemotaxis via a mechanism involving swelling of the cells. The project focused on the functional and molecular characterization of this mechanism. Using different experimental approaches in PMNs and cells closely related to PMNs (HL60 cells), we were able to identify that a protein called ATP1AL1 is responsible for this acid extruding mechanism. This molecule is related to the gastric acid pump, which serves to produce the acidic gastric juice. ATP1AL1 can be inhibited by drugs which are also in clinical use to block gastric acid secretion. This opens the possibility for the development of new therapeutic approaches for the treatment of inflammatory diseases.