Cell Physiol Biochem 2013;32(suppl 1):57-76 DOI: 10.1159/000356624 Published online: December 18, 2013 © 2013 S. Karger AG, Basel www.karger.com/cpb 57 Blanco et al.: Cell Water Volume and pH Changes Caused by NH 3 /NH 4 + Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry 1421-9778/13/0327-0057$38.00/0 Original Paper Accepted: December 02, 2013 This is an Open Access article licensed under the terms of the Creative Commons Attribution- NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only. Department of Pharmacology and Toxicology Wright State University, SOM, Room 063 MS, 3640 Colonel Glenn Hwy, Dayton, OH 45435 (USA) Fax+1 937 775-7221, E-Mail francisco.alvarez-leefmans@wright.edu Francisco J. Alvarez-Leefmans MD, Ph.D., Professor Parallel Changes in Intracellular Water Volume and pH Induced by NH 3 /NH 4 + Exposure in Single Neuroblastoma Cells Víctor M. Blanco a Martín S. Márquez b Francisco J. Alvarez-Leefmans Department of Pharmacology & Toxicology, Wright State University, Boonshoft School of Medicine, Dayton, OH, USA; a Present address: The Vontz Center for Molecular Studies, Division of Hematology/ Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA; b Present address: Division of Neuroscience, Instituto Nacional de Psiquiatría “Ramón de la Fuente”, México DF, México Key Words Ammonium • Ammonia • Isosmotic cell swelling • Isosmotic cell shrinkage • Isosmotic regulatory volume decrease • Isosmotic regulatory volume increase • Hyperammonemia • Brain edema Abstract Background: Increased blood levels of ammonia (NH 3 ) and ammonium (NH 4 + ), i.e. hyperammonemia, leads to cellular brain edema in humans with acute liver failure. The pathophysiology of this edema is poorly understood. This is partly due to incomplete understanding of the osmotic effects of the pair NH 3 /NH 4 + at the cellular and molecular levels. Cell exposure to solutions containing NH 3 /NH 4 + elicits changes in intracellular pH (pH i ), which can in turn affect cell water volume (CWV) by activating transport mechanisms that produce net gain or loss of solutes and water. The occurrence of CWV changes caused by NH 3 /NH 4 + has long been suspected, but the mechanisms, magnitude and kinetics of these changes remain unknown. Methods: Using fuorescence imaging microscopy we measured, in real time, parallel changes in pH i and CWV caused by brief exposure to NH 3 /NH 4 + of single cells (N1E-115 neuroblastoma or NG-108 neuroblastoma X glioma ) loaded with the fuorescent indicator BCECF. Changes in CWV were measured by exciting BCECF at its intracellular isosbestic wavelength (~438 nm), and pH i was measured ratiometrically. Results: Brief exposure to isosmotic solutions (i.e. having the same osmolality as that of control solutions) containing NH 4 Cl (0.5- 30 mM) resulted in a rapid, dose-dependent swelling, followed by isosmotic regulatory volume decrease (iRVD). NH 4 Cl solutions in which either extracellular [NH 3 ] or [NH 4 + ] was kept constant while the other was changed by varying the pH of the solution, demonstrated that [NH 3 ] o rather than [NH 4 + ] o is the main determinant of the NH 4 Cl-induced swelling. The iRVD response was sensitive to the anion channel blocker NPPB, and partly dependent on external Ca 2+ . Upon removal of NH 4 Cl, cells shrank and displayed isosmotic regulatory volume increase (iRVI). Regulatory volume responses could not be activated by comparable CWV changes