Parallel Changes in Intracellular Water Volume and pH Induced by NH3/NH4+ Exposure in Single Neuroblastoma Cells

Abstract

Background: Increased blood levels of ammonia (NH₃) and ammonium (NH₄⁺), 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 NH₃/NH₄⁺ at the cellular and molecular levels. Cell exposure to solutions containing NH₃/NH₄⁺ 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 NH₃/NH₄⁺ has long been suspected, but the mechanisms, magnitude and kinetics of these changes remain unknown. Methods: Using fluorescence imaging microscopy we measured, in real time, parallel changes in pH_i and CWV caused by brief exposure to NH₃/NH₄⁺ of single cells (N1E-115 neuroblastoma or NG-108 neuroblastoma X glioma ) loaded with the fluorescent 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 NH₄Cl (0.5- 30 mM) resulted in a rapid, dose-dependent swelling, followed by isosmotic regulatory volume decrease (iRVD). NH₄Cl solutions in which either extracellular [NH₃] or [NH₄⁺] was kept constant while the other was changed by varying the pH of the solution, demonstrated that [NH₃]_o rather than [NH₄⁺]_o is the main determinant of the NH₄Cl-induced swelling. The iRVD response was sensitive to the anion channel blocker NPPB, and partly dependent on external Ca²⁺. Upon removal of NH₄Cl, cells shrank and displayed isosmotic regulatory volume increase (iRVI). Regulatory volume responses could not be activated by comparable CWV changes produced by anisosmotic solutions, suggesting that membrane stretch or contraction by themselves are not sufficient to trigger these responses. Inhibition of glutamine synthetase partially blocked the NH₄Cl-induced swelling. Conclusions: A quantitative description of the osmotic changes produced by exposure to NH₃/NH₄⁺ in single neurons and glial cells shows that ∼35 to 45% of the initial cell swelling can be explained by intracellular accumulation of NH₄⁺ due to rapid permeation and protonation of NH₃. Another∼23% of the swelling can be accounted for by rapid glutamine accumulation. The results are discussed in terms of basic cell physiology and their potential relevance to the pathophysiology of hyperammonemic cellular brain edema.