Volume control is one of the evolutionarily oldest cellular homeostatic functions. It appeared when macromolecules were encased within a semipermeable plasma membrane, creating Donnan forces that tended to produce colloid-osmotic swelling. Animal cells lack rigid cell walls and are permeable to water. They evolved mechanisms for maintaining volume constant in the face of the Donnan effect, thus preventing osmotic swelling and lysis. Failure of these mechanisms compromises structural integrity and constancy of the intracellular milieu, causing osmotic disturbances in organ function that may be lethal, such as brain edema. Brain cells face unique and potentially severe challenges for cell volume homeostasis. Net accumulation or depletion of solutes may occur in neurons and glial cells as a consequence of neurotransmitter actions and nerve impulse activity, or in pathological conditions such as ischemia, trauma, seizures or metabolic disorders. Our long-term objective is to understand the mechanisms underlying cell volume control under normal and pathophysiological conditions in nerve and glial cells. The present proposal uses an in vitro model, at the single-cell level, to study mechanisms underlying short-term changes in cell water volume, intracellular pH, Ca2+ and organic osmolytes, elicited in neurons and glial cells by exposure to ammonia (NH3) and ammonium (NH4+). The clinical implications of this research stem from the fact that millimolar concentrations of NH3/NH4+ in arterial blood (hyperammonemia) are a key factor in producing brain edema characteristic of acute liver failure, a condition that may occur as a complication of viral hepatitis, toxic drug reactions, and some metabolic diseases. The pathogenesis of this edema is not understood in spite of being the main cause of death in acute liver failure. The short-term changes produced by NH3/NH4+ probably precede or cause the long-term changes occurring in brain tissue following acute hyperammonemia The proposed studies are as relevant for basic research as they are for pathophysiology; although the reciprocal interactions between pH1 and volume regulatory mechanisms have been recognized, concurrent measurements of pHi and cell water volume are lacking. To approach these issues we developed and validated optical methods based on fluorescence imaging and photometry, with unique time resolution (less than 1 second) and sensitivity (about1 percent), to measure simultaneously, in a single cell, changes in pHi (or [Ca2+]i) and water volume.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-SSS-P (01))
Program Officer
Hicks, Ramona R
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Wright State University
Schools of Medicine
United States
Zip Code
Blanco, Víctor M; Márquez, Martín S; Alvarez-Leefmans, Francisco J (2013) Parallel changes in intracellular water volume and pH induced by NH(3)/NH(4)(+) exposure in single neuroblastoma cells. Cell Physiol Biochem 32:57-76
Mao, Shihong; Garzon-Muvdi, Tomas; Di Fulvio, Mauricio et al. (2012) Molecular and functional expression of cation-chloride cotransporters in dorsal root ganglion neurons during postnatal maturation. J Neurophysiol 108:834-52
Rocha-Gonzalez, Hector I; Mao, Shihong; Alvarez-Leefmans, Francisco J (2008) Na+,K+,2Cl- cotransport and intracellular chloride regulation in rat primary sensory neurons: thermodynamic and kinetic aspects. J Neurophysiol 100:169-84
Ares, Gustavo R; Caceres, Paulo; Alvarez-Leefmans, Francisco J et al. (2008) cGMP decreases surface NKCC2 levels in the thick ascending limb: role of phosphodiesterase 2 (PDE2). Am J Physiol Renal Physiol 295:F877-87
Munoz, Alberto; Mendez, Pablo; DeFelipe, Javier et al. (2007) Cation-chloride cotransporters and GABA-ergic innervation in the human epileptic hippocampus. Epilepsia 48:663-73
Alvarez-Leefmans, Francisco J; Herrera-Perez, Jose J; Marquez, Martin S et al. (2006) Simultaneous measurement of water volume and pH in single cells using BCECF and fluorescence imaging microscopy. Biophys J 90:608-18
Granados-Soto, Vinicio; Arguelles, Carlos F; Alvarez-Leefmans, Francisco J (2005) Peripheral and central antinociceptive action of Na+-K+-2Cl- cotransporter blockers on formalin-induced nociception in rats. Pain 114:231-8
Hamann, Steffen; Herrera-Perez, Jose Jaime; Bundgaard, Magnus et al. (2005) Water permeability of Na+-K+-2Cl- cotransporters in mammalian epithelial cells. J Physiol 568:123-35
Marty, Serge; Wehrle, Rosine; Alvarez-Leefmans, Francisco Javier et al. (2002) Postnatal maturation of Na+, K+, 2Cl- cotransporter expression and inhibitory synaptogenesis in the rat hippocampus: an immunocytochemical analysis. Eur J Neurosci 15:233-45
Alvarez-Leefmans, F J; Leon-Olea, M; Mendoza-Sotelo, J et al. (2001) Immunolocalization of the Na(+)-K(+)-2Cl(-) cotransporter in peripheral nervous tissue of vertebrates. Neuroscience 104:569-82

Showing the most recent 10 out of 16 publications