In all animal cells, the presence of impermeant anions in the intracellular compartment creates Gibbs-Donnan forces which tend to produce colloid-osmotic swelling. Cells evolved mechanisms for maintaining their volume constant in isosmotic media, thus preventing osmotic swelling and lysis. Failure of these mechanisms underlies the pathophysiology of osmotic cell damage. Neurons face unique and potentially severe challenges for cell volume homeostasis. Net accumulation or depletion activity, or in pathological conditions such as ischemia, trauma, seizures or metabolic disorders. In spite of its clinical and therapeutic implications, knowledge on physiology and pathology of cell volume homeostasis in neuronal cells is scarce. This is a proposal to study the cellular and molecular mechanisms by which neuronal cells regulate and maintain their water volume upon changes in extracellular or intracellular osmolality elicited by external stimuli.
We aim to ascertain the role of ionized calcium (Ca2+) as an intracellular signal coupling external stimuli with effectors controlling cell volume decrease in cells swollen in hyposmotic media, and characterize pharmacologically the membrane transport systems effecting this response. In isosmotic medium, some hormones, peptides, and neurotransmitters, or inhibition of the Na+/K+ pump, produce neuronal shrinkage which seems to be mediated by an increase in [Ca2+]. We will also study the role of Ca2+ as mediator of these responses and the mechanisms leading to restoration of cell volume in isosmotic media. The consequences of inhibition of the Na+/K+ pump on osmotic balance and cell survival following acute Na+ loads, like those occurring under pathological conditions, will be determined. These studies will be conducted using, as in vitro models, murine neuronal cell lines and land snail neurons under strictly defined conditions. Changes in water volume and intracellular ion activities will be measured in single cells using fluorescent probes with newly developed optical methods. Transmembrane current will be studied with voltage clamp techniques.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS029227-06
Application #
2379663
Study Section
Neurology B Subcommittee 2 (NEUB)
Project Start
1991-04-01
Project End
2000-02-29
Budget Start
1997-03-01
Budget End
1998-02-28
Support Year
6
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Physiology
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
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

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