Francisco Javier Alvarez-Leefmans - US grants

DESCRIPTION: 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.

DESCRIPTION (provided by applicant): 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.