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.
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