Role of the plasma membrane calcium adenosine triphosphatase on domoate-induced intracellular acidification in primary cultures of cerebelar granule cells

Changes in intracellular pH (pHi) and cytosolic calcium concentration ([Ca2+]c) caused by the glutamate agonist domoate (DOM) were studied in single cultured mouse cerebellar granule cells (CGC) by using the fluorescent probes 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and simultaneous evaluation of cytosolic calcium concentration with the fluorescent dye Fura-2 acetoxymethyl ester (Fura-2 AM). DOM caused a concentration-dependent increase in [Ca2+]c and a concentration-dependent intracellular acidification of CGC. DOM-induced intracellular acidification was completely abolished by the use of Ca2+-free medium, suggesting that it was due mostly to an influx of extracellular calcium. The pHi decrease caused by DOM was also completely blocked in the presence of the AMPA/kainate receptor antagonist CNQX, indicating that the DOM-induced intracellular acidification was caused by DOM activation of the AMPA/kainate subtype of glutamate receptors. Different mechanisms that could be involved in DOM-induced pHi decrease, such as displacement of H+ by Ca2+ from a common intracellular binding site, DOM-induced alteration of pHi regulation mechanisms, and a possible acidification caused by DOM-induced increase of mitochondrial Ca2+ uptake, were excluded. DOM-induced intracellular acidification was completely prevented by inhibitors of the plasma membrane calcium adenosine triphosphatase (ATPase) (PMCA), including orthovanadate, lanthanum extracellular pH of 8.5, and the specific PMCA inhibitor caloxin 2A1. Our results therefore indicate that PMCA is involved in DOM-induced intracellular acidification in primary cultures of CGC. Simultaneous recording of [Ca2+]c and pHi indicates that the increase in intracellular calcium evoked by DOM will activate the calcium extrusion mechanisms through the calcium pump, which, in turn, will decrease intracellular pH by countertransport of H+ ions.
Ministerio de Ciencia y Tecnologı´a, Spain; Contract grant number: SAF2003-08765-C03-02; Contract grant number: REN2001-2959-C04-03; Contract grant number: REN2003-06598- C02-01; Contract grant number: AGL2004-08268-02-O2/ALI; Contract grant number: INIA CAL01-068; Contract grant number: SAF-FEDER 2003-0493; Contract grant sponsor: Xunta de Galicia, Spain; Contract grant number: PGIDT99INN26101; Contract grant number: PGIDIT03AL26101PR; Contract grant sponsor: Fondo de Investigaciones Sanitarias, Spain; Contract grant number: FISS REMA-G03-007; Contract grant sponsor: EU VIth Frame Program; Contract grant number: FOOD-CT-2004-06988 (BIOCOP); Contract grant number: FOODCT-2004-514055 (DETECTOX).