Effects of the marine phycotoxin palytoxin on neuronal pH in primary cultures of cerebellar granule cells

Abstract

Palytoxin (PTX) is a potent marine phycotoxin that binds to the Na,K-ATPase, converting this pump into an open channel. We have recently shown (Vale et al., 2006) that PTX causes an irreversible increase in the cytosolic calcium concentration ([Ca(2+)](c)) in primary cultures of cerebellar granule cells (CGC). In this work, we investigated the effect of PTX on the intracellular pH (pH(i)) in the same cellular model. PTX-induced changes in pH(i) were studied in CGC by using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). PTX caused an irreversible intracellular acidification of CGC. This acidification was due to an influx of extracellular calcium, inasmuch as it was completely abolished by the use of Ca(2+)-free medium. Different mechanisms that could be involved in the PTX-induced pH(i) decrease such as displacement of H(+) by Ca(2+) from a common intracellular binding site, PTX-induced alteration of pH(i) regulation mechanisms, and a possible acidification caused by an increase of mitochondrial Ca(2+) uptake by PTX were excluded. PTX-induced intracellular acidification was completely prevented by several inhibitors of the plasma membrane calcium ATPase (PMCA), including orthovanadate, lanthanum, high extracellular pH, and caloxin 2A1. Our results indicate that the PMCA is involved in the PTX-induced intracellular acidification in primary cultures of CGC. The PTX-evoked increase in [Ca(2+)](c) will activate the calcium extrusion mechanisms through the PMCA, which, in turn, will decrease pH(i) by countertransport of H(+) ions. The effect of PTX on neuronal pH could be a potential factor to contribute to the high cytotoxicity of this toxin in cultured cerebellar neurons.