Carbonic anhydrase involvement in the lysosomal system response to heavy metal exposure

Lysosomes are intracellular organelles with an acidic internal pH maintained by a V-ATPase H+ pump. Lysosomal reactions (alterations in lysosomal number and size and lysosomal destabilization) are involved in a number of physiological responses, including xenobiotic and heavy metal sequestration and detoxification. The aim of the study was to investigate the role of carbonic anhydrase, an ubiquitous enzyme catalyzing the hydration of CO2 to HCO2- and H+, in the lysosomal response to heavy metal exposure by using mussel (Mytilus galloprovincialis) digestive cells, characterized by a well developed lysosomal system, as experimental model. CdCl2 was used as reference toxicant. A novel microplate fluorimetric assay was developed on digestive cells in suspension, charged with the fluorescent acidotropic probe Lysosensor™ Green DND-189, for quantitating the number of lysosomes and their pH. In parallel, single cell analysis was performed by confocal microscopy. Digestive cells from Cd exposed animals showed a time and dose dependent increase of the probe fluorescence, suggesting a proliferation of the lysosomal compartment and decrease of the internal pH. This was confirmed by single cell confocal analysis. In animals in vivo exposed to acetazolamide, specific inhibitor of carbonic anhydrase, the metal induced fluorescence increase was dramatically inhibited. In conclusion, results indicate a key role of carbonic anhydrase in the lysosomal responses to metal exposure.