Background: Calcium is an important cellular mediator in numerous physiological processes. Under pathological conditions, large influxes of Ca²⁺ into mitochondria may occur, leading to activation of “cell-death” pathways. Although there are several methods by which to monitor mitochondrial Ca²⁺ [Ca²⁺]_m in vitro, current methods for quantifying [Ca²⁺]_m in the living animal are limited. Purpose: Herein, we describe a rapid and simple method by which to quantify the levels of total [Ca²⁺]_m in situ. Methods: The method relies on the presence of inhibitors of Ca²⁺ influx and efflux to prevent movement of Ca²⁺ during mitochondrial isolation followed by the lysing of the organelles to liberate the cation. Total [Ca²⁺]_m is quantified using the fluorescent probe Calcium Green 5-N. Results: A standard linear relationship between calcium green 5-N fluorescence signal intensity as a result of increasing concentrations of Cacl2 indicates that the amount of mitochondria in the assay mixture does not interfere with the fluorescent signal. A significant increase in Ca²⁺ was observed, presumably due to the release of both bound and free Ca²⁺ from the mitochondria in response to Triton X-100 exposure. The pH of samples from both DNP- and DMSO- treated animals remained unaltered after solubilization with Triton X-100. Conclusion: The method is sensitive to in vivo perturbations, as demonstrated by decreases in [Ca²⁺]_m following administration of the uncoupling agent 2, 4-dinitrophenol.

 

doi : 10.5214/ans.0972.7531.2009.160204

 

Competing interests: None.          Source of Funding: None

Received Date: 09 March 2009     Revised Date: 31 March 2009     Accepted Date: 18 April 2009