Caspofungin induces the release of Ca(2+) ions from internal stores by activating ryanodine receptor-dependent pathways in human tracheal epithelial cells

The antimycotic drug caspofungin is known to alter the cell function of cardiomyocytes and the cilia-bearing cells of the tracheal epithelium. The objective of this study was to investigate the homeostasis of intracellular Ca(2+) concentration ([Ca(2+)](i)) after exposure to caspofungin in isolated human tracheal epithelial cells. The [Ca(2+)](i) was measured using the ratiometric fluoroprobe FURA-2 AM. We recorded two groups of epithelial cells with distinct responses to caspofungin exposure, which demonstrated either a rapid transient rise in [Ca(2+)](i) or a sustained elevation of [Ca(2+)](i). Both patterns of Ca(2+) kinetics were still observed when an influx of transmembraneous Ca(2+) ions was pharmacologically inhibited. Furthermore, in extracellular buffer solutions without Ca(2+) ions, caspofungin exposure still evoked this characteristic rise in [Ca(2+)](i). To shed light on the origin of the Ca(2+) ions responsible for the elevation in [Ca(2+)](i) we investigated the possible intracellular storage of Ca(2+) ions. The depletion of mitochondrial Ca(2+) stores using 25 µM 2,4-dinitrophenol (DNP) did not prevent the caspofungin-induced rise in [Ca(2+)](i), which was rapid and transient. However, the application of caffeine (30 mM) to discharge Ca(2+) ions that were presumably stored in the endoplasmic reticulum (ER) prior to caspofungin exposure completely inhibited the caspofungin-induced changes in [Ca(2+)](i) levels. When the ER-bound IP(3) receptors were blocked by 2-APB (40 µM), we observed a delayed transient rise in [Ca(2+)](i) as a response to the caspofungin. Inhibition of the ryanodine receptors (RyR) using 40 µM ryanodine completely prevented the caspofungin-induced elevation of [Ca(2+)](i). In summary, caspofungin has been shown to trigger an increase in [Ca(2+)](i) independent from extracellular Ca(2+) ions by liberating the Ca(2+) ions stored in the ER, mainly via a RyR pathway.