Abstract
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 Ca2+ concentration ([Ca2+]i) after exposure to caspofungin in isolated human tracheal epithelial cells. The [Ca2+]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 [Ca2+]i or a sustained elevation of [Ca2+]i. Both patterns of Ca2+ kinetics were still observed when an influx of transmembraneous Ca2+ ions was pharmacologically inhibited. Furthermore, in extracellular buffer solutions without Ca2+ ions, caspofungin exposure still evoked this characteristic rise in [Ca2+]i. To shed light on the origin of the Ca2+ ions responsible for the elevation in [Ca2+]i we investigated the possible intracellular storage of Ca2+ ions. The depletion of mitochondrial Ca2+ stores using 25 µM 2,4-dinitrophenol (DNP) did not prevent the caspofungin-induced rise in [Ca2+]i, which was rapid and transient. However, the application of caffeine (30 mM) to discharge Ca2+ ions that were presumably stored in the endoplasmic reticulum (ER) prior to caspofungin exposure completely inhibited the caspofungin-induced changes in [Ca2+]i levels. When the ER-bound IP3 receptors were blocked by 2-APB (40 µM), we observed a delayed transient rise in [Ca2+]i as a response to the caspofungin. Inhibition of the ryanodine receptors (RyR) using 40 µM ryanodine completely prevented the caspofungin-induced elevation of [Ca2+]i. In summary, caspofungin has been shown to trigger an increase in [Ca2+]i independent from extracellular Ca2+ ions by liberating the Ca2+ ions stored in the ER, mainly via a RyR pathway.
Highlights
The antimycotic drug caspofungin is known to alter the cell function of cardiomyocytes and the ciliabearing cells of the tracheal epithelium
In order to find out, we investigated whether caspofungin changes the [ Ca2+]i in isolated human tracheal epithelial cells (HTEpC) and tried to elucidate the underlying regulatory mechanism
At the end of all the experiments, we applied a brief pulse of KCl (200 mM), and only the cells that responded to this vitality test by showing a significant increase in the FURA-2-fluorescence ratio were included for further statistical evaluation (Fig. 1)
Summary
The antimycotic drug caspofungin is known to alter the cell function of cardiomyocytes and the ciliabearing cells of the tracheal epithelium. Cilia beat continuously without external stimulation but can beat faster when necessary This elevated beating frequency depends on several interdepending signal transduction cascades, including changes in intracellular concentrations of C a2+ ions ([Ca2+]i)[3,4,5,6]. Mitochondria can buffer Ca2+ ions when [Ca2+]i exceeds a threshold of 500 nM and they slowly release Ca2+ into the cytosol when [Ca2+]i falls below the aforementioned threshold[9] It is still not known whether further intracellular Ca2+ stores such as lysosomes contribute to the regulation of [ Ca2+]i. In order to successfully treat mycotic infections in different organs, caspofungin has to reach therapeutic concentrations in many tissues or regions including the liver and the lower airways of the lungs that exceed plasma concentrations[11,12] This distribution pattern depends on the physiology of specific organs[13]. In order to find out, we investigated whether caspofungin changes the [ Ca2+]i in isolated human tracheal epithelial cells (HTEpC) and tried to elucidate the underlying regulatory mechanism
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