Role of mitochondria in modulation of spontaneous Ca2+ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethra

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit pacemaker activity that results from spontaneous Ca2+ waves. The purpose of this study was to investigate if this activity was influenced by Ca2+ uptake into mitochondria. Spontaneous Ca2+ waves were recorded using a Nipkow spinning disk confocal microscope and spontaneous transient inward currents (STICs) were recorded using the whole-cell patch clamp technique. Disruption of the mitochondrial membrane potential with the electron transport chain inhibitors rotenone (10 mu M) and antimycin A (5 mu M) abolished Ca2+ waves and increased basal Ca2+ levels. Similar results were achieved when mitochondria membrane potential was collapsed using the protonophores FCCP (0.2 mu M) and CCCP (1 mu M). Spontaneous Ca2+ waves were not inhibited by the ATP synthase inhibitor oligomycin (1 mu M), suggesting that these effects were not attributable to an effect on ATP levels. STICs recorded under voltage clamp at -60 mV were also inhibited by CCCP and antimycin A. Dialysis of cells with the mitochondrial uniporter inhibitor RU360 (10 mu M) also inhibited STICS. Stimulation of Ca2+ uptake into mitochondria using the plant flavonoid kaempferol (10 mu M) induced a series of propagating Ca2+ waves. The kaempferol-induced activity was inhibited by application of caffeine (10 mM) or removal of extracellular Ca2+, but was not significantly affected by the IP3 receptor blocker 2-APB (100 mu M). These data suggest that spontaneous Ca2+ waves in urethral ICC are regulated by buffering of cytoplasmic Ca2+ by mitochondria.