Abstract
In the present study we have investigated cytosolic and mitochondrial Ca(2+) signals in isolated mouse pancreatic acinar cells double-loaded with the fluorescent probes fluo-3 and rhod-2. Stimulation of pancreatic acinar cells with 500 nm acetylcholine caused release of Ca(2+) from intracellular stores and produced cytosolic Ca(2+) signals in form of Ca(2+) waves propagating from the luminal to the basal cell pole. The increase in the cytosolic Ca(2+) concentration was followed by Ca(2+) uptake into mitochondria. Between onset of cytosolic and mitochondrial Ca(2+) signals there was a delay of 10.7 +/- 0.4 s. Ca(2+) uptake into mitochondria could be inhibited with Ruthenium Red and carbonyl cyanide m-chlorophenylhydrazone, whereas 2,5-di-tert-butylhydroquinone, which inhibits sarco(endo)plasmic reticulum Ca(2+) ATPases, did not prevent Ca(2+) accumulation in mitochondria. Carbonyl cyanide m-chlorophenylhydrazone-induced Ca(2+) release from mitochondria could only be observed after a preceding stimulation of the cell with a physiological agonist or by treatment with 2, 5-di-tert-butylhydroquinone, indicating that under resting conditions mitochondria do not contain releasable Ca(2+) ions. Analysis of the propagation rate of acetylcholine-induced Ca(2+) waves revealed that inhibition of mitochondrial Ca(2+) uptake did not accelerate spreading of cytosolic Ca(2+) signals. Our experiments indicate that in the early phase of secretagogue-induced Ca(2+) signals, mitochondria behave as passive Ca(2+)-buffering elements and do not actively suppress spreading of Ca(2+) signals in pancreatic acinar cells.
Highlights
Stimulation of pancreatic acinar cells with acetylcholine (ACh),1 cholecystokinin, or bombesin causes secretion of chloride and enzymes across the apical cell membrane into the acinar lumen
In the present study we have investigated cytosolic and mitochondrial Ca2؉ signals in isolated mouse pancreatic acinar cells double-loaded with the fluorescent probes fluo-3 and rhod-2
To confirm that rhod-2 fluorescence did originate from mitochondria, pancreatic acinar cells were double-loaded with rhod-2/AM and MitoTrackerTM Green FM (n ϭ 6), an indicator that labels mitochondria regardless of mitochondrial membrane potentials [25]
Summary
Stimulation of pancreatic acinar cells with acetylcholine (ACh),1 cholecystokinin, or bombesin causes secretion of chloride and enzymes across the apical cell membrane into the acinar lumen. Since accumulation of rhod-2 in mitochondria could be demonstrated by double-staining with rhod-2 and MitoTrackerTM Green FM, we can conclude that our loading procedure for staining pancreatic acinar cells with rhod-2 and fluo-3 is suitable for monitoring changes in both [Ca2ϩ]cyt and [Ca2ϩ]m at the same time.
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