Abstract
Parotid acinar cells exhibit rapid cytosolic calcium signals ([Ca2+]i) that initiate in the apical region but rapidly become global in nature. These characteristic [Ca2+]i signals are important for effective fluid secretion, which critically depends on a synchronized activation of spatially separated ion fluxes. Apically restricted [Ca2+]i signals were never observed in parotid acinar cells. This is in marked contrast to the related pancreatic acinar cells, where the distribution of mitochondria has been suggested to contribute to restricting [Ca2+]i signals to the apical region. Therefore, the aim of this study was to determine the mitochondrial distribution and the role of mitochondrial Ca2+ uptake in shaping the spatial and temporal properties of [Ca2+]i signaling in parotid acinar cells. Confocal imaging of cells stained with MitoTracker dyes (MitoTracker Green FM or MitoTracker CMXRos) and SYTO dyes (SYTO-16 and SYTO-61) revealed that a majority of mitochondria is localized around the nucleus. Carbachol (CCh) and caged inositol 1,4,5-trisphosphate-evoked [Ca2+]i signals were delayed as they propagated through the nucleus. This delay in the CCh-evoked nuclear [Ca2+]i signal was abolished by inhibition of mitochondrial Ca2+ uptake with ruthenium red and Ru360. Likewise, simultaneous measurement of [Ca2+]i with mitochondrial [Ca2+] ([Ca2+]m), using fura-2 and rhod-FF, respectively, revealed that mitochondrial Ca2+ uptake was also inhibited by ruthenium red and Ru360. Finally, at concentrations of agonist that evoke[Ca2+]i oscillations, mitochondrial Ca2+ uptake, and a nuclear [Ca2+] delay, CCh also evoked a substantial increase in NADH autofluorescence. This autofluorescence exhibited a predominant perinuclear localization that was also sensitive to mitochondrial inhibitors. These data provide evidence that perinuclear mitochondria and mitochondrial Ca2+ uptake may differentially shape nuclear [Ca2+] signals but more importantly drive mitochondrial metabolism to generate ATP close to the nucleus. These effects may profoundly affect a variety of nuclear processes in parotid acinar cells while facilitating efficient fluid secretion.
Highlights
Regulation of both the spatial and temporal properties of intracellular Ca2ϩ ([Ca2ϩ]i)1 signals is known to underlie the specificity of stimulus-response coupling in a variety of cell types [1, 2]
Several reports have suggested that differential [Ca2ϩ] signals in the nucleus can be attributed to nuclear dye artifacts that are prevalent with single wavelength fluorescent dyes such as Oregon Green BAPTA 2 (OGB2) [17]
Because [Ca2ϩ] signals rapidly propagate from the apical region to the basal region of parotid acinar cells [5], boxes for analysis of the nuclear and non-nuclear basal region were strategically placed equidistant from the apical region to reveal any delay in the nucleus
Summary
Regulation of both the spatial and temporal properties of intracellular Ca2ϩ ([Ca2ϩ]i) signals is known to underlie the specificity of stimulus-response coupling in a variety of cell types [1, 2]. The major function of pancreatic acinar cells is the exocytosis of zymogen granules that can be activated by apically confined [Ca2ϩ]i signals [6], suggested to be the major physiological [Ca2ϩ]i signal evoked by threshold agonist concentrations [7]. These apically confined [Ca2ϩ]i signals are in part because of the distribution of mitochondria, which form a belt around the apically located zymogen granules [8, 9]. Mitochondrial Ca2ϩ uptake in this region differentially shapes nuclear [Ca2ϩ] signals and enhances metabolism and ATP generation close to the nucleus This may serve to differentially modulate a variety of processes within the nucleus
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