Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells

Abstract

Rat parotid acinar cells lacking zymogen granules were obtained by inducing granule discharge with the beta-adrenoceptor agonist isoproterenol. To assess whether zymogen granules are involved in the regulation of Ca(2+) signalling as intracellular Ca(2+) stores, changes in cytosolic free Ca(2+) ion concentration ([Ca(2+)](i)) were studied with imaging microscopy in fura-2-loaded parotid acinar cells lacking zymogen granules. The increase in [Ca(2+)](i) induced by muscarinic receptor stimulation was initiated at the apical pole of the acinar cells, and rapidly spread as a Ca(2+) wave towards the basolateral region. The magnitude of the [Ca(2+)](i) response and the speed of the Ca(2+) wave were essentially similar to those in control acinar cells containing zymogen granules. Western blot analysis of the inositol 1,4,5-trisphosphate receptor (IP(3)R) was performed on zymogen granule membranes and microsomes using anti-IP(3)R antibodies. The immunoreactivity of all three IP(3)Rs was clearly observed in the microsomal preparations. Although a weak band of IP(3)R type-2 was detected in the zymogen granule membranes, this band probably resulted from contamination by the endoplasmic reticulum (ER), because calnexin, a marker protein of the ER, was also detected in the same preparation. Furthermore, Western blotting and reverse transcriptase-PCR analysis failed to provide evidence for the expression of ryanodine receptors in rat parotid acinar cells, whereas expression was clearly detectable in rat skeletal muscle, heart and brain. These results suggest that zymogen granules do not have a critical role in Ca(2+) signalling in rat parotid acinar cells.

Organelles Containing Inositol Trisphosphate Receptor Type 2 in Adrenal Medullary Cells

“…However, the use of a similar approach to measure [Ca 2+ ] g in neurosecretory PC12 cells revealed that [Ca 2+ ] g was altered with a change in intracellular pH [33], but it did not noticeably respond to InsP 3 or cyclic ADP ribose [12]. Furthermore, the presence of InsP 3 Rs in zymogen granules has been disputed [19,20].…”

“…In pancreatic acinar cells, InsP 3 Rs in the apical pole were reported to be present in secretory granules [17,18]. However, later studies suggested that the InsP 3 -induced Ca 2+ release from a crude granule fraction might have come from the ER contaminated in the fraction [19,20]. These results led us to investigate the notion that the InsP 3 Rs at the cell periphery of AM cells may not be present in secretory granules.…”

Calcium dynamics in the secretory granules of neuroendocrine cells

“…Similarly, using a targeted aequorin to monitor granule [Ca 2+ ], no effect of InsP 3 was observed in MIN6 cells [20]. Regarding zymogen granules, InsP 3 R were not found using highly pure granule preparations [67] and the absence of zymogen granules did not modify the cytosolic Ca 2+ transients in rat parotid acinar cells [68]. Finally, in platelets, InsP 3 R are present in the dense tubular system (the equivalent of the ER) but not in the acidic granules [41].…”

Simultaneous monitoring of Ca2+ responses and salivary secretion in live animals reveals a threshold intracellular Ca2+ concentration for salivation

“…Chemically synthesized Ca 2+ indicators, such as fura-2 and fluo-3 (Grynkiewicz, Poenie, & Tsien, 1985;Minta, Kao, & Tsien, 1989), have been used extensively to monitor agonist-induced Ca 2+ responses in isolated salivary acinar cells (Bruce, Shuttleworth, Giovannucci, & Yule, 2002;Foskett & Melvin, 1989;Gray, 1989;Harmer, Smith, & Gallacher, 2005;Nezu et al, 2002;Tojyo, Tanimura, & Matsumoto, 1997 In previous studies, we expressed fluorescent protein-labelled Stim1 after gene transfer via retrograde ductal injection of adenovirus vectors through the orifice of the rat submandibular gland (SMG) (Morita et al, 2011(Morita et al, , 2013. We applied this method to express the ultrasensitive GECI YC-Nano50 (Horikawa et al, 2010) and succeeded in monitoring agonist-induced Ca 2+ responses of SMG acinar cells in live animals (Nezu, Morita, Tojyo, Nagai, & Tanimura, 2015).…”