Elsevier

Cell Calcium

Volume 43, Issue 5, May 2008, Pages 469-481
Cell Calcium

Functional coupling between ryanodine receptors, mitochondria and Ca2+ ATPases in rat submandibular acinar cells

https://doi.org/10.1016/j.ceca.2007.08.001Get rights and content

Abstract

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca2+ signals driven by inositol 1,4,5-trisphosphate receptors (IP3Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca2+ signal is propagated by a mechanism of Ca2+-induced Ca2+ release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca2+ signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca2+ concentration in the endoplasmic reticulum ([Ca2+]ER). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca2+ concentration ([Ca2+]i) suggesting fast local buffering of Ca2+ released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca2+ transient that was prevented by addition of Ca2+ chelator BAPTA but not EGTA. When mitochondrial Ca2+ uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca2+]i and substantially smaller Ca2+ release from the ER. Upon simultaneous inhibition of mitochondrial Ca2+ uptake and either plasmalemmal or ER Ca2+ ATPase, activation of RyRs caused a transient rise in [Ca2+]i. Collectively, our data suggest that Ca2+ released through RyRs is mostly “tunnelled” to mitochondria, while Ca2+ ATPases are responsible for the fast initial sequestration of Ca2+. Ca2+ uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca2+ ATPases is accomplished through strategic positioning of mitochondria close to both Ca2+ release sites in the ER and Ca2+ pumping sites of the plasmalemma and the ER.

Introduction

The exocrine acinar cells are highly morphologically and functionally polarized cellular systems in which a final step of stimulus–response coupling is the secretion of proteins, electrolytes and fluids [1], [2]. The main source of fluid and electrolyte enriched saliva is the submandibular gland [3], where secretion is induced by activation of muscarinic receptors, which trigger rapid increase of cytosolic Ca2+ concentration ([Ca2+]i). This increase in [Ca2+]i has a complex nature, resulting from 1,4,5-inositol trisphosphate receptors (IP3Rs)-mediated Ca2+ release from the endoplasmic reticulum (ER) followed by activation of Ca2+ influx through plasmalemmal store-operated Ca2+ channels (SOCs). In physiological settings, stimulation of exocrine cells with agonists leads to the generation of [Ca2+]i signals in the form of repetitive Ca2+ oscillations [4]. These [Ca2+]i signals localized in the apical pole and driven by IP3Rs rapidly become global due to generation of Ca2+ wave [5], [6], [7]. The global nature of Ca2+ signals is critical for the function of submandibular acinar cells because fluid secretion relies on a synchronized activation of spatially separated Ca2+-dependent ionic conductances represented by apical membrane Cl channels and basolateral membrane K+ channels [8], [9]. Generation of a Ca2+ wave critically depends on regenerative Ca2+ mobilization, because Ca2+ diffusing in the cytoplasm is limited by Ca2+ buffering systems [10]. In excitable cells, propagation of [Ca2+]i signal is often determined by a well-characterized mechanism of Ca2+-induced Ca2+ release (CICR) due to a wave of opening of ryanodine receptors (RyRs) [11], [12]. However, it is not clear if RyRs play the same role in non-excitable cells.

Expression of RyRs was immunocytochemically identified in salivary cells [13], [14], [15]. Caffeine, a RyRs activator, was shown to induce Ca2+-dependent K+ and C1 currents [16], and [Ca2+]i oscillations in exocrine acinar cells [17], [18], [19]. In addition, ryanodine (a RyR inhibitor), decreased the frequency of agonist-induced [Ca2+]i oscillations in parotid acinar cells [19], [20]. The CICR, activated by Ca2+ entry though SOCs, was also demonstrated in parotid acinar cells [21]. Nonetheless, caffeine in various concentrations failed to evoke global [Ca2+]i rise in exocrine acinar cells [17], [20], [21], [22], [23].

Recently, the mitochondria emerged as important regulators of intracellular Ca2+ signalling due to their ability for local Ca2+ buffering of Ca2+ released from the ER (for review see Refs. [10], [24]). However, mitochondria can play distinct roles in shaping Ca2+ signals in different types of exocrine acinar cells. In pancreatic acinar cells they confine apical [Ca2+]i signals by forming a belt around zymogen granules [25], while there is no definitive perigranular mitochondria in parotid acinar cells [26]. Furthermore rise in mitochondrial Ca2+ regulates local ATP production [27]. Several studies reported that mitochondria specifically accumulate Ca2+ released through the RyRs in different types of muscle cells [28], [29], [30]. Nevertheless, the presence of mitochondria interaction with RyRs in exocrine acinar cells remains unknown.

In the present paper we demonstrate that activation of RyRs in submandibular acinar cells induces transient Ca2+ release from the ER, which transforms into [Ca2+]i elevation only when mitochondrial and PMCA/SERCA-mediated Ca2+ uptake are inhibited. We also report that caffeine-induced activation of RyRs causes transient increase of Ca2+ concentration inside mitochondria occurring via microdomains of high [Ca2+]. Moreover mitochondrial Ca2+ uptake is critical for maintaining CICR in submandibular acinar cells. Ultrastructural data show close contacts between mitochondria and ER as well as specific localization of mitochondria between the plasmalemma and ER. We also propose a scheme describing interplay between RyR-mediated Ca2+ release and Ca2+ uptake by mitochondrial uniporter and plasma/ER membrane Ca2+ pumps.

Section snippets

Isolation of submandibular salivary gland acinar cells

Experiments were performed on male Wistar rats (6–7 weeks old, 100–150 g). Submandibular salivary gland acinar cells were isolated by collagenase treatment, as described previously [31]. All experiments were performed at room temperature (21–23 °C).

Measurement of [Ca2+]i in acinar cells

Freshly isolated acinar cells were loaded with fura-2/AM (5 μM) in extracellular solution supplemented with 0.02% pluronic F-127 for 30 min at 35 °C described elsewhere [31]. Dye loaded acinar cells were alternately excited with light at 360 and 390 nm

Changes in [Ca2+]ER upon activation of RyRs

To reveal functional RyR-mediated Ca2+ release from the ER of submandibular acinar cells, we directly measured changes in [Ca2+]ER. For this purpose we employed the low-affinity Ca2+-sensitive fluorescent dye mag-fura-2/AM that, under appropriate loading conditions, accumulates mainly in the ER Ca2+ stores of acinar and other cell types [34]. After loading, cells were subjected to mild plasma membrane permeabilization with β-escin. That allowed us to “clamp” [Ca2+] in the ICM surrounding the ER

Discussion

Here we demonstrate for the first time the availability of RyR-mediated operational CICR in submandibular cells and argue for a different role of RyRs in non-excitable cells. In salivary acinar cells the binding of IP3 to its receptor on the ER initiates Ca2+ release in the apical regions of the cell [43], [44]. After initiation, Ca2+ signal propagates throughout the cell as a Ca2+ wave [5], [6], [7]. In many excitable cells, e.g. in skeletal muscle and cardiac cells, propagation of the Ca2+

Acknowledgements

This work was supported by JDRF grant #1-2004-30 to N.V. and NATO reintegration grant, West-Ukrainian Research Foundation to N.F.

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