Extracellular ATP and P2Y2 receptors mediate intercellular Ca2+ waves induced by mechanical stimulation in submandibular gland cells: Role of mitochondrial regulation of store operated Ca2+ entry
Introduction
Intracellular Ca2+ signaling has a central role in the regulation of salivary gland cell function [1]. Once increased in the cytosol, Ca2+ activates various ion channels and transporters such as the Ca2+-activated K+ or Cl− channels, and Na+–K+–2Cl− transporters that are involved in saliva production and modification [1], [2], [3]. In a multi-cellular system, coordination of this Ca2+ signal between cells is important for synchronized and effective tissue function.
Intercellular Ca2+ wave (ICW) propagation has been reported in many different cell types such as respiratory tract epithelial [4], glial [5], aortic epithelial [6], liver epithelial [7], osteoblastic [8], and renin secreting juxtaglomerular [9] cells. Two main pathways are currently proposed as underling mechanisms of ICW propagation. One pathway is extracellular while the other is mediated through gap junction intercellular communication (GJIC). Two apposing connexin hemichannels form gap junctions between cells, which allow direct intercellular communication through passage of small signaling molecules such as Ca2+ and inositol 1,4,5-triphosphate (IP3) in the GJIC pathway. Previous studies in salivary gland cells indicate that GJIC is involved in the synchronization or propagation of Ca2+ signals. Muscarinic stimulation induced a synchronized Ca2+ signal among individual acinar cells that was disrupted by pretreatment with the gap junction uncoupler octanol in rat submandibular glands [10]. In blowfly salivary glands, IP3 microinjection induced propagation of a Ca2+ wave through gap junctions [11].
ATP and purinergic (P2) receptor dependent signaling form a common extracellular pathway that, like GJIC, also contributes to ICW propagation. Various stimuli like muscarinic receptor activation, mechanical stress, and hypoxic conditions cause a release of ATP via exocytosis or ion channels [12]. Two different subtypes of P2 receptors are involved in extracellular ATP-dependent signaling. P2X subtype receptors are Ca2+ permeable ion channels and the cytosolic Ca2+ increase upon P2X receptors activation is dependent on extracellular Ca2+. P2Y subtype receptors are G-protein coupled receptors that activate phospholipase C (PLC) which generates IP3. This IP3 induces a Ca2+ release via IP3 receptors in the endoplasmic reticulum (ER). Depletion of the ER Ca2+ store by IP3 receptor activation can further induce Ca2+ influx from the extracellular fluid through store operated Ca2+ channels (SOCC). Subtypes of both the P2X (P2X4, P2X7) and P2Y (P2Y1, P2Y2) receptors have been identified in different salivary gland cells [13].
Salivary glands experience repetitive mechanical stress during mastication. Myoepithelial cells, which contain myosin, contract to generate direct mechanical stimulation of salivary gland cells. Furthermore, mechanical stimulation, e.g. in chewing gum, has been suggested as an alternative treatment for xerostomia patients to increase saliva production [14], [15]. However, the detailed mechanisms underlying this cell signaling induced by mechanical stimulation are not clearly understood in salivary gland cells.
Here, we investigated the mechanism of ICW induced by mechanical stimulation in a monolayer of human submandibular gland (HSG) cells and in freshly isolated submandibular gland tissues using fluorescence Ca2+ imaging. The results below demonstrate that the propagation of Ca2+ waves from the mechanically stimulated cells to the neighboring cells relies on extracellular ATP-dependent signaling. Pharmacological characterization revealed that P2Y2 subtype receptors are involved in ICW. Our data also indicate that mechanosensitive maxi-anion channels are likely candidates for the ATP release pathway in mechanically stimulated cells. Both intracellular Ca2+ release from the ER and Ca2+ influx from the extracellular medium contribute to ICW. Finally, mitochondria were found to play an important role by actively regulating the Ca2+ mobilization pathway.
Section snippets
Cell culture
HSG cells were grown in MEM (minimum essential medium Eagle, Mediatec Inc.) containing 2 mM glutamine and supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin. Cells were maintained at 37 °C in a humidified 5% CO2 incubator and passed twice a week.
Preparation of native submandibular gland cells
Native submandibular gland cells were freshly prepared as previously described [16], [17] with some modifications. In brief, Sprague–Dawley rats were sedated by 100% CO2 and decapitated using a guillotine. Submandibular glands
Mechanical stimulation induces intercellular Ca2+ wave propagation in HSG cells
A monolayer of HSG cells was used to mimic the microenvironment of a salivary gland during mastication and investigate the intercellular Ca2+ wave (ICW) induced by mechanical stimulation. The cell clusters were 300–400 μm in diameter and contained 150–300 cells. Focal mechanical stimulation of single cells in the center of cell clusters was achieved by gently touching the target cell for less than 1 s with a sealed micropipette. The induced ICW was monitored by Fura-2 fluorescence ratio imaging.
Discussion
In the present study, the mechanism of intercellular Ca2+ wave (ICW) propagation induced by mechanical stimulation in HSG cells and freshly isolated submandibular gland cells was investigated (proposed model shown in Fig. 8). Extracellular ATP and P2Y2 receptor dependent signaling is central to the resulting Ca2+ wave propagation (Fig. 1, Fig. 2). Our data suggest that a mechanosensitive maxi-anion channel is a good candidate for the ATP release mechanism (Fig. 3). We found that both IP3
Conclusions
The present study enhances our understanding about how the extracellular ATP and P2Y2 receptor dependent signaling pathway mediates intercellular Ca2+ signaling, which synchronizes salivary gland cell function. Mitochondria were found to have a specific role in regulating the Ca2+ mobilization mechanism in salivary gland cells. Finally, this information identifies P2Y2 receptors as an alternative therapeutic target to alleviate dry mouth symptoms in many different oral pathologies related to
Acknowledgements
We thank Dan Malamud for providing HSG cells, Evgeny Pavlov for providing mtGFP, and Laurent Dejean and Oscar Teijido for critical discussions. This work was supported by NIH grants, RO1 DE14756 to David I. Yule and GM57249 to Kathleen W. Kinnally.
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