Initiation Site of Ca²⁺ Entry Evoked by Endoplasmic Reticulum Ca²⁺ Depletion in Mouse Parotid and Pancreatic Acinar Cells

The process of cellular Ca²⁺ signaling involves regulated changes in the concentration of Ca²⁺ in the cytoplasm ([Ca²⁺]_i) as well as other cellular compartments. A multitude of cellular processes are controlled through Ca²⁺ signaling and, in turn, a multitude of external cellular signals induce or regulate Ca²⁺ signaling. Because so many systems respond to or regulate Ca²⁺ signaling, it is not surprising that dysfunctions of various aspects of Ca²⁺ signaling pathways underlie several important diseases.1 When Ca²⁺ signaling is stimulated in a cell, Ca²⁺ enters the cytoplasm from one of two general sources: it is either released from intracellular Ca²⁺ stores or enters the cell across the plasma membrane. Both processes often occur simultaneously or sequentially. In all non-excitable cells, and some excitable cells, an important initiating step is the intracellular release of Ca²⁺ from internal stores by binding of a second messenger to its receptor in the endoplasmic reticulum (ER). Commonly, this messenger is inositol 1,4,5-trisphosphate (IP₃),2 but a number of other potential messengers have been discovered in recent years.3, 4 Ca²⁺ entry can be signaled by a variety of processes, including direct activation by surface receptors and activation by a variety of second messengers;5 however, the most commonly observed mechanism of regulated Ca²⁺ entry in non-excitable cells is a process knows as capacitative Ca²⁺ entry or store-operated Ca²⁺ entry (SOCE).6, 7 In many non-excitable cells, depletion of intracellular Ca²⁺ stores by IP₃ is the primary mechanism by which cell surface receptors activate Ca²⁺ influx. This phenomenon, which is termed capacitative Ca²⁺ entry,8 has been identified in the control of Ca²⁺ oscillations,9 secretion,10 and enzymatic regulation.11 Despite the wide range of processes in which SOCE is involved, the signal mechanism that couples store depletion to Ca²⁺ entry has not yet been fully identified.8 The exocrine acinar cells of the pancreas and the parotid gland are classic examples of non-excitable cells whose key physiological activities are known to be dependent on [Ca²⁺]_i signals that involve a component of Ca²⁺ entry.12, 13 To determine the site of initiation of store- operated Ca²⁺ entry in non-excitable cells, we investigated the initiation site of SOCE in parotid and pancreatic acinar cells. Our results suggest that the commencement site of SOCE has significance for the future study of the physiological importance of SOCE in parotid and pancreatic acinar cells.

In order to find the initiation sites of store-operated Ca²⁺ entry (SOCE) in mouse parotid and pancreatic acinar cells, we measured [Ca²⁺]_i increase as follows. The initial step to trigger the SOCE was conducted by depletion of intracellular Ca²⁺ stores. For this purpose, the endoplasmic reticulium (ER) in parotid and pancreatic acinar cells were depleted by 1µM thapsigargin (Tg), an inhibitor specific to sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA), in the absence of Ca²⁺. To confirm whether ER was definitely emptied by Tg, cells were stimulated with 1mM carbachol in a Ca²⁺ free state after depletion of ER. When a cellular response to carbachol was no longer observed, the cells were incubated with 7.5mM external calcium for 200 to 400 s so as to trigger SOCE. As a result, we confirmed that vacancy of ER is a kind of signal occurring Ca²⁺ entry (Fig. 1Aa and Ba. n = 5). However, this finding required us to determine whether the late [Ca²⁺]_i increases were accomplished across the plasma membrane. Thus, lanthanum (La³⁺), a non-specific blocker of plasma Ca²⁺ channel, was used to elucidate whether Tg-induced Ca²⁺ increases were indeed from extracellular sources. Both cell types were treated by 1µM Tg to deplete internal Ca²⁺ stores, and the cells were then treated with 1mM La³⁺ for 3 min prior to exposure 7.5mM external Ca²⁺. We found that the Ca²⁺ input from external Ca²⁺ had nearly disappeared in both mouse parotid and pancreatic acinar cells (Fig. 1Ab and Bb).

Fig. 1
Measurement of thapsigargin-induced [Ca²⁺]_i increases. A (a) and B (a): parotid and pancreatic acinar mouse cell responses, respectively, after depletion of internal Ca²⁺ stores with 1µM thapsigargin (Tg) in nominally Ca²⁺-free media. Cells were stimulated with 1mM carbachol, and then thapsigargin-induced [Ca²⁺]_i increases were measured by exposure to external 7.5mM Ca²⁺. A (b) and B (b): parotid and pancreatic acinar mouse cell responses, respectively, after the cells were treated with 1mM lanthanum before exposure to external high Ca²⁺. Each result was the Trepresentative of 5 independent experiments.

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Next, to identify the initiation sites of SOCE, the initiation site of Tg-induced Ca²⁺ entry was imaged with fura2 fluorescence. The Ca²⁺ response generated by the depletion of internal Ca²⁺ store was not initiated at apical membrane (AM), but rather basal membrane (BM) in parotid and pancreatic acinar cells (Fig. 2A and B, n = 5). In addition, the propagation direction of the initiated Ca²⁺ entry was from the BM to the AM. There were no differences in Ca²⁺ entry patterns between parotid acinar cells and pancreatic acinar cells: however, the time scores for Ca²⁺ entry were different between the two cell types. The half time value of Ca²⁺ entry (T1/2) in parotid acinar cells was 55.38 ± 6.63 sec following exposure to a 7.5mM Ca²⁺ solution, while the T1/2 in pancreatic acinar cells was 74.28 ± 6.80 sec., suggesting that the Ca²⁺ entry in parotid acinar cells was faster than that in pancreatic acinar cells.

Fig. 2
Measurement of the initiation site of Tg-induced [Ca2²⁺]_i increases. (A) and (B) parotid and pancreatic acinar mouse cell responses, respectively. Initiation site of Tg- induced [Ca²⁺]_i increases were imaged with fura2 fluorescence. (C) and (D) The cells were treated with 1mM lanthanum before exposure to external high Ca²⁺. In each transmission image, red and black circles shows apical and basal pole, respectively. Each result was the representative of 5 independent experiments.

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In the present work, we investigated the location of the initiation site of SOCE in mouse parotid and pancreatic acinar cells, as identification of this site may be helpful to identify the nature of SOCC. We showed that the store-regulated Ca²⁺ response initiated at BM of mouse parotid and pancreatic acinar cells after ER depletion by Tg and stimulation by external Ca²⁺. The mechanism by which Tg effectively empties intracellular Ca²⁺ stores in exocrine gland acinar cells is through its ability to completely suppress the SERCA.7 Moreover, the Ca²⁺ response from an external source was diminished when cells were treated with La³⁺ prior to exposure to external Ca²⁺, suggesting that Tg-induced Ca²⁺ entry is mediated by SOCE. Therefore, it is a notable discovery that SOCE initiation begins at the BM in the cells, since there are many Ca²⁺-signaling related proteins in the apical region in exocrine acinar cells.17

Exocrine acinar cells such as salivary gland acinar cells are structurally and functionally polarized between the basal region and apical region.18, 19 The receptors for secretagogues are localized in the basal region, whereas the apical region has a high density of secretory granules that are ultimately released by exocytosis at the apical plasma membrane.20 Many studies have shown that the increase in cytoplasmic free Ca²⁺ is initiated at the apical region and propagated to the basal region through a Ca²⁺ wave.21-23 Therefore, it is likely that in polarized exocrine cells, other Ca²⁺ signaling proteins together with SOCC exist in the basal region of the cells. Recently, a strong candidate and regulator of SOCC were reported as Orai1 and stromal- interaction molecule 1 (STIM1), respectively.24, 25 Therefore, it will be necessary to investigate the localization of Orai1 and STIM1 in exocrine gland acinar cells.

Our findings are important, as SOCCs play a fundamental role in both the immediate and long- term regulation of cells. It follows that unexpected perturbations in this process may have the potential for pathological outcomes, while planned pharmacological manipulations may find clinical utility in certain disease states. We look forward to increasing the body of information on the role of SOCCs in disease as well as in the therapy of disease.