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Originally published In Press as doi:10.1074/jbc.M314028200 on March 11, 2004

J. Biol. Chem., Vol. 279, Issue 20, 21511-21519, May 14, 2004
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Subpopulation of Store-operated Ca2+ Channels Regulate Ca2+-induced Ca2+ Release in Non-excitable Cells*

Jian Yao, Qin Li, Jin Chen, and Shmuel Muallem{ddagger}

From the Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9040

Received for publication, December 22, 2003 , and in revised form, February 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+-induced Ca2+ release (CICR) is a well characterized activity in skeletal and cardiac muscles mediated by the ryanodine receptors. The present study demonstrates CICR in the non-excitable parotid acinar cells, which resembles the mechanism described in cardiac myocytes. Partial depletion of internal Ca2+ stores leads to a minimal activation of Ca2+ influx. Ca2+ influx through this pathway results in an explosive mobilization of Ca2+ from the majority of the stores by CICR. Thus, stimulation of parotid acinar cells in Ca2+-free medium with 0.5 µM carbachol releases ~5% of the Ca2+ mobilizable by 1 mM carbachol. Addition of external Ca2+ induced the same Ca2+ release observed in maximally stimulated cells. Similar results were obtained by a short treatment with 2.5-10 µM cyclopiazonic acid, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase pump. The Ca2+ release induced by the addition of external Ca2+ was largely independent of IP3Rs because it was reduced by only ~30% by the inhibition of the inositol 1,4,5-trisphosphate receptors with caffeine or heparin. Measurements of Ca2+-activated outward current and [Ca2+]i suggested that most CICR triggered by Ca2+ influx occurred away from the plasma membrane. Measurement of the response to several concentrations of cyclopiazonic acid revealed that Ca2+ influx that regulates CICR is associated with a selective portion of the internal Ca2+ pool. The minimal activation of Ca2+ influx by partial store depletion was confirmed by the measurement of Mn2+ influx. Inhibition of Ca2+ influx with SKF96365or 2-aminoethoxydiphenyl borate prevented activation of CICR observed on addition of external Ca2+. These findings provide evidence for activation of CICR by Ca2+ influx in non-excitable cells, demonstrate a previously unrecognized role for Ca2+ influx in triggering CICR, and indicate that CICR in non-excitable cells resembles CICR in cardiac myocytes with the exception that in cardiac cells Ca2+ influx is mediated by voltage-regulated Ca2+ channels whereas in non-excitable cells Ca2+ influx is mediated by store-operated channels.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Agonist-evoked Ca2+ signaling entails the generation of IP31 to activate the IP3 receptors (IP3Rs) and release the Ca2+ stored in the endoplasmic reticulum (ER). Ca2+ release from the ER is followed by activation of store-operated Ca2+ channels (SOCs) in the plasma membrane and Ca2+ influx into the cells. Subsequently, the plasma membrane Ca2+ ATPase pump and the sarco/endoplasmic reticulum Ca2+ ATPase pump remove Ca2+ from the cytosol to stabilize [Ca2+]i at a steady state that is determined by the relative activities of Ca2+ influx and Ca2+ extrusion (1, 2). At physiological agonist concentrations, the Ca2+ signal is in the form of repetitive Ca2+ oscillations (1). In most cells, but particularly in polarized cells, the Ca2+ signal initiates at discrete sites and proceeds as a propagating Ca2+ wave (3-7). The various features and the proteins that mediate each phase of the Ca2+ signal that depends on IP3-mediated Ca2+ release have been extensively studied in many cell types and are reasonably well understood.

Another aspect of the Ca2+ signal that is poorly understood compared with IP3-mediated Ca2+ release is Ca2+-induced Ca2+ release (CICR) in non-excitable cells. CICR is mediated by the ryanodine receptors (RyRs), the function of which is well characterized in skeletal and cardiac muscles (8) where they play a central role in excitation-contraction coupling. The family of RyRs includes three isoforms that are thought to be expressed in a cell-specific manner: RyR1 is expressed primarily in skeletal muscle, RyR2 is expressed primarily in cardiac muscle, and RyR3 is expressed ubiquitously (8). The primary activator of RyRs in muscle cells is Ca2+. However, the source of the trigger pool of Ca2+ is cell-specific. In skeletal muscle, RyR1 directly interacts with the voltage-regulated L-type Ca2+ channel. The L-type channel functions as a voltage sensor that causes membrane depolarization to undergo a conformational change and activate RyR1. The initial Ca2+ released through RyR1 further activates RyR1 to result in the explosive Ca2+ release that mediates muscle contraction (8, 9). On the other hand, in cardiac muscle the L-type channel is not directly coupled to RyR2. Rather, the L-type channel is localized adjacent to RyR2. Membrane depolarization results in the activation of the L-type Ca2+ channel and Ca2+ influx. The incoming Ca2+ serves as the trigger to activate RyR2 in the cardiac sarcoplasmic reticulum and cause the explosive Ca2+ release and muscle contraction (8, 10).

Accumulating molecular, biochemical, and functional evidence in recent years indicated that non-muscle cells (11), including secretory acinar cells (5, 12-15), also express RyRs. However, their function and mechanism of regulation in non-muscle cells remain elusive. There is good evidence that RyRs in non-muscle cells are activated by the nucleotide cADPR (16, 17). In fact, activation of a Ca2+ signal by cADPR generally is accepted as evidence for the involvement of RyRs in generating the Ca2+ signal. Response to cADPR or the presence of RyRs was demonstrated in several secretory cells, including pancreatic acinar cells (12-15, 18, 19) and submandibular gland acinar and duct cells (15). The proposed main function of RyRs in these cells was to facilitate the propagation of the luminal-to-basal Ca2+ wave (18-21). All models to explain the role of RyRs in non-muscle cells depict them to be sensitized to Ca2+ by cADPR but, notably, to be activated by Ca2+ released from the IP3 pool. No other pool of Ca2+ is considered to participate in the regulation or activation of RyRs in non-muscle cells. The present work re-examined this premise in parotid acinar cells that have a robust CICR activity. We report that Ca2+ influx through SOCs is critical for Ca2+ release in parotid acinar cells and suggest that CICR in non-muscle cells occurs in a mechanism similar to that in cardiac myocytes, except that in cardiac myocytes Ca2+ influx is mediated by the L-type Ca2+ channel whereas in non-muscle cells Ca2+ influx is mediated by SOCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Cyclopiazonic acid and 1,4,5- and 2,4,5-IP3 were purchased from Alexis. Fura-2/AM was from Tef Labs (Austin, TX). 2-aminoethoxydiphenyl borate and SKF96365were from Calbiochem. Heparin was from Sigma. The standard bath solution A contained (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 10 HEPES (pH 7.4 with NaOH), 10 glucose, and 0.1% bovine serum albumin. Ca2+ concentration in this solution was adjusted between 0.5-7.5 mM by the addition of CaCl2. Ca2+-free solution was prepared by the addition of 0.2 mM EGTA to solution A. Solution A was supplemented with 10 mM sodium pyruvate, 0.02% soybean trypsin inhibitor, and 1 mg/ml bovine serum albumin to form the solution that was used for gland digestion and named PSA.

Preparation of Parotid Acini and Single Acinar Cells—Mouse parotid acini and single cells were prepared as described (22, 23). In brief, to prepare acini the parotid glands were washed with PSA and minced finely with dissection scissors. The minced tissue was transferred to a flask and suspended in PSA containing 3 mg/ml collagenase P (Roche Applied Science). The flask was gassed with 100% O2 and capped. After 2 min of digestion at 37 °C, the tissue was partially dispersed by pipetteting with a 5-ml plastic pipette tip, and digestion continued for an additional 4-5 min. The digested tissue was washed with PSA, filtered through a 75-µm nylon mesh to remove the large clamps, and the acini were loaded with 5 µM Fura-2 and kept on ice until use. To prepare single cells, mouse parotid glands were minced and washed once with Ca2+-free solution A. The minced tissue was incubated for 7 min at 37 °C with a solution containing 0.025% trypsin that was prepared by a 1:1 dilution of a trypsin/EDTA solution from Sigma with phosphate-buffered saline. Trypsin treatment was terminated by washing the cell with PSA containing 1.5 mg/ml soybean trypsin inhibitor, and the tissue was further digested for 20 min with 70 units/ml collagenase CLSPA (Worthington). The cells were washed with PSA and kept on ice until used.

[Ca2+]i Imaging—[Ca2+]i was measured by imaging Fura-2-loaded parotid acini as detailed before (6, 23). The Fura-2 fluorescence ratio was monitored at excitation wavelengths of 355 and 380 nm using a PTI image acquisition and analysis system. [Ca2+]i was determined from calibration of the signals by incubating the cells in medium containing 10 mM EGTA and 5 µM ionomycin and then medium containing ionomycin and 10 mM Ca2+ as detailed before (4-6). To measure Mn2+ influx, cells in Ca2+-free medium were stimulated for 2.5 min and then exposed to medium containing the agonists and 0.5 mM Mn2+. Fura-2 fluorescence quench was measured at an excitation wavelength of 360 mm. Results are given as the mean ± S.E. of the indicated number of experiments.

Electrophysiology—The whole cell configuration of the patch clamp technique was used to measure the Ca2+-activated Cl- or K+ current as a reporter of [Ca2+]i(5). The Ca2+-activated Cl- current was recorded at a holding potential of -60 mV, whereas the Ca2+-activated K+ current was recorded at a holding potential of 0 mV. The bath solution for current recording was solution A, and the pipette solution contained (in mM): 140 KCl, 1 MgCl2, 0.2 EGTA, 5 ATP, 10 HEPES (pH 7.3 with KOH) with or without cADPR and Heparin. Seals of 6-10 gigaohms were produced on the cell membrane, and the whole cell configuration was obtained by gentle suction or voltage pulses of 0.5 V for 0.3-1 ms. Recording started as soon as possible after establishing the whole cell configuration because the high concentrations of cADPR activated the current by rapidly releasing Ca2+ from internal stores. The patch clamp output (Axopatch-1B, Axon Instruments) was filtered at 20 Hz. Recording and analysis were performed with patch clamp 6 and a Digi-Data 1200 interface (Axon Instruments). Recordings were at room temperature.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CICR in Parotid Acinar Cells—CICR has been demonstrated in many non-muscle cells, yet regulation of this mode of Ca2+ release is not fully understood. To study regulation of CICR, we selected the parotid acinar cells for two reasons. First, there is ample evidence for expression (12, 15, 25) and function (12, 15, 24-26) of RyRs in these cells, or at least activation of Ca2+ release by cADPR. Second, as illustrated in Fig. 1, cADPR is capable of releasing the entire agonist-mobilizable Ca2+ pool. Thus, infusing parotid acinar cells with cADPR concentrations higher than 3 µM tended to induce broad oscillatory changes in the Ca2+-activated Cl- current (Fig. 1, C and D), a reporter of [Ca2+]i(5, 6, 18, 19). At a concentration of 1 µM cADPR induced either sporadic Ca2+ oscillations (3 cells) or a single broad peak (2 cells). Stimulation of cells infused with 10 µM or higher concentrations of cADPR with 1 mM carbachol resulted with a diminished response (4 cells). These results are similar to those found in submandibular acinar cells (5, 27) and are notably different from those reported in pancreatic acinar cells by several groups (5, 7, 18-20) in which cADPR was able to mobilize only a small fraction of the agonist-mobilizable pool. Hence, parotid acinar cells express RyRs mRNA and protein (12, 15, 25) and show robust response to cADPR (Fig. 1) (12, 15, 24-26) and are therefore suitable for studying the role of Ca2+ influx in activation of CICR.



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  		FIG. 1.
Ca2+ release by cADPR and effect of carbachol on [Ca2+]i Single parotid acinar cells (A-D) were used to measure the Ca2+-activated Cl- current. Pipette solutions contained 1 (A and B), 3 (C), or 10 µM (D) cADPR. Where indicated by the bars, the cells were stimulated with 1 mM carbachol. Small acinar clusters (5-8 cells) loaded with Fura-2 were used to measure [Ca2+]i (E and F). As indicated by the bars, the cells were stimulated with 1 mM carbachol and perfused with solutions containing 0, 1, or 2 mM CaCl2.

 
Another indication of robust CICR activity in parotid acinar cells was apparent from measurement of [Ca2+]i. Fig. 1E shows that when cells bathed in medium containing 1 mM Ca2+ were maximally stimulated with 1 mM carbachol there were two events of [Ca2+]i increase, an abrupt immediate increase and a secondary increase marked by an arrow. Fig. 1F shows that removal of external Ca2+ resulted in the reduction of [Ca2+]i to basal levels. Re-addition of external Ca2+ caused a rapid [Ca2+]i increase with an overshoot. Such protocols generally are used to demonstrate the contribution of Ca2+ influx to the agonist-evoked Ca2+ signal. However, the rapid and large [Ca2+]i increase seen on the addition of external Ca2+ is not typical of Ca2+ influx through SOCs (see below also). The abundance of RyRs in parotid acinar cells raised the possibility that the rapid increase in [Ca2+]i is caused by activation of CICR.

Two Events of Ca2+ Release—To clearly separate Ca2+ release and influx we measured the effect of extracellular Ca2+ on the Ca2+ signal evoked by carbachol. Fig. 2A shows that the removal of external Ca2+ reduced the extent and duration of the increase in [Ca2+]i. In the absence of external Ca2+, maximal stimulation increased [Ca2+]i from about 49 ± 6nM to only 186 ± 21 nM (n = 17). Subsequently, [Ca2+]i was rapidly reduced to the basal level such that the total amount of Ca2+ mobilized by the agonist (area under the curve) was reduced by nearly 90%. This indicates that in the absence of external Ca2+, and thus Ca2+ influx, agonist stimulation mobilizes only a fraction of the intracellular Ca2+ pool of parotid acinar cells. Ca2+ entry then was initiated by the addition of various concentrations of CaCl2 to the perfusion solution. Addition of 0.5 and 1.5 mM CaCl2 was followed by a slow [Ca2+]i increase. Once [Ca2+]i reached a certain level, an abrupt change in the rate on [Ca2+]i increase occurred (marked by an arrow), indicating a second event of Ca2+ release. The secondary Ca2+ release was very dramatic when the cells were exposed to an external CaCl2 concentration of 7.5 mM. Under these conditions [Ca2+]i increased to 435 ± 39 nM, which is approximately three times the level evoked by the agonist in the absence of external Ca2+ (n > 50 acini and >250 cells). Importantly, at the continued incubation with 7.5 mM Ca2+, the cells reduced [Ca2+]i to stabilize it at a plateau of about 165 ± 10 nM (n = 6) above resting levels.



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  		FIG. 2.
Effect of external Ca2+ on [Ca2+]i Acini perfused with Ca2+-free medium were stimulated with 1 mM carbachol (A and C) and were then perfused alternately with media containing 0.5, 1.5, 7.5, or 0 mM Ca2+ (A) or alternately with media containing 7.5 or 0 mM Ca2+ (C). The acini in B were incubated in Ca2+-free medium and were treated with 100 µM CPA for about 20 min and then were exposed to a medium containing 100 µM CPA and 7.5 mM Ca2+.

 
The response in Fig. 2A raised the question of whether 7.5 mM external Ca2+ increased [Ca2+]i primarily by the activation of CICR to release Ca2+ from internal stores or whether parotid acinar cells have a very robust Ca2+ influx that undergo partial desensitization/inactivation. This question can be addressed by complete depletion of the stores prior to the addition of external Ca2+. To do so, the cells were incubated with 100 µM of the sarco/endoplasmic reticulum Ca2+ ATPase pump inhibitor cyclopiazonic acid (CPA) for about 20 min. Fig. 2B shows that addition of 7.5 mM external Ca2+ to such treated cells only slowly increased [Ca2+]i, and [Ca2+]i stabilized at ~138 ± 11 nM (n = 5) above the resting level. In an additional protocol, cells stimulated with 1 mM carbachol were repeatedly and alternately exposed to 7.5 and 0 mM external CaCl2 until no explosive increase in Ca2+ was observed. Fig. 2C shows that the addition of 7.5 mM external Ca2+ could elicit several events of rapid, transient [Ca2+]i increases (2-3 in various experiments), after which the addition of 7.5 mM external Ca2+ resulted in a slow [Ca2+]i increase that by the fourth addition stabilized at 142 ± 16 nM (n = 8) above the resting level.

It is evident that the type of signal illustrated in Fig. 2 and described below is likely caused by activation of CICR by Ca2+ entering the cells through the plasma membrane. This can be by activation of the RyRs and perhaps the IP3Rs because both are activated by Ca2+ (see below). Well characterized pharmacological agents that modify the activity of the RyRs in muscle cells are caffeine and ryanodine. However, in agreement with previous work in parotid acinar cells (26, 28, 29), we were unable to see any stimulation of Ca2+ release by using up to 20 mM caffeine and inhibition or stimulation of CICR by treating the cells with up to 250 µM ryanodine. Although previous work reported inhibition of cADPR-mediated Ca2+ release by ryanodine in parotid cells, all the effects were observed with microsomes (29, 30). Therefore, to determine the nature of the Ca2+ signal evoked by 7.5 mM external Ca2+, we proceeded to determine the role of Ca2+ release and Ca2+ influx in this signal and then examined the contribution of RyRs and IP3Rs to the observed signal.

Minimal Store Depletion Is Sufficient to Activate a Large Ca2+ Release—The next issue we addressed is the relationship between the extent of cell stimulation, Ca2+ release from internal stores, and the explosive [Ca2+]i increase evoked by 7.5 mM external Ca2+. For this, parotid acini were incubated in a Ca2+-free medium and stimulated with carbachol concentrations ranging from 0.1 µM to1mM. Two min later, the cells were exposed to media containing the same carbachol concentrations and 7.5 mM Ca2+. Remarkably, stimulating the cells with only 0.5 µM carbachol was sufficient to evoke the same [Ca2+]i increase by 7.5 mM external Ca2+ as that observed when the cells were stimulated with 1 mM carbachol (Fig. 3A). The concentration of carbachol sufficient to trigger the explosive [Ca2+]i increase by high external Ca2+ varied somewhat between experiments. When the cells were stimulated with 0.1, 0.5, and 1.5 µM carbachol, 7.5 mM Ca2+ induced the explosive [Ca2+]i increase in 1/9, 7/13 and 25/25 acini, respectively, and was the same as that induced by stimulated with 1 mM carbachol in the same experiments.



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  		FIG. 3.
The extent of pool depletion needed to activate CICR. A, acini incubated in Ca2+-free medium were stimulated with 0 (solid gray trace, control), 0.1 (dashed gray trace), 0.5 (solid black trace), or 15 µM carbachol (dotted blavk trace) for 2.5 min before exposure to media containing the same carbachol concentration and 7.5 mM Ca2+. B, the cells were incubated in Ca2+-free medium containing 1 µM CPA for 2.5 (dotted rectangle), 5 (dashed gray rectangle), 7.5 (solid gray rectangle), or 10 min (solid black rectangle) before exposure to media containing 7.5 mM Ca2+ (filled black bars in each experiment).

 
The minimal agonist stimulation needed to activate Ca2+ release by 7.5 mM external CaCl2 indicates that either minimal cell stimulation or minimal depletion of the internal Ca2+ pool is sufficient to activate CICR. To distinguish between these possibilities and to examine the possible role of SOCs in allowing the effect of external CaCl2, we tested the ability of treatment with a low concentration of CPA to activate the Ca2+ release by high external CaCl2. The results of these experiments are shown in Fig. 3B. For these experiments we selected 1 µM CPA, which only partially inhibits the sarco/endoplasmic reticulum Ca2+ ATPase pumps to slowly deplete the ER Ca2+ store. Incubating parotid acini in Ca2+-free medium containing 1 µM CPA between 2-10 min resulted in a progressively higher increase in [Ca2+]i on the addition of 7.5 mM external Ca2+. Hence, the explosive Ca2+ release observed on the addition of external Ca2+ is not dependent on cell stimulation but, rather, on the Ca2+ release induced by cell stimulation. Furthermore, minimal depletion of the stores by either cell stimulation or treatment with CPA is sufficient to allow the Ca2+ release evoked by addition of external Ca2+.

It was of interest to determine the extent of store depletion needed to activate the pathway that mediates the effect of high external Ca2+. This can be estimated fairly well by treating the cells with CPA. First, we measured the ability of incubation with different concentrations of CPA at a set period of time to activate the Ca2+ release by 7.5 mM external Ca2+. Fig. 4A shows that treatment with CPA was as effective as stimulation with carbachol in allowing the Ca2+ release observed on the addition of high external Ca2+. In addition, incubation with 100 µM CPA for a short period of time caused only a small [Ca2+]i increase in cells incubated in Ca2+-free media. Furthermore, incubating acini in Ca2+-free media with 2.5 µM CPA for 2 min was sufficient to allow the explosive Ca2+ release caused by 7.5 mM external Ca2+, an effect that was observed in all acini treated with 2.5 µM CPA for 2 min or longer (n = 45 acini, >180 cells). Fig. 4B shows how the extent of store depletion by this and higher concentrations of CPA was estimated. The acini were incubated for 2 min with 2.5, 10, or 100 µM CPA and then the stores were completely discharged by exposing the cells to CPA and 1 mM carbachol. It is evident that during the 2-min incubation all concentrations of CPA mobilized only a small fraction of the pool. Furthermore, from the reduced response to carbachol plus CPA, we determined that mobilization of less than 5% of the pool was sufficient to maximally activate the pathway that mediated the effect of 7.5 mM external Ca2+.



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  		FIG. 4.
Relationship between store depletion by CPA and CICR. A, acini in Ca2+-free medium were treated for 2 min with 0 (solid gray trace, control), 1 (dotted gray trace), 2.5 (solid black trace), 10 (dotted black trace), or 100 µM CPA (dashed black trace) before exposure to media containing 7.5 mM CaCl2 and then to Ca2+-free media. B, to estimate the extent of store depletion, acini in Ca2+-free media were treated for 2 min with 2.5 (dotted black trace), 10 (solid black trace), or 100 µM CPA (solid gray trace) before exposure to Ca2+-free media containing 100 µM CPA and 1 mM carbachol. After reduction of [Ca2+]i to basal levels, the acini were exposed to media containing 7.5 and then 0 mM Ca2+.

 
Contribution of IP3Rs and RyRs to CICR—Two processes can contribute to the CICR. Both the RyRs (9, 10) and IP3Rs (31) are activated by Ca2+ with the type 3 IP3Rs particularly sensitive to Ca2+ concentrations between 25-250 nM (31). To distinguish between the contributions of each process, we used inhibitors of the IP3Rs because the pharmacological agents that modify the activity of the RyRs in muscle cells had no effect on CICR in parotid acini. The lack of activation by caffeine provided us with the first compound because caffeine was shown to inhibit the IP3Rs in a reversible manner (7, 32). This is further shown in Fig. 5A for parotid acini stimulated with carbachol. In preliminary experiments we determined the minimal concentration of caffeine required to inhibit Ca2+ release evoked by 15 µM carbachol to minimize nonspecific effect of caffeine, which is a potent inhibitor of IP3 production in secretory cells (33). Fig. 5A shows that when added before cell stimulation 2.5 mM caffeine prevented the carbachol-induced Ca2+ release and the explosive Ca2+ increase triggered by 7.5 mM external Ca2+. Washing away the caffeine reversed the inhibition although exposure to 7.5 mM external Ca2+ resulted in a slower rate of Ca2+ increase. As shown in Fig. 5B, the cells were first stimulated with carbachol to deplete the stores and activate Ca2+ influx and then were exposed to 2.5 mM caffeine. Although caffeine reduced the rate, it did not prevent the Ca2+ increase in response to 7.5 mM external Ca2+. The slow rate of Ca2+ increase in Fig. 5, A and B may suggest the contribution of IP3Rs to CICR or the effects of caffeine on the stimulated state (33). This was examined by testing the effect of caffeine on the response observed in cells treated with CPA because CPA depletes the stores in an IP3-independent manner. Incubating the cells with 2.5 mM caffeine had no effect on the Ca2+ increase evoked by 7.5 mM external Ca2+ in CPA-treated cells. Fig. 5C shows that 10 mM caffeine reduced the response to 7.5 mM external Ca2+ by about 26 ± 5% (n = 5) in acini treated with 10 µM CPA.



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  		FIG. 5.
CICR does not require active IP3Rs. A and B, acini in Ca2+-free medium were incubated with 2.5 mM caffeine before (A) or after (B) stimulation with 15 µM carbachol and then alternately in Ca2+-free media and media containing 7.5 mM Ca2+. Where indicated, caffeine was removed from the perfusion solutions. C, acini were treated with 10 µM CPA for 2 min before incubation with 10 mM caffeine in media containing 0 or 7.5 mM Ca2+, as indicated. D and E, the Ca2+-activated K+ current was measured in single cells. D, the cells were exposed to media containing 7.5 Ca2+ before incubation in Ca2+-free media containing 10 µM CPA and then were exposed to media containing CPA and 7.5 mM Ca2+. E, the pipette solution contained 200 µg/ml heparin. Treatment with CPA began shortly after break-in and the cells were exposed to 7.5 mM external Ca2+ as indicated by the bar.

 
Another inhibitor of the IP3Rs is heparin (34). To test the effect of heparin it was infused into the cells through a patch pipette, and the activity of the Ca2+-activated K+ channel was measured (Fig. 5, D and E). Exposing non-stimulated cells to 7.5 mM external Ca2+ had minimal effect on the current. Treating the cells with 10 µM CPA in Ca2+-free media failed to activate any current, and the addition of 7.5 mM external Ca2+ resulted in an abrupt but modest increase in the outward current (Fig. 5D). Infusing the cells with 200 µg/ml heparin for 7-8 min completely inhibited the response to carbachol stimulation (not shown) but reduced the response to 7.5 mM external Ca2+ in CPA-treated cells by only 33 ± 5% (n = 4). Together, the results in Fig. 5 indicate that the majority of the CICR was independent of Ca2+ release mediated by the IP3Rs. The modest effects of caffeine and heparin can be caused by the nonspecific effect of the blockers or a small Ca2+ release from the IP3 pool that required sensitization of the IP3Rs by Ca2+.

Site of CICR—The modest activation of K+ current in response to the addition of 7.5 mM external Ca2+ to cells treated with CPA (Fig. 5) was unexpected in view of the robust Ca2+ increase caused by the addition of 7.5 mM external Ca2+ (Figs. 3 and 4). At least two mechanisms can explain this discrepancy. The first possibility is that the current was measured in single cells whereas the [Ca2+]i was measured in acini. It is possible that the ratio of [Ca2+]i increase in the presence and absence of external Ca2+ is different in a single cell than in acini. Second, it is possible that the robust [Ca2+]i increase caused by CICR was slower than the initial Ca2+ release evoked by carbachol and occurs largely away from the plasma membrane. To distinguish between these possibilities, we measured [Ca2+]i and the Ca2+-activated K+ current in single cells stimulated with carbachol. Fig. 6A shows that stimulation of cells incubated in Ca2+-free media with 2.5 µM or 1 mM carbachol resulted in a rapid and a large increase in the current. Noticeably, exposing the cells to 7.5 mM external Ca2+ resulted in a small increase in the current. Similar results were obtained with 4 cell preparations (and at least 8 experiments under each condition) and additional experiments with different carbachol concentrations between 1-100 µM. Furthermore, essentially the same results were obtained by measuring the Ca2+-activated Cl- current, except that the response to 7.5 mM external Ca2+ was even smaller relative to the initial response in the absence of external Ca2+.



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  		FIG. 6.
Ca2+-activated K+ current and [Ca2+]i in single parotid acinar cells. A, single parotid acinar cells in Ca2+-free medium were stimulated with 2.5 µM (left trace)or 1 mM carbachol (right trace). After return of the outward current to base line, the external Ca2+ was increased to 7.5 mM. B, a single cell loaded with Fura-2 was used to monitor [Ca2+]i. The cell was first incubated in Ca2+-free media and stimulated with 1 mM carbachol (upper images). After the return of [Ca2+]i to base line, the cell was exposed to media containing 1 mM carbachol and 7.5 mM Ca2+ (lower images). Images recorded at selective periods during the experiment are shown. The first and last images of each sequence are marked in yellow 1 and 2, and the time of their acquisition is marked at the traces. The traces in a and b show the relative increase of fluorescence at the apical (brown) and basal (green) poles. In c, the trace of [Ca2+]i increase at the apical pole caused by carbachol stimulation (blue) was scaled up and superimposed with the trace of [Ca2+]i increase caused by the addition of 7.5 Ca2+ to illustrate the different rates of Ca2+ increase. Both traces are drawn at the same time scale. B, upper left, the first upper image shows the bright field image.

 
An example of the Ca2+ signal observed in single parotid acinar cells is shown in Fig. 6B. Carbachol stimulation of cells in Ca2+-free media resulted in a luminal-to-basal Ca2+ wave (Fig. 6B, top panel). As reported before (26, 35), the agonist-evoked Ca2+ wave in parotid acini was faster than that in pancreatic acinar cells with the maximal increase in [Ca2+]i attained within 0.8 s of stimulation. Subsequent exposure of the cells to media containing 7.5 mM Ca2+ resulted in a slower but higher [Ca2+]i increase (Fig. 6, traces b and c). In six experiments the [Ca2+]i increase induced by 7.5 mM external Ca2+ was 2.56- ± 0.36-fold higher than the initial increase measured in the absence of external Ca2+. Interestingly, the Ca2+ release evoked by 7.5 mM external Ca2+ also tended to start at the apical pool. However, it did not occur as a propagating luminal-to-basal Ca2+ wave. Rather, the rate of [Ca2+]i increase was slow and occurred in other parts of the cell shortly after the initial increase at the apical pole. This suggests that Ca2+ influx starts at the apical pole but takes place in all regions of the basolateral membrane and that Ca2+ entering the cell at all sites can trigger CICR, including Ca2+ entering at the IP3Rs-poor basal pole. The combined results in Fig. 6 are consistent with the idea that the Ca2+ increase caused by CICR occurs mainly at sites far from the plasma membrane such that it does not activate the Cl- and K+ channels. Dissociation between a detectable [Ca2+]i increase and activation of the K+ channel was reported before (36) and was attributed to a rapid initial agonist-stimulated Ca2+ increase next to the plasma membrane.

Ca2+ Influx through SOCs Is Essential for CICR Triggered by Addition of External Ca2+—The need to stimulate the cells with carbachol or to treat them with CPA in order to observe the Ca2+ release evoked by the addition of external Ca2+ suggests the activation of Ca2+ influx by the agonist and CPA. Obviously, the extent of Ca2+ influx activated by partial store depletion could not be determined from Ca2+ removal and re-addition protocols because of the activation of the CICR. An alternative procedure to estimate the activity of SOCs is to measure the unidirectional influx of Mn2+. Most SOCs channels admit Mn2+, and Mn2+ is not a substrate for the sarco/endoplasmic reticulum Ca2+ ATPase and plasma membrane Ca2+ ATPase pumps so that Mn2+ influx accurately reports the plasma membrane permeability to divalent ions (37). The results of Mn2+ influx measurement are given in Fig. 7. Maximal store depletion by treating the cells with 1 mM carbachol and 25 µM CPA increased Mn2+ influx ~5-fold. Stimulation with 0.5 µM carbachol or treatment with 2.5 µM CPA for 2.5 min increased Mn2+ influx by only 7 ± 3% (n = 3) -fold, highlighting the fact that selective activation of Ca2+ influx is required to activate the CICR.



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  		FIG. 7.
Measurement of Mn2+ influx. Acini in Ca2+-free medium were incubated for 2.5 min with 0 (control), 2.5 µM CPA, 0.5 µM carbachol, or 25 µM CPA and 1 mM carbachol (Car), as indicated next to each trace, before exposure to Ca2+-free medium containing the same agonists and 0.5 mM Mn2+ to measure Mn2+ influx. The first derivatives of the slopes were used to obtain the rate of Mn2+ influx.

 
If Ca2+ influx is obligatory for the Ca2+ release evoked by the addition of high external Ca2+, then inhibition of the influx should inhibit the Ca2+ release. This was tested using two SOCs blockers, SKF96365and 2-aminoethoxydiphenyl borate. Fig. 8A shows that incubating the cells with 20 and 40 µM SKF96365partially or maximally, respectively, inhibited the [Ca2+]i increase observed on the addition of external CaCl2 and that at both concentrations the inhibition was reversible. 2APB was reported to inhibit both IP3-mediated Ca2+ release and Ca2+ influx by SOCs (38) but not CICR by RyRs (39). Using the skeletal muscle cell line C2C12, we confirmed that 2ABP does not inhibit Ca2+ release evoked by caffeine or by depolarization, whereas it inhibited the Ca2+ signal evoked by stimulation of the P2Y2 receptors.2 As with SKF96365 incubating the cells with 20 and 40 µM 2APB partially or maximally inhibited the [Ca2+]i increase triggered by the addition of 7.5 mM external Ca2+, indicating that Ca2+ influx and [Ca2+]i increase are essential to activate the explosive increase in [Ca2+]i that is caused by CICR.



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  		FIG. 8.
Inhibition of Ca2+influx inhibits CICR. Acini in Ca2+-free medium were treated with 10 µM CPA to activate SOCs and then were incubated with 0 (A and B, control, thin solid trace), 20 µM SKF96365(A, dotted trace), 40 µM SKF96365(A, thick solid trace), 20 µM 2APB (B, dotted trace) or 40 µM 2APB (B, thick solid trace) before perfusion with media containing CPA, the respective inhibitor concentration, and 7.5 mM Ca2+. Finally, blockers were washed by perfusing the acini with media containing CPA and 7.5 mM Ca2+.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The widespread expression of RyRs in non-muscle cells (11-15) indicates that CICR by activation of RyRs is central to agonist-evoked Ca2+ signaling. Furthermore, selective activation of CICR by G protein-coupled receptors appears to be part of the regulatory repertoire (7) that is used to generate receptor-specific Ca2+ signals (2). Therefore, it is important to understand the activation mechanism of CICR and its regulation in non-muscle cells. Whereas the RyRs mediating CICR in skeletal (RyR1) and cardiac (RyR2) cells are well established (8-10), it is still a matter of debate which RyRs are expressed in non-muscle cells and even whether they mediate the CICR. For example, using the same molecular and biochemical approaches, it was reported that pancreatic acinar cells express RyR2 but not RyR1 and RyR3 (14) or all three RyRs isoforms (13). Similarly, parotid acinar cells were reported to express only an RyR1 isoform (25) or only RyR3 (15). Knowledge of the RyR isoforms and their role in Ca2+ signaling is equally poor in other non-muscle cells, which makes it impossible to know with certainty the molecular identity of the protein mediating CICR in non-muscle cells.

The pharmacology of CICR in non-muscle cells is equally confusing and is the subject of conflicting reports. Although caffeine activates RyRs in muscle and chromaffin cells, most studies failed to show caffeine-mediated Ca2+ release in non-muscle cells that responded well to cADPR. Similarly, in the present work we were not able to see any effect of caffeine on [Ca2+]i by itself. In fact caffeine is a good inhibitor of IP3-mediated Ca2+ release (33) and was used in several studies to distinguish between Ca2+ release mediated by IP3 and by cADPR (7, 32). In the present work we confirmed inhibition of agonist-evoked Ca2+ release by caffeine and used this property to show that most of the CICR was not mediated by the IP3Rs (Fig. 5). Similar uncertainties exist with ryanodine. Most studies on parotid acini of inhibition by ryanodine of Ca2+ release induced by cADPR used permeabilized cells or microsomes (24, 29, 30). One study reported inhibition of large Ca2+ oscillations induced by thapsigargin by 10-50 µM ryanodine but failed to find inhibition of the response to the agonist (28). A recent report showed that 500 µM ryanodine was required to inhibit the Ca2+ oscillations riding on the top of a Ca2+ plateau evoked by low carbachol concentration without affecting the plateau (26). We were unable to inhibit any part of the Ca2+ signal observed under all protocols used in the present work. Therefore, we conclude that the CICR in parotid acinar cells is not very sensitive to either caffeine of ryanodine. However, we do argue that CICR is mediated by a channel separate from the IP3 receptor channels because it was equally well activated by treatment with a low concentration of CPA and inhibited by only 30% by inhibition of the IP3Rs with caffeine or heparin. Whether it is mediated by a classical RyR remains to be established.

A consensus does exist of activation of CICR by cADPR in non-muscle cells and that cADPR is a ligand that directly or indirectly activates the RyRs (16). Based on the response to cADPR, many cell types, including parotid acinar cells, do have a CICR mechanism. Furthermore, it appears that at least in parotid acinar cells, CICR plays a prominent role in agonist-evoked Ca2+ mobilization. Thus, in the absence of external Ca2+, the agonist-evoked [Ca2+]i increase is markedly diminished and is short lasting (Figs. 1, 2, 3). This was largely caused by the elimination of CICR because maximal Ca2+ influx through SOCs account for only a small fraction of the agonist-evoked [Ca2+]i increase measured in the presence of external Ca2+ (Fig. 2). Thus, the majority of Ca2+ release in parotid acini is caused by CICR. The robustness of CICR in parotid acinar cells also is evident from the effect of cADPR. In most experiments 10 µM cADPR mobilized almost the entire intracellular Ca2+ pool as concluded from the small effect of carbachol on Ca2+-activated Cl- current in cells incubated with 10 µM cADPR (Fig. 1C). Similar findings were reported in submandibular acinar cells by us (5) and others (27), although higher concentrations of cADPR were needed in the study of Harmer et al. (27). The potent CICR mechanism in parotid acini may be of particular physiological significance in these cells. Analysis of Ca2+ waves in pancreatic and parotid acini revealed that the threshold Ca2+ release that triggers a propagating wave is much lower in parotid than in pancreatic acinar cells and that at modest stimulation the wave propagated about 8 times faster in parotid than in pancreatic acinar cells (35). The increased sensitivity to stimulus intensity was attributed to a 4-fold higher expression of IP3Rs (35). The prominent CICR likely is the mechanism behind the faster Ca2+ wave propagation in parotid acinar cells.

The most notable finding of the present work is that Ca2+ influx through SOCs plays a critical role in the activation of CICR in parotid acinar cells. This conclusion is supported by several findings: (a) removal of external Ca2+ markedly reduced the amplitude and the amount of Ca2+ mobilized in response to agonist stimulation, an effect that is not simply caused by removal of the contribution of Ca2+ influx to the [Ca2+]i increase (Figs. 1 and 2); (b) activation of SOCs by receptor-independent depletion of the stores with CPA was sufficient to activate CICR; (c) inhibition of Ca2+ influx through SOCs by SKF96365or 2APB inhibited the activation of CICR by the addition of high external Ca2+. Although these blockers are not specific and have additional effects beside the inhibition of SOCs, they were shown to inhibit SOCs in many cell types. Because most CICR is independent of IP3Rs, 2-aminoethoxydiphenyl borate does not inhibit CICR by RyRs, and the major acute effect of SKF9636 is inhibition of Ca2+ influx, the results with the blockers are consistent with a critical role of Ca2+ influx through SOCs for activation of CICR. Together, our results indicate that Ca2+ influx through SOCs activates the CICR mechanism to mediate most of the [Ca2+]i increase observed during agonist stimulation of parotid acinar cells.

It is of note that although Ca2+ release from the IP3 pool (measured in cells stimulated in the absence of external Ca2+) increases [Ca2+]i to higher levels than Ca2+ influx through SOCs (for example, see Fig. 2, A and C), it was not sufficient to activate CICR. This indicates that not "all [Ca2+]i is the same" and Ca2+ influx through SOCs specifically activates CICR. This conclusion received further support with the finding that Ca2+ release from the IP3 pool strongly activated the Ca2+-activated Cl- and K+ channels, whereas CICR caused by subsequent Ca2+ influx increased [Ca2+]i about 2.5-fold higher than Ca2+ release from the IP3 pool but only modestly activated the channels. These findings suggest that most CICR occurred away from the plasma membrane and further emphasis the tight association between Ca2+ influx and CICR. Compartmentalization of Ca2+ release events in parotid acini were reported before as a lag in detection in Ca2+ increase relative to detection of the Ca2+-activated K+ current (36).

An important feature of the Ca2+ influx that activates CICR is that it is mediated by a selective subpopulation of SOCs channel. Thus, activation of at most 5% of maximal SOCs activity was sufficient to mediate the Ca2+ influx needed to activate CICR. This population of SOCs channels was regulated by a small portion of the ER Ca2+ pool. These findings are similar to two previous reports showing that a selective 5% of the IP3 pool governs regulation of Icrac (40, 41). Such a relationship between Ca2+ and the SOCs and CICR channels requires that both channels exist in close proximity in cellular microdomains. Such an arrangement is well established for Ca2+ signaling complexes in many cell types including secretory cells (2, 6) and is the hallmark of excitation-contraction coupling (8-10). Similar to the situation in muscle, it is likely that only a subpopulation of the CICR channels is coupled to SOCs and that Ca2+ released from these stores then serves to activate CICR in adjacent stores and to propagate a Ca2+ wave. In accord with such a mechanism, cADPR-mediated Ca2+ release appears to be essential for the propagation of Ca2+ waves (19-21, 42). The properties of CICR as demonstrated here indicate that CICR in cardiac myocytes is a specialized mechanism of a more general mechanism found in non-muscle cells. That is, regulation of CICR by a selective population of SOCs channels and the obligatory dependence of CICR on Ca2+ influx through SOCs is equivalent to CICR in cardiac myocytes with the exception that in cardiac myocytes CICR is activated by Ca2+ influx through L-type Ca2+ channels whereas in parotid acinar cells Ca2+ influx occurs through SOCs. If this finding can be extended to other non-muscle cells, it will generalize the mechanism of regulation of CICR and highlight the role of SOCs in shaping the agonist-evoked Ca2+ signal.


   FOOTNOTES
 
* This work was supported by National Institutes of Heath Grants DE12309 and DK38938 and Cystic Fibrosis Foundation Grant MUALLE01G0. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9040. Tel.: 214-648-2593; Fax: 214-648-8879; E-mail: SHMUEL.MUALLEM{at}utsouthwestern.edu.

1 The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; ER, endoplasmic reticulum; SOC, store-operated Ca2+ channel; CICR, Ca2+-induced Ca2+ release; RyRs, ryanodine receptors; cADPR, cyclic ADP-ribose; CPA, cyclopiazonic acid. Back

2 K. Kiselyov and S. Muallem, unpublished observation. Back



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 ABSTRACT
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 DISCUSSION
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