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Inositol 1,4,5-trisphosphate receptors (InsP3Rs) modulate Ca2+ release from intracellular Ca2+ store and are extensively expressed in the membrane of endoplasmic/sarcoplasmic reticulum and Golgi. Although caffeine and 2-aminoethoxydiphenyl borate (2-APB) have been widely used to block InsP3Rs, the use of these is limited due to their multiple actions. In the present study, we examined and compared the ability of caffeine and 2-APB as a blocker of Ca2+ release from intracellular Ca2+ stores and Ca2+ entry through store-operated Ca2+ (SOC) channel in the mouse pancreatic acinar cell. Caffeine did not block the Ca2+ entry, but significantly inhibited carbamylcholine (CCh)-induced Ca2+ release. In contrast, 2-APB did not block CCh-induced Ca2+ release, but remarkably blocked SOC-mediated Ca2+ entry at lower concentrations. In permeabilized acinar cell, caffeine had an inhibitory effect on InsP3-induced Ca2+ release, but 2-APB at lower concentration, which effectively blocked Ca2+ entry, had no inhibitory action. At higher concentrations, 2-APB has multiple paradoxical effects including inhibition of InsP3-induced Ca2+ release and direct stimulation of Ca2+ release. Based on the results, we concluded that caffeine is useful as an inhibitor of InsP3R, and 2-APB at lower concentration is considered a blocker of Ca2+ entry through SOC channels in the pancreatic acinar cell.
Intracellular Ca2+ controls a vast array of cellular functions like as contraction, secretion, cell growth, and proliferation [1-3]. The concentration of [Ca2+]i could be raised by Ca2+ entry from extracellular space through membrane bound receptor-operated Ca2+ (ROC) channels or store-operated Ca2+ (SOC) channels, and Ca2+ release from intracellular stores through inositol 1,4,5-trisphosphate receptors (InsP3Rs) or ryanodine receptors [3-6]. Widely expressed InsP3Rs in various cells can be specifically activated using a membrane-permeant InsP3 ester , but the using of inhibiting compound is still limited because of the non-specific actions.
Heparin is one of the most commonly used InsP3R antagonists, but it has multiple actions including uncoupling G-protein signaling and activation ryanodine receptors . Additionally, heparin should be used by injection or infusion into the cell due to its non-permeable characteristics even though it has been suggested that low molecular weight heparin may cross the plasma membrane and thus inhibit InsP3Rs in the intact cell. Although xestospongins, another InsP3R antagonist, have been used frequently to prove the InsP3-induced Ca2+ release in the intact cell, the mechanism of action has not been fully elucidated at the present time. Furthermore this compound is expensive, is slow to act, and has not been universally successful .
Presently, the most widely used cell permeable antagonists of InsP3Rs in the intact cell are caffeine and 2-aminoethoxydiphenyl borate (2-APB) despite their some non-specific actions. Caffeine easily permeates the plasma membrane and can effectively inhibit InsP3Rs in the intact cell. However, one of the side actions of caffeine itself is an increased Ca2+ release from internal stores through ryanodine receptor activation . 2-APB has been subsequently used to evaluate the contribution of InsP3Rs in the generation of Ca2+ signals. In an initial study, 2-APB evoked concentration-dependent inhibition of InsP3-induced Ca2+ release from the mouse cerebellar membrane . The inhibitory mechanism of 2-APB to InsP3Rs is unclear, since it does not directly block InsP3 binding site [11-13]. Recently, it has been reported that 2-APB failed to inhibit InsP3-induced Ca2+ release in the some cell types, and consistently blocked the Ca2+ entry through SOC channel [14-18].
Despite the lack of specificity of caffeine and 2-APB to InsP3Rs, they remain as widely-used inhibitors of intracelluar Ca2+ signaling. It is still important to elucidate their cellular targets and cell specific action because different cells have different Ca2+ mobilizing mechanisms. Therefore, we examined and compared the ability of caffeine and 2-APB in mouse pancreatic acinar cell in the present study. Here, we report that only caffeine potently blocks the InsP3-induced Ca2+ release, while 2-APB in low concentration works as a blocker of store-operated Ca2+ entry channel in this cell.
Small clusters of pancreatic acinar cells were isolated by collagenase digestion as described previously . Briefly, the pancreas was removed from freely fed male Balb/C mice after CO2 asphyxiation and cervical dislocation. The dissected tissue was enzymatically digested with type-II collagenase in DMEM containing 0.1% bovine serum albumin and 1 mg/ml soybean trypsin inhibitor for 30 min followed gentle agitation. Acinar cells were filtered using 100 µm nylon mesh and then centrifuged at 75 g. After washing twice, cells were resuspended in HEPES-buffered physiological saline solution (HEPES-PSS) containing 5.5 mM glucose, 137 mM NaCl, 0.56 mM MgCl2, 4.7 mM KCl, 1 mM Na2HPO4, 10 mM HEPES (pH 7.4), 1.28 CaCl2, and 1% (w/v) bovine serum albumin until ready for use. All experimental procedures were carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.
For measurement of [Ca2+]i, isolated acinar cells were loaded with Ca2+-sesitive dye, 2 µM fura-2/AM for 30 min at room temperature. Fura-2 loaded cells were mounted on a glass coverslip at a bottom of a perfusion chamber. Cells were continuously superfused with HEPES-PSS at a flow rate of 1 ml/min using an electronic controlled perfusion system (Warner Instrument, Hamden, CT, USA). Global Ca2+ imaging was performed using an inverted Olympus IX71 microscope through a 40× fluorescence objective lens. Cells were excited alternately with light at 340 nm and 380 nm, using a Polychrome V monochrometer (Till Photonics, Pleasanton, CA, USA). Fluorescence images were captured at an emitted wavelength of 510 nm using a cooled charged-coupled device Cool-SNAP HQ2 camera (Photometrics, Tuscon, AZ, USA).
Cells were loaded with 10 µM furaptra/AM for 30 min at room temperature. Acinar cells were then allowed to adhere to Cell-Tak (BD Biosciences, San Jose, CA, USA)-coated coverslips at the bottom of a small volume perfusion chamber. Cells were permeabilized by superfusion for 1~2 min with β-escin (40 µM) in intracellular medium containing 125 mM KCl, 19 mM NaCl, 10 mM HEPES, and 1 mM EGTA (pH 7.3). Permeabilized cells were washed in intracellular medium without β-escin for 15 min to facilitate removal of cytosolic dye. Cells were superfused in intracellular medium containing 0.650 µM CaCl2 (free [Ca2+]=200 nM), 1.4 mM MgCl2, and 3 mM Na2ATP to activate sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and to load the intracellular Ca2+ stores. The free [Ca2+] was maintained at a constant 200 nM throughout all experimental maneuvers. The emission of the dye above 505 nm following excitation at 340 nm and 380 nm was recorded using a TILL Photonics imaging system.
Carbamylcholine, caffeine, β-escin, and other chemicals for making buffers were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). 2-APB was purchased from Tocris Bioscience (Ballwin, MO, USA). Thapsigargin was purchased from Calbiochem (San Diego, CA, USA). Fura 2-AM and furaptra-AM were purchased from TefLabs Inc. (Austin, TX, USA). Inositol 1,4,5-trisphosphate (InsP3) was purchased from Biomol Research Laboratories (Plymouth, PA, USA).
Results were presented as mean±S.E. Data were analyzed using the Student t test. Rates of Ca2+ release were estimated from these responses by fitting the initial 10 sec period of decreasing fluorescence to a single exponential function using the Origin program as described previously . Differences were considered significant when the p value was less than 0.05.
Inhibitory effects of caffeine and 2-APB on lower CCh-induced [Ca2+]i changes were examined in intact pancreatic acinar small clusters. A 300 nM carbamylcholine (CCh) induced a base-up [Ca2+]i oscillation and this pattern was sustained continuously in the presence of 1.28 mM [Ca2+]o. Both caffeine (20 mM) and 2-APB (30 µM) effectively blocked sustained [Ca2+]i oscillation (Fig. 1A), and its inhibitory effects were dose-dependent (Fig. 1B and 1C). However, the recovery patterns after washout of these drugs were remarkably different. The initial Ca2+ peak of oscillation was completely recovered after withdrawal of caffeine, while that was not fully recovered after removal of 2-APB. Since [Ca2+]i oscillation could be abolished by reduced Ca2+ entry from extracellular space as well as reduced Ca2+ release from intracellular Ca2+ store, follow-up experiments were performed to define which pathways were blocked by caffeine and 2-APB, respectively.
Supraphysiological concentration of CCh has known to make two compartments of Ca2+ signals, initial peak and sustained plateau, in the normal [Ca2+]o . It is well known that the initial peak is the result of Ca2+ release from intracellular store, and a sustained plateau is due to the consecutive store-operated Ca2+ entry . To define what pathways are blocked by caffeine and 2-APB, the next experiments were performed. Caffeine (30 mM) completely blocked [Ca2+]i elevation by 4.60±0.75% of control value (Fig. 2A). Transient [Ca2+]i rise after washout of caffeine and CCh may be due to the action of remained InsP3 in the cell. The inhibitory effect of caffeine on initial [Ca2+]i peak was dose dependent (Fig. 2C). On the other hand, 2-APB (30 µM) has no effect on initial Ca2+ peak, but it significantly blocked sustained Ca2+ plateau (Fig. 2B and 2C). These above results suggest that the inhibitory effect of caffeine on [Ca2+]i signaling may be resulted from inhibiting Ca2+ release and that of 2-APB due to Ca2+ entry block. To further clarify this issue, we tested the effects of caffeine and 2-APB on thapsigargin-induced Ca2+ entry.
Perfusion of thapsigargin, a sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, resulted in marked [Ca2+]i rise followed by decline to basal value without [Ca2+]o. Reintroduction of 1.28 mM of Ca2+ elevated [Ca2+]i , which may be due to activation of store-operated Ca2+ (SOC) channel. This SOC channel-mediated Ca2+ entry was not blocked by caffeine, but dose-dependently blocked by 2-APB (Fig. 3A and 3B). As shown in Fig. 3C, 30 µM 2-APB maximally inhibited SOC channel-medicated Ca2+ entry (46.21±6.06% of control value). The recovery of Ca2+ entry was not observed after removal of 2-APB.
To determine the inhibitory effects of caffeine and 2-APB on InsP3-induced calcium release, we performed the experiment in the permeabilized pancreatic acinar cell using β-escin. After re-filling of internal Ca2+ stores by MgATP in the presence of 200 nM of Ca2+, submaximal (0.5 µM) or maximal (3 µM) concentrations of InsP3 were perfused with or without caffeine and 2-APB, respectively. Caffeine (20 mM) significantly inhibited submaximal and maximal InsP3-induced Ca2+ release rate by 65.0% and 51.6% of control values, respectively (Fig. 4A and 4C), while 2-APB (30 µM) failed to inhibit Ca2+ release induced by both concentrations of InsP3 (Fig. 4B and 4C). These results strongly indicate that caffeine is a useful blocker of InsP3-induced Ca2+ release in the mouse pancreatic acinar cell.
Although low concentrations of 2-APB (below 30 µM) had no effect on CCh-induced initial Ca2+ peak in intact cell and InsP3-induced calcium release in permeabilized cell, high concentrations of 2-APB (above 100 µM) itself at markedly increased [Ca2+]i in the intact cell. As shown in Fig. 5A, the patterns of [Ca2+]i elevation are very different according to the perfused concentrations of 2-APB; 100 µM of 2-APB produced a slightly progressive [Ca2+]i increase, 300 µM of 2-APB produced a transient peak-type [Ca2+]i increase, and 1 mM of 2-APB produced a sustained [Ca2+]i increase. In the permeabilized cell, 100 µM of 2-APB directly enhanced Ca2+ release from internal stores by itself (Fig. 5B). Paradoxically, 2-APB reduced InsP3-induced Ca2+ release by 74.7% of control value (Fig. 5B and 5C). We could not test the inhibitory effect of 300 µM of 2-APB on InsP3-induced Ca2+ release in permeabilized cell, because it rapidly accelerated Ca2+ release from internal stores in the present study (data not shown). From the above results, we suggest that high (~100 µM) concentrations of 2-APB may need careful use, because this compound has paradoxical, multiple effects on intracellular Ca2+ mobilization.
Caffeine has been widely used in studies to demonstrate the mechanism of InsP3Rs-mediated Ca2+ release from internal store in various cells, including human B lymphocytes, retinal ganglionic cells, pyramidal neurons, and pancreatic acinar cells [22-25]. Its high plasma membrane permeability, relatively low cost, and lack of effect on Ca2+ entry from extracellular space make it more useful than other antagonists, such as xestospongins or heparin. CCh, an acetylcholine analogue, has known to make specific [Ca2+]i signals in pancreatic acinar cells in a dose-dependent manner. Lower CCh evoked continuous Ca2+ oscillation, and higher CCh induced initial Ca2+ peak and sustained plateau in the presence of normal extracellular Ca2+. CCh-induced initial Ca2+ mobilization has been recognized as a result of Ca2+ release from internal stores, because those are essentially unaffected by removal of extracellular Ca2+, and those are not observed by store depletion using thapsigargin or cyclopiazonic acid (CPA), SERCA inhibitors . We found that caffeine blocked lower CCh-induced Ca2+ oscillation, as well as higher CCh-induced initial [Ca2+]i peak on a dose-dependent in intact cell. In addition, caffeine inhibited InsP3-induced Ca2+ release from internal store in permeabilized cell. Present data provide that caffeine is very effective blocker of InsP3-induced Ca2+ release from internal store in pancreatic acinar cell. A different pathway that increases [Ca2+]i is SOC channel-mediated Ca2+ entry. Many underlying mechanisms linking intracellular Ca2+ store and Ca2+ entry have been investigated. A proposed mechanism is conformational coupling between the ER membrane and the plasma membrane by Orai and TRP-family [25-28]. In this study, [Ca2+]i was increased when Ca2+ was reintroduced after thapsigargin treatment within free extracellular Ca2+. This [Ca2+]i elevation may be due to Ca2+ entry via the SOC channel. In contrary to a blocking effect of InsP3-induced Ca2+ release, caffeine failed to inhibit SOC channel-mediated Ca2+ entry. These results indicate that caffeine effectively antagonizes the InsP3R-medicated Ca2+ release from intracellular stores without an effect on the SOC channel-mediated Ca2+ entry in this cell.
In highly RyRs-expressed cells, such as skeletal muscle, cardiac muscle, and neurons, caffeine is known as an activator of RyRs. Caffeine itself increases [Ca2+]i and this [Ca2+]i rise is blocked by RyR antagonist [8,29]. Caffeine increases both the open time and open probability of RyR in a cooperative manner with both Ca2+ and ATP, and therefore enhances the affinity of RyR for the physiological activator . However, caffeine does not elicit Ca2+ release in the pancreatic acinar cell, actually inhibits the secretagogue-induced Ca2+ signals through an inhibitory action to InsP3-medicated Ca2+ release from internal store like the observed results in the present study [31,32]. InsP3Rs have a well-defined localization in the pancreatic acinar cell, but a few RyRs distribute diffusely in the basal aspect of the cell. These studies suggest that RyRs play a limited role in the propagation of Ca2+ signals from the initially released trigger zone to basal aspects of the pancreatic acinar cell . In the present study, caffeine at any dose did not enhance [Ca2+]i when treated alone. This result, together with presumably much lower numbers of RyRs in pancreatic acinar cell, probably explains the absence of caffeine-induced Ca2+ release in this cell. Therefore, caffeine could be a very useful tool in the study of InsP3Rs-mediated Ca2+ release in the pancreatic acinar cell due to lack of effects on RyR-medicated Ca2+ release and on SOC channel-mediated Ca2+ entry.
2-Aminoethoxydiphenyl borate (2-APB) was first introduced as an InsP3R antagonist. Unlike xestospongins or caffeine, which can modulate both InsP3Rs and RyRs, 2-APB may not have any effects on RyRs-mediated Ca2+ release from internal store . Therefore, 2-APB has been subsequently used in many studies to prove the contribution of InsP3Rs in the generation of [Ca2+]i signals. Although 2-APB has been used as an InsP3R antagonist, there are some reports that 2-APB has different effects according to the cell types and concentrations used. In the smooth muscle cell, 2-APB inhibits contractile response to InsP3-generating stimulus, whereas that triggered by KCl-induced depolarization was unaffected, suggesting that there is no effect on voltage-operated Ca2+ (VOC) entry . 2-APB has been also known as an inhibitor of SOC channels, which is important to internal Ca2+ store refilling [14-18]. Although many results suggest that 2-APB reduces [Ca2+]i through blockage of stored Ca2+ release or Ca2+ entry, there are several reports that 2-APB enhances [Ca2+]i through inhibition of SERCA activity or activation of Ca2+ leak from internal stores [11,12,35]. It remains controversial as to the active mechanism of 2-APB according to the experiments. In our study, 30 µM of 2-APB, which effectively blocked SOC channel-mediated Ca2+ entry, but showed no effect on higher CCh-induced initial Ca2+ peak in intact cell and on InsP3-induced Ca2+ release in permeabilized cell. On the other hand, 100 µM of 2-APB, had paradoxical multiple effects that enhance [Ca2+]i in the intact cell, stimulate Ca2+ release from stores in the permeabilized cell, and inhibit InsP3-induced Ca2+ release in the permeabilized cell. These findings indicate that low dose (~30 µM) of 2-APB could be used as a SOC inhibitor in this cell without any effect on InsP3R, but high dose of (100~ µM) of 2-APB must be used with caution due to the multiple actions on SOC channel, SERCA, and InsP3R.
Cytosolic Ca2+ is mobilized from two closely coupled components, rapid release of Ca2+ stored in the endoplasmic reticulum followed by slowly developing extracellular Ca2+ entry [26,27]. G protein-coupled receptors, through activation of phospholipase C, generate InsP3 which interact with InsP3Rs on the ER. The InsP3Rs serve as Ca2+ channels to release stored Ca2+ and generate the initial Ca2+ signal in pancreatic acinar cell . Pharmacological discrimination of these channels activity is important because a number of reports show that each channel influences the activity of the others. Unfortunately, common blockers have non-selective ability on these channels, which makes the interpretion of their roles and activities difficult. Although caffeine and 2-APB have a benefit due to the high plasma membrane permeability and the relatively low cost, their utility as universal inhibitors of InsP3R is limited due to the multiple responses according to cell types. From the present study, we could suggest that caffeine is useful as a reversible inhibitor of InsP3-mediated Ca2+ release channel, and at lower concentrations; 2-APB is a considerable tool as a poorly reversible inhibitor of SOC-mediated Ca2+ entry channels in the pancreatic acinar cell. Moreover, high concentration of 2-APB must be used with caution due to the actions on multiple targets and the paradoxical responses of [Ca2+]i signals.
This work was supported by Myung-Gok Research Fund of Konyang University in 2006.