Dominant Role of Sarcoendoplasmic Reticulum Ca2+-ATPase Pump in Ca2+ Homeostasis and Exocytosis in Rat Pancreatic -Cells
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《内分泌学杂志》
Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
Abstract
The exocytosis of insulin-containing granules from pancreatic -cells is tightly regulated by changes in cytosolic Ca2+ concentration ([Ca2+]i). We investigated the role of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pump, Na+/Ca2+ exchanger, and plasma membrane Ca2+-ATPase pump in the Ca2+ dynamics of single rat pancreatic -cells. When the membrane potential was voltage clamped at –70 mV (in 3 mM glucose at 22 or 35 C), SERCA pump inhibition dramatically slowed (4-fold) cytosolic Ca2+ clearance and caused a sustained rise in basal [Ca2+]i via the activation of capacitative Ca2+ entry. SERCA pump inhibition increased (1.8-fold) the amplitude of the depolarization-triggered Ca2+ transient at approximately 22 C. Inhibition of the Na+/Ca2+ exchanger or plasma membrane Ca2+-ATPase pump had only minor effects on Ca2+ dynamics. Simultaneous measurement of [Ca2+]i and exocytosis (with capacitance measurement) revealed that SERCA pump inhibition increased the magnitude of depolarization-triggered exocytosis. This enhancement in exocytosis was not due to the slowing of the cytosolic Ca2+ clearance but was closely correlated to the increase in the peak of the depolarization-triggered Ca2+ transient. When compared at similar [Ca2+]i with controls, the rise in basal [Ca2+]i during SERCA pump inhibition did not cause any enhancement in the magnitude of the ensuing depolarization-triggered exocytosis. Therefore, we conclude that in rat pancreatic -cells, the rapid uptake of Ca2+ by SERCA pump limits the peak amplitude of depolarization-triggered [Ca2+]i rise and thus controls the amount of insulin secretion.
Introduction
THE INTRACELLULAR Ca2+ signal plays a key role in the regulation of insulin secretion from pancreatic -cells during stimulation by glucose and other secretagogues. The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pump is responsible for removing Ca2+ from the cytosol into intracellular Ca2+ stores, and inhibition of the SERCA pump leads to emptying of intracellular Ca2+ stores. Other than the replenishment of intracellular Ca2+ stores, recent studies suggest that the SERCA pump may have an important role in the regulation of the depolarization-triggered Ca2+ signal. In the mouse pancreatic -cell, the time course of decay of the Ca2+ signal after a short (<10 sec) KCl depolarization was monotonic, and the SERCA pump was the dominant Ca2+ clearance mechanism (1). With long (30 sec) KCl depolarization, the rapid decay of the Ca2+ signal in mouse pancreatic -cell was followed by a slow increase that arose from Ca2+ release from the endoplasmic reticulum (ER) (1, 2, 3). In mouse pancreatic islets, inhibition of the SERCA pump increased the amplitude of the glucose-triggered [Ca2+]i oscillations and abolished the slow release of Ca2+ from the ER (4, 5). Similar changes in Ca2+ signal have also been observed in islets isolated from mice deficient in SERCA3 (the SERCA isoform that is expressed predominantly in mouse pancreatic -cells) (4). The correlation between the SERCA pump inhibitor mediated changes in the pattern of the Ca2+ signal and insulin secretion is not fully understood. Nevertheless, when single or clusters of mouse pancreatic -cells were stimulated by short KCl depolarization, their secretory response (monitored with carbon fiber amperometry) was enhanced by SERCA pump inhibitors (1). Interestingly, in islets of SERCA3-deficient mice, despite the faster decay of the islet Ca2+ signal (triggered by long KCl depolarization), the glucose-stimulated insulin secretion appeared to be better than those from the wild type (4).
Other than mouse islets, rat pancreatic islets are frequently used in the study of -cell stimulus-secretion coupling. Rat and human islets, but not mouse islets, exhibit a rising second phase of insulin secretion (6). In contrast to mouse pancreatic -cells, much less is known about the role of SERCA pump in rat pancreatic -cells. When stimulated by glucose, rat pancreatic -cells exhibited sustained [Ca2+]i elevations, instead of the oscillatory Ca2+ signal that was typically observed in mouse pancreatic -cells (7). A previous study on rat pancreatic -cells stimulated with long (approximately 10 min) KCl depolarization, suggested that Na+/Ca2+ exchanger (NCX) was the major Ca2+ clearance mechanism (8). On the other hand, in mouse pancreatic -cells, both the NCX and plasma membrane Ca2+-ATPase (PMCA) pumps have only minor contributions to Ca2+ clearance (1, 9). The discrepancy in the role of NCX in the Ca2+ dynamics between rat and mouse pancreatic -cells has been attributed to differences in the level of mRNA transcription as well as the expression of various splice variants of NCX between the two species (10).
In view of the possible differences in Ca2+ signal and secretory response between the mouse and rat pancreatic -cells, we examined the influence of the SERCA, NCX, and PMCA pumps in the regulation of Ca2+ homeostasis in single rat pancreatic -cells. Using the whole-cell patch clamp technique to deliver short trains of depolarization to activate voltage-gated Ca2+ entry, we found that the SERCA pump has an important role in regulating the amplitude of the depolarization-triggered Ca2+ transient, the time course of Ca2+ clearance from the cytosol after depolarization as well as the basal [Ca2+]i in rat pancreatic -cells. On the other hand, inhibition of the NCX or PMCA pump caused only a small slowing of cytosolic Ca2+ clearance. To understand precisely how these changes in the pattern of depolarization triggered Ca2+ signal after SERCA pump inhibition affect insulin secretion, we monitored simultaneously [Ca2+]i and exocytosis (capacitance measurement) from individual rat pancreatic -cells. Our results revealed that the rapid uptake of Ca2+ by the SERCA pump limited the magnitude of the exocytotic response in rat pancreatic -cells. In contrast, the slowing of the decay of the Ca2+ signal and the rise in basal [Ca2+]i during SERCA pump inhibition had no immediate effect on the exocytotic response.
Materials and Methods
Chemicals
Indo-1 K+ salt and indo-1-AM were from Teflabs (Austin, TX). Thapsigargin, 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ), 2-aminoethoxydiphenyl borate (2-APB) were from Calbiochem (San Diego, CA). Culture medium, serum, and antibiotics were from Life Technologies, Inc. (Burlington, Ontario, Canada). 2-[4-[(2,5-difluorophenyl)methoxy] phenoxy]-5-ethoxyaniline (SEA0400) was from Taisho Pharmaceutical Co. (gift from Dr. Peter Light). All other chemicals were from Sigma (Oakville, Ontario, Canada).
Cell preparation
Rat pancreases were removed from male Sprague Dawley rats (150–200 g) killed with halothane in accordance with the standards of the Canadian Council on Animal Care. The pancreases were cut into small pieces and then shaken at 37 C for 12 min with Hanks’ buffered salt solution containing collagenase (type V; 1.2 mg/ml) and DNase (type II; 5 μg/ml) (11). Islets were purified with a discontinuous Ficoll gradient (25, 23, 21.5, and 11.5%) and then handpicked under a dissecting microscope. Single cells were obtained by incubating the islets in trypsin solution (L-1-tosylamide-2-phenylethyl chloromethyl ketone treated; 0.0025 mg/ml) for 4 min at 37 C, followed by gentle trituration. Isolated cells were plated on glass coverslips coated with poly-L-lysine (0.1 mg/ml) and kept in RPMI 1640 culture medium containing 11 mM glucose, 10% fetal bovine serum, 50 μg/ml streptomycin, and 50 IU/ml penicillin. Experiments were performed in cells maintained in standard culture condition (37 C, 5% CO2) for 1–3 d. Because rat pancreatic -cells are larger in size in comparison with other islet cells (12) and high glucose stimulates [Ca2+]i rise in these cells (our unpublished observations), we select individual -cells based on cell size.
Solutions
The standard bath solutions contained (in mM): 150 NaCl, 2.5 KCl, 5 CaCl2, 1 MgCl2, 3 glucose, and 10 Na-HEPES (pH 7.4). In experiments involving inhibition of the PMCA pump, the pH of the standard bath solution was raised to 8.8. For experiments involving Ca2+-free extracellular solution, Ca2+ in the standard bath solution was replaced by 1 mM EGTA and the concentration of MgCl2 was raised to 3 mM. For experiments involving Na+-free solution, NaCl in the standard solution was replaced with N-methyl-glucamine-Cl. For experiments involving capacitance measurement at 22 C, Ca2+ in the standard bath solution was raised to 15 mM. The whole-cell pipette solution contained (in mM): 135 Cs-aspartate, 20 tetraethylammonium-Cl, 20 Cs-HEPES, 2.5 MgCl2, 5 Na2-ATP, 0.1 GTP, and 0.1 indo-1 (pH 7.4).
Measurement of [Ca2+]i
[Ca2+]i was measured fluorometrically using the Ca2+ indicator, indo-1, at room temperature (22 C) or high temperature (35 C). In all experiments involving high temperature, the bath was constantly perfused with extracellular solution that was warmed by a heated water jacket. Except for experiments involving Mn2+ quench, the Ca2+ indicator, indo-1 K+ salt was dialyzed into the cell via the whole-cell patch pipette. Details of the [Ca2+]i measurement were as described previously (13). Briefly, indo-1 in single rat -cell was excited by 365 nm (band-pass filtered) light delivered from a HBO 100 W mercury lamp via a x40, 1.3 NA UV fluor oil objective (Nikon, Mississauga, Ontario, Canada). To reduce fluorescence from the pipette, light collection was restricted to a spot of approximately 25 μM by inserting a 1-mm pinhole before a x5 projection lens. Photon counts were collected at 405 and 500 nm by two photomultiplier tubes (H3460-04; Hamamatsu, Bridgewater, NJ) and then translated into logic signals counted simultaneously by a CYCTM-10 counter card (Cyber Research Inc., Branford, CT) in an IBM-compatible computer. In each experiment, the background counts (from the tip of the pipette and the cell) were measured after forming a cell-attached seal and then subsequently subtracted. [Ca2+]i was calculated from the ratio (R) of fluorescence at 405 and 500 nm, using the following equation (14):
(1)
where Rmin is the fluorescence ratio of Ca2+-free indicator and Rmax is the ratio of Ca2+-bound indicator. K is a constant that was determined empirically. Calibrations were determined from single rat pancreatic -cells dialyzed with one of the three pipette solutions as described previously (15). Rmin was measured in cells loaded with (in mM): 52 K-aspartate, 10 KCl, 50 K-EGTA, 0.1 indo-1, and 50 K-HEPES (pH 7.4); and Rmax was measured in cells loaded with (in mM): 136 K-aspartate, 15 CaCl2, 0.1 indo-1, and 50 K-HEPES (pH 7.4). K was calculated from the equation above using R values obtained from cells loaded with (in mM): 60 K-aspartate; 50 K-HEPES; 20 K-EGTA; 15 CaCl2; and 0.1 indo-1 (pH 7.4), which had a calculated free Ca2+ concentration of 212 and 170 nM at 22 and 35 C, respectively (16).
Measurement of extracellular Ca2+ influx using Mn2+ quenching
Mn2+ enters into the cell via Ca2+-permeable pathways and causes quenching (reduction) in indo-1 fluorescence (both at 405 and 500 nm). Because the fluorescence at 405 nm (F405) is not sensitive to changes in [Ca2+]i (near the isosbestic wavelength of indo-1), the rate of decrease in F405 by Mn2+ reflects the rate of extracellular Ca2+ influx (17, 18). On the other hand, [Ca2+]i is determined by the ratio of F405/F500 (as described above). Rise in [Ca2+]i results in decreases in F500. Mn2+ quenches both F405 and F500 to the same extent and thus has no effect on the ratio of F405 to F500 and [Ca2+]i. Therefore, the Mn2+ quenching experiments allow us to monitor simultaneously [Ca2+]i and extracellular Ca2+ influx. In experiments involving Mn2+ quenching, cells were incubated with 5 μM indo-1 AM in standard bath solution for 15–20 min at 37 C and then in dye-free solution for 5–10 min. The fluorescence at 405 nm was then monitored during exposure of the cells to the standard bath solution with 0.2 mM Mn2+ added. All Mn2+ experiments were conducted at approximately 22 C.
Electrophysiology
Single -cells were voltage clamped with the whole-cell, gigaseal method (19) with an EPC-7 amplifier (List-Electronic, Darmstadt, Germany). The pipettes were made from hematocrit glass (VWR Scientific Canada Ltd., London, Ontario, Canada), and the resistance was 2–4 M after filling with the pipette solution. The cell membrane potential was held at –70 mV (d.c.) and a train (five to seven steps) of depolarization (150 msec in duration) to +10 mV was delivered at 2.5 Hz to trigger [Ca2+]i rise. The peak of the depolarization-triggered [Ca2+]i rise varied between 0.5 and 3 μM. A –10 mV correction for junction potential was applied throughout.
Capacitance measurement of exocytotic response
Exocytosis was measured as increases in membrane capacitance (Cm) that result from the addition of granule membrane to cell membrane (20). Individual -cells were whole-cell voltage clamped at –80 mV (d.c.) with an EPC-9 amplifier (List-Electronic), and a train (10–15 steps) of depolarization (200 or 300 msec in duration) to +10 mV was delivered to trigger [Ca2+]I rise, and exocytosis. Cm was measured at high temporal resolution with a separate dual-phase lock-in amplifier by superimposing an 800-Hz sinusoid of 30 mV peak-to-peak amplitude onto the holding potential as previously described (21). Values of [Ca2+]i and Cm were first recorded on VCR tapes with a NeuroData PCM recorder (Neuro Data Instruments Corp., New York, NY) and digitized later.
Calculations and statistics
Functions in the Microcal Origin program 6.0 (Origin Lab Corp., Northampton, MA) were used for all statistical and curve fitting procedures. The time constant of Ca2+ clearance was estimated by fitting the decay of the Ca2+ signal with a single exponential. The rate of Mn2+ quench was calculated from the slopes of the linear fit to individual segments of the fluorescence at 405 nm. A Student’s t test was used in comparisons of mean values between two populations of cells. Any difference with P < 0.05 was considered statistically significant and was marked with an asterisk in the figures. All values shown were means ± SEM.
Results
Inhibition of SERCA pump dramatically affects Ca2+ homeostasis
One difficulty in examining the effect of SERCA pump inhibition on the amplitude of the depolarization triggered Ca2+ signal is to separate the action of the SERCA pump inhibitors on the voltage-gated Ca2+ channels (VGCCs) from that on Ca2+ uptake. Among the SERCA pump inhibitors, cyclopiazonic acid was reported to enhance VGCCs, but BHQ was shown to have inhibitory action (22). On the other hand, thapsigargin was reported to have both enhancing and inhibitory action on VGCCs (22). In view of this, we used BHQ in our first series of experiments. Because BHQ is known to inhibit VGCCs, any rise in the depolarization-triggered Ca2+ signal in the presence of BHQ must be related to the inhibition of the SERCA pump uptake of Ca2+.
In these experiments, an individual rat pancreatic -cell was whole-cell voltage clamped at –70 mV. A train (five steps) of depolarization (+10 mV; 150 msec; 2.5 Hz) was delivered before and during BHQ (10 μM) application. The short train of depolarization allowed us to examine the time course of the decay of the Ca2+ transient without any interference from the slow Ca2+ release from the ER (1). The cell was continuously supplied with 5 mM ATP via the whole-cell pipette, thus ensuring a constant supply of ATP to maintain the activities of the various Ca2+-ATPases in the cell. In the example shown in Fig. 1A, the basal [Ca2+]i in control condition at approximately 22 C was approximately 0.2 μM, and a train of depolarization raised [Ca2+]i to approximately 1.7 μM. After the termination of the depolarization, [Ca2+]i decayed monotonically and rapidly to the resting level. Application of BHQ raised basal [Ca2+]i to approximately 0.6 μM. After the BHQ-mediated [Ca2+]i rise reached a plateau, the same train of depolarization was delivered, and it raised [Ca2+]i to approximately 2.8 μM. Thus, even with the increase in basal [Ca2+]i by BHQ (0.4 μM) taken into account, the amplitude of the depolarization-triggered Ca2+ transient in the presence of BHQ was still approximately 0.7 μM higher than that elicited in the same cell before BHQ exposure. In 33 cells examined at approximately 22 C (Fig. 1B), the average basal [Ca2+]i was 0.20 ± 0.02 μM, and BHQ raised the basal [Ca2+]i to 0.76 ± 0.08 μM (an average increase of 0.57 ± 0.07 μM). An elevation of basal [Ca2+]i by BHQ (from 0.14 ± 0.02 to 0. 35 ± 0.04 μM) was also observed in cells recorded at approximately 35 C (n = 21; Fig. 1B).
The action of BHQ on the amplitude of the depolarization-triggered Ca2+ transient is summarized in Fig. 1C. Due to the variability of the amplitude of the depolarization-triggered Ca2+ transient among the rat pancreatic -cells, the amplitude of the depolarization-triggered Ca2+ transient in the presence of BHQ was normalized to that elicited from the same cell before BHQ exposure. To separate the effect of BHQ on basal [Ca2+]i from that on the depolarization-triggered Ca2+ transient, the amplitude of the Ca2+ transient in BHQ was measured as the difference between the basal [Ca2+]i in the presence of BHQ before depolarization and that of the peak of the Ca2+ transient. Consistent with a previous report on the inhibitory action of BHQ on VGCCs (22), the amplitude of the Ca2+ current (measured from the first step of the depolarization) in the presence of BHQ (at 22 C) was reduced by 25 ± 9% (n = 9). Note that despite of the VGCC reduction, BHQ increased the amplitude of the depolarization-triggered Ca2+ transient in 17 of 19 cells examined at approximately 22 C. On average, BHQ increased the amplitude of the depolarization-triggered Ca2+ transient at approximately 22 C by 1.77 ± 0.18-fold (n = 19). At approximately 35 C, however, BHQ did not cause any significant increase in the amplitude of the depolarization-triggered Ca2+ transient (Fig. 1C). This is probably due to the large reduction of VGCCs in the presence of BHQ at high temperature (46 ± 6%; n = 12).
In Fig. 1D, we compared the kinetics of the Ca2+ signal at approximately 22 C by superimposing the depolarization-triggered Ca2+ transients before and after BHQ exposure (same record as in Fig. 1A). For comparison, the amplitude of the Ca2+ transient in control was scaled up to match that of the Ca2+ transient evoked in BHQ. Note that BHQ dramatically slowed the decay phase of the Ca2+ signal (the time constant of cytosolic Ca2+ clearance increased from 0.98 to 10.4 sec). In 20 cells examined at approximately 22 C, the time constant of cytosolic Ca2+ clearance in control was 1.59 ± 0.11 sec, and BHQ increased the time constant by 4.2-fold to 6.72 ± 0.83 sec (Fig. 1E). The rate of cytosolic Ca2+ clearance in rat -cells increased by approximately 2.5-fold when the temperature was elevated to approximately 35 C (time constant of cytosolic Ca2+ clearance in control cells was 0.63 ± 0.05 sec). Note that at high temperature, BHQ caused a similar slowing in Ca2+ clearance, and the time constant of Ca2+ clearance in BHQ increased by 3.8-fold to 2.36 ± 0.25 sec (Fig. 1E). The effects of BHQ on Ca2+ homeostasis were reversible. As shown in Fig. 1A, after BHQ removal, the basal [Ca2+]i returned to the control level and the train of depolarization evoked a smaller Ca2+ transient, which decayed with a faster time constant. These results suggest that SERCA pumps have important roles in Ca2+ homeostasis in rat pancreatic -cell.
Inhibition of NCX causes a small slowing of cytosolic Ca2+ clearance
Using similar experimental procedures, we examined the influence of NCX inhibition on rat pancreatic -cells. We first tested the actions of SEA0400, a newly developed NCX inhibitor (23). As shown in the example in Fig. 2A, at approximately 22 C, SEA0400 (1 μM) caused a very small elevation (0.09 μM) in basal [Ca2+]i, and the time constant of cytosolic Ca2+ clearance increased from 1.2 to 1.84 sec. Note that there was no apparent change in the amplitude of the depolarization-triggered Ca2+ transient. In 14 cells tested with SEA0400 at approximately 22 C, there was no significant increase in basal [Ca2+]i (Fig. 2B) or the amplitude of the depolarization-triggered Ca2+ transient (Fig. 2C). For the same cells, SEA0400 increased the time constant of Ca2+ clearance by approximately 35% (from 1.20 ± 0.10 to 1.62 ± 0.14 sec; Fig. 2D). In a recent study, intracellular ATP (3 mM) was reported to antagonize the action of SEA0400 (24). Because our pipette solution contained 5 mM ATP, it is possible that SEA0400 might be less effective in inhibiting NCX. Therefore, we further examined the role of NCX in rat pancreatic -cells by inhibiting NCX with a Na+-free extracellular solution. For cells recorded at approximately 22 C, the removal of extracellular Na+ caused only a small slowing (20%) in Ca2+ clearance (from 2.0 ± 0.15 to 2.41 ± 0.22 sec; n = 4), and neither the basal [Ca2+]i nor the amplitude of the depolarization-triggered Ca2+ transient was affected. When the same experiment was repeated at approximately 35 C, a small slowing in cytosolic Ca2+ clearance (35%; Fig. 2D) and a small rise (0.1 μM) in basal [Ca2+]i (Fig. 2B) were detected after the removal of extracellular Na+. Thus, under our experimental conditions (short depolarization and continuous supply of ATP via the whole-cell pipette), NCX has a much smaller influence in the Ca2+ homeostasis of rat pancreatic -cells when compared with the SERCA pump.
Inhibition of PMCA pump causes a small slowing of cytosolic Ca2+ clearance
To examine whether the PMCA pump contributes to Ca2+ homeostasis in rat pancreatic -cells, we reduced PMCA activities by exposing the cells to an extracellular solution with pH 8.8. This experimental approach is based on the Ca2+/H+ exchanger property of the PMCA pump, and by lowering the extracellular H+ concentration, the activities of the PMCA pump can be reduced (25). Such manipulation has been used successfully to reduce PMCA pump activities in mouse pancreatic -cells (1) and rat pituitary corticotropes (26). An example of such experiment is shown in Fig. 3. Note that at both approximately 22 and approximately 35 C, inhibition of PMCA pump caused only a small slowing of cytosolic Ca2+ clearance (27 and 34%; Fig. 3D). At high temperature, PMCA inhibition caused a small rise in basal [Ca2+]i as well as the amplitude of the Ca2+ transient. Thus, PMCA activities have minor roles in the Ca2+ dynamics of rat -cells.
SERCA pump inhibition evokes capacitative Ca2+ entry
In mouse pancreatic -cells, SERCA pump inhibition has been reported to cause depolarization and activation of voltage-gated Ca2+ entry (2, 27, 28, 29). However, in our experiments (Fig. 1), the cell membrane potential was held at -70 mV. Thus, the BHQ-mediated rise in basal [Ca2+]i could not be due to depolarization. To examine the source of the BHQ-mediated rise in basal [Ca2+]i, we compared the action of BHQ on basal [Ca2+]i with or without extracellular Ca2+. In the experiment shown in Fig. 4A, the cell was voltage clamped at -70 mV and exposed to two consecutive BHQ challenges. In the presence of 5 mM extracellular Ca2+, application of BHQ elevated the basal [Ca2+]i by approximately 0.2 μM. After the removal of BHQ, [Ca2+]i returned to the control level. The bath solution was then switched to a Ca2+-free solution (no added Ca2+, and 1 mM EGTA was included to chelate any contaminating Ca2+). This resulted in a small reduction in the basal [Ca2+]i. A second BHQ challenge in the absence of extracellular Ca2+ elicited only a very small [Ca2+]i rise (0.03 μM). The small BHQ-mediated [Ca2+]i rise during the second challenge was not due to any desensitization of the BHQ response because a subsequent BHQ challenge in the presence of 5 mM extracellular Ca2+ still raised [Ca2+]i by approximately 0.2 μM (data not shown). The experimental results obtained from 12 cells are summarized in Fig. 4B. On average, the BHQ-mediated [Ca2+]i rise was 0.06 ± 0.02 μM in the absence of extracellular Ca2+, much smaller than that evoked in the presence of extracellular Ca2+ (0.30 ± 0.06 μM) for the same batch of cells. This result suggests that a major component of the BHQ-mediated rise in basal [Ca2+]i requires extracellular Ca2+ influx. Consistent with this, Fig. 4C shows that the removal of extracellular Ca2+ reversibly reduced the BHQ-mediated [Ca2+]i rise. When extracellular Ca2+ was added back to the bath solution, the BHQ-mediated [Ca2+]i was restored to its previous level. Similar results were observed in five of five cells.
One possible explanation for the strong dependence on extracellular Ca2+ during the BHQ-mediated rise in basal [Ca2+]i is that SERCA pump inhibition causes depletion of Ca2+ from intracellular stores, which in turn activates capacitative Ca2+ entry (30, 31). To examine whether the BHQ-mediated [Ca2+]i rise is associated with the activation of extracellular Ca2+ influx pathways, we used the Mn2+ quench technique (26) to simultaneously monitor [Ca2+]i and Mn2+ entry (see explanation in Materials and Methods). A representative example of this experiment is shown in Fig. 5, A and B. Single rat pancreatic -cells were loaded with indo-1 AM and then perfused with the standard bath solution. Note that in this experiment, the cell membrane potential was unclamped. Application of Mn2+ (0.2 mM) resulted in a small and slow decrease in F405, reflecting a basal leakage of Mn2+ into the cell (Fig. 5B). The basal [Ca2+]i, however, was not affected by this Mn2+ leakage (Fig. 5A). Application of BHQ (10 μM) stimulated a rise in [Ca2+]i (Fig. 5A). Note that the BHQ-mediated [Ca2+]i rise was accompanied by an acceleration in the rate of decrease of F405 (Fig. 5B). After BHQ removal, the rate of F405 decrease slowed down and [Ca2+]i decayed to the resting level. The result of this experiment is summarized in Fig. 5C. In 21 cells examined, the basal rate of Mn2+ quench (reflected by the decrease in F405) was 0.24 ± 0.04 arbitrary units (AU)–1, and BHQ accelerated the rate by approximately 2.2-fold to 0.52 ± 0.07 AU–1. Thus, the BHQ-mediated rise in basal [Ca2+]i in rat pancreatic -cells was closely accompanied by an increase in extracellular Ca2+ influx. The above experiment was then repeated with diazoxide (0.2 mM) in the bath. In seven cells hyperpolarized with diazoxide, the basal rate of Mn2+ quench was 0.27 ± 0.06 AU–1, and BHQ increased the rate by 2.2-fold to 0.60 ± 0.11 AU–1 (Fig. 5C). The similar increase in the rate of Mn2+ quench by BHQ between the unclamped cells and hyperpolarized cells suggests that membrane depolarization did not have any major contribution to the BHQ-mediated increase in extracellular Ca2+ influx.
To examine whether the BHQ-mediated extracellular Ca2+ influx involves activation of capacitative Ca2+ entry channels, we used 2-APB, a blocker of capacitative Ca2+ entry (32, 33, 34). Figure 6A shows that the rate of Mn2+ quench in BHQ was slowed by 2-APB (50 μM). In nine cells examined, BHQ increased the rate of Mn2+ quench from 0.16 ± 0.05 to 0.53 ± 0.10 AU–1, and 2-APB reduced the rate to 0.26 ± 0.04 AU–1 (Fig. 5B). As shown in Fig. 6C, in cells voltage clamped at –70 mV, the BHQ-mediated rise in basal [Ca2+]i was also reduced by 2-APB (seven of nine cells). Overall, our results indicate that the majority of the BHQ-mediated rise in basal [Ca2+]i was due to the activation of capacitative Ca2+ entry. The small BHQ-mediated [Ca2+]i rise in the absence of extracellular Ca2+ was probably due to the leakage of Ca2+ from the intracellular stores.
SERCA pump inhibition enhances depolarization- triggered exocytosis
Our results above show that SERCA pump inhibition in rat pancreatic -cells could dramatically affect the shape of the depolarization-triggered Ca2+ signal. To examine how these changes in the pattern of the Ca2+ signal affect exocytosis (and hence insulin secretion), we compared the depolarization-triggered Ca2+ signal and the resultant exocytosis (measured as increases in membrane capacitance) in the same cell before and after SERCA pump inhibition. We first performed this set of experiments at approximately 22 C because there was a clear increase in the amplitude of the depolarization-triggered Ca2+ transient in BHQ (Fig. 1C). At this temperature, a train of depolarization (in the presence of 15 mM extracellular Ca2+) was needed to evoke a robust exocytotic response. Figure 7A shows a representative example of such an experiment. The cell was whole-cell voltage clamped at –80 mV and the resting [Ca2+]i was 0.22 μM in control condition (Fig. 7A). A train of depolarization was delivered to trigger [Ca2+]i rise and exocytosis. After the termination of the fourth step of depolarization when [Ca2+]i rose to approximately 0.74 μM, the Cm increased by approximately 11 fF. After the sixth depolarizing step, [Ca2+]i reached approximately 1 μM, and the subsequent depolarizing steps maintained [Ca2+]i near this level. Note that each of the subsequent depolarizing steps still evoked additional increase in membrane capacitance. At the end of the train of depolarization, [Ca2+]i started to decay, and there was no further increase in Cm. Overall, the cumulative Cm increase elicited by the train of depolarization in this cell was 73 fF. The exocytosis of a single granule in mouse pancreatic -cell has been assumed to contribute approximately 2 fF (35). Because the granules in rat and mouse pancreatic -cells are similar in size (36), the 73 fF increase reflects the release of approximately 36 granules. After the return of [Ca2+]i to the basal level, the same cell was exposed to BHQ (10 μM). Basal [Ca2+]i rose from 0.22 to 0.43 μM, and the same train of depolarization was then delivered (Fig. 7B). In the presence of BHQ, [Ca2+]i rose to approximately 1 μM after the termination of the fourth depolarizing step, and the membrane capacitance increased by approximately 28 fF. At the end of the train, [Ca2+]i rose to approximately 1.8 μM, and the cumulative Cm increase was 190 fF (2.6-fold of that triggered under control condition; Fig. 7A).
Figure 7, C and D, shows that enhancement of depolarization-triggered exocytosis could also be observed with thapsigargin, another SERCA pump inhibitor. In the example shown in Fig. 7C, under control conditions, the train of depolarization elevated [Ca2+]i to only approximately 0.7 μM and did not trigger any significant increase in Cm. When the same cell was exposed to thapsigargin (1 μM), the resting [Ca2+]i rose from 0.19 to 0.37 μM, and the same train of depolarization raised [Ca2+]i to approximately 1.6 μM, resulting in a cumulative Cm increase of 257 fF. Note that in this cell, after the eighth depolarizing step, although [Ca2+]i remained elevated near 1.6 μM, subsequent depolarizing steps caused only small additional increase in membrane capacitance. This probably reflects the exhaustion of the readily releasable pool (RRP) of granules during the first eight steps of the train of depolarization, and the subsequent slow mobilization of additional granules. Figure 7 also shows that exocytosis typically stopped with the termination of the train of depolarization, even though [Ca2+]i remained elevated. Thus, during SERCA pump inhibition, the slowing of Ca2+ clearance after the train of depolarization is unlikely to contribute to the enhancement in exocytosis. Instead, the overall increase in the peak of the depolarization-triggered Ca2+ transient during SERCA pump inhibition appeared to trigger more exocytosis.
To address this possibility, we examined whether the enhancement in exocytotic response was correlated to the overall changes in the peak of the depolarization-triggered Ca2+ transient. The experimental protocol was identical with the examples shown in Fig. 7. Because the depolarization-triggered [Ca2+]i rise typically reached a maximum before the termination of the train of depolarization, we measured the cumulative Cm at the time that the depolarization-triggered Ca2+ transient reached its peak (e.g. eighth step in Fig. 7A). For each cell, the difference in cumulative Cm before and after SERCA pump inhibition was plotted against the difference in the peak value of the depolarization-triggered Ca2+ transients. Figure 8A shows the results from five cells challenged with BHQ and four cells challenged with thapsigargin at approximately 22 C. Note that in three of the nine cells, there was a decrease in the peak value of the depolarization-triggered Ca2+ rise after SERCA pump inhibition (reflected by the negative values in the change in peak [Ca2+]i), probably due to rundown in VGCCs. For these three cells, the exocytotic response in the presence of SERCA pump inhibitor was also smaller than the controls. On the other hand, for cells with a larger increase in the peak value of the depolarization-triggered Ca2+ transient during SERCA pump inhibition, there was a bigger increase in the exocytotic response. As shown in Fig. 8A, there was a strong correlation between the increase in the peak value of the depolarization-triggered Ca2+ transient by SERCA pump inhibitor and the enhancement in exocytotic response at approximately 22 C (r = 0.985; P < 0.0001).
Figure 8A also shows the exocytotic response obtained from cells challenged with BHQ at approximately 35 C. At this temperature, exocytosis could be readily triggered by depolarization in the presence of 5 mM extracellular Ca2+. Thus, all the experiments involving exocytotic response at approximately 35 C were performed in 5 mM extracellular Ca2+. As described earlier, there was a larger decrease in VGCCs in the presence of BHQ at high temperature. Thus, only five of the 13 cells recorded at approximately 35 C have a higher peak Ca2+ transient in the presence of BHQ. Nevertheless, there was still a good correlation between the change in the peak value of the depolarization-triggered Ca2+ transient by SERCA pump inhibitor and the change in exocytotic response at approximately 35 C (r = 0.855; P < 0.0002).
In mouse pancreatic -cells, elevation of basal [Ca2+]i has been reported to potentiate Ca2+-dependent exocytosis (via calmodulin kinase II), even when the amplitude of the depolarization-triggered Ca2+ transient was unchanged (37). Because SERCA pump inhibition causes elevation of basal [Ca2+]i (Fig. 1) in rat pancreatic -cells, it is possible that such mechanism may also contribute to the enhancement in exocytotic response during SERCA pump inhibition. To address this, we plotted in Fig. 8B the amount of exocytosis triggered in rat pancreatic -cells at approximately 22 or approximately 35 C when [Ca2+]i was elevated to different levels during a train of depolarization. To examine whether the elevated basal [Ca2+]i affected exocytosis independently from the increase in the peak of the depolarization-triggered Ca2+ transient, we compared the amount of exocytosis triggered with or without SERCA pump inhibitor at similar [Ca2+]i. Note that although SERCA pump inhibition increased the basal [Ca2+]i in these cells (by 0.34 μM at 22 C and 0.17 μM at 35 C), at comparable [Ca2+]i, SERCA pump inhibition did not cause any significant increase in the amount of exocytosis over the control (Fig. 8B). Thus, in rat pancreatic -cells, the increase in the amplitude of the depolarization-triggered Ca2+ transient during SERCA pump inhibition is the major mechanism underlying the enhancement of exocytosis.
Discussion
The present study examines the contribution of SERCA, NCX, and PMCA pumps to the Ca2+ dynamics of rat pancreatic -cells, at both approximately 22 and approximately 35 C. Under control conditions (in 3 mM glucose), the time constant of cytosolic Ca2+ clearance after a short train of depolarization in the rat pancreatic -cells at approximately 22 C was approximately 1.6 sec (Fig. 1E), reflecting an overall clearance rate constant of 0.63 sec–1. At approximately 35 C, the time constant of Ca2+ clearance decreased to 0.63 sec, and the overall clearance rate constant increased to 1.59 sec–1, a value comparable with that described in the mouse pancreatic -cells [1 sec–1 at 35 C and in 15 mM glucose (1)]. Thus, raising the temperature from approximately 22 to approximately 35 C increases the rate of cytosolic Ca2+ clearance in rat pancreatic -cells. Note that, however, elevation of temperature did not affect the relative contributions of SERCA, NCX, and PMCA pumps to Ca2+ homoeostasis. At both approximately 22 and approximately 35 C, the SERCA pump is the dominant mechanism of cytosolic Ca2+ clearance in rat pancreatic -cells. Inhibition of the SERCA pump at the two temperatures increased the time constant of cytosolic Ca2+ clearance in rat -cells to approximately 6.7 and approximately 2.36 sec, respectively (Fig. 1E), suggesting that all non-SERCA Ca2+ clearance mechanisms (including the NCX and PMCA pumps, mitochondria) have a combined clearance rate constant of 0.15 sec–1 at approximately 22 C and 0.42 sec–1 at approximately 35 C. This indicates that, at both temperatures, the non-SERCA Ca2+ clearance mechanisms in rat pancreatic -cell contribute only approximately 25% of the overall Ca2+ clearance. Therefore, similar to what has been reported in mouse pancreatic -cells at 35 C (1), the SERCA pump is the dominant Ca2+ clearance mechanism in rat pancreatic -cells.
Our study also shows that NCX has minor contributions to Ca2+ homeostasis in rat pancreatic -cells. At both approximately 22 and approximately 35 C, inhibition of NCX did not affect the amplitude of the depolarization-triggered Ca2+ transient (Fig. 2C) but caused a small (30%) slowing in the rate of cytosolic Ca2+ clearance (Fig. 2D). This finding is in contrast to a previous study in rat pancreatic -cells that showed NCX inhibition with antisense oligos reduced the amplitude of the KCl- or tolbutamide-triggered Ca2+ transient at approximately 37 C by 28 or 40% and slowed Ca2+ clearance by 72 or 40%, respectively (8). The discrepancies between the two studies could not be due to a difference in temperature because our study showed clearly that raising the temperature from approximately 22 to approximately 35 C did not cause any increase in the contribution of NCX to the regulation of Ca2+ dynamics in rat pancreatic -cells (Fig. 2). Instead, we suggest that the discrepancy may be related to a rundown in the cellular ATP level in the study with antisense oligos (8). Note that in the current study, a short train of depolarization (total duration < 1.5 sec) was applied to elicit a transient [Ca2+]i rise, and ATP was continuously supplied to the cell via the whole-cell pipette. In contrast, the study with antisense oligos (8) used a 10-min application of KCl to elicit [Ca2+]i rise in intact cells. Because the function of SERCA pump requires constant supply of ATP, it is possible that after a prolonged [Ca2+]i rise, the cellular ATP level may become low and thus reducing the SERCA pump activity. Under this condition, the NCX may become the dominant Ca2+ clearance mechanism.
Consistent with the previous report in mouse pancreatic -cells (at 35 C) (1), we found that the PMCA pump makes only minor contribution to the Ca2+ dynamics in rat pancreatic -cells. At both approximately 22 and approximately 35 C, inhibition of the PMCA pump slowed cytosolic Ca2+ clearance by approximately 30% (Fig. 3D). At approximately 35 C, the PMCA pump inhibition also caused a significant increase in the basal [Ca2+]i (Fig. 3B) as well as the amplitude of the depolarization-triggered Ca2+ transient (Fig. 3C). Thus, it is possible that the activity of the PMCA pump is slightly more prominent at high temperatures.
Insulin secretion from pancreatic -cells is known to be highly dependent on temperature (38, 39). This temperature dependence has been attributed to the sensitivity of the replenishment of the RRP granules to temperature (40). In the current study, we also found that raising the temperature from approximately 22 to approximately 35 C in rat pancreatic -cells dramatically increased the exocytotic response, even when compared at similar [Ca2+]i (Fig. 8B). Nevertheless, the influence of SERCA pump inhibition on exocytosis was similar at the two temperatures. Despite the dramatic slowing in cytosolic Ca2+ clearance during SERCA pump inhibition, we found that the enhancement of the exocytotic response in rat pancreatic -cells was not directly related to the slower decay in the depolarization-triggered Ca2+ signal. Note that after the termination of the train of depolarization (at either approximately 22 or 35 C), there was no additional increase in membrane capacitance, even though [Ca2+]i remained elevated. This observation suggests that in rat pancreatic -cells, the secretory granules may be in close proximity to the VGCCs such that the local [Ca2+] near the granules was much higher than the average cytosolic [Ca2+] (41, 42). After the closure of VGCCs, the spatial Ca2+ gradient rapidly dissipates and the drop in local [Ca2+] near the granules terminates exocytosis. Although the slower Ca2+ clearance during SERCA pump inhibition in rat pancreatic -cells has no immediate effect on exocytosis, elevated basal [Ca2+]i has been shown to act directly as well as indirectly via protein kinase C activation to increase the size of RRP in bovine chromaffin cells at 20–25 C (43). A recent study in rat pancreatic -cells (44) has also shown that protein kinase C activation at 30–32 C increase the size of RRP. Whether the slower Ca2+ clearance during SERCA pump inhibition in rat pancreatic -cells also affects the replenishment of granules in the RRP awaits future studies.
Our study shows that acute inhibition of the SERCA pump in rat pancreatic -cell can dramatically potentiate secretion at both approximately 22 and approximately 35 C. This result is in agreement with a previous finding that showed glucose-triggered insulin secretion in rat pancreatic islets (at 37 C) was potentiated by thapsigargin (45). We also found that at approximately 22 and approximately 35 C, the increase in the peak of the depolarization-triggered Ca2+ transient during SERCA pump inhibition is the major mechanism underlying the enhancement of exocytosis (Fig. 8A). An increase in the amplitude of the depolarization-triggered Ca2+ transient in the presence of the SERCA pump inhibitor has also been reported in mouse islets (at 37 C) (4) as well as single mouse pancreatic -cells (at 35 C) (1). In view of the inhibitory action of BHQ on VGCCs (22), the approximately 1.8-fold increase in the amplitude of depolarization-triggered Ca2+ transient of rat pancreatic -cells by BHQ at approximately 22 C (Fig. 1) was probably underestimated. Nevertheless, this finding suggests that the SERCA pump can rapidly uptake Ca2+ during extracellular Ca2+ entry via VGCCs, thus limiting the amplitude of the depolarization-triggered Ca2+ transient. The potentiating action of the SERCA pump inhibitor on exocytosis is particularly dramatic in cells in which the depolarization-triggered Ca2+ transient was initially below the threshold for triggering of exocytosis (e.g. Fig. 7C). Under this condition, the increase in the amplitude of the depolarization-triggered Ca2+ transient during SERCA pump inhibition could turn the exocytotic response from none to a robust one (Fig. 7D).
Interestingly, we found that SERCA pump inhibition in rat pancreatic -cells at both approximately 22 and approximately 35 C caused a sustained rise in basal [Ca2+]i (Fig. 1), which was much larger than that reported in the single mouse pancreatic -cells (31–37 C) (1, 27, 46). Our findings suggest that the rise in basal [Ca2+]i did not increase the magnitude of the ensuing exocytotic response. As shown in Fig. 8B, when compared at similar [Ca2+]I in both approximately 22 and approximately 35 C, cells with higher basal [Ca2+]i (after SERCA pump inhibition) did not exhibit any potentiation of the exocytotic response. Our result is different from a previous study in mouse pancreatic -cells that showed elevation of basal [Ca2+]i at 33 C increased the size of RRP and thus potentiated the exocytotic response (37). Because our experiment was already conducted at higher temperature (35 C), the lack of potentiation of the exocytotic response with the elevated basal [Ca2+]i could not be due to the temperature sensitivity of the replenishment of the RRP. One major difference between the two studies is that the whole-cell pipette solution in our study did not contain any cAMP and our bath solution did not contain any forskolin. Because cAMP has been reported to increase the size of RRP in rat pancreatic -cells (44), it is possible that a low cellular cAMP level may reduce the enhancing effect of elevated basal [Ca2+]i on exocytosis. Nevertheless, this elevation in basal [Ca2+]i acts in concert with the change in the amplitude of the depolarization-triggered Ca2+ transient to create an overall increase in the peak value of the Ca2+ transient during SERCA pump inhibition, thus potentiating the exocytotic response at both approximately 22 and approximately 35 C (Fig. 8A).
Inhibition of the SERCA pump is known to cause depletion of ER Ca2+ stores because the continuous leakage of Ca2+ from the ER is no longer balanced by the SERCA pump-mediated reuptake of Ca2+. In the absence of extracellular Ca2+, SERCA pump inhibition in rat pancreatic -cells resulted in only a very small [Ca2+]i rise (0.06 μM; Fig. 4B), suggesting that the ER has a small Ca2+ reserve, which can be rapidly depleted. The basal [Ca2+]i rise induced by SERCA pump inhibition was much larger in the presence of extracellular Ca2+, suggesting that Ca2+ depletion from the ER in turn activates capacitative Ca2+ entry into the cell. Consistent with this, the BHQ-mediated rise in [Ca2+]i was reduced by 2-APB, a blocker of capacitative Ca2+ entry (Fig. 6C). Although 2-APB has been reported to decrease inositol 1,4,5-triphosphate (IP3)-mediated intracellular Ca2+ release, inhibit Ca2+ pumps and reduce mitochondrial Ca2+ uptake (32, 33), our observation that the BHQ-mediated increase in the rate of Mn2+ quench was reduced by 2-APB (Fig. 6, A and B) supports the notion that capacitative Ca2+ entry is the major mechanism underlying the BHQ-mediated rise in basal [Ca2+]i in rat pancreatic -cells.
A small capacitative Ca2+ entry has also been reported in mouse pancreatic -cell (17, 18). Note also that the capacitative Ca2+ entry in the rat pancreatic -cell is not caused by depolarization as the cells were held at hyperpolarized potentials (with voltage clamp or diazoxide). This observation is different from the mouse pancreatic -cells in which the emptying of ER Ca2+ store was reported to activate a depolarizing current that in turn activates voltage-gated Ca2+ entry (2, 27, 29). The major function of the robust capacitative Ca2+ entry observed in the rat pancreatic -cells is probably related to the refilling of the ER Ca2+ stores. Pancreatic -cells possess multiple intracellular Ca2+ stores, including the IP3-sensitive stores (47), ryanodine-sensitive stores (48, 49), nicotinic acid adenine dinucleotide phosphate stores (50, 51), and atypical Ca2+-induced Ca2+ release stores (52). The filling of these Ca2+ stores may not all involve capacitative Ca2+ entry. For example, the activation of ryanodine receptor in a rat insulinoma cell line (S-5 cells) was reported to trigger an extracellular Ca2+ influx, which was different from that triggered by the SERCA pump inhibitors (48). Nevertheless, in mouse pancreatic -cells, the emptying of the IP3-sensitive stores by cholinergic agonist has been reported to trigger capacitative Ca2+ entry (17, 18). Because cholinergic agonist also triggers ER Ca2+ release in rat pancreatic -cells (53), it is likely that the capacitative Ca2+ entry observed in our study is important in maintaining the cholinergic response in these cells. Moreover, sustained depletion of ER Ca2+ stores has been linked to ER stress and cell apoptosis in pancreatic -cells (54, 55). Thus, the refilling of ER stores by capacitative Ca2+ entry may be important for -cell survival.
Overall, our study shows that the SERCA pump is the dominant Ca2+ clearance mechanisms in rat pancreatic -cells. We found that elevation of temperature increased the rate of cytosolic Ca2+ clearance as well as the amplitude of the exocytotic response. However, temperature elevation did not alter the relative contributions of the SERCA, NCX, and PMCA pumps to Ca2+ homeostasis. At both room temperature (22 C) and physiological temperature (35 C), SERCA pumps accounted for approximately 75% of the total Ca2+ clearance in rat pancreatic -cells. SERCA pump inhibition resulted in a larger amplitude of the depolarization-triggered Ca2+ transient, and more exocytosis. SERCA pump inhibition also activated capacitative Ca2+ entry for replenishment of intracellular Ca2+ stores. These multiple effects of SERCA pump inhibition underscore the physiological importance of the SERCA pump in rat pancreatic -cells. Modulation of SERCA pump activities may be an important mechanism for regulation of insulin secretion.
Acknowledgments
The authors thank Dr. Ray Rajotte for advice on isolation of rat pancreatic -cells, Dr. Frederick Tse for comments on the manuscript, and Dr. Peter Light for suggestions on the NCX experiments and the gift of SEA0400.
Footnotes
This work was supported by grants from the Canadian Institute of Health Research and the Alberta Heritage Foundation for Medical Research (AHFMR). A.T. is an AHFMR Senior Scholar.
The authors have no conflict of interest.
First Published Online December 8, 2005
Abbreviations: 2-APB, 2-Aminoethoxydiphenyl borate; AU, arbitrary unit; BHQ, 2,5-di-(t-butyl)-1,4-hydroquinone; [Ca2+]i, intracellular Ca2+ concentration; Cm, in membrane capacitance; ER, endoplasmic reticulum; F405, fluorescence at 405 nm; IP3, inositol 1,4,5-triphosphate; NCX, Na+/Ca2+ exchanger; PMCA, plasma membrane Ca2+-ATPase; R, ratio; RRP, readily releasable pool; SEA0400, 2-[4-[(2,5-difluorophenyl)methoxy] phenoxy]-5-ethoxyaniline; SERCA, sarcoendoplasmic reticulum Ca2+-ATPase; VGCC, voltage-gated Ca2+ channel.
Accepted for publication November 30, 2005.
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Abstract
The exocytosis of insulin-containing granules from pancreatic -cells is tightly regulated by changes in cytosolic Ca2+ concentration ([Ca2+]i). We investigated the role of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pump, Na+/Ca2+ exchanger, and plasma membrane Ca2+-ATPase pump in the Ca2+ dynamics of single rat pancreatic -cells. When the membrane potential was voltage clamped at –70 mV (in 3 mM glucose at 22 or 35 C), SERCA pump inhibition dramatically slowed (4-fold) cytosolic Ca2+ clearance and caused a sustained rise in basal [Ca2+]i via the activation of capacitative Ca2+ entry. SERCA pump inhibition increased (1.8-fold) the amplitude of the depolarization-triggered Ca2+ transient at approximately 22 C. Inhibition of the Na+/Ca2+ exchanger or plasma membrane Ca2+-ATPase pump had only minor effects on Ca2+ dynamics. Simultaneous measurement of [Ca2+]i and exocytosis (with capacitance measurement) revealed that SERCA pump inhibition increased the magnitude of depolarization-triggered exocytosis. This enhancement in exocytosis was not due to the slowing of the cytosolic Ca2+ clearance but was closely correlated to the increase in the peak of the depolarization-triggered Ca2+ transient. When compared at similar [Ca2+]i with controls, the rise in basal [Ca2+]i during SERCA pump inhibition did not cause any enhancement in the magnitude of the ensuing depolarization-triggered exocytosis. Therefore, we conclude that in rat pancreatic -cells, the rapid uptake of Ca2+ by SERCA pump limits the peak amplitude of depolarization-triggered [Ca2+]i rise and thus controls the amount of insulin secretion.
Introduction
THE INTRACELLULAR Ca2+ signal plays a key role in the regulation of insulin secretion from pancreatic -cells during stimulation by glucose and other secretagogues. The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) pump is responsible for removing Ca2+ from the cytosol into intracellular Ca2+ stores, and inhibition of the SERCA pump leads to emptying of intracellular Ca2+ stores. Other than the replenishment of intracellular Ca2+ stores, recent studies suggest that the SERCA pump may have an important role in the regulation of the depolarization-triggered Ca2+ signal. In the mouse pancreatic -cell, the time course of decay of the Ca2+ signal after a short (<10 sec) KCl depolarization was monotonic, and the SERCA pump was the dominant Ca2+ clearance mechanism (1). With long (30 sec) KCl depolarization, the rapid decay of the Ca2+ signal in mouse pancreatic -cell was followed by a slow increase that arose from Ca2+ release from the endoplasmic reticulum (ER) (1, 2, 3). In mouse pancreatic islets, inhibition of the SERCA pump increased the amplitude of the glucose-triggered [Ca2+]i oscillations and abolished the slow release of Ca2+ from the ER (4, 5). Similar changes in Ca2+ signal have also been observed in islets isolated from mice deficient in SERCA3 (the SERCA isoform that is expressed predominantly in mouse pancreatic -cells) (4). The correlation between the SERCA pump inhibitor mediated changes in the pattern of the Ca2+ signal and insulin secretion is not fully understood. Nevertheless, when single or clusters of mouse pancreatic -cells were stimulated by short KCl depolarization, their secretory response (monitored with carbon fiber amperometry) was enhanced by SERCA pump inhibitors (1). Interestingly, in islets of SERCA3-deficient mice, despite the faster decay of the islet Ca2+ signal (triggered by long KCl depolarization), the glucose-stimulated insulin secretion appeared to be better than those from the wild type (4).
Other than mouse islets, rat pancreatic islets are frequently used in the study of -cell stimulus-secretion coupling. Rat and human islets, but not mouse islets, exhibit a rising second phase of insulin secretion (6). In contrast to mouse pancreatic -cells, much less is known about the role of SERCA pump in rat pancreatic -cells. When stimulated by glucose, rat pancreatic -cells exhibited sustained [Ca2+]i elevations, instead of the oscillatory Ca2+ signal that was typically observed in mouse pancreatic -cells (7). A previous study on rat pancreatic -cells stimulated with long (approximately 10 min) KCl depolarization, suggested that Na+/Ca2+ exchanger (NCX) was the major Ca2+ clearance mechanism (8). On the other hand, in mouse pancreatic -cells, both the NCX and plasma membrane Ca2+-ATPase (PMCA) pumps have only minor contributions to Ca2+ clearance (1, 9). The discrepancy in the role of NCX in the Ca2+ dynamics between rat and mouse pancreatic -cells has been attributed to differences in the level of mRNA transcription as well as the expression of various splice variants of NCX between the two species (10).
In view of the possible differences in Ca2+ signal and secretory response between the mouse and rat pancreatic -cells, we examined the influence of the SERCA, NCX, and PMCA pumps in the regulation of Ca2+ homeostasis in single rat pancreatic -cells. Using the whole-cell patch clamp technique to deliver short trains of depolarization to activate voltage-gated Ca2+ entry, we found that the SERCA pump has an important role in regulating the amplitude of the depolarization-triggered Ca2+ transient, the time course of Ca2+ clearance from the cytosol after depolarization as well as the basal [Ca2+]i in rat pancreatic -cells. On the other hand, inhibition of the NCX or PMCA pump caused only a small slowing of cytosolic Ca2+ clearance. To understand precisely how these changes in the pattern of depolarization triggered Ca2+ signal after SERCA pump inhibition affect insulin secretion, we monitored simultaneously [Ca2+]i and exocytosis (capacitance measurement) from individual rat pancreatic -cells. Our results revealed that the rapid uptake of Ca2+ by the SERCA pump limited the magnitude of the exocytotic response in rat pancreatic -cells. In contrast, the slowing of the decay of the Ca2+ signal and the rise in basal [Ca2+]i during SERCA pump inhibition had no immediate effect on the exocytotic response.
Materials and Methods
Chemicals
Indo-1 K+ salt and indo-1-AM were from Teflabs (Austin, TX). Thapsigargin, 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ), 2-aminoethoxydiphenyl borate (2-APB) were from Calbiochem (San Diego, CA). Culture medium, serum, and antibiotics were from Life Technologies, Inc. (Burlington, Ontario, Canada). 2-[4-[(2,5-difluorophenyl)methoxy] phenoxy]-5-ethoxyaniline (SEA0400) was from Taisho Pharmaceutical Co. (gift from Dr. Peter Light). All other chemicals were from Sigma (Oakville, Ontario, Canada).
Cell preparation
Rat pancreases were removed from male Sprague Dawley rats (150–200 g) killed with halothane in accordance with the standards of the Canadian Council on Animal Care. The pancreases were cut into small pieces and then shaken at 37 C for 12 min with Hanks’ buffered salt solution containing collagenase (type V; 1.2 mg/ml) and DNase (type II; 5 μg/ml) (11). Islets were purified with a discontinuous Ficoll gradient (25, 23, 21.5, and 11.5%) and then handpicked under a dissecting microscope. Single cells were obtained by incubating the islets in trypsin solution (L-1-tosylamide-2-phenylethyl chloromethyl ketone treated; 0.0025 mg/ml) for 4 min at 37 C, followed by gentle trituration. Isolated cells were plated on glass coverslips coated with poly-L-lysine (0.1 mg/ml) and kept in RPMI 1640 culture medium containing 11 mM glucose, 10% fetal bovine serum, 50 μg/ml streptomycin, and 50 IU/ml penicillin. Experiments were performed in cells maintained in standard culture condition (37 C, 5% CO2) for 1–3 d. Because rat pancreatic -cells are larger in size in comparison with other islet cells (12) and high glucose stimulates [Ca2+]i rise in these cells (our unpublished observations), we select individual -cells based on cell size.
Solutions
The standard bath solutions contained (in mM): 150 NaCl, 2.5 KCl, 5 CaCl2, 1 MgCl2, 3 glucose, and 10 Na-HEPES (pH 7.4). In experiments involving inhibition of the PMCA pump, the pH of the standard bath solution was raised to 8.8. For experiments involving Ca2+-free extracellular solution, Ca2+ in the standard bath solution was replaced by 1 mM EGTA and the concentration of MgCl2 was raised to 3 mM. For experiments involving Na+-free solution, NaCl in the standard solution was replaced with N-methyl-glucamine-Cl. For experiments involving capacitance measurement at 22 C, Ca2+ in the standard bath solution was raised to 15 mM. The whole-cell pipette solution contained (in mM): 135 Cs-aspartate, 20 tetraethylammonium-Cl, 20 Cs-HEPES, 2.5 MgCl2, 5 Na2-ATP, 0.1 GTP, and 0.1 indo-1 (pH 7.4).
Measurement of [Ca2+]i
[Ca2+]i was measured fluorometrically using the Ca2+ indicator, indo-1, at room temperature (22 C) or high temperature (35 C). In all experiments involving high temperature, the bath was constantly perfused with extracellular solution that was warmed by a heated water jacket. Except for experiments involving Mn2+ quench, the Ca2+ indicator, indo-1 K+ salt was dialyzed into the cell via the whole-cell patch pipette. Details of the [Ca2+]i measurement were as described previously (13). Briefly, indo-1 in single rat -cell was excited by 365 nm (band-pass filtered) light delivered from a HBO 100 W mercury lamp via a x40, 1.3 NA UV fluor oil objective (Nikon, Mississauga, Ontario, Canada). To reduce fluorescence from the pipette, light collection was restricted to a spot of approximately 25 μM by inserting a 1-mm pinhole before a x5 projection lens. Photon counts were collected at 405 and 500 nm by two photomultiplier tubes (H3460-04; Hamamatsu, Bridgewater, NJ) and then translated into logic signals counted simultaneously by a CYCTM-10 counter card (Cyber Research Inc., Branford, CT) in an IBM-compatible computer. In each experiment, the background counts (from the tip of the pipette and the cell) were measured after forming a cell-attached seal and then subsequently subtracted. [Ca2+]i was calculated from the ratio (R) of fluorescence at 405 and 500 nm, using the following equation (14):
(1)
where Rmin is the fluorescence ratio of Ca2+-free indicator and Rmax is the ratio of Ca2+-bound indicator. K is a constant that was determined empirically. Calibrations were determined from single rat pancreatic -cells dialyzed with one of the three pipette solutions as described previously (15). Rmin was measured in cells loaded with (in mM): 52 K-aspartate, 10 KCl, 50 K-EGTA, 0.1 indo-1, and 50 K-HEPES (pH 7.4); and Rmax was measured in cells loaded with (in mM): 136 K-aspartate, 15 CaCl2, 0.1 indo-1, and 50 K-HEPES (pH 7.4). K was calculated from the equation above using R values obtained from cells loaded with (in mM): 60 K-aspartate; 50 K-HEPES; 20 K-EGTA; 15 CaCl2; and 0.1 indo-1 (pH 7.4), which had a calculated free Ca2+ concentration of 212 and 170 nM at 22 and 35 C, respectively (16).
Measurement of extracellular Ca2+ influx using Mn2+ quenching
Mn2+ enters into the cell via Ca2+-permeable pathways and causes quenching (reduction) in indo-1 fluorescence (both at 405 and 500 nm). Because the fluorescence at 405 nm (F405) is not sensitive to changes in [Ca2+]i (near the isosbestic wavelength of indo-1), the rate of decrease in F405 by Mn2+ reflects the rate of extracellular Ca2+ influx (17, 18). On the other hand, [Ca2+]i is determined by the ratio of F405/F500 (as described above). Rise in [Ca2+]i results in decreases in F500. Mn2+ quenches both F405 and F500 to the same extent and thus has no effect on the ratio of F405 to F500 and [Ca2+]i. Therefore, the Mn2+ quenching experiments allow us to monitor simultaneously [Ca2+]i and extracellular Ca2+ influx. In experiments involving Mn2+ quenching, cells were incubated with 5 μM indo-1 AM in standard bath solution for 15–20 min at 37 C and then in dye-free solution for 5–10 min. The fluorescence at 405 nm was then monitored during exposure of the cells to the standard bath solution with 0.2 mM Mn2+ added. All Mn2+ experiments were conducted at approximately 22 C.
Electrophysiology
Single -cells were voltage clamped with the whole-cell, gigaseal method (19) with an EPC-7 amplifier (List-Electronic, Darmstadt, Germany). The pipettes were made from hematocrit glass (VWR Scientific Canada Ltd., London, Ontario, Canada), and the resistance was 2–4 M after filling with the pipette solution. The cell membrane potential was held at –70 mV (d.c.) and a train (five to seven steps) of depolarization (150 msec in duration) to +10 mV was delivered at 2.5 Hz to trigger [Ca2+]i rise. The peak of the depolarization-triggered [Ca2+]i rise varied between 0.5 and 3 μM. A –10 mV correction for junction potential was applied throughout.
Capacitance measurement of exocytotic response
Exocytosis was measured as increases in membrane capacitance (Cm) that result from the addition of granule membrane to cell membrane (20). Individual -cells were whole-cell voltage clamped at –80 mV (d.c.) with an EPC-9 amplifier (List-Electronic), and a train (10–15 steps) of depolarization (200 or 300 msec in duration) to +10 mV was delivered to trigger [Ca2+]I rise, and exocytosis. Cm was measured at high temporal resolution with a separate dual-phase lock-in amplifier by superimposing an 800-Hz sinusoid of 30 mV peak-to-peak amplitude onto the holding potential as previously described (21). Values of [Ca2+]i and Cm were first recorded on VCR tapes with a NeuroData PCM recorder (Neuro Data Instruments Corp., New York, NY) and digitized later.
Calculations and statistics
Functions in the Microcal Origin program 6.0 (Origin Lab Corp., Northampton, MA) were used for all statistical and curve fitting procedures. The time constant of Ca2+ clearance was estimated by fitting the decay of the Ca2+ signal with a single exponential. The rate of Mn2+ quench was calculated from the slopes of the linear fit to individual segments of the fluorescence at 405 nm. A Student’s t test was used in comparisons of mean values between two populations of cells. Any difference with P < 0.05 was considered statistically significant and was marked with an asterisk in the figures. All values shown were means ± SEM.
Results
Inhibition of SERCA pump dramatically affects Ca2+ homeostasis
One difficulty in examining the effect of SERCA pump inhibition on the amplitude of the depolarization triggered Ca2+ signal is to separate the action of the SERCA pump inhibitors on the voltage-gated Ca2+ channels (VGCCs) from that on Ca2+ uptake. Among the SERCA pump inhibitors, cyclopiazonic acid was reported to enhance VGCCs, but BHQ was shown to have inhibitory action (22). On the other hand, thapsigargin was reported to have both enhancing and inhibitory action on VGCCs (22). In view of this, we used BHQ in our first series of experiments. Because BHQ is known to inhibit VGCCs, any rise in the depolarization-triggered Ca2+ signal in the presence of BHQ must be related to the inhibition of the SERCA pump uptake of Ca2+.
In these experiments, an individual rat pancreatic -cell was whole-cell voltage clamped at –70 mV. A train (five steps) of depolarization (+10 mV; 150 msec; 2.5 Hz) was delivered before and during BHQ (10 μM) application. The short train of depolarization allowed us to examine the time course of the decay of the Ca2+ transient without any interference from the slow Ca2+ release from the ER (1). The cell was continuously supplied with 5 mM ATP via the whole-cell pipette, thus ensuring a constant supply of ATP to maintain the activities of the various Ca2+-ATPases in the cell. In the example shown in Fig. 1A, the basal [Ca2+]i in control condition at approximately 22 C was approximately 0.2 μM, and a train of depolarization raised [Ca2+]i to approximately 1.7 μM. After the termination of the depolarization, [Ca2+]i decayed monotonically and rapidly to the resting level. Application of BHQ raised basal [Ca2+]i to approximately 0.6 μM. After the BHQ-mediated [Ca2+]i rise reached a plateau, the same train of depolarization was delivered, and it raised [Ca2+]i to approximately 2.8 μM. Thus, even with the increase in basal [Ca2+]i by BHQ (0.4 μM) taken into account, the amplitude of the depolarization-triggered Ca2+ transient in the presence of BHQ was still approximately 0.7 μM higher than that elicited in the same cell before BHQ exposure. In 33 cells examined at approximately 22 C (Fig. 1B), the average basal [Ca2+]i was 0.20 ± 0.02 μM, and BHQ raised the basal [Ca2+]i to 0.76 ± 0.08 μM (an average increase of 0.57 ± 0.07 μM). An elevation of basal [Ca2+]i by BHQ (from 0.14 ± 0.02 to 0. 35 ± 0.04 μM) was also observed in cells recorded at approximately 35 C (n = 21; Fig. 1B).
The action of BHQ on the amplitude of the depolarization-triggered Ca2+ transient is summarized in Fig. 1C. Due to the variability of the amplitude of the depolarization-triggered Ca2+ transient among the rat pancreatic -cells, the amplitude of the depolarization-triggered Ca2+ transient in the presence of BHQ was normalized to that elicited from the same cell before BHQ exposure. To separate the effect of BHQ on basal [Ca2+]i from that on the depolarization-triggered Ca2+ transient, the amplitude of the Ca2+ transient in BHQ was measured as the difference between the basal [Ca2+]i in the presence of BHQ before depolarization and that of the peak of the Ca2+ transient. Consistent with a previous report on the inhibitory action of BHQ on VGCCs (22), the amplitude of the Ca2+ current (measured from the first step of the depolarization) in the presence of BHQ (at 22 C) was reduced by 25 ± 9% (n = 9). Note that despite of the VGCC reduction, BHQ increased the amplitude of the depolarization-triggered Ca2+ transient in 17 of 19 cells examined at approximately 22 C. On average, BHQ increased the amplitude of the depolarization-triggered Ca2+ transient at approximately 22 C by 1.77 ± 0.18-fold (n = 19). At approximately 35 C, however, BHQ did not cause any significant increase in the amplitude of the depolarization-triggered Ca2+ transient (Fig. 1C). This is probably due to the large reduction of VGCCs in the presence of BHQ at high temperature (46 ± 6%; n = 12).
In Fig. 1D, we compared the kinetics of the Ca2+ signal at approximately 22 C by superimposing the depolarization-triggered Ca2+ transients before and after BHQ exposure (same record as in Fig. 1A). For comparison, the amplitude of the Ca2+ transient in control was scaled up to match that of the Ca2+ transient evoked in BHQ. Note that BHQ dramatically slowed the decay phase of the Ca2+ signal (the time constant of cytosolic Ca2+ clearance increased from 0.98 to 10.4 sec). In 20 cells examined at approximately 22 C, the time constant of cytosolic Ca2+ clearance in control was 1.59 ± 0.11 sec, and BHQ increased the time constant by 4.2-fold to 6.72 ± 0.83 sec (Fig. 1E). The rate of cytosolic Ca2+ clearance in rat -cells increased by approximately 2.5-fold when the temperature was elevated to approximately 35 C (time constant of cytosolic Ca2+ clearance in control cells was 0.63 ± 0.05 sec). Note that at high temperature, BHQ caused a similar slowing in Ca2+ clearance, and the time constant of Ca2+ clearance in BHQ increased by 3.8-fold to 2.36 ± 0.25 sec (Fig. 1E). The effects of BHQ on Ca2+ homeostasis were reversible. As shown in Fig. 1A, after BHQ removal, the basal [Ca2+]i returned to the control level and the train of depolarization evoked a smaller Ca2+ transient, which decayed with a faster time constant. These results suggest that SERCA pumps have important roles in Ca2+ homeostasis in rat pancreatic -cell.
Inhibition of NCX causes a small slowing of cytosolic Ca2+ clearance
Using similar experimental procedures, we examined the influence of NCX inhibition on rat pancreatic -cells. We first tested the actions of SEA0400, a newly developed NCX inhibitor (23). As shown in the example in Fig. 2A, at approximately 22 C, SEA0400 (1 μM) caused a very small elevation (0.09 μM) in basal [Ca2+]i, and the time constant of cytosolic Ca2+ clearance increased from 1.2 to 1.84 sec. Note that there was no apparent change in the amplitude of the depolarization-triggered Ca2+ transient. In 14 cells tested with SEA0400 at approximately 22 C, there was no significant increase in basal [Ca2+]i (Fig. 2B) or the amplitude of the depolarization-triggered Ca2+ transient (Fig. 2C). For the same cells, SEA0400 increased the time constant of Ca2+ clearance by approximately 35% (from 1.20 ± 0.10 to 1.62 ± 0.14 sec; Fig. 2D). In a recent study, intracellular ATP (3 mM) was reported to antagonize the action of SEA0400 (24). Because our pipette solution contained 5 mM ATP, it is possible that SEA0400 might be less effective in inhibiting NCX. Therefore, we further examined the role of NCX in rat pancreatic -cells by inhibiting NCX with a Na+-free extracellular solution. For cells recorded at approximately 22 C, the removal of extracellular Na+ caused only a small slowing (20%) in Ca2+ clearance (from 2.0 ± 0.15 to 2.41 ± 0.22 sec; n = 4), and neither the basal [Ca2+]i nor the amplitude of the depolarization-triggered Ca2+ transient was affected. When the same experiment was repeated at approximately 35 C, a small slowing in cytosolic Ca2+ clearance (35%; Fig. 2D) and a small rise (0.1 μM) in basal [Ca2+]i (Fig. 2B) were detected after the removal of extracellular Na+. Thus, under our experimental conditions (short depolarization and continuous supply of ATP via the whole-cell pipette), NCX has a much smaller influence in the Ca2+ homeostasis of rat pancreatic -cells when compared with the SERCA pump.
Inhibition of PMCA pump causes a small slowing of cytosolic Ca2+ clearance
To examine whether the PMCA pump contributes to Ca2+ homeostasis in rat pancreatic -cells, we reduced PMCA activities by exposing the cells to an extracellular solution with pH 8.8. This experimental approach is based on the Ca2+/H+ exchanger property of the PMCA pump, and by lowering the extracellular H+ concentration, the activities of the PMCA pump can be reduced (25). Such manipulation has been used successfully to reduce PMCA pump activities in mouse pancreatic -cells (1) and rat pituitary corticotropes (26). An example of such experiment is shown in Fig. 3. Note that at both approximately 22 and approximately 35 C, inhibition of PMCA pump caused only a small slowing of cytosolic Ca2+ clearance (27 and 34%; Fig. 3D). At high temperature, PMCA inhibition caused a small rise in basal [Ca2+]i as well as the amplitude of the Ca2+ transient. Thus, PMCA activities have minor roles in the Ca2+ dynamics of rat -cells.
SERCA pump inhibition evokes capacitative Ca2+ entry
In mouse pancreatic -cells, SERCA pump inhibition has been reported to cause depolarization and activation of voltage-gated Ca2+ entry (2, 27, 28, 29). However, in our experiments (Fig. 1), the cell membrane potential was held at -70 mV. Thus, the BHQ-mediated rise in basal [Ca2+]i could not be due to depolarization. To examine the source of the BHQ-mediated rise in basal [Ca2+]i, we compared the action of BHQ on basal [Ca2+]i with or without extracellular Ca2+. In the experiment shown in Fig. 4A, the cell was voltage clamped at -70 mV and exposed to two consecutive BHQ challenges. In the presence of 5 mM extracellular Ca2+, application of BHQ elevated the basal [Ca2+]i by approximately 0.2 μM. After the removal of BHQ, [Ca2+]i returned to the control level. The bath solution was then switched to a Ca2+-free solution (no added Ca2+, and 1 mM EGTA was included to chelate any contaminating Ca2+). This resulted in a small reduction in the basal [Ca2+]i. A second BHQ challenge in the absence of extracellular Ca2+ elicited only a very small [Ca2+]i rise (0.03 μM). The small BHQ-mediated [Ca2+]i rise during the second challenge was not due to any desensitization of the BHQ response because a subsequent BHQ challenge in the presence of 5 mM extracellular Ca2+ still raised [Ca2+]i by approximately 0.2 μM (data not shown). The experimental results obtained from 12 cells are summarized in Fig. 4B. On average, the BHQ-mediated [Ca2+]i rise was 0.06 ± 0.02 μM in the absence of extracellular Ca2+, much smaller than that evoked in the presence of extracellular Ca2+ (0.30 ± 0.06 μM) for the same batch of cells. This result suggests that a major component of the BHQ-mediated rise in basal [Ca2+]i requires extracellular Ca2+ influx. Consistent with this, Fig. 4C shows that the removal of extracellular Ca2+ reversibly reduced the BHQ-mediated [Ca2+]i rise. When extracellular Ca2+ was added back to the bath solution, the BHQ-mediated [Ca2+]i was restored to its previous level. Similar results were observed in five of five cells.
One possible explanation for the strong dependence on extracellular Ca2+ during the BHQ-mediated rise in basal [Ca2+]i is that SERCA pump inhibition causes depletion of Ca2+ from intracellular stores, which in turn activates capacitative Ca2+ entry (30, 31). To examine whether the BHQ-mediated [Ca2+]i rise is associated with the activation of extracellular Ca2+ influx pathways, we used the Mn2+ quench technique (26) to simultaneously monitor [Ca2+]i and Mn2+ entry (see explanation in Materials and Methods). A representative example of this experiment is shown in Fig. 5, A and B. Single rat pancreatic -cells were loaded with indo-1 AM and then perfused with the standard bath solution. Note that in this experiment, the cell membrane potential was unclamped. Application of Mn2+ (0.2 mM) resulted in a small and slow decrease in F405, reflecting a basal leakage of Mn2+ into the cell (Fig. 5B). The basal [Ca2+]i, however, was not affected by this Mn2+ leakage (Fig. 5A). Application of BHQ (10 μM) stimulated a rise in [Ca2+]i (Fig. 5A). Note that the BHQ-mediated [Ca2+]i rise was accompanied by an acceleration in the rate of decrease of F405 (Fig. 5B). After BHQ removal, the rate of F405 decrease slowed down and [Ca2+]i decayed to the resting level. The result of this experiment is summarized in Fig. 5C. In 21 cells examined, the basal rate of Mn2+ quench (reflected by the decrease in F405) was 0.24 ± 0.04 arbitrary units (AU)–1, and BHQ accelerated the rate by approximately 2.2-fold to 0.52 ± 0.07 AU–1. Thus, the BHQ-mediated rise in basal [Ca2+]i in rat pancreatic -cells was closely accompanied by an increase in extracellular Ca2+ influx. The above experiment was then repeated with diazoxide (0.2 mM) in the bath. In seven cells hyperpolarized with diazoxide, the basal rate of Mn2+ quench was 0.27 ± 0.06 AU–1, and BHQ increased the rate by 2.2-fold to 0.60 ± 0.11 AU–1 (Fig. 5C). The similar increase in the rate of Mn2+ quench by BHQ between the unclamped cells and hyperpolarized cells suggests that membrane depolarization did not have any major contribution to the BHQ-mediated increase in extracellular Ca2+ influx.
To examine whether the BHQ-mediated extracellular Ca2+ influx involves activation of capacitative Ca2+ entry channels, we used 2-APB, a blocker of capacitative Ca2+ entry (32, 33, 34). Figure 6A shows that the rate of Mn2+ quench in BHQ was slowed by 2-APB (50 μM). In nine cells examined, BHQ increased the rate of Mn2+ quench from 0.16 ± 0.05 to 0.53 ± 0.10 AU–1, and 2-APB reduced the rate to 0.26 ± 0.04 AU–1 (Fig. 5B). As shown in Fig. 6C, in cells voltage clamped at –70 mV, the BHQ-mediated rise in basal [Ca2+]i was also reduced by 2-APB (seven of nine cells). Overall, our results indicate that the majority of the BHQ-mediated rise in basal [Ca2+]i was due to the activation of capacitative Ca2+ entry. The small BHQ-mediated [Ca2+]i rise in the absence of extracellular Ca2+ was probably due to the leakage of Ca2+ from the intracellular stores.
SERCA pump inhibition enhances depolarization- triggered exocytosis
Our results above show that SERCA pump inhibition in rat pancreatic -cells could dramatically affect the shape of the depolarization-triggered Ca2+ signal. To examine how these changes in the pattern of the Ca2+ signal affect exocytosis (and hence insulin secretion), we compared the depolarization-triggered Ca2+ signal and the resultant exocytosis (measured as increases in membrane capacitance) in the same cell before and after SERCA pump inhibition. We first performed this set of experiments at approximately 22 C because there was a clear increase in the amplitude of the depolarization-triggered Ca2+ transient in BHQ (Fig. 1C). At this temperature, a train of depolarization (in the presence of 15 mM extracellular Ca2+) was needed to evoke a robust exocytotic response. Figure 7A shows a representative example of such an experiment. The cell was whole-cell voltage clamped at –80 mV and the resting [Ca2+]i was 0.22 μM in control condition (Fig. 7A). A train of depolarization was delivered to trigger [Ca2+]i rise and exocytosis. After the termination of the fourth step of depolarization when [Ca2+]i rose to approximately 0.74 μM, the Cm increased by approximately 11 fF. After the sixth depolarizing step, [Ca2+]i reached approximately 1 μM, and the subsequent depolarizing steps maintained [Ca2+]i near this level. Note that each of the subsequent depolarizing steps still evoked additional increase in membrane capacitance. At the end of the train of depolarization, [Ca2+]i started to decay, and there was no further increase in Cm. Overall, the cumulative Cm increase elicited by the train of depolarization in this cell was 73 fF. The exocytosis of a single granule in mouse pancreatic -cell has been assumed to contribute approximately 2 fF (35). Because the granules in rat and mouse pancreatic -cells are similar in size (36), the 73 fF increase reflects the release of approximately 36 granules. After the return of [Ca2+]i to the basal level, the same cell was exposed to BHQ (10 μM). Basal [Ca2+]i rose from 0.22 to 0.43 μM, and the same train of depolarization was then delivered (Fig. 7B). In the presence of BHQ, [Ca2+]i rose to approximately 1 μM after the termination of the fourth depolarizing step, and the membrane capacitance increased by approximately 28 fF. At the end of the train, [Ca2+]i rose to approximately 1.8 μM, and the cumulative Cm increase was 190 fF (2.6-fold of that triggered under control condition; Fig. 7A).
Figure 7, C and D, shows that enhancement of depolarization-triggered exocytosis could also be observed with thapsigargin, another SERCA pump inhibitor. In the example shown in Fig. 7C, under control conditions, the train of depolarization elevated [Ca2+]i to only approximately 0.7 μM and did not trigger any significant increase in Cm. When the same cell was exposed to thapsigargin (1 μM), the resting [Ca2+]i rose from 0.19 to 0.37 μM, and the same train of depolarization raised [Ca2+]i to approximately 1.6 μM, resulting in a cumulative Cm increase of 257 fF. Note that in this cell, after the eighth depolarizing step, although [Ca2+]i remained elevated near 1.6 μM, subsequent depolarizing steps caused only small additional increase in membrane capacitance. This probably reflects the exhaustion of the readily releasable pool (RRP) of granules during the first eight steps of the train of depolarization, and the subsequent slow mobilization of additional granules. Figure 7 also shows that exocytosis typically stopped with the termination of the train of depolarization, even though [Ca2+]i remained elevated. Thus, during SERCA pump inhibition, the slowing of Ca2+ clearance after the train of depolarization is unlikely to contribute to the enhancement in exocytosis. Instead, the overall increase in the peak of the depolarization-triggered Ca2+ transient during SERCA pump inhibition appeared to trigger more exocytosis.
To address this possibility, we examined whether the enhancement in exocytotic response was correlated to the overall changes in the peak of the depolarization-triggered Ca2+ transient. The experimental protocol was identical with the examples shown in Fig. 7. Because the depolarization-triggered [Ca2+]i rise typically reached a maximum before the termination of the train of depolarization, we measured the cumulative Cm at the time that the depolarization-triggered Ca2+ transient reached its peak (e.g. eighth step in Fig. 7A). For each cell, the difference in cumulative Cm before and after SERCA pump inhibition was plotted against the difference in the peak value of the depolarization-triggered Ca2+ transients. Figure 8A shows the results from five cells challenged with BHQ and four cells challenged with thapsigargin at approximately 22 C. Note that in three of the nine cells, there was a decrease in the peak value of the depolarization-triggered Ca2+ rise after SERCA pump inhibition (reflected by the negative values in the change in peak [Ca2+]i), probably due to rundown in VGCCs. For these three cells, the exocytotic response in the presence of SERCA pump inhibitor was also smaller than the controls. On the other hand, for cells with a larger increase in the peak value of the depolarization-triggered Ca2+ transient during SERCA pump inhibition, there was a bigger increase in the exocytotic response. As shown in Fig. 8A, there was a strong correlation between the increase in the peak value of the depolarization-triggered Ca2+ transient by SERCA pump inhibitor and the enhancement in exocytotic response at approximately 22 C (r = 0.985; P < 0.0001).
Figure 8A also shows the exocytotic response obtained from cells challenged with BHQ at approximately 35 C. At this temperature, exocytosis could be readily triggered by depolarization in the presence of 5 mM extracellular Ca2+. Thus, all the experiments involving exocytotic response at approximately 35 C were performed in 5 mM extracellular Ca2+. As described earlier, there was a larger decrease in VGCCs in the presence of BHQ at high temperature. Thus, only five of the 13 cells recorded at approximately 35 C have a higher peak Ca2+ transient in the presence of BHQ. Nevertheless, there was still a good correlation between the change in the peak value of the depolarization-triggered Ca2+ transient by SERCA pump inhibitor and the change in exocytotic response at approximately 35 C (r = 0.855; P < 0.0002).
In mouse pancreatic -cells, elevation of basal [Ca2+]i has been reported to potentiate Ca2+-dependent exocytosis (via calmodulin kinase II), even when the amplitude of the depolarization-triggered Ca2+ transient was unchanged (37). Because SERCA pump inhibition causes elevation of basal [Ca2+]i (Fig. 1) in rat pancreatic -cells, it is possible that such mechanism may also contribute to the enhancement in exocytotic response during SERCA pump inhibition. To address this, we plotted in Fig. 8B the amount of exocytosis triggered in rat pancreatic -cells at approximately 22 or approximately 35 C when [Ca2+]i was elevated to different levels during a train of depolarization. To examine whether the elevated basal [Ca2+]i affected exocytosis independently from the increase in the peak of the depolarization-triggered Ca2+ transient, we compared the amount of exocytosis triggered with or without SERCA pump inhibitor at similar [Ca2+]i. Note that although SERCA pump inhibition increased the basal [Ca2+]i in these cells (by 0.34 μM at 22 C and 0.17 μM at 35 C), at comparable [Ca2+]i, SERCA pump inhibition did not cause any significant increase in the amount of exocytosis over the control (Fig. 8B). Thus, in rat pancreatic -cells, the increase in the amplitude of the depolarization-triggered Ca2+ transient during SERCA pump inhibition is the major mechanism underlying the enhancement of exocytosis.
Discussion
The present study examines the contribution of SERCA, NCX, and PMCA pumps to the Ca2+ dynamics of rat pancreatic -cells, at both approximately 22 and approximately 35 C. Under control conditions (in 3 mM glucose), the time constant of cytosolic Ca2+ clearance after a short train of depolarization in the rat pancreatic -cells at approximately 22 C was approximately 1.6 sec (Fig. 1E), reflecting an overall clearance rate constant of 0.63 sec–1. At approximately 35 C, the time constant of Ca2+ clearance decreased to 0.63 sec, and the overall clearance rate constant increased to 1.59 sec–1, a value comparable with that described in the mouse pancreatic -cells [1 sec–1 at 35 C and in 15 mM glucose (1)]. Thus, raising the temperature from approximately 22 to approximately 35 C increases the rate of cytosolic Ca2+ clearance in rat pancreatic -cells. Note that, however, elevation of temperature did not affect the relative contributions of SERCA, NCX, and PMCA pumps to Ca2+ homoeostasis. At both approximately 22 and approximately 35 C, the SERCA pump is the dominant mechanism of cytosolic Ca2+ clearance in rat pancreatic -cells. Inhibition of the SERCA pump at the two temperatures increased the time constant of cytosolic Ca2+ clearance in rat -cells to approximately 6.7 and approximately 2.36 sec, respectively (Fig. 1E), suggesting that all non-SERCA Ca2+ clearance mechanisms (including the NCX and PMCA pumps, mitochondria) have a combined clearance rate constant of 0.15 sec–1 at approximately 22 C and 0.42 sec–1 at approximately 35 C. This indicates that, at both temperatures, the non-SERCA Ca2+ clearance mechanisms in rat pancreatic -cell contribute only approximately 25% of the overall Ca2+ clearance. Therefore, similar to what has been reported in mouse pancreatic -cells at 35 C (1), the SERCA pump is the dominant Ca2+ clearance mechanism in rat pancreatic -cells.
Our study also shows that NCX has minor contributions to Ca2+ homeostasis in rat pancreatic -cells. At both approximately 22 and approximately 35 C, inhibition of NCX did not affect the amplitude of the depolarization-triggered Ca2+ transient (Fig. 2C) but caused a small (30%) slowing in the rate of cytosolic Ca2+ clearance (Fig. 2D). This finding is in contrast to a previous study in rat pancreatic -cells that showed NCX inhibition with antisense oligos reduced the amplitude of the KCl- or tolbutamide-triggered Ca2+ transient at approximately 37 C by 28 or 40% and slowed Ca2+ clearance by 72 or 40%, respectively (8). The discrepancies between the two studies could not be due to a difference in temperature because our study showed clearly that raising the temperature from approximately 22 to approximately 35 C did not cause any increase in the contribution of NCX to the regulation of Ca2+ dynamics in rat pancreatic -cells (Fig. 2). Instead, we suggest that the discrepancy may be related to a rundown in the cellular ATP level in the study with antisense oligos (8). Note that in the current study, a short train of depolarization (total duration < 1.5 sec) was applied to elicit a transient [Ca2+]i rise, and ATP was continuously supplied to the cell via the whole-cell pipette. In contrast, the study with antisense oligos (8) used a 10-min application of KCl to elicit [Ca2+]i rise in intact cells. Because the function of SERCA pump requires constant supply of ATP, it is possible that after a prolonged [Ca2+]i rise, the cellular ATP level may become low and thus reducing the SERCA pump activity. Under this condition, the NCX may become the dominant Ca2+ clearance mechanism.
Consistent with the previous report in mouse pancreatic -cells (at 35 C) (1), we found that the PMCA pump makes only minor contribution to the Ca2+ dynamics in rat pancreatic -cells. At both approximately 22 and approximately 35 C, inhibition of the PMCA pump slowed cytosolic Ca2+ clearance by approximately 30% (Fig. 3D). At approximately 35 C, the PMCA pump inhibition also caused a significant increase in the basal [Ca2+]i (Fig. 3B) as well as the amplitude of the depolarization-triggered Ca2+ transient (Fig. 3C). Thus, it is possible that the activity of the PMCA pump is slightly more prominent at high temperatures.
Insulin secretion from pancreatic -cells is known to be highly dependent on temperature (38, 39). This temperature dependence has been attributed to the sensitivity of the replenishment of the RRP granules to temperature (40). In the current study, we also found that raising the temperature from approximately 22 to approximately 35 C in rat pancreatic -cells dramatically increased the exocytotic response, even when compared at similar [Ca2+]i (Fig. 8B). Nevertheless, the influence of SERCA pump inhibition on exocytosis was similar at the two temperatures. Despite the dramatic slowing in cytosolic Ca2+ clearance during SERCA pump inhibition, we found that the enhancement of the exocytotic response in rat pancreatic -cells was not directly related to the slower decay in the depolarization-triggered Ca2+ signal. Note that after the termination of the train of depolarization (at either approximately 22 or 35 C), there was no additional increase in membrane capacitance, even though [Ca2+]i remained elevated. This observation suggests that in rat pancreatic -cells, the secretory granules may be in close proximity to the VGCCs such that the local [Ca2+] near the granules was much higher than the average cytosolic [Ca2+] (41, 42). After the closure of VGCCs, the spatial Ca2+ gradient rapidly dissipates and the drop in local [Ca2+] near the granules terminates exocytosis. Although the slower Ca2+ clearance during SERCA pump inhibition in rat pancreatic -cells has no immediate effect on exocytosis, elevated basal [Ca2+]i has been shown to act directly as well as indirectly via protein kinase C activation to increase the size of RRP in bovine chromaffin cells at 20–25 C (43). A recent study in rat pancreatic -cells (44) has also shown that protein kinase C activation at 30–32 C increase the size of RRP. Whether the slower Ca2+ clearance during SERCA pump inhibition in rat pancreatic -cells also affects the replenishment of granules in the RRP awaits future studies.
Our study shows that acute inhibition of the SERCA pump in rat pancreatic -cell can dramatically potentiate secretion at both approximately 22 and approximately 35 C. This result is in agreement with a previous finding that showed glucose-triggered insulin secretion in rat pancreatic islets (at 37 C) was potentiated by thapsigargin (45). We also found that at approximately 22 and approximately 35 C, the increase in the peak of the depolarization-triggered Ca2+ transient during SERCA pump inhibition is the major mechanism underlying the enhancement of exocytosis (Fig. 8A). An increase in the amplitude of the depolarization-triggered Ca2+ transient in the presence of the SERCA pump inhibitor has also been reported in mouse islets (at 37 C) (4) as well as single mouse pancreatic -cells (at 35 C) (1). In view of the inhibitory action of BHQ on VGCCs (22), the approximately 1.8-fold increase in the amplitude of depolarization-triggered Ca2+ transient of rat pancreatic -cells by BHQ at approximately 22 C (Fig. 1) was probably underestimated. Nevertheless, this finding suggests that the SERCA pump can rapidly uptake Ca2+ during extracellular Ca2+ entry via VGCCs, thus limiting the amplitude of the depolarization-triggered Ca2+ transient. The potentiating action of the SERCA pump inhibitor on exocytosis is particularly dramatic in cells in which the depolarization-triggered Ca2+ transient was initially below the threshold for triggering of exocytosis (e.g. Fig. 7C). Under this condition, the increase in the amplitude of the depolarization-triggered Ca2+ transient during SERCA pump inhibition could turn the exocytotic response from none to a robust one (Fig. 7D).
Interestingly, we found that SERCA pump inhibition in rat pancreatic -cells at both approximately 22 and approximately 35 C caused a sustained rise in basal [Ca2+]i (Fig. 1), which was much larger than that reported in the single mouse pancreatic -cells (31–37 C) (1, 27, 46). Our findings suggest that the rise in basal [Ca2+]i did not increase the magnitude of the ensuing exocytotic response. As shown in Fig. 8B, when compared at similar [Ca2+]I in both approximately 22 and approximately 35 C, cells with higher basal [Ca2+]i (after SERCA pump inhibition) did not exhibit any potentiation of the exocytotic response. Our result is different from a previous study in mouse pancreatic -cells that showed elevation of basal [Ca2+]i at 33 C increased the size of RRP and thus potentiated the exocytotic response (37). Because our experiment was already conducted at higher temperature (35 C), the lack of potentiation of the exocytotic response with the elevated basal [Ca2+]i could not be due to the temperature sensitivity of the replenishment of the RRP. One major difference between the two studies is that the whole-cell pipette solution in our study did not contain any cAMP and our bath solution did not contain any forskolin. Because cAMP has been reported to increase the size of RRP in rat pancreatic -cells (44), it is possible that a low cellular cAMP level may reduce the enhancing effect of elevated basal [Ca2+]i on exocytosis. Nevertheless, this elevation in basal [Ca2+]i acts in concert with the change in the amplitude of the depolarization-triggered Ca2+ transient to create an overall increase in the peak value of the Ca2+ transient during SERCA pump inhibition, thus potentiating the exocytotic response at both approximately 22 and approximately 35 C (Fig. 8A).
Inhibition of the SERCA pump is known to cause depletion of ER Ca2+ stores because the continuous leakage of Ca2+ from the ER is no longer balanced by the SERCA pump-mediated reuptake of Ca2+. In the absence of extracellular Ca2+, SERCA pump inhibition in rat pancreatic -cells resulted in only a very small [Ca2+]i rise (0.06 μM; Fig. 4B), suggesting that the ER has a small Ca2+ reserve, which can be rapidly depleted. The basal [Ca2+]i rise induced by SERCA pump inhibition was much larger in the presence of extracellular Ca2+, suggesting that Ca2+ depletion from the ER in turn activates capacitative Ca2+ entry into the cell. Consistent with this, the BHQ-mediated rise in [Ca2+]i was reduced by 2-APB, a blocker of capacitative Ca2+ entry (Fig. 6C). Although 2-APB has been reported to decrease inositol 1,4,5-triphosphate (IP3)-mediated intracellular Ca2+ release, inhibit Ca2+ pumps and reduce mitochondrial Ca2+ uptake (32, 33), our observation that the BHQ-mediated increase in the rate of Mn2+ quench was reduced by 2-APB (Fig. 6, A and B) supports the notion that capacitative Ca2+ entry is the major mechanism underlying the BHQ-mediated rise in basal [Ca2+]i in rat pancreatic -cells.
A small capacitative Ca2+ entry has also been reported in mouse pancreatic -cell (17, 18). Note also that the capacitative Ca2+ entry in the rat pancreatic -cell is not caused by depolarization as the cells were held at hyperpolarized potentials (with voltage clamp or diazoxide). This observation is different from the mouse pancreatic -cells in which the emptying of ER Ca2+ store was reported to activate a depolarizing current that in turn activates voltage-gated Ca2+ entry (2, 27, 29). The major function of the robust capacitative Ca2+ entry observed in the rat pancreatic -cells is probably related to the refilling of the ER Ca2+ stores. Pancreatic -cells possess multiple intracellular Ca2+ stores, including the IP3-sensitive stores (47), ryanodine-sensitive stores (48, 49), nicotinic acid adenine dinucleotide phosphate stores (50, 51), and atypical Ca2+-induced Ca2+ release stores (52). The filling of these Ca2+ stores may not all involve capacitative Ca2+ entry. For example, the activation of ryanodine receptor in a rat insulinoma cell line (S-5 cells) was reported to trigger an extracellular Ca2+ influx, which was different from that triggered by the SERCA pump inhibitors (48). Nevertheless, in mouse pancreatic -cells, the emptying of the IP3-sensitive stores by cholinergic agonist has been reported to trigger capacitative Ca2+ entry (17, 18). Because cholinergic agonist also triggers ER Ca2+ release in rat pancreatic -cells (53), it is likely that the capacitative Ca2+ entry observed in our study is important in maintaining the cholinergic response in these cells. Moreover, sustained depletion of ER Ca2+ stores has been linked to ER stress and cell apoptosis in pancreatic -cells (54, 55). Thus, the refilling of ER stores by capacitative Ca2+ entry may be important for -cell survival.
Overall, our study shows that the SERCA pump is the dominant Ca2+ clearance mechanisms in rat pancreatic -cells. We found that elevation of temperature increased the rate of cytosolic Ca2+ clearance as well as the amplitude of the exocytotic response. However, temperature elevation did not alter the relative contributions of the SERCA, NCX, and PMCA pumps to Ca2+ homeostasis. At both room temperature (22 C) and physiological temperature (35 C), SERCA pumps accounted for approximately 75% of the total Ca2+ clearance in rat pancreatic -cells. SERCA pump inhibition resulted in a larger amplitude of the depolarization-triggered Ca2+ transient, and more exocytosis. SERCA pump inhibition also activated capacitative Ca2+ entry for replenishment of intracellular Ca2+ stores. These multiple effects of SERCA pump inhibition underscore the physiological importance of the SERCA pump in rat pancreatic -cells. Modulation of SERCA pump activities may be an important mechanism for regulation of insulin secretion.
Acknowledgments
The authors thank Dr. Ray Rajotte for advice on isolation of rat pancreatic -cells, Dr. Frederick Tse for comments on the manuscript, and Dr. Peter Light for suggestions on the NCX experiments and the gift of SEA0400.
Footnotes
This work was supported by grants from the Canadian Institute of Health Research and the Alberta Heritage Foundation for Medical Research (AHFMR). A.T. is an AHFMR Senior Scholar.
The authors have no conflict of interest.
First Published Online December 8, 2005
Abbreviations: 2-APB, 2-Aminoethoxydiphenyl borate; AU, arbitrary unit; BHQ, 2,5-di-(t-butyl)-1,4-hydroquinone; [Ca2+]i, intracellular Ca2+ concentration; Cm, in membrane capacitance; ER, endoplasmic reticulum; F405, fluorescence at 405 nm; IP3, inositol 1,4,5-triphosphate; NCX, Na+/Ca2+ exchanger; PMCA, plasma membrane Ca2+-ATPase; R, ratio; RRP, readily releasable pool; SEA0400, 2-[4-[(2,5-difluorophenyl)methoxy] phenoxy]-5-ethoxyaniline; SERCA, sarcoendoplasmic reticulum Ca2+-ATPase; VGCC, voltage-gated Ca2+ channel.
Accepted for publication November 30, 2005.
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