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Dominant Role of Sarcoendoplasmic Reticulum Ca²⁺-ATPase Pump in Ca²⁺ Homeostasis and Exocytosis in Rat Pancreatic ß-Cells

Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Address all correspondence and requests for reprints to: Amy Tse, 9-70 Medical Sciences Building, Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. E-mail: amy.tse{at}ualberta.ca.

	Abstract

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The exocytosis of insulin-containing granules from pancreatic ß-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 ß-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 ß-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

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THE INTRACELLULAR Ca²⁺ signal plays a key role in the regulation of insulin secretion from pancreatic ß-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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-cells, much less is known about the role of SERCA pump in rat pancreatic ß-cells. When stimulated by glucose, rat pancreatic ß-cells exhibited sustained [Ca²⁺]_i elevations, instead of the oscillatory Ca²⁺ signal that was typically observed in mouse pancreatic ß-cells (7). A previous study on rat pancreatic ß-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 ß-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 ß-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 ß-cells, we examined the influence of the SERCA, NCX, and PMCA pumps in the regulation of Ca²⁺ homeostasis in single rat pancreatic ß-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 ß-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 ß-cells. Our results revealed that the rapid uptake of Ca²⁺ by the SERCA pump limited the magnitude of the exocytotic response in rat pancreatic ß-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

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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 ß-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 ß-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 ß-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 R_min is the fluorescence ratio of Ca²⁺-free indicator and R_max is the ratio of Ca²⁺-bound indicator. K* is a constant that was determined empirically. Calibrations were determined from single rat pancreatic ß-cells dialyzed with one of the three pipette solutions as described previously (15). R_min 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 R_max 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 ß-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 (C_m) that result from the addition of granule membrane to cell membrane (20). Individual ß-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. C_m 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 C_m 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

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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 ß-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).

View larger version (23K): [in this window] [in a new window]   FIG. 1. Action of SERCA pump inhibitor on [Ca2+]i homeostasis in single rat pancreatic ß-cell. A, BHQ (10 µM) increased basal [Ca2+]i and the amplitude of the depolarization-triggered Ca2+ transient. The cell was voltage clamped at –70 mV at approximately 22 C and a train of depolarization (indicated by arrow) was delivered before and during exposure to BHQ (10 µM) as well as after BHQ removal. B, Summary of the effect of BHQ on basal [Ca2+]i at approximately 22 (n = 33) and approximately 35 C (n = 21). C, Summary of the effect of BHQ on the amplitude of the depolarization-triggered Ca2+ transient (measured as the difference between the basal [Ca2+]i before depolarization and the peak of the depolarization-triggered Ca2+ transient) at approximately 22 (n = 19) and approximately 35 C (n = 21). The amplitude of the depolarization-triggered Ca2+ transient was normalized to that elicited in the same cell before BHQ exposure. D, BHQ slowed the decay of the Ca2+ signal. Superimposed traces of depolarization-triggered Ca2+ transient before and in the presence of BHQ. For comparison, the amplitude of the Ca2+ transient in control was scaled up to match that in the presence of BHQ. Note the dramatic difference in the time course of the decay of the Ca2+ transient (same cell as in A). E, Summary of the effect of BHQ on the time constant of the decay of the Ca2+ transient at approximately 22 (n = 20) and 35 C (n = 21). The time constant was obtained by fitting the decay phase of the Ca2+ signal with a single exponential. Con, Control.

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 ß-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 ß-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 ß-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 ß-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 ß-cells when compared with the SERCA pump.

View larger version (25K): [in this window] [in a new window]   FIG. 2. Effect of NCX inhibitin on [Ca2+]i homeostasis. A, Application of SEA0400 (1 µM) slightly elevated basal [Ca2+]i, but the amplitude of the depolarization-triggered Ca2+ transient was unaffected. The cell was held at –70 mV at approximately 22 C and a train of depolarization (indicated by arrow) was delivered before and after exposure to SEA0400. Summary of the effects of SEA0400 (at 22 C; n = 14) and removal of extracellular Na+ (at 35 C; n = 14) on basal [Ca2+]i (B), the amplitude of depolarization-triggered Ca2+ transient (C), and the time constant of Ca2+ clearance (D). Con, Control.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Effect of PMCA pump inhibition on [Ca2+]i homeostasis. A, When the pH of the bath solution was raised to 8.8, there was a small elevation in basal [Ca2+]i and an increase in the amplitude of the depolarization-triggered Ca2+ transient. The cell was held at –70 mV (at 22 C), and a train of depolarization (indicated by arrow) was delivered before and in the presence of the pH 8.8 solution. Summary of the effects of PMCA inhibition on basal [Ca2+]i (B), the amplitude of depolarization-triggered Ca2+ transient (C), and the time constant of Ca2+ clearance (D) at approximately 22 (n = 6) and approximately 35 C (n = 12). Con, Control.

View larger version (19K): [in this window] [in a new window]   FIG. 4. The BHQ-mediated rise in basal [Ca2+]i was dependent on extracellular Ca2+. A, In the presence of extracellular Ca2+ (5 mM), BHQ evoked a rise in basal [Ca2+]i and increased the amplitude of the depolarization-triggered Ca2+ transient. After the removal of extracellular Ca2+, BHQ evoked a very small [Ca2+]i rise. The cell was voltage clamped at –70 mV. The arrows indicate the delivery of a train of depolarization. B, Summary of the BHQ-mediated rise of basal [Ca2+]i in the absence or presence of extracellular Ca2+ (n = 12). C, The removal of extracellular Ca2+ reversed the BHQ-mediated increase in basal [Ca2+]i (representative of five cells). The cell was held at –70 mV. All experiments were at approximately 22 C.

View larger version (20K): [in this window] [in a new window]   FIG. 5. The BHQ-mediated rise in basal [Ca2+]i was due to an increase in extracellular Ca2+ influx. A and B, Simultaneous monitoring of [Ca2+]i and Mn2+ quench. The BHQ-mediated rise in basal [Ca2+]i was accompanied by an acceleration in the rate of Mn2+ quench, reflecting an increase in extracellular Ca2+ influx. The rate of Mn2+ quench was estimated from the slope of the linear fit to segments of F405 (solid line). C, Summary of the effect of BHQ on the rate of Mn2+ quench in cells whose membrane potential were unclamped (n = 21) and cells whose membrane potential were hyperpolarized by diazoxide (0.2 mM; n = 7). All experiments were at approximately 22 C.

View larger version (22K): [in this window] [in a new window]   FIG. 6. The emptying of the ER by BHQ stimulated capacitative Ca2+ entry. A, The increase in the rate of Mn2+ quench during BHQ was reversed by 2-APB (50 µM), a blocker of capacitative Ca2+ influx. B, Summary of the effect of 2-APB on the rate of Mn2+ quench in the presence of BHQ (n = 9) (same experimental protocol as in A). C, The BHQ-mediated rise in basal [Ca2+]i could be reversed by 2-APB (representative of seven cells). The cell was voltage clamped at –70 mV. All experiments were at approximately 22 C.

View larger version (30K): [in this window] [in a new window]   FIG. 7. SERCA pump inhibition enhanced the depolarization-triggered exocytotic response. A and B, The depolarization-triggered [Ca2+]i rise and exocytosis (Cm) in a cell before (control) and after BHQ exposure. Note that in the presence of BHQ, the basal [Ca2+]i was elevated, and the same train of depolarization triggered a larger Ca2+ transient and more exocytosis. C and D, Thapsigargin also caused an increase in the amplitude of the depolarization-triggered Ca2+ transient and exocytosis. The depolarization-triggered [Ca2+]i rise and exocytosis (Cm) in a cell before (control) and after thapsigargin (1 µM) exposure.

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 C_m 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 C_m 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).

View larger version (19K): [in this window] [in a new window]   FIG. 8. The increase in the peak value of the depolarization-triggered Ca2+ transient during SERCA pump inhibition is the major mechanism underlying the enhancement of exocytosis. A, The enhancement of the depolarization-triggered exocytotic response during SERCA pump inhibition was closely correlated to the increase in the peak of the depolarization-triggered Ca2+ transient. Summary of the SERCA pump inhibitor-mediated change in the peak values of the depolarization-triggered Ca2+ transient and the corresponding change in exocytotic response at approximately 22 (n = 9) and approximately 35 C (n = 13). The dashed line is the linear regression to the data at approximately 22 C (r = 0.985; P < 0.0001), and the solid line is the linear regression to the data at approximately 35 C (r = 0.855; P < 0.0002). B, Although SERCA pump inhibition elevated basal [Ca2+]i, the amount of exocytosis triggered in the presence of SERCA pump inhibitor was similar to the control when compared at similar [Ca2+]i level. Each data point was averaged from three to seven cells.

In mouse pancreatic ß-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 ß-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 ß-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 ß-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

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The present study examines the contribution of SERCA, NCX, and PMCA pumps to the Ca²⁺ dynamics of rat pancreatic ß-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 ß-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 ß-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 ß-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 ß-cells. Inhibition of the SERCA pump at the two temperatures increased the time constant of cytosolic Ca²⁺ clearance in rat ß-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 ß-cell contribute only approximately 25% of the overall Ca²⁺ clearance. Therefore, similar to what has been reported in mouse pancreatic ß-cells at 35 C (1), the SERCA pump is the dominant Ca²⁺ clearance mechanism in rat pancreatic ß-cells.

Our study also shows that NCX has minor contributions to Ca²⁺ homeostasis in rat pancreatic ß-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 ß-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 ß-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 ß-cells (at 35 C) (1), we found that the PMCA pump makes only minor contribution to the Ca²⁺ dynamics in rat pancreatic ß-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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-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 ß-cells.

A small capacitative Ca²⁺ entry has also been reported in mouse pancreatic ß-cell (17, 18). Note also that the capacitative Ca²⁺ entry in the rat pancreatic ß-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 ß-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 ß-cells is probably related to the refilling of the ER Ca²⁺ stores. Pancreatic ß-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 ß-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 ß-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 ß-cells (54, 55). Thus, the refilling of ER stores by capacitative Ca²⁺ entry may be important for ß-cell survival.

Overall, our study shows that the SERCA pump is the dominant Ca²⁺ clearance mechanisms in rat pancreatic ß-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 ß-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 ß-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 ß-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; C_m, 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.

Received August 11, 2005.

Accepted for publication November 30, 2005.

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