[Ca2+]i-reducing action of cAMP in rat pancreatic beta -cells: involvement of thapsigargin-sensitive stores

Kazuro Yaekura1,2 and Toshihiko Yada1,3

1 Department of Physiology and 2 First Department of Internal Medicine, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890; and 3 Laboratory of Intracellular Metabolism, National Institute for Physiological Sciences, Okazaki 444, Japan

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, we examined the ability of adenosine 3',5'-cyclic monophosphate (cAMP) to reduce elevated levels of cytosolic Ca2+ concentration ([Ca2+]i) in pancreatic beta -cells. [Ca2+]i and reduced pyridine nucleotide, NAD(P)H, were measured in rat single beta -cells by fura 2 and autofluorescence microfluorometry. Sustained [Ca2+]i elevation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibutyryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activator forskolin (10 µM), and an incretin glucagon-like peptide-1-(7-36) amide (10-9 M), as well as by glucose (16.7 mM). The [Ca2+]i-reducing effects of cAMP were greater at elevated glucose (8.3-16.7 mM) than at basal glucose (2.8 mM). An inhibitor of protein kinase A (PKA), H-89, counteracted [Ca2+]i-reducing effects of cAMP but not those of glucose. Okadaic acid, a phosphatase inhibitor, at 10-100 nM also reduced sustained [Ca2+]i elevation in a concentration-dependent manner. Glucose, but not DBcAMP, increased NAD(P)H in beta -cells. [Ca2+]i-reducing effects of cAMP were inhibited by 0.3 µM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+ pump. In contrast, [Ca2+]i-reducing effects of cAMP were not altered by ryanodine, an ER Ca2+-release inhibitor, Na+-free conditions, or diazoxide, an ATP-sensitive K+ channel opener. In conclusion, the cAMP-PKA pathway reduces [Ca2+]i elevation by sequestering Ca2+ in thapsigargin-sensitive stores. This process does not involve, but is potentiated by, activation of beta -cell metabolism. Together with the known [Ca2+]i-increasing action of cAMP, our results reveal dual regulation of beta -cell [Ca2+]i by the cAMP-signaling pathway and by a physiological incretin.

cytosolic Ca2+ concentration; Ca2+ sequestration; endoplasmic reticulum; protein kinase A; glucagon-like peptide-1; glucose; adenosine 3',5'-cyclic monophosphate

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

INSULIN RELEASE IN RESPONSE to glucose and other secretagogues is triggered by an increase in cytosolic Ca2+ concentration ([Ca2+]i) in pancreatic islet beta -cells (25, 33). The [Ca2+]i signaling in response to glucose exhibits a dynamic change: an initial decrease, subsequent increase, and oscillation of [Ca2+]i (14). In the presence of stimulatory glucose, a rise in adenosine 3',5'-cyclic monophosphate (cAMP), an important regulator of islet beta -cell functions (25, 33), also elicits [Ca2+]i oscillation (8, 12, 14). Simultaneous measurements of [Ca2+]i and insulin release in mouse islets revealed that [Ca2+]i oscillations are causally linked to oscillations of insulin release (3, 4, 10). We speculate that the time-coordinated [Ca2+]i signaling, including oscillations, is produced by an interplay between two mechanisms, one that increases and another that decreases [Ca2+]i. Although the former mechanism has been well elucidated in beta -cells, the latter is yet poorly understood.

It is known that cAMP increases [Ca2+]i in a glucose-dependent manner by enhancing Ca2+ influx through the L-type Ca2+ channel (8, 12, 16, 31, 35), in which activation of an Na+-permeable channel has been suggested (16). In our previous study, which was designed to determine whether cAMP has a direct Ca2+ channel agonist action, we examined the effect of cAMP-increasing agents on sustained [Ca2+]i elevation induced by high KCl. cAMP not only failed to further increase [Ca2+]i (38), it rather, to our surprise, reduced the elevated [Ca2+]i. It was also previously reported that cAMP reduces the glucose-induced [Ca2+]i increase in suspension of islet cells (28). In the present study, we attempted to elucidate the underlying mechanism by which cAMP-increasing agents reduce [Ca2+]i elevation. We also examined the effect of glucagon-like peptide-1 (GLP-1), a physiological incretin that stimulates the cAMP-signaling pathway (16, 23, 32, 35). [Ca2+]i and reduced pyridine nucleotide, NAD(P)H, as an indicator of energy metabolism (9, 21, 24), were measured in rat pancreatic beta -cells. We found that cAMP attenuates [Ca2+]i elevation by promoting Ca2+ sequestration in the endoplasmic reticulum (ER) in pancreatic beta -cells and that GLP-1 mimics the cAMP effect.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Preparation of single islet cells and selection of beta -cells. Islets of Langerhans were isolated, by collagenase digestion, from Wistar rats aged 8-12 wk. Animals were anesthetized by intraperitoneal injection of pentobarbital sodium at 80 mg/kg. The abdomen was opened, and collagenase (3 mg/ml) dissolved in 6 ml of 5 mM Ca2+-containing Krebs-Ringer bicarbonate buffer (KRB) solution was injected into the common bile duct at the distal end after ligation of the duct proximal to the pancreas. The pancreas was dissected out and incubated at 37°C for 17 min. Islets were collected and immediately dispersed into single cells in Ca2+-free KRB. The single cells were plated on coverslips and maintained in short-term culture for up to 3 days in Eagle's minimum essential medium containing 5.6 mM glucose supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin at 37°C in a 95% air-5% CO2 atmosphere. During the culture period, no appreciable change was observed in the frequency or the pattern of [Ca2+]i responses to glucose, tolbutamide, KCl, hormones, or cAMP-increasing agents.

beta -Cells were selected among single islet cells according to the previously reported procedure (34, 35). Briefly, single islet cells that had a diameter of 12.5-17.5 µm on coverslips and responded to glucose (8.3 or 16.7 mM) and tolbutamide (300 mM) with increases in [Ca2+]i were found to be immunocytochemically positive for insulin (37). Data were taken from the cells that fulfilled these morphological and physiological criteria for beta -cells.

Solutions and chemicals. Measurements were carried out in KRB solution composed of (in mM) 129 NaCl, 5.0 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.0 CaCl2, 1.2 MgSO4, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid at pH 7.4 supplemented with 0.1% bovine serum albumin. Na+-free KRB was made by replacement of Na+ with equimolar N-methyl-D-glucamine (NMDG; Nacalai Tesque, Kyoto, Japan). Fura 2 and fura 2-acetoxymethyl ester (AM) were obtained from Dojin Chemical (Kumamoto, Japan), and dibutyryl cAMP (DBcAMP) was obtained from Boehringer Mannheim (Indianapolis, IN). H-89 [N-[2-(p-bromocinnamylamine)ethyl]-5-isoquinoline-sulfonamide] was provided by Dr. H. Hidaka and also obtained from Seikagaku Kogyo (Tokyo, Japan). All other chemicals were from Sigma (St. Louis, MO).

Measurements of [Ca2+]i. [Ca2+]i was measured by dual-wavelength fura 2 microfluorometry combined with imaging, as previously reported (36, 37). Briefly, cells on coverslips were incubated with 1 µM fura 2-AM for 30 min at 37°C in KRB containing 2.8 mM glucose. Cells were then mounted in a chamber and superfused at a rate of 1 ml/min at 37°C in KRB. Cells were excited at 340 and 380 nm alternately every 2.5 s, emission signals at 510 nm (F340 and F380, respectively) were detected with an intensified charge-coupled device camera, and ratio (F340/F380) images were produced by an Argus-50 system (Hamamatsu Photonics, Hamamatsu, Japan). Ratio values were converted to [Ca2+]i according to calibration curves.

Measurements of NAD(P)H. Autofluorescence of NAD(P)H in two to eight beta -cells in cluster was measured under superfusion conditions identical to those used for [Ca2+]i measurements. Cells were excited at 360 nm every 1 s, and emission signals through a 470 ± 20-nm band-pass filter were detected by a high-sensitivity photomultiplier using a P101 system (Nikon, Tokyo, Japan).

Criteria for [Ca2+]i-reducing response and determination of response amplitude. The amplitude of [Ca2+]i reduction in response to cAMP-increasing agents was determined at the time point of maximal reduction of [Ca2+]i. When the KCl-induced [Ca2+]i elevation was not flat but mildly declining or increasing at a stable rate, an extrapolated curve was drawn based on the [Ca2+]i levels before addition and 5-10 min after washing out of cAMP agents, and the maximal deviation from the extrapolated curve was taken as the amplitude of [Ca2+]i reduction. Only the deviation that was >0.1 ratio (F340/F380) unit from the extrapolated curve and which took place in a clear shape within 5 min upon exposure to cAMP agents was considered as the response.

Statistical analyses. All data are presented as means ± SE (n = number of observations). The statistical analysis was carried out by Student's t-test. Differences were considered statistically significant when P < 0.05.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

KCl-induced sustained [Ca2+]i elevation and its attenuation by cAMP-increasing agents and GLP-1. To examine whether cAMP has an ability to reduce [Ca2+]i elevation, [Ca2+]i was clamped at high levels and the effect of cAMP was examined. At a basal glucose concentration of 2.8 mM, KCl at a depolarizing concentration (25 mM) induced a sharp initial peak followed by a sustained elevation of [Ca2+]i in single beta -cells (Fig. 1). The level of the sustained [Ca2+]i elevation either stayed constant or changed slowly and mildly at a stable rate. The KCl-induced sustained elevation of [Ca2+]i was reduced by membrane-permeable cAMP analogs 5 mM DBcAMP (Fig. 1A and Table 1) and 1 mM 8-bromo-cAMP (data not shown), by 10 µM forskolin (Fig. 1B), and by 1 nM GLP-1-(7-36) amide (GLP-1) (Fig. 1C). DBcAMP, forskolin, and GLP-1 evoked [Ca2+]i reduction in ~80% of the single beta -cells. When DBcAMP was combined with 0.3 mM 3-isobutyl-1-methylxanthine, the percentage of cells showing [Ca2+]i reduction was further increased to ~90%. The reduction of [Ca2+]i started 0.5-1 min after administration of the cAMP-increasing agents, peaked at 2-5 min, and then gradually declined or stayed at the peak. In the presence of glucose concentrations that are stimulatory for the beta -cell [Ca2+]i and insulin release (8.3 and 16.7 mM) and in the absence of 25 mM KCl, these cAMP-increasing agents and hormone evoked increases in [Ca2+]i, thereby exhibiting a dual effect on [Ca2+]i (Fig. 1).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Adenosine 3',5'-cyclic monophosphate (cAMP)-increasing agents reduce sustained elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by high K+ at basal glucose as well as increasing [Ca2+]i at stimulatory glucose in single beta -cells. KCl (25 mM) induced a sustained elevation of [Ca2+]i in single beta -cells. Five milimolar dibutyryl cAMP (DBcAMP) (A), 10 µM forskolin (B), and 1 nM glucagon-like peptide-1 (GLP-1)-(7-36) amide (C) attenuated the sustained [Ca2+]i elevation induced by high K+ at 2.8 mM glucose (G2.8). These agents also increased [Ca2+]i in the presence of 8.3 mM glucose (G8.3) and at normal K+. [Ca2+]i responses to G8.3 and 300 µM tolbutamide (Tolb) are also shown. Glucose concentration is indicated at top. Bars above tracing specify period of exposure to agents specified. Dotted lines indicate beginning of exposure to cAMP-increasing agents. Results are representative of 17 of 21 single beta -cells examined in A, 20 of 28 cells in B, and 16 of 21 cells in C.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   [Ca2+]i reduction in response to DBcAMP and okadaic acid, a phosphatase inhibitor, and effect of H-89, a PKA inhibitor

Glucose dependence of [Ca2+]i-reducing effects of cAMP. At stimulatory glucose (8.3 and 16.7 mM) concentrations, DBcAMP and forskolin reduced KCl-induced [Ca2+]i elevation to a greater extent than at 2.8 mM (Fig. 2, A and B). The amplitude of [Ca2+]i reduction was significantly increased when glucose concentration was raised (Fig. 3).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   cAMP-increasing agents reduce sustained [Ca2+]i elevation induced by high K+ at stimulatory glucose in single beta -cells. In the presence of a moderate elevation of [Ca2+]i induced by 8.3 and 16.7 mM glucose, 25 mM KCl produced a further elevation of [Ca2+]i in a sustained manner in single beta -cells. Five milimolar DBcAMP (A) and 10 µM forskolin (B) attenuated the sustained [Ca2+]i elevation induced by high K+. Magnitude of attenuation of [Ca2+]i elevation was greater at stimulatory glucose than at basal glucose (see Fig. 1). Results are representative of 23 of 26 cells in A and 28 of 30 cells in B.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 3.   Glucose concentration dependence of [Ca2+]i-reducing action of cAMP in single beta -cells. Amplitude of reduction of high K+-induced [Ca2+]i elevation by 5 mM DBcAMP (solid bars) and 10 µM forskolin (hatched bars) at 2.8, 8.3, and 16.7 mM glucose in single beta -cells. Experimental protocol is the same as in Figs. 1 and 2. No. of cells examined given at top of bars. * P < 0.001 vs. DBcAMP at G2.8, # P < 0.005 vs. forskolin at G2.8, and ** P < 0.001 vs. DBcAMP at G2.8 and P < 0.01 vs. DBcAMP at G8.3.

[Ca2+]i-reducing effects of cAMP, glucose, and a phosphatase inhibitor, and selective inhibition of cAMP effects by a PKA inhibitor. The KCl-induced [Ca2+]i elevation was attenuated both by DBcAMP and by high glucose (16.7 mM) in single beta -cells (Fig. 4A). After the same cells had been treated for 20 min with 40 µM H-89, an inhibitor of protein kinase A (PKA), the amplitude of [Ca2+]i reduction by DBcAMP was significantly suppressed, whereas that by glucose was unchanged (Fig. 4A and Table 1). H-89 at 10 µM also suppressed the effect of DBcAMP to reduce [Ca2+]i to a lesser extent (data not shown). In control experiments, when DBcAMP was successively administered two times, the cells exhibited [Ca2+]i-reducing responses twice without appreciable change in their amplitudes (Fig. 4B). Okadaic acid, an inhibitor of phosphatase, at 10-100 nM also attenuated the KCl-induced [Ca2+]i elevation in a concentration-dependent manner (Fig. 4, C and D, and Table 1). Okadaic acid at 100 nM, a maximal concentration, evoked [Ca2+]i reduction in a similar pattern and with a similar amplitude to that induced by DBcAMP (Fig. 4D and Table 1).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 4.   Distinct properties between cAMP- vs. glucose-induced [Ca2+]i reduction. A: in presence of 25 mM KCl and sustained [Ca2+]i elevation, 5 mM DBcAMP and 16.7 mM glucose (G16.7) were administered in pulses in a single beta -cell (left). The cell was then treated for 20 min with 40 µM H-89, a protein kinase A (PKA) inhibitor (arrow), followed by the recording shown on right. Note that [Ca2+]i-reducing effect of cAMP, but not that of glucose, was markedly suppressed by a PKA inhibitor. Treatment with H-89 had no effect on subsequent [Ca2+]i responses to stimulatory glucose (8.3 mM) and tolbutamide (300 µM). B: in presence of 25 mM KCl and sustained [Ca2+]i elevation, successive administration of 5 mM DBcAMP evoked [Ca2+]i-reducing responses two times sequentially without appreciable change in their amplitudes in a single beta -cell. C: okadaic acid (OA), an inhibitor of phosphatase, at 10 and 30 nM attenuated the KCl-induced [Ca2+]i elevation in a concentration-dependent manner in a single beta -cell. D: OA at 100 nM attenuated the 25 mM KCl-induced sustained [Ca2+]i elevation in a manner similar to 5 mM DBcAMP in a single beta -cell. Treatment with OA had no effect on subsequent [Ca2+]i responses to stimulatory glucose and tolbutamide. E: effects of DBcAMP and glucose on NAD(P)H levels in beta -cells. NAD(P)H autofluorescence at 470 nm in 3 beta -cells in cluster is expressed as a percentage relative to the signal at the resting state with 2.8 mM glucose before KCl addition. Note that 16.7 mM glucose, but not 5 mM DBcAMP, increased fluorescence. Glucose concentration was 2.8 mM unless otherwise indicated in A-E. Results shown are representative of 13 of 17 cells in A, 8 of 12 cells in B, 25 of 28 cells in C, and 26 of 28 cells in D, whereas they represent 4 similar experiments in E.

Effects of cAMP and glucose on NAD(P)H levels in beta -cells. Because the metabolic activity is known to control the level of [Ca2+]i (7, 25), we next measured intracellular NAD(P)H as an indicator of the metabolic activity. The NAD(P)H autofluorescence from beta -cells at basal glucose (2.8 mM) was not significantly altered by 25 mM KCl. In the presence of 25 mM KCl, DBcAMP produced no change, whereas 16.7 mM glucose rapidly and markedly increased fluorescence (Fig. 4E).

Effects of inhibitors of ER Ca2+ pump and Ca2+-release channel on [Ca2+]i-reducing action of cAMP. In the presence of 0.3 µM thapsigargin (TG), an inhibitor of the ER Ca2+ pump (20, 30), KCl increased [Ca2+]i to a level somewhat higher than that of control. cAMP-increasing agents failed to reduce the elevated levels of [Ca2+]i in the majority of single beta -cells (Fig. 5); thus the fraction of cells responding to cAMP-increasing agents with [Ca2+]i reduction was dramatically reduced (for the response to DBcAMP, 21.7% with TG vs. 76.6% in control) (Table 2). The amplitude of the [Ca2+]i reduction in the responding cells was also markedly attenuated [19.7 ± 2.5 nM (n = 5) with TG vs. 94.4 ± 4.1 nM (n = 23) in control, P < 0.0001] (Table 2). [Ca2+]i-reducing effects of glucose (8.3 and 16.7 mM) were also inhibited by TG, confirming previous reports (6, 13). In contrast, 20 µM ryanodine, an inhibitor of the ryanodine-sensitive Ca2+-release channel in ER, affected neither the level of the KCl-induced [Ca2+]i elevation nor the cAMP action to attenuate this level (Fig. 6A and Table 2).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5.   [Ca2+]i-reducing action of cAMP is inhibited by an endoplasmic reticulum (ER) Ca2+ pump blocker in single beta -cells. Effect of 5 mM DBcAMP on the 25 mM KCl-induced [Ca2+]i elevation in absence and presence of 0.3 µM thapsigargin (TG) in single beta -cells. At time when administration of DBcAMP was started (indicated by dashed lines), [Ca2+]i level was lower in the test (with TG) than in control (A), whereas it was higher in the test than in control in B. Note that [Ca2+]i-reducing effect was inhibited by TG, irrespective of whether the [Ca2+]i level at beginning of DBcAMP administration in the test was lower or higher than in control. After treatment with TG, cells responded to 8.3 mM glucose and 300 µM tolbutamide with increases in [Ca2+]i. These results, showing inhibition of [Ca2+]i-reducing effect of cAMP by TG, are representative of 18 of 23 cells.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Percentage of cells responding to cAMP with [Ca2+]i reduction and mean amplitude of [Ca2+]i reduction in single beta -cells under conditions that alter endoplasmic reticulum and plasma membrane Ca2+ transport


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of conditions that modify ER Ca2+ release, plasma membrane Na+/Ca2+ exchange, and ATP-sensitive K+ channel (KATP) on [Ca2+]i-reducing action of cAMP in single beta -cells. A: in presence of 20 µM ryanodine, 10 µM forskolin reduced sustained [Ca2+]i elevation induced by 25 mM KCl in a single beta -cell. B and C: under Na+-free conditions, in which Na+ was replaced with N-methyl-D-glucamine, 5 mM DBcAMP reduced the sustained [Ca2+]i elevation induced by 25 mM KCl in a manner similar to control with normal Na+ at both 2.8 mM (B) and 16.7 mM glucose (C) in single beta -cells. D: in presence of 0.4 mM diazoxide (DZ), a KATP opener, 5 mM DBcAMP reduced the sustained [Ca2+]i elevation induced by 25 mM KCl in a single beta -cell. E: sustained [Ca2+]i elevation induced by 1 mM tolbutamide, a KATP blocker, was also reduced by 5 mM DBcAMP in a single beta -cell. Glucose concentration was 2.8 mM in A, B, D, and E, whereas it was 16.7 mM in C. Results shown are representative of 16 of 18 cells in A, 21 of 27 cells in B, 17 of 21 cells in C, 20 of 28 cells in D, and 11 of 17 cells in E.

Effects of inhibition or activation of plasma membrane Na+/Ca2+ exchange and KATP on [Ca2+]i-reducing action of cAMP. It has been shown that the plasma membrane Na+/Ca2+ exchange serves as a Ca2+ extrusion mechanism in beta -cells (15) and plays a greater role at elevated glucose than at basal glucose in the regulation of [Ca2+]i (13, 38). Therefore, we assessed an involvement of Na+/Ca2+ exchange in the cAMP-induced [Ca2+]i reduction at basal and elevated glucose concentrations by examining the effect of Na+-free conditions that inhibit Na+/Ca2+ exchange. Na+-free conditions were achieved by replacement of extracellular Na+ with NMDG. Under Na+-free conditions, high K+-induced sustained [Ca2+]i elevation was attenuated by DBcAMP in a manner similar to control conditions, and this was observed at both basal and elevated glucose concentrations (Fig. 6, B and C, and Table 2).

It is also possible that cAMP reduces [Ca2+]i by influencing the ATP-sensitive K+ channel (KATP), which is the major determinant of the beta -cell resting membrane potential (2). This possibility was examined by testing the effect of cAMP under conditions in which KATP was fixed in either a maximally open or closed state. A sustained [Ca2+]i elevation induced by KCl plus the KATP opener diazoxide at 0.4 mM, a concentration that fully opens this channel (2, 10), was attenuated by DBcAMP (Fig. 6D and Table 2) and by forskolin (data not shown). The KATP blocker tolbutamide at 1 mM, a supramaximal concentration for complete closure of this channel (2, 9), induced sustained elevation of [Ca2+]i, which was attenuated by DBcAMP (Fig. 6E). These results indicate that neither Na+/Ca2+ exchange nor KATP is involved in the [Ca2+]i-reducing action of cAMP.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study has shown that membrane-permeable cAMP analogs, forskolin and GLP-1, agents that increase cAMP, all reduce sustained [Ca2+]i elevation induced by high K+ and tolbutamide, thereby revealing the [Ca2+]i-reducing action of cAMP in pancreatic beta -cells. The [Ca2+]i reduction by cAMP was abolished or markedly inhibited by TG at the concentration that specifically inhibits glucose-induced [Ca2+]i decrease, a process mediated by Ca2+ sequestration in ER (13). It is well established that TG inhibits the ER and sarcoplasmic reticulum (SR) Ca2+-ATPase (Ca2+ pump) activity without influencing the plasma membrane Ca2+-ATPase and Na+-K+-ATPase in a variety of preparations (20, 30). In contrast, [Ca2+]i-reducing effects of cAMP were observed similarly in the absence and presence of ryanodine. Therefore, it is unlikely that cAMP attenuates [Ca2+]i elevation by inhibiting the ryanodine-sensitive Ca2+ release from ER. The [Ca2+]i reduction could, alternatively, be due to alteration of ion transport systems in the beta -cell plasma membrane. Na+/Ca2+ exchange functions as a Ca2+ extrusion mechanism in beta -cells (15, 38). However, [Ca2+]i-reducing effects of cAMP were unaltered under Na+-free conditions, irrespective of whether the glucose concentration was basal or stimulatory. Therefore, the cAMP-induced [Ca2+]i reduction is not accounted for by stimulation of Na+/Ca2+ exchange at the plasma membrane. Another possibility is that cAMP reduces [Ca2+]i via influencing KATP, the channel that mainly determines the resting membrane potential of beta -cells. In the present study, however, in the presence of the KATP opener diazoxide at 0.4 mM, a supermaximal concentration that fully opens this channel (2), the KCl-induced [Ca2+]i elevation was also reduced by cAMP. Likewise, the KATP blocker tolbutamide at 1 mM, a concentration that fixes this channel at the completely closed state (13), induced sustained [Ca2+]i elevation, and it was also attenuated by cAMP. Thus KATP appears not to be involved in [Ca2+]i-reducing effects of cAMP. Taken together, it is concluded that [Ca2+]i-reducing effects of cAMP are due primarily to stimulation of Ca2+ sequestration in the TG-sensitive ER in beta -cells.

Glucose also stimulates Ca2+ sequestration in the TG-sensitive ER in beta -cells, accounting for the glucose-induced initial decrease in [Ca2+]i (6, 11, 13, 26, 27, 36). It is likely that the glucose-induced [Ca2+]i reduction is due to activation of beta -cell metabolism for the following reasons. Glucose is metabolized by beta -cells, resulting in production of ATP from ADP (2); the ATP/ADP ratio is a regulatory factor of the Ca2+-pumping ATPase; and the steady-state [Ca2+]i varies inversely with the ATP/ADP ratio in the permeabilized insulinoma cells (7). It is possible that activation of beta -cell metabolism is also involved in the cAMP-induced [Ca2+]i reduction. In the present study, however, the NAD(P)H level in beta -cells, an indicator of metabolism, was not significantly altered by cAMP, whereas it was markedly increased by high glucose, as previously reported (9, 24). In contrast, the [Ca2+]i-reducing effect of cAMP, but not that of high glucose, was inhibited by pretreatment with the PKA inhibitor H-89. Furthermore, okadaic acid, an inhibitor of phosphatase, mimicked cAMP-increasing agents to reduce the KCl-induced [Ca2+]i elevation. These findings indicate that cAMP and glucose reduce [Ca2+]i elevation by distinct mechanisms and that PKA-mediated phosphorylation may be involved in the [Ca2+]i-reducing action of cAMP.

Although the [Ca2+]i-reducing effect of cAMP does not involve activation of beta -cell metabolism, it was substantially enhanced as the glucose concentration was increased. Thus interactive effects of cAMP and glucose were demonstrated. This interaction may be accounted for by the following hypothetical mechanism. The cAMP-dependent phosphorylation pathway stimulates Ca2+ sequestration in ER by upregulating the active form of Ca2+-ATPase (Ca2+ pump); because Ca2+-ATPase is an ATP-dependent enzyme, higher ATP levels at higher glucose concentrations make more Ca2+-ATPase in the active form, thereby allowing a larger potentiation of the Ca2+-ATPase activity by cAMP. However, precise mechanisms for the interaction between cAMP and glucose in reducing [Ca2+]i are yet to be elucidated.

Because the [Ca2+]i-reducing action of cAMP is blocked by an ER Ca2+ pump inhibitor and a PKA inhibitor and mimicked by a phosphatase inhibitor, a possible involvement of PKA-mediated phosphorylation in the TG-sensitive Ca2+ sequestration in ER is suggested. It has been shown that glucagon stimulates Ca2+ uptake in ER of liver cells (1, 5), cAMP mimics this effect (1, 29), and Ca2+-pumping activity is enhanced by PKA (22). In cardiac muscles, phospholamban, a membrane-associated protein in SR, is a substrate of PKA, and the ratio of phosphorylated to unphosphorylated phospholamban correlates with the Ca2+ transport enzyme activity and the rate of Ca2+ uptake in SR (17, 18, 22). However, phospholamban or related proteins have not yet been demonstrated in pancreas. The mechanisms that link PKA to the ER Ca2+ sequestration in pancreatic beta -cells remain to be investigated.

It should be noted that administration of cAMP-increasing agents induced large transients of [Ca2+]i in 6.6% of beta -cells (data not shown). These [Ca2+]i transients took place usually before, and occasionally during, the [Ca2+]i reduction. The duration of a transient was 30-120 s. A previous study (19) reported that cAMP induces the pronounced transients of [Ca2+]i in glucose- or KCl-stimulated beta -cells and suggested a mechanism by which cAMP sensitizes the inositol 1,4,5-trisphosphate (IP3) receptor to stimulate mobilization of intracellular Ca2+. Therefore, our [Ca2+]i transients could reflect the IP3-mediated Ca2+ mobilization. However, the time resolution of our [Ca2+]i measurements may not be fast enough to detect short transients evoked by IP3. It should be emphasized that [Ca2+]i-reducing effects of cAMP were observed irrespective of whether the [Ca2+]i transients occurred. A link between the cAMP-induced [Ca2+]i reduction and [Ca2+]i transients remains unknown.

The [Ca2+]i-reducing action of cAMP may play a role in optimizing the amplitude of [Ca2+]i signals, thereby finely controlling Ca2+-dependent functions in beta -cells. It is also suggested that the [Ca2+]i-reducing action of cAMP takes part in buffering an excessive rise in [Ca2+]i, a putative cytotoxic signal which leads to beta -cell dysfunction and death. On the other hand, it is well established that cAMP increases [Ca2+]i in a glucose-dependent manner in rat beta -cells. Thus the present study reveals a dual action of cAMP on [Ca2+]i. The dual function of the cAMP-signaling pathway may play a role in producing frequency-coded [Ca2+]i signals, such as oscillations of [Ca2+]i, in pancreatic beta -cells.

    ACKNOWLEDGEMENTS

We thank Dr. Geoffrey W. G. Sharp for carefully reading the manuscript. We are grateful to Dr. H. Hidaka at Nagoya University for kindly providing H-89 and to Drs. M. Kakei and H. Tanaka at Kagoshima University for encouragement during this study.

    FOOTNOTES

This research was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan (to T. Yada).

Address for reprint requests: T. Yada, Dept. of Physiology, Kagoshima Univ. School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890, Japan.

Received 25 June 1997; accepted in final form 27 October 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Andia-Waltenbaugh, A. M., A. Lam, L. Hummel, and N. Friedmann. Characterization of the hormone-sensitive Ca2+ uptake activity of the hepatic endoplasmic reticulum. Biochim. Biophys. Acta 630: 165-175, 1980[Medline].

2.   Ashcroft, F. M., and P. Rorsman. Electrophysiology of the pancreatic beta -cell. Prog. Biophys. Mol. Biol. 54: 87-143, 1989[Medline].

3.   Bergsten, P. Slow and fast oscillations of cytoplasmic Ca2+ in pancreatic islets correspond to pulsatile insulin release. Am. J. Physiol. 268 (Endocrinol. Metab. 31): E282-E287, 1995[Abstract/Free Full Text].

4.   Bergsten, P., E. Grapengiesser, E. Gylfe, A. Tengholm, and B. Hellman. Synchronous oscillations of cytoplasmic Ca2+ and insulin release in glucose-stimulated pancreatic islets. J. Biol. Chem. 269: 8749-8753, 1994[Abstract/Free Full Text].

5.   Bygrave, F. L., and C. J. Tranter. The subcellular location, maturation and response to increased plasma glucagon of ruthenium red-insensitive calcium-ion transport in rat liver. Biochem. J. 174: 1021-1030, 1978[Medline].

6.   Chow, R. H., P. E. Lund, S. Loser, U. Panten, and E. Gylfe. Coincidence of early glucose-induced depolarization with lowering of cytoplasmic Ca2+ in mouse pancreatic beta -cells. J. Physiol. (Lond.) 485: 607-617, 1995[Abstract].

7.   Corkey, B. E., J. T. Deeney, M. C. Glennon, F. M. Matschinsky, and M. Prentki. Regulation of steady-state free Ca2+ levels by the ATP/ADP ratio and orthophosphate in permeabilized RINm5F insulinoma cells. J. Biol. Chem. 263: 4247-4253, 1988[Abstract/Free Full Text].

8.   Fournier, L., J. F. Whitfield, J. L. Schwartz, and N. Begin-Heick. Cyclic AMP triggers large [Ca2+]i oscillations in glucose-stimulated beta -cells from ob/ob mice. J. Biol. Chem. 269: 1120-1124, 1994[Abstract/Free Full Text].

9.   Gilon, P., and J. C. Henquin. Influence of membrane potential changes on cytoplasmic Ca2+ concentration in an electrically excitable cell, the insulin-secreting pancreatic B-cell. J. Biol. Chem. 267: 20713-20720, 1992[Abstract/Free Full Text].

10.   Gilon, P., R. M. Shepherd, and J. C. Henquin. Oscillations of secretion driven by oscillations of cytoplasmic Ca2+ as evidenced in single pancreatic islets. J. Biol. Chem. 268: 22265-22268, 1993[Abstract/Free Full Text].

11.   Grapengiesser, E., E. Gylfe, and B. Hellman. Dual effect of glucose on cytoplasmic Ca2+ in single pancreatic beta -cells. Biochem. Biophys. Res. Commun. 150: 419-425, 1988[Medline].

12.   Grapengiesser, E., E. Gylfe, and B. Hellman. Cyclic AMP as a determinant for glucose induction of fast Ca2+ oscillations in isolated pancreatic beta -cells. J. Biol. Chem. 266: 12207-12210, 1991[Abstract/Free Full Text].

13.   Hamakawa, N., and T. Yada. Interplay of glucose-stimulated Ca2+ sequestration and acetylcholine-induced Ca2+ release at the endoplasmic reticulum in rat pancreatic beta -cells. Cell Calcium 17: 21-31, 1995[Medline].

14.   Hellman, B., E. Gylfe, E. Grapengiesser, P. E. Lund, and A. Berts. Cytoplasmic Ca2+ oscillations in pancreatic beta -cells. Biochim. Biophys. Acta 1113: 295-305, 1992[Medline].

15.   Herchuelz, A., and P. Lebrun. A role for Na/Ca exchange in the pancreatic B cell. Studies with thapsigargin and caffeine. Biochem. Pharmacol. 45: 7-11, 1993[Medline].

16.   Holz, G. G., C. A. Leech, and J. F. Habener. Activation of a cAMP-regulated Ca2+ signaling pathway in pancreatic beta -cells by the insulinotropic hormone glucagon-like peptide-1. J. Biol. Chem. 270: 17749-17757, 1995[Abstract/Free Full Text].

17.   Kasinathan, C., Z. C. Xu, and M. A. Kirchberger. Differences in calcium uptake in native cardiac microsomes are correlated with the ratio of unphosphorylated to phosphorylated phospholamban as determined by Western blot analysis. Biochem. Biophys. Res. Commun. 157: 1296-1301, 1988[Medline].

18.   Kirchberger, M. A., M. Tada, and A. Katz. Adenosine 3',5'-monophosphate-dependent protein kinase-catalyzed phosphorylation reaction and its relationship to Ca2+ transport in cardiac sarcoplasmic reticulum. J. Biol. Chem. 249: 6166-6173, 1974[Abstract/Free Full Text].

19.   Liu, Y. J., E. Grapengiesser, E. Gylfe, and B. Hellman. Crosstalk between the cAMP and inositol trisphosphate-signalling pathways in pancreatic beta -cells. Arch. Biochem. Biophys. 334: 295-302, 1996[Medline].

20.   Lytton, J., M. Westlin, and M. R. Hanley. Thapsigargin inhibits the sarcoplasmic and endoplasmic reticulum Ca-ATPase family of calcium pumps. J. Biol. Chem. 266: 17067-17071, 1991[Abstract/Free Full Text].

21.   Malaisse, W. J., A. Sener, A. Herchuelz, and J. C. Hutton. Insulin release: the fuel hypothesis. Metabolism 28: 373-386, 1979[Medline].

22.   Missiaen, L., F. Wuytack, L. Raeymaekers, H. D. Smedt, G. Droogmans, S. D. Jaegere, and R. Casteels. Intracellular Messengers. Oxford, UK: Pergamon, 1993, p. 347-405.

23.   Ørskov, C. Glucagon-like peptide-1, a new hormone of the entero-insular axis. Diabetologia 35: 701-711, 1992[Medline].

24.   Pralong, W. F., C. Bartley, and C. B. Wollheim. Single islet beta -cells stimulation by nutrients: relationship between pyridine nucleotides, cytosolic Ca2+ and secretion. EMBO J. 9: 53-60, 1990[Abstract].

25.   Prentki, M., and F. M. Matschinsky. Ca2+, cAMP, and phospholipid-derived messengers in coupling mechanisms of insulin secretion. Physiol. Rev. 67: 1185-1248, 1987[Free Full Text].

26.   Roe, M. W., R. J. Mertz, M. E. Lancaster, J. F. Worley, and I. D. Dukes. Thapsigargin inhibits the glucose-induced decrease of intracellular Ca2+ in mouse islets of Langerhans. Am. J. Physiol. 266 (Endocrinol. Metab. 29): E852-E862, 1994[Abstract/Free Full Text].

27.   Roe, M. W., L. H. Philipson, C. J. Frangakis, A. Kuznetsov, R. J. Mertz, M. E. Lancaster, B. Spencer, J. F. Worley, and I. D. Dukes. Defective glucose-dependent endoplasmic reticulum Ca2+ sequestration in diabetic mouse islets of Langerhans. J. Biol. Chem. 269: 18279-18282, 1994[Abstract/Free Full Text].

28.   Rorsman, P., and H. Abrahamsson. Cyclic AMP potentiates glucose-induced insulin release from mouse pancreatic islets without increasing cytosolic free Ca2+. Acta Physiol. Scand. 125: 639-647, 1985[Medline].

29.   Tayler, W. M., P. Reinhart, N. H. Hunt, and F. L. Bygrave. Role of 3',5'-cyclic AMP in glucagon-induced stimulation of ruthenium red-insensitive calcium transport in an endoplasmic reticulum-rich fraction of rat liver. FEBS Lett. 12: 92-96, 1980.

30.   Thastrup, O., P. J. Cullen, B. K. Drobak, M. R. Hanley, and A. P. Dawson. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc. Natl. Acad. Sci. USA 87: 2466-2470, 1990[Abstract].

31.   Wang, J. L., J. A. Corbett, C. A. Marshall, and M. L. McDaniel. Glucose-induced insulin secretion from purified beta -cells. A role for modulation of Ca2+ influx by cAMP- and protein kinase C-dependent signal transduction pathways. J. Biol. Chem. 268: 7785-7791, 1993[Abstract/Free Full Text].

32.   Wang, Z., R. M. Wang, A. A. Owji, D. M. Smith, M. A. Ghayei, and S. R. Bloom. Glucagon-like peptide-1 is a physiological incretin in rat. J. Clin. Invest. 95: 417-421, 1995[Medline].

33.   Wollheim, C. B., and G. W. G. Sharp. Regulation of insulin release by calcium. Physiol. Rev. 61: 914-973, 1981[Free Full Text].

34.   Yada, T., N. Hamakawa, and K. Yaekura. Two distinct modes of Ca2+ signalling by ACh in rat pancreatic beta -cells: concentration, glucose dependence and Ca2+ origin. J. Physiol. (Lond.) 488: 13-24, 1995[Abstract].

35.   Yada, T., K. Itoh, and M. Nakata. Glucagon-like peptide-1-(7-36)amide and a rise in cyclic adenosine 3',5'-monophosphate increase cytosolic free Ca2+ in rat pancreatic beta -cells by enhancing Ca2+ channel activity. Endocrinology 133: 1685-1692, 1993[Abstract].

36.   Yada, T., M. Kakei, and H. Tanaka. Single pancreatic beta -cells from normal rats exhibit an initial decrease and subsequent increase in cytosolic free Ca2+ in response to glucose. Cell Calcium 13: 69-76, 1992[Medline].

37.   Yada, T., M. Sakurada, K. Ihida, M. Nakata, F. Murata, A. Arimura, and M. Kikuchi. Pituitary adenylate cyclase activating polypeptide is an extraordinarily potent intra-pancreatic regulator of insulin secretion from islet beta -cells. J. Biol. Chem. 269: 1290-1293, 1994[Abstract/Free Full Text].

38.   Yaekura, K., M. Kakei, and T. Yada. cAMP-signalling pathway acts in selective synergism with glucose or tolbutamide to increase cytosolic Ca2+ in rat pancreatic beta -cells. Diabetes 45: 295-301, 1996[Abstract].


AJP Cell Physiol 274(2):C513-C521
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society