Dual effect of nitric oxide on cytosolic Ca2+ concentration and insulin secretion in rat pancreatic beta -cells

Yukiko Kaneko, Tomohisa Ishikawa, Satoshi Amano, and Koichi Nakayama

Department of Pharmacology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka City, Shizuoka 422-8526, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
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In isolated rat pancreatic beta -cells, the nitric oxide (NO) donor NOC-7 at 1 µM reduced the amplitude of the oscillations of cytosolic Ca2+ concentration ([Ca2+]c) induced by 11.1 mM glucose, and at 10 µM terminated them. In the presence of NG-nitro-L-arginine (L-NNA), however, NOC-7 at 0.5 and 1 µM increased the amplitude of the [Ca2+]c oscillations, although the NO donor at 10 µM still suppressed them. Aqueous NO solution also had a dual effect on the [Ca2+]c oscillations. The soluble guanylate cyclase inhibitor LY-83583 and the cGMP-dependent protein kinase inhibitor KT5823 inhibited the stimulatory effect of NO, and 8-bromo-cGMP increased the amplitude of the [Ca2+]c oscillations. Patch-clamp analyses in the perforated configuration showed that 8-bromo-cGMP inhibited whole cell ATP-sensitive K+ currents in the isolated rat pancreatic beta -cells, suggesting that the inhibition by cGMP of ATP-sensitive K+ channels is, at least in part, responsible for the stimulatory effect of NO on the [Ca2+]c oscillations. In the presence of L-NNA, the glucose-induced insulin secretion from isolated islets was facilitated by 0.5 µM NOC-7, whereas it was suppressed by 10 µM NOC-7. These results suggest that NO facilitates glucose-induced [Ca2+]c oscillations of beta -cells and insulin secretion at low concentrations, which effects are mediated by cGMP, whereas NO inhibits them in a cGMP-independent manner at high concentrations.

islets of Langerhans; calcium oscillations; guanosine 3',5'-cyclic monophosphate; NOC-7; glucose


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INTRODUCTION
METHODS
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NITRIC OXIDE (NO) produced from L-arginine by NO synthase (NOS) is an important regulator of various physiological and pathological functions in various types of cells. In pancreatic islets, a large amount of NO generated by inducible NOS (iNOS) has been postulated to be involved in beta -cell degeneration in the process of insulin-dependent diabetes mellitus (1, 6, 22). On the other hand, recent immunostaining studies clearly demonstrated the presence of constitutive NOS (cNOS), which is activated by Ca2+ and calmodulin, in rat and mouse pancreatic islet cells, i.e., neuronal NOS in alpha -, beta -, and delta -cells (3, 25) and endothelial NOS in alpha -and delta -cells (37). Furthermore, direct biochemical evidence for the cNOS enzyme activity was obtained from isolated islets of mice (33). These findings suggest a physiological involvement of NO in the regulation of the hormone secretion from pancreatic islet cells.

By binding to iron in the heme at the active site of soluble guanylate cyclase (sGC), NO activates the enzyme, which results in elevation of the cGMP level. Given that the enzyme activities of guanylate cyclase (16) and cGMP-dependent protein kinase have been demonstrated in pancreatic islets (23), it is plausible that NO exerts its effect partly through the elevation of cGMP level. Several studies have shown that a rise in the cGMP level in beta -cells stimulates insulin secretion (15, 26). We previously reported that an aqueous NO solution as well as 8-bromo-cGMP elevates the cytosolic Ca2+ concentration ([Ca2+]c) at 7.0 mM glucose in isolated rat beta -cells (29). Thus NO is predicted to facilitate glucose-induced insulin secretion via the stimulation of cGMP formation.

However, the physiological role of NO produced by cNOS in insulin secretion from beta -cells is now a matter of controversy. For instance, nonselective NOS inhibitors, such as NG-nitro-L-arginine (L-NNA) and NG-nitro-L-arginine methyl ester (L-NAME), have been shown to inhibit insulin secretion induced by L-arginine, which is a substrate of NOS, or by 3 mM glucose in the HIT-T15 beta -cell line (10, 34) and in isolated rat islets (27, 37), suggesting a stimulatory effect of NO on insulin secretion. In contrast, several other groups have reported that NOS inhibitors increase the L-arginine- or glucose-induced insulin secretion from isolated islets of mice (2, 18, 31, 33) and rats (38). Moreover, controversial data showing stimulatory (10, 24, 27, 34, 41) and inhibitory (4, 9, 31, 35, 38) effects of aqueous NO solution or NO donors, such as 3-morpholinosydnonimine (SIN-1), sodium nitroprusside, and hydroxylamine, on insulin secretion have also been presented.

We could not rule out that a part of the controversial data hitherto reported concerning the effect of NO on the glucose-induced insulin secretion is due to differences in the concentration of glucose or NO used: In most studies showing an inhibitory effect of NO on the insulin secretion, high concentrations of glucose such as 11.1, 16.7, and 20 mM or millimolar concentrations of NO donors were used for the analyses (4, 5, 9, 12, 35). A large amount of NO could be produced by high concentrations of glucose in beta -cells (34); however, the endogenously produced NO has been ignored in all the studies that investigated the effect of exogenously applied NO on the insulin secretion. The aim of the present study was to resolve the controversy by focusing on the concentration of exogenously applied NO and on endogenous NO produced by glucose. Our results provide evidence for a concentration-dependent dual effect of NO, i.e., a stimulatory effect at low concentrations and an inhibitory one at high concentrations, on the [Ca2+]c of beta -cells and insulin secretion.


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Drugs. The following drugs were used: 8-bromo-cGMP, KT5823, L-NNA, and tolbutamide (Sigma, St. Louis, MO); LY-83583 (6-anilino-5,8-quinolinedione; Affinity Bioreagents, Golden, CO); streptomycin and penicillin (Meiji Seika, Tokyo, Japan); thapsigargin (Wako, Osaka, Japan); and fura 2-acetoxymethyl ester (fura 2-AM) and NOC-7 (1-hydroxy-2-oxo-3-[N-methyl-3-aminopropyl]-3-methyl-1-triazene; Dojindo Laboratories, Kumamoto, Japan).

LY-83583 and NOC-7 were first dissolved in ethanol as a 20 mM stock solution and in 1 M NaOH as a 1 M solution, respectively, and were stored at -20°C for later use. The stock solutions were further diluted in HEPES-buffered Krebs (HK) solution (in mM: 129 NaCl, 5.0 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.0 CaCl2, 1.2 MgSO4, 2.8 glucose, and 10 HEPES; pH 7.4 with NaOH) just before administration. 8-Bromo-cGMP, L-NNA, and tolbutamide were directly dissolved in the HK solution before each experiment. Control studies performed with each vehicle alone showed that there were no apparent effects of each vehicle at the concentrations used in any of the protocols. A saturated solution of NO, the concentration of which was estimated to be 2 mM, was made by applying NO gas to distilled, degassed water according to the method described previously (29) and kept on ice. Aqueous NO solution at 2 or 20 µM was made by diluting the saturated solution with the HK solution just before its application.

Measurements of [Ca2+]c. Pancreatic beta -cells were isolated from male Wistar rats (9-12 wk old, 200-300 g; SLC, Hamamatsu, Japan) by the collagenase digestion technique as described previously (29). The dispersed beta -cells were plated on coverslips and cultured for 1 day in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin.

[Ca2+]c was measured in fura 2-loaded cells by using dual-wavelength fluorometry as described previously (29). Briefly, the cells were loaded with 2 µM fura 2-AM for 30 min at 37°C. They were then mounted in a chamber on the stage of an inverted microscope (Diaphot TMD 300; Nikon, Tokyo, Japan) and were continuously superfused with HK solution maintained at 37°C by use of a peristaltic pump at a flow rate of 1 ml/min. [Ca2+]c was measured with an Argus-50/CA system (Hamamatsu Photonics, Hamamatsu, Japan), with alternating excitation of cells at 340 ± 10 and 380 ± 10 nm and monitoring of the resultant emission at 510 ± 20 nm. Pairs of 340- and 380-nm fluorescence images were captured every 10 s and were converted to 340/380 ratio images after subtraction of the background fluorescence. The 340/380 ratio was used to indicate the relative [Ca2+]c because of the many uncertainties involved in fura 2 calibration (21). The amplitude and frequency of [Ca2+]c oscillations were determined by measuring nadir to peak height and peak-to-peak interval, respectively. Drugs were applied in the superfusing HK solution. beta -Cells were selected according to the procedure described previously; i.e., single cells that responded to 7.0 mM glucose and 0.3 mM tolbutamide with increases in [Ca2+]c were considered to be beta -cells (14). We also confirmed by immunostaining with an anti-insulin antibody (Neo Markers, Fremont, CA) that most of the living cells isolated by this dispersion technique from rat islets were beta -cells (data not shown).

All NOC-7 solutions and 2 µM NO solution were freshly prepared and changed every 5 min, and 20 µM NO solution every 2 min, because NOC-7 and NO are unstable substances. The NO concentration in the superfusing solutions in the chamber on the stage of the microscope was measured with an NO sensor (ISO-NO Mark II Nitric Oxide Meter; World Precision Instruments, Sarasota, FL).

Measurements of ion currents. Whole cell currents in isolated rat pancreatic beta -cells were measured in the perforated configuration of the patch-clamp technique as described previously (20). Patch pipettes were pulled from borosilicate glass capillaries and fire-polished before use. The pipette resistance (when filled with the pipette solution) was 1-2 MOmega , and the current recording was performed when the series resistance was <10 MOmega . The bathing solution contained (in mM) 135 NaCl, 5 KCl, 1.2 CaCl2, 1.2 MgCl2, 5.5 glucose, and 10 HEPES (pH 7.4 with NaOH). The pipette solution contained (in mM) 100 K-aspartate, 40 KCl, 10 HEPES, and 270 µg/ml amphotericin B (pH 7.2 with KOH). Macroscopic currents were recorded using a patch-clamp amplifier (Axopatch 1-D; Axon Instruments, Foster City, CA). Data were filtered at 1 kHz, digitized at 2 kHz, and stored in a computer by using pCLAMP 6.0 software with an analog-to-digital converter (TL-1; Axon Instruments). The holding potential was -80 mV, and currents were evoked by 200-ms voltage ramps from -120 to -20 mV. The experiments were performed at room temperature.

Measurements of insulin secretion. Rat pancreatic islets isolated by the collagenase digestion technique were cultured for 1-2 days in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin. Next, the islets were preincubated for 30 min at 37°C in HK solution containing 11.1 mM glucose and 5 mM L-NNA, and then batches of 10 islets were incubated for 10 min in 1 ml of the same solution with 0, 0.5, or 10 µM NOC-7. At the end of the incubation, 700 µl of the incubation medium were collected and kept at -70°C for later assay. Insulin released into the medium was measured by use of an enzyme immunoassay kit for rat insulin (Amersham Pharmacia Biotech, Amersham, UK) and was expressed as nanograms per islet.

Statistics. Data are expressed as means ± SE. The effects of treatment were analyzed with paired or unpaired Student's t-test as appropriate. A probability of P < 0.05 was accepted as the level of statistical significance.


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Effects of NO on [Ca2+]c of beta -cells. In isolated rat pancreatic beta -cells, [Ca2+]c was low and stable at 2.8 mM glucose. Elevating the glucose concentration to 7.0 mM, which is known to cause a slight release of insulin, induced an initial decrease and subsequent transient increase in the [Ca2+]c, followed by a sustained, moderate elevation of the [Ca2+]c, as described previously (29). At 11.1 mM glucose, which is known to stimulate insulin secretion, most of the beta -cells showed [Ca2+]c oscillations (Fig. 1). These [Ca2+]c oscillations were completely abolished by the Ca2+ channel blocker nicardipine (1 µM; data not shown), suggesting that they were caused by Ca2+ influx through L-type voltage-operated Ca2+ channels. The exposure to 1 µM NOC-7 for 30 min decreased the amplitude of the [Ca2+]c oscillations in about 55% of the beta -cells tested (Fig. 1A). Only 15% of the beta -cells responded to 1 µM NOC-7 with an increase in the amplitude of the [Ca2+]c oscillations (data not shown). On the other hand, 10 µM NOC-7 almost terminated the [Ca2+]c oscillations at 11.1 mM glucose in all of the beta -cells tested (Fig. 1B).


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Fig. 1.   Effect of 1 (A) or 10 µM (B) NOC-7 on cytosolic Ca2+ concentration ([Ca2+]c) oscillations of beta -cells at 11.1 mM glucose. Tracings in A and B are representative of 40 and 81 cells from 4 and 3 experiments, respectively.

Effects of NO on [Ca2+]c of beta -cells in the presence of L-NNA. It had been shown previously (10, 34, 39) that glucose could stimulate endogenous NO production in rat pancreatic islets and in the HIT-T15 beta -cell line. Thus endogenously released NO might inherently affect the [Ca2+]c response to exogenously applied NO. Therefore, we reexamined the effects of NOC-7 on the [Ca2+]c oscillations in the presence of the NOS inhibitor L-NNA. After the treatment with 1 mM L-NNA for longer than 30 min, 11.1 mM glucose still evoked [Ca2+]c oscillations. Under this condition, 0.5 and 1 µM NOC-7 significantly increased the amplitude of the [Ca2+]c oscillations in about 50% of the beta -cells tested (Figs. 2A and 3A) and caused no apparent changes in the rest of the beta -cells (data not shown). The frequency of the [Ca2+]c oscillations was not affected by NOC-7 (Fig. 3B). In contrast, 10 µM NOC-7 still suppressed the [Ca2+]c oscillations even in the presence of 1 mM L-NNA in all of the beta -cells tested (Fig. 2B). The decomposed products of NOC-7, which were prepared by incubating NOC-7 in the HK solution at 37°C for 1 h, showed no apparent effects on the [Ca2+]c oscillations (data not shown). When 0.5, 1, and 10 µM NOC-7 solutions were superfused, the concentration of NO in the chamber on the stage of the microscope was in the range of 10-30, 30-50, and 160-200 nM, respectively.


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Fig. 2.   Effect of 0.5 (A) or 10 µM (B) NOC-7 on [Ca2+]c oscillations of beta -cells at 11.1 mM glucose after treatment with NG-nitro-L-arginine (L-NNA). The cells were pretreated with 1 mM L-NNA for >30 min. Tracings in A and B are representative of 35 and 112 cells, respectively, from 3 experiments.



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Fig. 3.   Effects of 0.5 µM NOC-7 and 1 mM 8-bromo-cGMP (8-BrcGMP) on the amplitude (A) and frequency (B) of [Ca2+]c oscillations of beta -cells at 11.1 mM glucose after treatment with L-NNA. The amplitude and frequency of [Ca2+]c oscillations were measured between 15 and 20 min after the application of the drugs and were averaged. Each value was normalized to 100% of the corresponding control that was obtained by averaging the values between 10 and 20 min before the application of the drugs. The cells were pretreated with 5 mM L-NNA for >30 min. Values are means ± SE for the data of 35 and 49 cells that responded to NOC-7 and 8-BrcGMP, respectively, from 3 experiments. **P < 0.01 vs. control.

Similarly, aqueous NO solution had a concentration-dependent dual effect on the [Ca2+]c oscillations. Superfusion with 2 µM aqueous NO solution increased the amplitude of the [Ca2+]c oscillations (Fig. 4A). In contrast, superfusion with 20 µM aqueous NO solution decreased the amplitude of the [Ca2+]c oscillations (Fig. 4B). When 2 and 20 µM aqueous NO solutions were superfused, the concentration of NO in the chamber on the stage of the microscope was in the range of 10-20 and 60-100 nM, respectively.


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Fig. 4.   Effect of 2 (A) or 20 µM (B) aqueous NO solution on [Ca2+]c oscillations of beta -cells at 11.1 mM glucose after treatment with L-NNA. The cells were pretreated with 5 mM L-NNA for >30 min. Tracings in A and B are representative of 71 and 125 cells, respectively, from 3 experiments.

Mechanism for the dual effect of NO on [Ca2+]c of beta -cells. The involvement of cGMP in the dual effect of NOC-7 was investigated. The stimulatory effect of 0.5 µM NOC-7 on the [Ca2+]c oscillations in the presence of L-NNA was abolished by LY-83583 (3 µM), an inhibitor of sGC (Fig. 5A). In contrast, the inhibition by 10 µM NOC-7 of the [Ca2+]c oscillations was not affected by LY-83583 (3 µM; Fig. 5B). 8-Bromo-cGMP (1 mM; Fig. 5C) increased the amplitude of the [Ca2+]c oscillations without affecting their frequency (Fig. 3). Furthermore, KT5823 (0.5 µM), an inhibitor of cGMP-dependent protein kinase (PKG), abolished the stimulatory effect of 0.5 µM NOC-7 on the [Ca2+]c oscillations in the presence of L-NNA (Fig. 5D). These results suggest that cGMP and PKG are involved in the stimulatory effect of NO, whereas the inhibitory effect is cGMP-independent.


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Fig. 5.   Effects of 0.5 (A and D) or 10 µM (B) NOC-7 or 1 mM 8-BrcGMP (C) on [Ca2+]c oscillations of beta -cells at 11.1 mM glucose. LY-83583 (3 µM; A and B), an inhibitor of soluble guanylate cyclase, and KT5823 (0.5 µM; D), an inhibitor of cGMP-dependent protein kinase, were present as indicated. The beta -cells were pretreated with 1 mM L-NNA for >30 min. Tracings in A, B, C, and D are representative of 44, 44, 49, and 50 cells, respectively, from 2-4 experiments.

Figure 6 shows the effect of 8-bromo-cGMP on whole cell currents of isolated beta -cells evoked by voltage ramps from -120 to -20 mV. Diazoxide (0.1 mM), an ATP-sensitive K+ (KATP) channel opener, increased whole cell currents, and tolbutamide (0.1 mM), a KATP channel blocker, abolished the currents activated by diazoxide (Fig. 6A). Tolbutamide-sensitive currents were estimated from the difference between currents in the presence of diazoxide and those in the presence of diazoxide plus tolbutamide (Fig. 6C). The current-voltage relationship for the difference currents was essentially linear, suggesting that the currents were voltage independent. The reversal potential of the difference currents was -84.5 ± 1.6 mV (n = 4), which was comparable with the estimated value of the potassium equilibrium potential (-89 mV). Similar to tolbutamide, 8-bromo-cGMP (1 mM) inhibited the currents activated by diazoxide (0.1 mM; Fig. 6B). As shown in Fig. 6C, the current-voltage relationship for the 8-bromo-cGMP-sensitive currents, which were estimated from the difference between currents in the presence of diazoxide and those in the presence of diazoxide plus 8-bromo-cGMP, was also linear, and its reversal potential was -85.5 ± 2.5 mV (n = 4).


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Fig. 6.   Effect of 8-BrcGMP on whole cell ATP-sensitive K+ (KATP) currents in isolated beta -cells. Whole cell currents were evoked by 200-ms voltage ramps from -120 to -20 mV. Diazoxide (Diazo; 0.1 mM) increased whole cell currents evoked by the ramps (A and B). The currents activated by diazoxide were almost abolished and largely suppressed by tolbutamide (Tolb; 0.1 mM) (A) and 8-BrcGMP (cGMP; 1 mM) (B), respectively. C: average current-voltage relationship for the whole cell currents sensitive to tolbutamide (open circle ) or 8-BrcGMP (). The currents were estimated from the difference between currents in the presence of diazoxide and those in the presence of diazoxide plus tolbutamide or 8-BrcGMP. The amplitude of the different currents was measured in 10-mV increments. Values are means ± SE for the data of 4 cells.

Effects of NO on insulin secretion. We finally examined how the concentration-dependent dual effect of NO on the [Ca2+]c of beta -cells acted on the insulin secretion from beta -cells. It is well established that glucose induces biphasic insulin secretion, i.e., the first phase, where the insulin secretion rapidly increases and decreases during the first 5-10 min, and the second phase, which characteristically shows a sustained increase in insulin secretion (11). For comparison with the effects of NOC-7 on the [Ca2+]c oscillations, we investigated effects of the NO donor on the second phase of the insulin secretion induced by 11.1 mM glucose. In the presence of 5 mM L-NNA, 0.5 µM NOC-7 significantly facilitated the insulin secretion induced by 11.1 mM glucose, whereas 10 µM NOC-7 suppressed the hormone secretion (Fig. 7).


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Fig. 7.   Effect of 0.5 (A) or 10 µM (B) NOC-7 on insulin secretion from isolated pancreatic islets. Insulin secretion was measured after a 10-min incubation in the absence (control) and presence of NOC-7 at 11.1 mM glucose. The islets were preincubated with 5 mM L-NNA for 30 min. Data are presented as means ± SE of values from 4 experiments. *P < 0.05 vs. control.


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There is considerable controversy as to the effects of NO on insulin secretion. Several research groups including ours have suggested that NO serves to increase insulin secretion (10, 24, 26, 29, 34, 41), whereas other researchers have proposed that the main effect of NO is to suppress insulin secretion (5, 9, 31, 36, 39). The finding in the present study provides at least one explanation for this controversy. The data shown here indicate a concentration-dependent dual effect of NO on the [Ca2+]c of isolated rat pancreatic beta -cells.

In the presence of L-NNA, the amplitude of the [Ca2+]c oscillations induced by 11.1 mM glucose was increased when 0.5 and 1 µM NOC-7 and 2 µM aqueous NO solution were superfused. In contrast, the [Ca2+]c oscillations were suppressed when 10 µM NOC-7 and 20 µM aqueous NO solution were superfused. These results are in good agreement with the recent study showing that the NO donor hydroxylamine has a concentration-dependent dual effect on [Ca2+]c oscillations of beta -cells isolated from ob/ob mice (13). There is an intimate correlation between [Ca2+]c of beta -cells and insulin secretion (11, 17). It is also suggested that an increase in the amplitude of the [Ca2+]c oscillations induced by glucose results in the augmentation of insulin secretion (7, 28). Hence, the present results obtained with the [Ca2+]c measurement suggest that NO stimulates insulin secretion at low concentrations, whereas it inhibits the hormone secretion at high concentrations. This hypothesis was supported by the direct measurement of insulin secretion in the present study, which demonstrated that under the condition where NO production was inhibited by L-NNA, the insulin secretion induced by 11.1 mM glucose was facilitated by 0.5 µM NOC-7, whereas it was suppressed by 10 µM NOC-7. The concentration range of NO in the chamber, which was measured with the NO sensor, was between 10 and 50 nM when 0.5 or 1 µM NOC-7 or 2 µM aqueous NO solution was superfused and was higher than 60 nM when 10 µM NOC-7 or 20 µM aqueous NO solution was superfused. It is suggested, therefore, that low concentrations of NO, lower than 50 nM, facilitate glucose-induced [Ca2+]c oscillations of beta -cells and insulin secretion, whereas high concentrations of NO, higher than 60 nM, suppress them.

NO serves numerous biological functions, some of which are mediated by the ability of NO to activate sGC, resulting in cGMP formation (19, 30). The stimulatory effect of NOC-7 on [Ca2+]c was likely to have been mediated by cGMP, because similar [Ca2+]c responses were produced by 8-bromo-cGMP and the stimulatory effect of NOC-7 was abolished by the sGC inhibitor LY-83583. Furthermore, the facilitatory effect of NOC-7 was also eliminated by the PKG inhibitor KT5823, suggesting the involvement of PKG in the effect. Thus our data suggest that low concentrations of NO potentiate the glucose-induced insulin secretion via the production of cGMP followed by the activation of PKG. This is in good accordance with previous studies showing that cGMP stimulated insulin secretion from rat pancreatic islets (4, 15, 38).

The mechanism for the stimulatory effect of cGMP on [Ca2+]c oscillations of beta -cells was further investigated by the measurement of whole cell membrane currents. The patch-clamp experiment clearly showed that 8-bromo-cGMP inhibited the potassium-selective, voltage-independent currents activated by diazoxide, a KATP channel opener. We confirmed that the currents were also blocked by tolbutamide, a KATP channel blocker. These results suggest that cGMP inhibits KATP channels of beta -cells. In line with this, 8-bromo-cGMP has recently been shown to decrease the activity of single KATP currents recorded from cell-attached patches in mouse beta -cells (32). Inhibition of KATP channels would lead to membrane depolarization, which is thought to be an important mechanism for the potentiation of [Ca2+]c oscillations. Thus our results support the idea that inhibition of KATP channels is involved in the facilitation by cGMP of insulin secretion from rat pancreatic islets.

Even in the presence of L-NNA, 10 µM NOC-7 caused an appreciable inhibition of the glucose-induced [Ca2+]c oscillations. This result is in good agreement with those of previous studies showing that NO inhibited insulin secretion (5, 9, 31, 36, 39). The inhibition by NO of the glucose-induced [Ca2+]c responses appeared to be independent of cGMP, because the inhibitory effect of NOC-7 was not affected by the sGC inhibitor LY-83583 and 8-bromo-cGMP had only stimulatory effects on the [Ca2+]c oscillations. These results are also consistent with previous studies suggesting that the inhibition of insulin secretion by NO donors such as SIN-1 and sodium nitroprusside is independent of cGMP (8, 15, 38). This cGMP-independent mechanism appears to be relevant for the inhibition by NO of islet function. One possible mechanism is that NO hyperpolarizes the beta -cell membrane by opening KATP channels, which is brought about by a reduction in the ATP/ADP ratio through suppression of mitochondrial aconitase (40) or phosphofructokinase (38), or through a depolarization of the mitochondrial membrane potential (12).

The effects of NOC-7 on the [Ca2+]c oscillations were somewhat complicated. In the absence of L-NNA, 1 µM NOC-7 inhibited the [Ca2+]c oscillations. Conversely, in the presence of the NOS inhibitor, the dominant effect of 0.5 or 1 µM NOC-7 was rather stimulatory on the [Ca2+]c oscillations. Because glucose produces NO in pancreatic islets in a concentration-dependent manner (10, 34, 39), it can be estimated that glucose at 11.1 mM produces a large amount of NO. The NO produced by 11.1 mM glucose possibly induced both the cGMP-independent inhibitory and the cGMP-mediated stimulatory effects on the [Ca2+]c oscillations, and the former effect might have overwhelmed the latter one. Under this condition, it is likely that the exogenously applied NO inevitably acted in an inhibitory manner on the [Ca2+]c oscillations.

The concentration-dependent dual effect of NO on the [Ca2+]c of beta -cells was further supported by the experiments measuring insulin secretion. The present study clearly demonstrates that in the presence of L-NNA, the insulin secretion induced by 11.1 mM glucose is facilitated by 0.5 µM NOC-7, whereas it is inhibited by 10 µM NOC-7. These results suggest that NO has a dual effect on the insulin secretion, i.e., stimulation at low concentrations and inhibition at high concentrations. Concerning the effects of NO, NO donors, or NOS inhibitors on insulin secretion, a number of conflicting data have been reported as described in the Introduction. The controversy would be explained, at least in part, by the concentration-dependent dual effect of NO on insulin secretion. In most studies showing an inhibitory effect of NO on the insulin secretion, a relatively high concentration of NO donors or aqueous NO solution was used, e.g., 1 mM hydroxylamine (5, 9), 0.1-1 mM sodium nitroprusside (4), 0.1 or 1 mM SIN-1 (9, 35), and 20 µM aqueous NO solution (12). Here, it should also be noted that glucose by itself produces NO in pancreatic islets (10, 39). Although several researchers showed an inhibitory effect of NO on insulin secretion in the presence of high concentrations of glucose such as 11.1, 16.7, and 20 mM, they did not consider the influence of NO endogenously produced by such high concentrations of glucose on the insulin secretion (4, 5, 9, 35, 39). As mentioned above, 11.1 mM glucose is likely to produce a large enough amount of NO to inhibit insulin secretion.

It has been demonstrated that neuronal NOS, which is activated by Ca2+ and calmodulin, resides in rat pancreatic beta -cells (3, 25). Insulin secretagogues, such as acetylcholine and glucagon-like peptide-1, elevate the [Ca2+]c of beta -cells, whereas exposure of beta -cells to catecholamines, i.e., epinephrine and norepinephrine, or to somatostatin released from pancreatic delta -cells decreases their [Ca2+]c (11). It is conceivable, therefore, that not only glucose but also numerous other substances contributing to insulin secretion participate in the regulation of NO production by increasing or decreasing [Ca2+]c of beta -cells in vivo.

In summary, NO exerts both stimulatory and inhibitory effects on [Ca2+]c and insulin secretion in rat pancreatic beta -cells depending on its concentration. The dual effect of NO was shown to be attributable to apparently different mechanisms: a cGMP-mediated mechanism for the stimulatory effect and a cGMP-independent one for the inhibitory effect. As a consequence, we propose that endogenously released NO functions as a physiological insulinotropic substance at concentrations lower than 50 nM, whereas it takes part in the negative feedback system in insulin secretion at higher concentrations.


    ACKNOWLEDGEMENTS

We thank E. Iwasaki for technical assistance.


    FOOTNOTES

This study was supported by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science.

Address for reprint requests and other correspondence: T. Ishikawa, Dept. of Pharmacology, School of Pharmaceutical Sciences, Univ. of Shizuoka, 52-1 Yada, Shizuoka City, Shizuoka 422-8526, Japan (E-mail:ishikat{at}u-shizuoka-ken.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 15, 2003;10.1152/ajpcell.00223.2002

Received 16 May 2002; accepted in final form 8 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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