Tolbutamide and diazoxide modulate phospholipase C-linked Ca2+ signaling and insulin secretion in beta -cells

Christof Schöfl, Julia Börger, Thilo Mader, Mark Waring, Alexander von zur Mühlen, and Georg Brabant

Abteilung Klinische Endokrinologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany


    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Arginine vasopressin (AVP), bombesin, and ACh increase cytosolic free Ca2+ and potentiate glucose-induced insulin release by activating receptors linked to phospholipase C (PLC). We examined whether tolbutamide and diazoxide, which close or open ATP-sensitive K+ channels (KATP channels), respectively, interact with PLC-linked Ca2+ signals in HIT-T15 and mouse beta -cells and with PLC-linked insulin secretion from HIT-T15 cells. In the presence of glucose, the PLC-linked Ca2+ signals were enhanced by tolbutamide (3-300 µM) and inhibited by diazoxide (10-100 µM). The effects of tolbutamide and diazoxide on PLC-linked Ca2+ signaling were mimicked by BAY K 8644 and nifedipine, an activator and inhibitor of L-type voltage-sensitive Ca2+ channels, respectively. Neither tolbutamide nor diazoxide affected PLC-linked mobilization of internal Ca2+ or store-operated Ca2+ influx through non-L-type Ca2+ channels. In the absence of glucose, PLC-linked Ca2+ signals were diminished or abolished; this effect could be partly antagonized by tolbutamide. In the presence of glucose, tolbutamide potentiated and diazoxide inhibited AVP- or bombesin-induced insulin secretion from HIT-T15 cells. Nifedipine (10 µM) blocked both the potentiating and inhibitory actions of tolbutamide and diazoxide on AVP-induced insulin release, respectively. In glucose-free medium, AVP-induced insulin release was reduced but was again potentiated by tolbutamide, whereas diazoxide caused no further inhibition. Thus tolbutamide and diazoxide regulate both PLC-linked Ca2+ signaling and insulin secretion from pancreatic beta -cells by modulating KATP channels, thereby determining voltage-sensitive Ca2+ influx.

phospholipase C; intracellular calcium; insulin secretion; adenosine 5'-triphosphate-sensitive potassium channels


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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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THE CONTROL OF insulin secretion is a multifactorial and highly interconnected process involving nutritional and nonnutritional factors. Glucose stimulates insulin secretion by an increase in the cytosolic ratio of ATP to ADP, which inhibits ATP-sensitive K+ channels (KATP channels), thereby causing membrane depolarization, activation of voltage-sensitive Ca2+ influx, and a rise in the cytosolic free Ca2+ concentration ([Ca2+]i) that triggers insulin secretion (19, 22, 38). In addition, glucose further augments insulin secretion by a KATP channel-independent pathway, i.e., by enhancing the stimulatory effect of Ca2+ on the secretory process (7, 29, 38). Neurotransmitters and hormones that activate the Ca2+-phosphoinositide (PI) pathway potentiate glucose-induced insulin release and thus may be of major relevance to the regulation of insulin secretion (27, 38, 40). ACh, arginine vasopressin (AVP), and bombesin, which activate the Ca2+-PI signaling pathway, cause a rise in [Ca2+]i and stimulate insulin secretion from both normal and transformed beta -cells (6, 14, 17, 24, 25, 27, 30, 32, 34, 40). The generation of Ca2+ signals by phospholipase C (PLC)-linked hormones requires inositol 1,4,5-trisphosphate (IP3)-linked mobilization of Ca2+ from intracellular stores and voltage-sensitive and -insensitive Ca2+ influx from the outside (17, 30, 32). The actions of PLC-linked agonists on [Ca2+]i and on insulin secretion are glucose dependent, further demonstrating the interactive regulation of insulin secretion (10, 18, 27, 40). Sulfonylurea drugs, which are widely used drugs in the treatment of non-insulin-dependent diabetes mellitus, and diazoxide, which has been used for many years to control hypoglycemia caused by inappropriate insulin secretion, modulate insulin release by regulating KATP channel activity (23). Sulfonylureas, which much like a rise in extracellular glucose, inhibit KATP channels and cause membrane depolarization, activation of voltage-sensitive Ca2+ influx, and a rise in [Ca2+]i that initiates insulin release (3, 23, 33). Diazoxide, by contrast, activates KATP channels, thereby causing membrane hyperpolarization, inhibition of Ca2+ influx through voltage-sensitive Ca2+ channels (VSCC), and inhibition of insulin secretion (3, 23, 33). Thus control of insulin release by sulfonylureas or diazoxide is thought to be mainly caused by its own regulatory actions on KATP channels and by augmenting or opposing the effects of glucose on KATP channel activity. However, given the pivotal role of KATP channels in determining the membrane potential and thereby controling voltage-sensitive Ca2+ influx in beta -cells, sulfonylurea- or diazoxide-induced modulation of KATP channel activity may also interact with the intracellular Ca2+ signal evoked by PLC-linked hormones, thereby regulating insulin release in response to these agonists. This may be relevant to our understanding of the pharmacological actions of sulfonylureas and diazoxide, and the role of KATP channels in PLC-linked control of beta -cell function, and may elucidate some of the mechanisms underlying the glucose dependency of PLC-linked Ca2+ signaling and insulin secretion. In the present study, we therefore investigated the effects of tolbutamide and diazoxide on the cytosolic Ca2+ signal and insulin secretion evoked by the PLC-linked agonists AVP and bombesin and the ACh analog carbachol. [Ca2+]i was measured in single fura 2-loaded HIT-T15 and normal mouse beta -cells. Insulin release was determined from cell populations of HIT-T15 cells that respond to various secretagogues, including glucose (12, 28).


    MATERIALS AND METHODS
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HIT-T15 cell culture. The HIT-T15 cells were kindly provided by Dr. W. Knepel (Göttingen, Germany). The cells were grown in RPMI 1640 medium containing 10 mM glucose supplemented with 10% FCS (vol/vol), 100 units of penicillin/ml, and 100 µg streptomycin/ml at 37°C in 5% CO2 and 95% air (vol/vol). All experiments were performed with cells from passage 65 to 86.

Preparation of islet beta -cells. NMRI mice were housed in a temperature-controlled room with a 12:12-h light-dark cycle and had ad libitum access to standard chow and water. They were treated in accordance with all guidelines and regulations of our institutional animal care and use committee. The islets of Langerhans were isolated from female NMRI mice aged 8-12 wk by collagenase digestion. To obtain dispersed cells, islets were incubated for 10 min in Ca2+-free medium [135 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 3 mM glucose, 10 mM NaHEPES, 100 units of penicillin/ml, 100 µg streptomycin/ml, and 1% BSA (wt/vol) gassed with 100% O2 (vol/vol), pH 7.4] with gentle pipetting through a siliconized glass pipette until the islets disappeared. Islet cells were washed, resuspended in RPMI 1640 medium containing 5.5 mM glucose supplemented with 10% FCS (vol/vol), 100 units of penicillin/ml, and 100 µg streptomycin/ml, allowed to attach to glass coverslips, and maintained in a short culture for up to 2 days at 37°C in 5% CO2 and 95% air (vol/vol).

Measurement of [Ca2+]i. HIT-T15 cells or primary islet cells subcultured on coverslips were loaded with 5 µM fura 2-AM for 30 min at 37°C in medium containing 130 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.5 mM CaCl2, 10 mM glucose, 20 mM HEPES, 2% BSA (wt/vol), and 0.1% Pluronic F-127 (wt/vol) gassed with 100% O2 (vol/vol), pH 7.4. After being loaded, the coverslips were washed, mounted in a temperature-controlled superfusion chamber (37°C), and placed on the stage of a Zeiss Axiovert IM 135 equipped with a ×40 Achrostigmat oil immersion objective (Zeiss, Jena, Germany). The chamber was superfused with the same buffer as that used for measuring insulin release at a flow rate of 0.75 ml/min. Ca2+ measurements were done on cells of average size and healthy appearance (round in shape, no membrane blebs). Fura 2 fluorescence from a single cell was recorded with a dual excitation spectrofluorometer system (Deltascan 4000; Photon Technology Instruments, Wedel, Germany). [Ca2+]i were calculated according to the formula [Ca2+]i = Kd × B × (R - Rmin)/(Rmax - R), where R is the ratio of fluorescence, and the dissociation constant (Kd) = 225 nM (9). The ratio at saturating Ca2+ concentration (Rmax), the ratio of fluorescence at zero Ca2+ (Rmin), and the ratio of the fluorescence intensity at 380 nm at zero and saturated Ca2+ concentrations (B) are constants that were determined in the superfusion chamber from solutions containing fura 2-free acid (1 µM) and various concentrations of free Ca2+ (data not shown). All records have been corrected for autofluorescence of unloaded cells at each wavelength before the ratio was used.

Insulin secretion. HIT-T15 cells were subcultured in six-well plates at a density of 2 × 106 cells/well. After 2 days, the culture medium was removed, and the cells were washed with medium containing 130 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.5 mM CaCl2, 10 mM glucose, 20 mM HEPES, and 0.1% BSA (wt/vol) gassed with 100% O2 (vol/vol), pH 7.4. The cells were then preincubated for 1 h with or without glucose (10 mM) and were washed again, and fresh medium (2 ml) was added with the respective test agents. After 15 min, the supernatant was removed, and the insulin concentration was measured by RIA using a commercial kit (Kabi Diagnostics Pharmacia, Uppsala, Sweden). During the experiments, the cells were kept at 37°C.

Materials. Fura 2-AM and Pluronic F-127 were purchased from Molecular Probes (Eugene, OR), RPMI 1640, penicillin, and streptomycin were from Life Technologies (Berlin, Germany), collagenase was from Boehringer (Mannheim, Germany), thapsigargin was from Calbiochem (Bad Soden, Germany), and AVP and the other substances were from Sigma Chemical (Munich, Germany). Nifedipine and BAY K 8544 were provided by Bayer (Leverkusen, Germany). Stock solutions were prepared in water or as follows: AVP (100 µM) in 0.01 M HCl, tolbutamide (30 mM) in 150 mM NaOH, diazoxide (10 mM) and thapsigargin (5 mM) in DMSO, nifedipine and BAY K 8644 (5 mM) in ethanol.

Statistics. Unless representative tracings are shown, values are means ± SE. Statistical analysis was performed using Student's t-test for paired or unpaired data when two samples were compared. Multiple comparisons were assessed by ANOVA followed by the Student-Newman-Keuls test. P < 0.05 was considered as significantly different.


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Effects of tolbutamide and diazoxide on PLC-linked Ca2+ signals in HIT-T15 cells. In HIT-T15 cells [Ca2+]i averaged 138 ± 2 nM (n = 61) in the presence of glucose (10 mM). The sulfonylurea drug tolbutamide (30 µM) increased [Ca2+]i by 40 ± 6 nM in 26 of 36 cells. In the remaining 10 cells, tolbutamide (30 µM) caused no changes in [Ca2+]i. The tolbutamide-induced rise in [Ca2+]i could be blocked by verapamil (50 µM; data not shown). AVP (1 nM) and bombesin (200 pM) caused repetitive Ca2+ transients, as reported previously (Figs. 1 and 2; see Refs. 30-32). In the absence of AVP or bombesin, no Ca2+ transients were observed. Tolbutamide (3-300 µM) enhanced the frequency of the AVP- or bombesin-induced Ca2+ transients in 13 of 17 cells tested (Fig. 1, A and B). Tolbutamide (3-300 µM) increased the frequency of the AVP (1 nM)-induced Ca2+ transients from 0.74 ± 0.12 to 1.41 ± 0.13 min-1 (n = 7 cells, P < 0.001) and of the bombesin (200 pM)-induced Ca2+ transients from 0.51 ± 0.06 to 1.41 ± 0.16 min-1 (n = 6 cells, P < 0.001). As depicted in Fig. 1A, the acceleration of the PLC-linked Ca2+ transients by tolbutamide was concentration dependent at the level of an individual cell. In 4 of 17 cells, tolbutamide (30 or 300 µM) switched the AVP- or bombesin-induced Ca2+ signal to a plateau-like rise in [Ca2+]i with cessation of the Ca2+ transients (Fig. 1B). Tolbutamide (300 µM) affected the AVP- or bombesin-induced Ca2+ transients in eight of eight cells, although not all of the cells responded to lower concentrations of tolbutamide (3 or 30 µM). The actions of tolbutamide (3-300 µM) on the AVP- or bombesin-induced Ca2+ transients were reversible in all cells tested (Fig. 1, A and B). Diazoxide (30 or 100 µM) decreased [Ca2+]i by 16 ± 5 nM (n = 5 of 12 cells; data not shown) in the presence of glucose (10 mM). In the remaining seven cells, diazoxide (30 or 100 µM) caused no changes in [Ca2+]i. In 11 of 14 cells, diazoxide (10-100 µM) reduced the frequency and sometimes the amplitude of the AVP- or bombesin-induced Ca2+ transients, whereas in 3 of 14 cells, the Ca2+ transients ceased in the presence of diazoxide (Fig. 1, C and D). Diazoxide (100 µM, which was the highest concentration used) either reduced the frequency or eliminated the AVP- or bombesin-induced Ca2+ transients in all cells tested (9 cells), but 3 cells did not respond to lower concentrations of diazoxide (10 or 30 µM). The actions of diazoxide (10-100 µM) on the AVP- or bombesin-induced Ca2+ transients were fully reversible in all cells tested and could be reversed by the addition of increasing concentrations of tolbutamide (n = 13 cells, Fig. 1, C and D). When glucose was removed from the superfusion medium, the amplitude and the frequency of the AVP-induced Ca2+ transients dropped, and the Ca2+ transients finally ceased after ~5-60 min (Fig. 1, E and F; n = 8 cells). In some cells (4 of 8 cells), however, this effect of glucose deprivation was preceded by a transient increase in the frequency of the AVP-induced Ca2+ transients and a rise in the intertransient [Ca2+]i (Fig. 1E). Reexposure of cells to glucose (10 mM) led to the generation of AVP-induced Ca2+ transients (Fig. 1, E and F). The addition of tolbutamide (3-300 µM) partly reversed the inhibitory effect of glucose deprivation on the AVP-induced Ca2+ transients in five of eight cells (Fig. 1E) while in the remaining three cells tolbutamide up to a concentration of 300 µM was without any effect (Fig. 1F). In cells that had been pretreated for 30-60 min in glucose-free medium, tolbutamide (30 µM) caused a plateau-like increase in [Ca2+]i by 65 ± 13 nM (n = 6 of 10 cells).


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Fig. 1.   Effect of tolbutamide and diazoxide on arginine vasopressin (AVP)- or bombesin-induced Ca2+ transients in single HIT-T15 cells. A and B: increasing concentrations of tolbutamide reversibly increased frequency and sometimes amplitude of AVP- and bombesin-induced Ca2+ transients or switched the Ca2+ transients to a plateau-like rise in cytosolic Ca2+ concentration ([Ca2+]i) in the presence of glucose (10 mM). C: increasing concentrations of diazoxide reversibly decreased the frequency and sometimes the amplitude of AVP-induced Ca2+ transients or stopped them in the presence of glucose (10 mM). D: decrease in the frequency of the phospholipase C (PLC)-linked Ca2+ transients by diazoxide in the presence of glucose (10 mM) could be reversed by increasing concentrations of tolbutamide. E and F: in glucose-free medium the frequency and amplitude of the AVP-induced Ca2+ transients dropped, and the Ca2+ transients finally ceased. In a subset of cells, this was preceded by a transient increase in the frequency of the AVP-induced Ca2+ transients and a rise in the intertransient [Ca2+]i (E). Tolbutamide partly reversed the inhibitory effect of glucose deprivation on the AVP-induced Ca2+ transients in 5 of 8 cells (E), whereas in the remaining 3 cells tolbutamide up to a concentration of 300 µM was without any effect (F). Bars indicate the presence of the respective agents in the superfusion medium. See text for average values.



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Fig. 2.   Effect of BAY K 8644 and verapamil on AVP-induced Ca2+ transients in single HIT-T15 cells. A: effect of BAY K 8644 (1 µM) on AVP-induced Ca2+ transients in the presence of glucose (10 mM). B: effect of verapamil (50 µM) on AVP-induced Ca2+ transients in the presence of glucose (10 mM). C: effect of verapamil (50 µM) on tolbutamide (30 µM)-induced potentiation of AVP-induced Ca2+ transients in the presence of glucose (10 mM). Bars indicate the presence of the respective agents in the superfusion medium. See text for average values.

Mechanisms of the tolbutamide- and diazoxide-induced changes of PLC-linked Ca2+ signals in HIT-T15 cells. The effects of tolbutamide and diazoxide on the PLC-linked Ca2+ transients could be mimicked by the VSCC agonist BAY K 8644 and the VSCC antagonist verapamil, respectively (Fig. 2, A and B). BAY K 8644 (1 µM) reversibly increased the frequency and amplitude of the PLC-linked Ca2+ transients and led to a plateau-like rise in [Ca2+]i in six of six cells. Verapamil (50 µM) either reduced the frequency and/or amplitude of the AVP- and bombesin-induced Ca2+ transients in 10 of 18 cells by 55 ± 16 and 18 ± 9%, respectively, or stopped them in 8 of 18 cells. In addition, verapamil (50 µM) inhibited the tolbutamide-induced enhancement of the PLC-linked Ca2+ transients in three of three cells (Fig. 2C). In Ca2+-free medium, AVP caused one or two Ca2+ transients due to mobilization of internal Ca2+ (data not shown and Ref. 32). As shown in Table 1, neither tolbutamide (30 µM) nor diazoxide (100 µM) changed the amplitude of the Ca2+ transients or the amount of internally released Ca2+, as judged by the area under the curve above basal. Thapsigargin (2 µM), which is a major tool to study capacitative Ca2+ entry (35), caused a biphasic rise in [Ca2+]i with an initial peak reflecting mobilization of internal Ca2+ and a secondary plateau phase that is caused by influx of Ca2+ through L-type VSCC and non-L-type Ca2+ channels, most likely voltage-insensitive Ca2+ channels (data not shown and Refs. 30 and 32). To eliminate Ca2+ influx through L-type VSCC, HIT-T15 cells were stimulated with thapsigargin (2 µM) in the presence of verapamil (50 µM). Neither tolbutamide (30 µM) nor diazoxide (100 µM) affected the initial peak or plateau of the thapsigargin (2 µM)-induced Ca2+ signal (Table 1).

                              
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Table 1.   Effect of tolbutamide and diazoxide on intracellular Ca2+ mobilization by AVP in Ca2+-free medium (EGTA) and on thapsigargin-induced rises in [Ca2+]i in the presence of verapamil and extracellular Ca2+ in HIT-T15 cells

Effects of tolbutamide and diazoxide on PLC-linked Ca2+ signals in mouse beta -cells. In mouse beta -cells, [Ca2+]i was 101 ± 5 nM (n = 32) in the presence of glucose (6 mM). Carbachol (3 µM), which stimulates muscarinic receptors coupled to the Ca2+-PI signaling pathway, elicited a biphasic rise in [Ca2+]i with an initial peak followed by a sustained plateau in most cells (Fig. 3). In some cells, however, repetitive Ca2+ transients were observed in response to carbachol (3 µM; data not shown). Carbachol (3 µM) increased [Ca2+]i by 327 ± 47 and 58 ± 14 nM at its peak or plateau (measured after 5 min), respectively (n = 18). Reexposure of cells to carbachol (3 µM) after a washout period of 30 min caused a nearly identical Ca2+ response (96 ± 4% of the initial peak and plateau; n = 9; Fig. 3A). To assess the effect of tolbutamide on the carbachol-induced Ca2+ signal, tolbutamide (1 µM) was added to the perfusion medium 5 min before the second stimulation with carbachol (3 µM). Tolbutamide (1 µM) increased [Ca2+]i by 325 ± 170 nM in four of eight cells, whereas in the remaining four cells tolbutamide (1 µM) caused no changes in [Ca2+]i. Pretreatment with tolbutamide (1 µM) enhanced the carbachol (3 µM)-induced Ca2+ signal when compared with the first stimulation in the same cell (Fig. 3C). In the presence of tolbutamide (1 µM), the carbachol (3 µM)-induced peak increase and plateau rise in [Ca2+]i amounted to 196 ± 44 and 359 ± 155% of the control stimulation (n = 8; P < 0.05). Diazoxide (100 µM) caused no changes in [Ca2+]i in the presence of glucose (6 mM). Pretreatment for 5 min with diazoxide (100 µM) reduced the carbachol (3 µM)-induced peak increase and plateau rise in [Ca2+]i by 69 ± 13 and 68 ± 18% (n = 4; P < 0.02; Fig. 3E), respectively, when compared with the control stimulation in the same cell. Preincubation of mouse beta -cells for 30-60 min in glucose-free medium reduced basal [Ca2+]i to 86 ± 3 nM (n = 11). Under these glucose-free conditions, the carbachol (3 µM)-induced Ca2+ response was much smaller than in the presence of glucose (6 mM) and amounted to 33 ± 10 and 10 ± 4 nM at its peak or plateau, respectively (n = 11; Fig. 3B). Pretreatment with tolbutamide (10 µM) for 5 min, which by itself caused no changes in [Ca2+]i, significantly enhanced the Ca2+ signal evoked by a second stimulation with carbachol (3 µM) in the same cells (Fig. 3B). The carbachol (3 µM)-induced peak Ca2+ response was 123 ± 54 nM, and the plateau rise was 46 ± 31 nM (n = 4; P < 0.05).


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Fig. 3.   Effect of tolbutamide, diazoxide, BAY K 8644, and nifedipine on carbachol-induced Ca2+ signals in single mouse beta -cells. A: effect of repetitive carbachol (3 µM) stimulation on [Ca2+]i in the same cell in the presence of glucose (6 mM). The cell was perfused for 30 min with medium between the two stimulations. B: effect of tolbutamide (10 µM) on the carbachol (3 µM)-induced Ca2+ signal in glucose-free medium. C: effect of tolbutamide (1 µM) on the carbachol (3 µM)-induced Ca2+ signal in the presence of glucose (6 mM). D: effect of BAY K 8644 (1 µM) on the carbachol (3 µM)-induced Ca2+ signal in the presence of glucose (6 mM). E: effect of diazoxide (100 µM) on the carbachol (3 µM)-induced Ca2+ signal in the presence of glucose (6 mM). F: effect of nifedipine (10 µM) on the carbachol (3 µM)-induced Ca2+ signal in the presence of glucose (6 mM). Bars indicate the presence of the respective agents in the superfusion medium. Tolbutamide, diazoxide, BAY K 8644, and nifedipine were added to the perfusion medium 5 min before the second stimulation with carbachol (3 µM). See text for average values.

Mechanisms of the tolbutamide- and diazoxide-induced changes of PLC-linked Ca2+ signals in mouse beta -cells. The effects of tolbutamide and diazoxide on the carbachol (3 µM)-induced increase in [Ca2+]i in 6 mM glucose could be mimicked by BAY K 8644 and the VSCC antagonist nifedipine (Fig. 3, D and F). BAY K 8644 (1 µM), which caused a small rise in [Ca2+]i by 6 ± 2 nM (n = 7) in 6 mM glucose, increased the carbachol (3 µM)-induced peak increase and plateau rise in [Ca2+]i to 421 ± 127 and 515 ± 154% of the control stimulation (n = 7; P < 0.05; Fig. 3D). Nifedipine (10 µM), which like diazoxide (100 µM) had no effect on [Ca2+]i in the presence of glucose (6 mM), inhibited the carbachol (3 µM)-induced peak increase and plateau rise in [Ca2+]i by 61 ± 12 and 42 ± 22% (n = 8; P < 0.05; Fig. 3F), respectively. As shown in Table 2, neither tolbutamide (30 µM) nor diazoxide (100 µM) affected the carbachol (10 µM)-induced Ca2+ signal in Ca2+-free medium or the thapsigargin (2 µM)-induced Ca2+ influx through non-L-type Ca2+ channels.

                              
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Table 2.   Effect of tolbutamide and diazoxide on intracellular Ca2+ mobilization by carbachol in Ca2+-free medium (EGTA) and on thapsigargin-induced rises in [Ca2+]i in the presence of verapamil and extracellular Ca2+ in mouse beta -cells

Effects of tolbutamide and diazoxide on PLC-linked insulin secretion from HIT-T15 cells. In the presence of glucose (10 mM), insulin secretion was 46 ± 5 µU/ml (n = 24) during 15 min static incubation from populations of HIT-T15 cells and was enhanced by tolbutamide (30 µM), AVP (1 nM), and bombesin (100 pM; Fig. 4A). When tolbutamide (30 µM) and AVP (1 nM) or bombesin (100 pM) were added together, the insulin secretory response to the combined agonists was greater than additive (Fig. 4A). Diazoxide (30 µM) inhibited insulin release in the presence of glucose (10 mM) by 20 ± 9% (n = 12; data not significant) and diminished the AVP (1 nM)- and bombesin (100 pM)-induced insulin secretion (Fig. 4A). Nifedipine (10 µM) reduced insulin secretion in the presence of glucose (10 mM) by 32 ± 4% (n = 16; P < 0.05) and inhibited AVP-induced insulin secretion by 39 ± 2% (n = 24; P < 0.001). Neither tolbutamide (30 µM) nor diazoxide (30 µM) affected basal insulin secretion or AVP-stimulated insulin secretion in the presence of nifedipine (10 µM), as depicted in Fig. 4B. When HIT-T15 cells had been preincubated for 60 min in glucose-free medium and were kept at zero glucose during the static incubation period, insulin release amounted to 35 ± 5 µU/ml (n = 12), which was somewhat but not significantly lower than in the presence of glucose (10 mM). Stimulation with glucose (10 mM) for 15 min in HIT-T15 cells that had previously been kept in zero glucose for 30-60 min resulted in a significant increase in insulin secretion from 31 ± 2 µU/ml (n = 29) to 51 ± 4 µU/ml (n = 23, P < 0.009), demonstrating that the HIT-T15 cells used are responsive to glucose. Under glucose-free conditions, the stimulation of insulin release by tolbutamide (30 µM) and AVP (1 nM) was significantly reduced (Fig. 4C). However, insulin secretion in response to the combination of tolbutamide (30 µM) and AVP (1 nM) was again greater than additive, although the overall secretory response was lower than in experiments carried out in the presence of 10 mM glucose (Fig. 4A). In the absence of glucose, diazoxide (30 µM) neither affected basal nor AVP (1 nM)-induced insulin secretion from HIT-T15 cells (Fig. 4C).


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Fig. 4.   Effects of tolbutamide and diazoxide on AVP (1 nM)- or bombesin (100 pM)-induced insulin secretion with or without glucose and in the presence of nifedipine. Insulin secretion from HIT-T15 cells was determined as described in MATERIALS AND METHODS. A: effect of tolbutamide (30 µM) and diazoxide (30 µM) on AVP (1 nM)- or bombesin (100 pM)-induced insulin secretion in the presence of glucose (10 mM). B: effect of nifedipine (10 µM) on the tolbutamide (30 µM)- and diazoxide (30 µM)-induced potentiation and inhibition of AVP (1 nM)-induced insulin secretion in the presence of glucose (10 nM). Nifedipine (10 µM) was added to the cells 5 min before the respective stimulus and was present throughout the experiment. C: effect of tolbutamide (30 µM) and diazoxide (30 µM) on AVP (1 nM)-induced insulin secretion in the absence of glucose. Values are means ± SE of 3-5 independent experiments performed in quadruplicate.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we demonstrate that tolbutamide and diazoxide, which close and open KATP channels, respectively, critically regulate PLC-linked Ca2+ signaling in clonal HIT-T15 and primary mouse beta -cells. In HIT-T15 cells, tolbutamide caused a concentration-dependent increase in the frequency of the AVP- or bombesin-induced Ca2+ transients in the presence of high extracellular glucose (10 mM). At intermediate or high concentrations of tolbutamide (30 and 300 µM), the oscillatory Ca2+ signal switched to a plateau-like rise in [Ca2+]i. Conversely, diazoxide, which activates KATP channels, reduced the frequency and sometimes the amplitude of the Ca2+ transients and stopped them completely in some cells, as described previously (16). Qualitatively similar results were obtained in mouse beta -cells where in the presence of glucose (6 mM) tolbutamide and diazoxide potentiated or inhibited the carbachol-induced biphasic increase in [Ca2+]i, respectively. Because BAY K 8644, which increases the open probability of L-type VSCC, and the L-type VSCC antagonists verapamil and nifedipine mimicked the actions of tolbutamide and diazoxide, respectively, their effects on PLC-linked Ca2+ signals could be explained by modulation of voltage-sensitive Ca2+ influx.

In the presence of elevated glucose, PLC-linked agonists by themselves cause membrane depolarization and activation of Ca2+ influx through VSCC, which is necessary for the sustained generation of PLC-linked Ca2+ signals in beta -cells, as demonstrated by the actions of the VSCC blockers in this study and as reported previously (16, 30-32). However, the mechanisms, underlying the depolarizing action of PLC-linked agonists in beta -cells are incompletely understood. In RINm5F cells, AVP causes membrane depolarization directly (21), and activation of protein kinase C has been shown to cause membrane depolarization by closure of KATP channels (37). In HIT-T15 cells and in normal mouse beta -cells, no such direct effects of PLC-linked agonists on KATP channels have been reported (6, 13). It rather appears that emptying of intracellular Ca2+ stores activates a depolarizing current, which enhances Ca2+ influx through VSCC in beta -cells (39). In addition, PLC-linked agonists may inhibit Ca2+-activated K+ channels, thereby augmenting glucose-induced electrical activity and voltage-sensitive Ca2+ influx (36). Tolbutamide and diazoxide, by modulating KATP channel activity, may therefore indirectly enhance or oppose the depolarizing mechanisms activated by PLC-linked agonists, thereby tuning voltage-sensitive Ca2+ influx activated by PLC-linked agonists.

The mechanisms by which Ca2+ influx through VSCC could modulate PLC-linked Ca2+ signals are yet unclear but may involve modulation of IP3-production and/or the IP3-linked Ca2+ release process (1, 2). This can even occur without changing the average [Ca2+]i as observed here where the changes in PLC-linked Ca2+ signals caused by the addition of tolbutamide or diazoxide were not usually accompanied by concomitant changes in baseline Ca2+. Rather, such limited Ca2+ influx may cause local changes of Ca2+ at the microdomains adjacent to the Ca2+ regulatory sites of the IP3 receptor, thereby determining the threshold for IP3-mediated Ca2+ release (8). Such a mechanism could well explain the influence of voltage-sensitive Ca2+ influx on PLC-linked Ca2+ transient frequency and amplitude in HIT-T15 cells and on the carbachol-induced Ca2+ signal in mouse beta -cells where not only the plateau of the Ca2+ signal, which is dependent on Ca2+ influx from the outside, but also the initial peak, which mainly reflects internal Ca2+ mobilization, is affected by tolbutamide and diazoxide. Evidence for a direct interaction of tolbutamide or diazoxide with IP3-induced Ca2+ mobilization could not be found. Likewise, as judged by the actions of thapsigargin in the presence of L-type VSCC blockers, capacitative Ca2+ entry through non-L-type Ca2+ channels, which also contributes to sustained PLC-linked Ca2+ signals in beta -cells (16, 30, 32), is not altered by tolbutamide or diazoxide. Thus it appears that tolbutamide and diazoxide regulate PLC-linked Ca2+ signals predominantly if not exclusively by modulating KATP channel activity, thereby determining membrane potential and voltage-sensitive Ca2+ influx.

Physiologically, KATP channel activity is controlled by the cytosolic ratio of ATP to ADP, which in turn is determined by the ambient glucose concentration. In glucose-free medium, which leads to a decrease in the cytosolic ratio of ATP to ADP, activation of KATP channels, and membrane hyperpolarization, the frequency and amplitude of AVP-induced Ca2+ transients decreased and finally ceased in HIT-T15 cells, and in mouse beta -cells the carbachol-induced Ca2+ signal was greatly reduced, confirming the glucose dependency of PLC-linked Ca2+ signaling in beta -cells (10, 18, 27, 40). In the absence of glucose, tolbutamide restored AVP-induced Ca2+ transients in some but not all HIT-T15 cells and potentiated the Ca2+ signal elicited by carbachol in mouse beta -cells. This indicates that glucose-dependent membrane predepolarization and facilitation of voltage-sensitive Ca2+ influx, which is required for further membrane depolarization and activation of VSCC by PLC-linked signals (18), may contribute to the glucose dependency of PLC-linked Ca2+ signals in beta -cells (10, 18, 27, 40). However, other sites appear to exist whereby glucose-dependent processes regulate PLC-linked Ca2+ signals. Diazoxide even at high concentrations stopped the Ca2+ transients only in a small subset of cells, and tolbutamide only partly restored the PLC-linked Ca2+ signals in glucose-free medium. Although not tested here, glucose has been shown to enhance production of IP3 in response to receptor activation (40) and to be a prerequisite for the reuptake of Ca2+ into the IP3-sensitive Ca2+ pool (11), thus providing additional mechanisms for the glucose dependency of the effects of PLC-linked agonists on [Ca2+]i in beta -cells.

In HIT-T15 cells, tolbutamide stimulated insulin secretion and potentiated insulin release in response to AVP and bombesin, whereas diazoxide reduced basal insulin release, albeit not significantly, and inhibited PLC-linked insulin secretion in the presence of glucose (10 mM). The actions of tolbutamide and diazoxide on PLC-linked insulin secretion largely paralleled their effects on PLC-linked Ca2+ signaling and were similar to the effects of VSCC activation or inhibition reported previously (31). This suggests that tolbutamide and diazoxide critically determine insulin secretion in response to PLC-linked hormones by regulating PLC-linked Ca2+ signaling through modulation of voltage-sensitive Ca2+ influx. Recently, it has been proposed that sulfonylureas and diazoxide, in addition to their actions on KATP channels, could modulate insulin secretion by altering the efficacy of Ca2+ on the exocytotic process (4, 5, 26). However, such a KATP channel-independent pathway does not appear to be involved in the actions of tolbutamide or diazoxide on PLC-linked insulin secretion, since in the presence of nifedipine tolbutamide failed to potentiate AVP-induced insulin secretion and diazoxide did not further inhibit AVP-induced insulin secretion. This is consistent with a recent report showing convincingly that both agents influence insulin secretion by changing the concentration but not the action of cytoplasmic Ca2+ in mouse beta -cells (20).

In the absence of glucose, insulin release was somewhat lower than from cells chronically exposed to glucose (10 mM), and AVP-induced insulin secretion was largely inhibited, confirming the glucose-dependent action of PLC-linked agonists on insulin secretion both from transformed and normal beta -cells (40). This may involve modulation of KATP channels, since diazoxide inhibited AVP-induced insulin secretion in the presence of glucose (10 mM) to a similar degree and since AVP-induced insulin secretion was potentiated by tolbutamide in the absence of glucose. However, in glucose-free medium, the tolbutamide-induced insulin secretion was lower despite similar effects on [Ca2+]i in the presence or absence of glucose, and the combined effect of tolbutamide and AVP on insulin secretion was reduced. This points to additional sites by which glucose could enhance PLC-linked insulin secretion, such as the release process itself (15, 38).

In summary, we demonstrate here that tolbutamide and diazoxide, by modulating KATP channel activity and thereby controlling Ca2+ influx through VSCC, critically regulate Ca2+ signaling and insulin secretion elicited by PLC-linked agonists in beta -cells. This might contribute to the stimulatory and inhibitory actions on insulin secretion by sulfonylureas and diazoxide when used as therapeutic agents. Furthermore, besides their well-accepted role in glucose-mediated Ca2+ signaling and insulin secretion, KATP channels may be similarly important for the regulation of insulin release in response to neurohumoral signals activating the Ca2+-PI signaling pathway in beta -cells. Modulation of KATP channels appears to be a major mechanism for the glucose-dependent action of PLC-linked agonists on [Ca2+]i and insulin secretion. This may be important for the integration of metabolic and neurohumoral signals under conditions where the glucose concentration is high and beta -cells are exposed to PLC-linked stimuli.


    ACKNOWLEDGEMENTS

We are grateful to Petra Wübbolt, Natalie Wittner, and Daniela Biegert for excellent technical assistance.


    FOOTNOTES

This work was supported by DFG Grant Scho 466/1-3.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C. Schöfl, Abteilung Klinische Endokrinologie, Medizinische Hochschule, 30623 Hannover, Germany (E-mail: schefl.christof{at}mh-hannover.de).

Received 27 April 1999; accepted in final form 8 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 278(4):E639-E647
0193-1849/00 $5.00 Copyright © 2000 the American Physiological Society




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