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Endocrinology Vol. 140, No. 12 5516-5523
Copyright © 1999 by The Endocrine Society

ARTICLES

Ca2+/Calmodulin Inhibition and Phospholipase C-Linked Ca2+ Signaling in Clonal ß-Cells1

Christof Schöfl, Thilo Mader, Claudia Krämer, Mark Waring, Peter Krippeit-Drews, Klaus Prank, Alexander von zur Mühlen, Gisela Drews and Georg Brabant

Abteilung für Klinische Endokrinologie, Medizinische Hochschule Hannover (C.S., T.M., M.W., K.P., A.v.z.M., G.B.), 30623 Hannover; and Pharmazeutisches Institut, Eberhard-Karls-Universität Tubingen (C.K., P.K.-D., G.D.), 72076 Tubingen, Germany

Address all correspondence and requests for reprints to: Dr. Christof Schöfl, Abteilung Klinische Endokrinologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neurotransmitters and hormones, such as arginine vasopressin (AVP) and bombesin, evoke frequency-modulated repetitive Ca2+ transients in insulin-secreting HIT-T15 cells by binding to receptors linked to phospholipase C (PLC). The role of calmodulin (CaM)-dependent mechanisms in the generation of PLC-linked Ca2+ transients was investigated by use of the naphthalenesulfonamide CaM antagonists W-7 and W-13 and their dechlorinated control analogs W-5 and W-12. W-7 (10–30 µM) and W-13 (30–100 µM), but not W-5 (100 µM) and W-12 (300 µM), reversibly inhibited the AVP- and bombesin-induced Ca2+ transients. As the generation of PLC-linked Ca2+ transients requires mobilization of internal Ca2+ and Ca2+ influx through voltage-sensitive (VSCC) and -insensitive (VICC) Ca2+ channels, the effects of the W compounds on these processes were further investigated. First, W-7 dose dependently diminished K+ (45 mM)-induced Ca2+ signals (IC50, ~25 µM), and W-13 (100 µM) reduced the K+ (45 mM)-induced [Ca2+]i rise by about 40–60%, whereas W-5 (100 µM) and W-12 (300 µM) had no effect. In addition, W-7 (100 µM) inhibited whole cell Ca2+ currents in mouse ß-cells by about 60%. Second, pretreatment of cells (5 min) with W-7 (30 µM), but not W-5 (30 µM), inhibited agonist-induced internal Ca2+ mobilization by about 75% in Ca2+-free medium. Neither W-7 (30 µM) nor W-5 (30 µM) affected AVP (100 nM)-stimulated formation of IP3. Third, capacitative Ca2+ influx through VICC activated by thapsigargin (2 µM) in the presence of verapamil (50 µM) was inhibited by W-7 (30 µM) but not by W-5 (30 µM). As all of the W compound effects corresponded well to their reported anticalmodulin activity, a specific anticalmodulin action can be assumed. Thus, Ca2+ via activation of CaM-dependent processes could provide positive feedback on the generation of PLC-linked Ca2+ transients in HIT-T15 cells. This appears to involve CaM-dependent regulation of both mobilization of internal Ca2+ and Ca2+ influx through VSCC and VICC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN RELEASE from pancreatic ß-cells is stimulated by a variety of nutrients and nonnutritional factors (1, 2, 3). All insulin secretagogues elevate cytosolic free Ca2+ ([Ca2+]i), and numerous studies have shown that Ca2+ plays a key role in triggering insulin secretion (2). Neurotransmitters and hormones, such as acetylcholine, arginine vasopressin (AVP) or bombesin, that activate the Ca2+-phosphoinositide (PI) pathway cause a rise in [Ca2+]i and stimulate insulin secretion from normal and transformed ß-cells in the presence of glucose (2, 3, 4, 5, 6, 7, 8). The generation of Ca2+ signals by PLC-linked hormones requires inositol 1,4,5-trisphosphate (IP3)-mediated mobilization of intracellular Ca2+ and influx of Ca2+ from the outside through voltage-sensitive (VSCC) and voltage-insensitive (VICC) Ca2+ channels in hamster insulinoma tumor (HIT-T15) cells (9, 10, 11). In HIT-T15 cells and in primary ß-cells, PLC-linked agonists at low, near-physiological concentrations cause repetitive Ca2+ transients whose frequency is determined by the extracellular agonist concentration while the amplitude remains constant (9, 10, 11, 12, 13, 14). This indicates that the cytosolic Ca2+ signal evoked by PLC-linked agonists might be primarily frequency encoded. As transient rises in [Ca2+]i increase the rate of exocytosis from single HIT-T15 and primary ß-cells, a functional role of PLC-linked Ca2+ transients can be assumed (15, 16). Although the basic features of PLC-linked Ca2+ transients have been elucidated in recent years, the mechanisms underlying the PLC-linked Ca2+ transients are still unknown. Various positive and negative feedback mechanisms, usually assumed to involve Ca2+ itself, have been described and used to model transient Ca2+ oscillations in a number of cell systems (17). Ca2+-dependent regulatory mechanisms are often mediated by the activation of the ubiquitous cellular protein calmodulin (CaM), which, in turn, activates a range of cellular proteins, such as CaM-dependent protein kinase II (CaM kinase II) (18, 19). Several lines of evidence suggest that CaM and/or CaM kinase II could be involved in the regulation of PLC-linked Ca2+ signals (20, 21, 22, 23, 24, 25). To investigate the role of CaM activity in PLC-linked Ca2+ signals in ß-cells, the effects of naphthalenesulfonamide anticalmodulin agents, the W compounds, on AVP- and bombesin-induced Ca2+ signals were studied in single fura-2-loaded, insulin-secreting HIT-T15 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
HIT-T15 cell culture
HIT-T15 cells were provided by Dr. Knepel (Göttingen, Germany). The cells were grown in RPMI 1640 medium containing 10 mM glucose supplemented with 10% FCS (vol/vol), 100 U 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 65–86.

Preparation of islet ß-cells
Patch-clamp experiments were performed with single ß-cells obtained from pancreata of fed female NMRI mice (25–30 g) killed by cervical dislocation. Islets were isolated by collagenase digestion of the pancreas. Islets cells were dispersed in Ca2+-free medium and cultured for up to 4 days in RPMI 1640 medium supplemented with 10% FCS, 1000 U penicillin/ml, and 100 µg streptomycin/ml (26).

Measurement of [Ca2+]i
HIT-T15 cells cultured on coverslips were loaded with 5 µM fura-2/AM for 30 min at 37 C. The loading buffer was as follows: 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 acid (wt/vol), gassed with 100% O2 (vol/vol), pH 7.4. Primary islet cells were loaded in medium containing 140 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 0.5 mM glucose, and 10 mM HEPES, pH 7.4. After loading, the coverslips were washed, mounted in a temperature-controlled superfusion chamber (37 C), and placed on the stage of a Carl Zeiss Axiovert IM 135 equipped with a 40x Achrostigmat oil immersion objective (Carl Zeiss, Jena, Germany). The chamber was superfused with the same buffer as that used for fura-2 loading with 0.1% BSA (wt/vol) and without pluronic acid. The flow rate was 0.75–2 ml/min. [Ca2+]i was measured in cells of average size and healthy appearance (round in shape, no membrane blebs). To identify primary ß-cells, islet cells were shortly perfused with medium containing 0.5 mM glucose and thereafter treated with 6 mM glucose. Only cells that exhibited a typical glucose-induced decrease in [Ca2+]i were considered to be ß-cells and chosen for the Ca2+ experiments. Fura-2 fluorescence from a single cell was recorded with a dual excitation spectrofluorometer system (Deltascan 4000, Photon Technology Instruments, Wedel, Germany). [Ca2+]i was calculated according to the formula [Ca2+]i = Kd x B x (R - Rmin)/(Rmax -R), where Kd = 225 nM (27), and Rmax, Rmin, and 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).

Measurement of whole cell currents
Ca2+ currents from single ß-cells were recorded with the perforated patch technique with 150–250 µM nystatin in the patch pipette. Cells were bathed in a solution containing 115 mM NaCl, 20 mM tetraethylammonium chloride, 1.2 mM MgCl2, 10 mM CaCl2, 0.1 mM tolbutamide, 15 mM glucose, and 10 mM HEPES, pH 7.4, adjusted with NaOH. The pipette solution was composed of 70 mM Cs2SO4, 10 mM NaCl, 10 mM KCl, 7 mM MgCl2, and 10 mM HEPES, pH 7.4, adjusted with NaOH. The holding potential was -70 mV. Currents were elicited by 50-msec pulses to 0 mV. Measurements were started when the perforation of the patch resulted in a series resistance less than 50 M{Omega}. The experiments were carried out at 25 C.

Measurement of IP3
HIT-T15 cells grown in 35-mm petri dishes were preincubated for 30 min in Krebs-Ringer Henseleit buffer without BSA at 37 C. During the last 5 min of the preincubation period, W-7 (30 µM), W-5 (30 µM), or the respective solvent was added to the cells. Then the cells were stimulated with or without AVP (100 nM). The incubation was stopped after 15 sec by adding 300 µl ice-cold trichloroacetic acid (2 mM) to reach a final concentration of 0.5 mM. The petri dishes were put on ice for 15 min. Then the cells were detached, and the samples were transferred to Eppendorf tubes and again kept on ice for 2 h. After centrifugation at 7500 x g for 5 min at 4 C, the supernatants were extracted three times with water-saturated diethyl ether, subsequently neutralized by addition of 250 µl NaHCO3 (65 mM), and stored at -20 C until IP3 was evaluated by RRA as previously described (28). The pellet was resuspended in 1 ml 0.1 N NaOH, and the protein content was determined by the Bio-Rad Laboratories, Inc., method (Richmond, CA).

Materials
Fura-2/AM was purchased from Molecular Probes, Inc. (Eugene, OR), verapamil was provided by Knoll Pharmaceutical Co. (Ludwigshafen, Germany), and nifedipine was obtained from Bayer Corp. (Leverkusen, Germany). RPMI 1640, penicillin, and streptomycin were purchased from Life Technologies, Inc. (Berlin, Germany); collagenase was obtained from Boehringer Mannheim (Mannheim, Germany); thapsigargin, W-7, W-5, W-13, and W-12 were obtained from Calbiochem (Bad Soden, Germany); and AVP and the other substances were purchased from Sigma Chemical Co. (Munich, Germany) or Merck & Co., Inc. (Darmstadt, Germany). Stock solutions were prepared in water or as follows: AVP, 100 µM in 0.01 N HCl; thapsigargin, 5 mM in dimethylsulfoxide; and nifedipine, 10 mM in ethanol.

Statistics
Unless representative tracings are shown, values are the mean ± SEM. 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 significantly different.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CaM antagonists inhibit PLC-linked Ca2+signals
In the presence of glucose (10 mM) [Ca2+]i amounted to 150 ± 2 nM (n = 378 cells) in HIT-T15 cells. AVP (1 nM) (Figs. 1Go and 4Go) and bombesin (200 pM; not shown) induced repetitive Ca2+ transients as reported previously (9, 11). In the presence of AVP (1 nM) or bombesin (200 pM), the [Ca2+]i rose by 137 ± 7 nM (n = 47) and 106 ± 9 nM (n = 18) during each Ca2+ transient, respectively. The mean frequency of the Ca2+ transients elicited by AVP (1 nM) or bombesin (200 pM) was 0.63 ± 0.04 min-1 (n = 47) and 0.44 ± 0.03 min-1 (n = 18), respectively. The amplitude and frequency of the Ca2+ transients in response to the same agonist concentration varied from cell to cell (Figs. 1Go and 4Go). This could be explained by heterogeneity of single cells regarding the individual expression and activity state of membrane receptors and other elements of the Ca2+-PI signaling pathway. The CaM antagonists W-7 (10–30 µM) and W-13 (100–300 µM) reduced the frequency and amplitude of the AVP- or bombesin-induced Ca2+ transients and stopped them in 9 of 14 cells (Fig. 1Go). There was no obvious correlation between Ca2+ transient frequency and their inhibition by the CaM antagonists, as W-7 and W-13 were equally effective regardless of whether Ca2+ transient frequency was low or high (Fig. 1Go, A and C). The inhibitory effect of the CaM antagonists was fully reversible in all cells tested (Fig. 1Go). The control compounds W-5 (100 µM) and W-12 (300 µM), which are chlorine-deficient analogs of W-7 and W-13, respectively, with greatly reduced anticalmodulin activity, caused no changes in the AVP- or bombesin-induced repetitive Ca2+ transients (Fig. 1Go; n = 8 cells). To exclude that the inhibitory action of the calmodulin antagonists on PLC-linked Ca2+ signals is an HIT-T15 cell-specific phenomenon, the effects of W-7 and W-5 on carbachol-induced Ca2+ signals in primary mouse ß-cells were investigated. Like AVP and bombesin in HIT-T15 cells, carbachol activates muscarinic receptors coupled to the Ca2+-PI signaling pathway in primary ß-cells (1, 2, 3). In primary mouse ß-cells [Ca2+]i amounted to 68 ± 4 nM (n = 18 cells) in the presence of glucose (6 mM). Carbachol (10 µM) elicited a biphasic rise in [Ca2+]i, with an initial peak followed by a sustained plateau (Fig. 1Go, E and F). Carbachol (10 µM) increased [Ca2+]i by 216 ± 41 and 21 ± 6 nM at its peak or plateau (measured after 4 min), respectively (n = 10). Pretreatment with W-7 (30 µM) for 5 min, which by itself had no effect on [Ca2+]i (not shown), significantly inhibited the carbachol (10 µM)-induced Ca2+ signal (Fig. 1EGo). In the presence of W-7 (30 µM), the carbachol (10 µM)-induced Ca2+ peak and plateau were 58 ± 14 and 3 ± 1 nM, respectively (n = 4; P < 0.05 vs. control). After pretreatment with W-5 (30 µM), however, the carbachol (10 µM)-induced Ca2+ response was mainly preserved (Fig. 1FGo) and amounted to 183 ± 48 and 15 ± 4 nM at its peak or plateau (n = 4), which was not significantly different from the control value.



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  		Figure 1. CaM antagonists inhibit PLC-linked Ca2+ signals in HIT-T15 and primary mouse ß-cells. Effects of W-7 (A), W-5 (B), W-13 (C), and W-12 (D) on AVP-induced Ca2+ transients in single HIT-T15 cells. Identical results were obtained with bombesin (200 pM) as the agonist (not shown). Bars indicate the presence of the respective agents in the superfusion medium. Representative tracings of at least 4 cells are shown. Effects of W-7 (E) and W-5 (F) on the carbachol-induced increase in [Ca2+]i in primary mouse ß-cells. The thin line denotes the intracellular Ca2+ response elicited by carbachol (10 µM) in a control cell. The thick line depicts the intracellular Ca2+ response elicited by carbachol (10 µM) in the presence of W-7 (30 µM) and W-5 (30 µM), respectively. W-7 and W-5 were added 5 min before the carbachol stimulation. Representative tracings of 4 or 10 cells are shown. For average values, see text.

 


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  		Figure 4. Effects of verapamil (A) and W-7 (B) and nifedipine (C) in the presence of verapamil on AVP-induced Ca2+ transients in HIT-T15 cells. A, Effect of verapamil (50 µM) on AVP (1 nM)-induced Ca2+ transients. B, Effect of W-7 (30 µM) in the presence of verapamil (50 µM) on AVP-induced Ca2+ transients. C, Effect of nifedipine (10 µM) in the presence of verapamil (50 µM) on AVP-induced Ca2+ transients. Bars indicate the presence of the respective agents in the superfusion medium. Representative tracings of 4–13 cells, respectively, are shown.

 
CaM antagonists inhibit Ca2+ influx through VSCC
In HIT-T15 cells, membrane depolarization by K+ (45 mM) caused a biphasic rise in [Ca2+]i, with an initial peak followed by a sustained plateau. K+ (45 mM) increased [Ca2+]i by 173 ± 7 and 104 ± 4 nM at its peak or plateau (measured after 5 min), respectively (n = 120). Reexposure of the cells to K+ (45 mM) after 30 min elicited a nearly identical Ca2+ response (85 ± 6% and 84 ± 9% of the initial peak and plateau; n = 16; Fig. 2AGo). To investigate the effects of CaM antagonists on voltage-sensitive Ca2+ influx, cells were pretreated for 5 min with the respective antagonist before the second stimulation with K+ (45 mM). W-7 (10–100 µM), but not W-13 (100 µM), W-5 (100 µM), or W-12 (100 µM), slowly increased [Ca2+]i by 42 ± 15 nM in a subset of cells (13 of 20 cells; not shown). W-7 caused a concentration-dependent inhibition of the K+ (45 mM)-induced rise in [Ca2+]i, with an estimated IC50 of 27.5 µM for the peak and 19.2 µM for the plateau, respectively (Fig. 2DGo). W-13 (100 µM), which is less potent, inhibited the K+ (45 mM)-induced Ca2+ response by 41 ± 2% and 64 ± 10% at the peak and plateau, respectively (n = 4; Fig. 2DGo). W-5 (100 µM) and W-12 (100 µM) caused no significant changes in the K+ (45 mM)-induced Ca2+ signal (Fig. 2DGo). To investigate whether calmodulin antagonists can affect Ca2+ influx when VSCC are already open, the cells were first stimulated with K+ (45 mM), and W-7 (100 µM) or W-5 (100 µM) was then added during the plateau phase (Fig. 2CGo). Under these conditions W-7 (100 µM), but not W-5 (100 µM), caused a gradual decrease in the Ca2+ signal back to basal levels, whereas VSCC blockers of the L type, such as verapamil (50 µM) or nifedipine (10 µM), immediately decreased [Ca2+]i. The time constant for the rate of fall of the Ca2+ signal was 62 ± 7 sec (n = 5) for W-7 (100 µM), which was significantly longer than the time constants of 10 ± 2 sec (n = 5; P < 0.001) and 12 ± 2 sec (n = 5; P < 0.002) for nifedipine (10 µM) or verapamil (50 µM), respectively. The effect of W-7 on voltage-sensitive Ca2+ influx was further assessed by measuring whole cell Ca2+ currents in primary mouse ß-cells. As shown in Fig. 3Go, W-7 (100 µM) gradually inhibited Ca2+ currents elicited every 15 sec by short (50-msec) depolarizing voltage steps from the holding potential of -70 to 0 mV. The Ca2+ currents decreased by about 60% from -118 ± 9 to -50 ± 12 pA in the presence of W-7 (100 µM) in 4 cells (P < 0.005). This inhibitory effect was partially reversible by the wash-out of W-7. W5 (100 µM) slightly decreased Ca2+ currents by 28 ± 14% (n = 6; P < 0.01). Next, we investigated whether inhibition of Ca2+ influx through VSCC underlies the inhibitory action of the CaM antagonists on AVP- or bombesin-induced Ca2+ transients in HIT-T15 cells. Verapamil (50 µM), which inhibited the high K+ (45 mM)-induced increase in [Ca2+]i by 93 ± 7% (n = 8), reduced the frequency and/or amplitude of the AVP- and bombesin-induced Ca2+ transients in 13 of 22 cells by 51 ± 15% and 20 ± 7%, respectively. In 9 of 20 cells, verapamil (50 µM) stopped the agonist-induced Ca2+ transients (Fig. 4AGo). In cells that still generated Ca2+ transients in the presence of verapamil (50 µM) W-7 (30 µM) either stopped the AVP- or bombesin-induced Ca2+ transients (4 of 6 cells) or further reduced the frequency and amplitude of the Ca2+ transients (2 of 6 cells), as depicted in Fig. 4BGo. Nifedipine (10 µM), by contrast, caused no further inhibition of the AVP-induced Ca2+ transients under these conditions (Fig. 4CGo). Thus, the effect of W-7 (30 µM) under these conditions was not caused by further inhibition of Ca2+ influx through VSCC. This indicates that CaM antagonists interact with other mechanisms involved in the generation of PLC-linked Ca2+ transients besides their inhibitory action on voltage-sensitive Ca2+ influx.



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  		Figure 2. Effects of CaM antagonists on the K+ (45 mM)-induced [Ca2+]i rise in HIT-T15 cells. A, Effect of double stimulation with high K+ (45 mM) on [Ca2+]i in a control cell. The second K+ (45 mM) stimulation (thick line) was performed 30 min after the first stimulation and elicited an almost identical Ca2+ response. A representative tracing of 16 cells is shown. For average values, see text. B, Addition of W-7 (30 µM) to the perfusion medium 5 min before the second K+ (45 mM) stimulation caused a marked inhibition of the K+ (45 mM)-induced [Ca2+]i rise (thick line) compared with the first stimulation (thin line) in the same cell. A representative tracing of 8 cells. For average values, see D. C, Time course of the W-7 (100 µM)- and nifedipine (10 µM)-induced inhibition of the K+ (45 mM)-induced [Ca2+]i rise. Arrows indicate the addition of the respective agent to the superfusion medium. Representative tracings of 5 cells each are shown. D, Dose-response curve for W-7 and effects of W-13 (100 µM), W-5 (100 µM), and W-12 (100 µM) on the K+ (45 mM)-induced peak and plateau rise in [Ca2+]i. {Delta} [Ca2+]i (percentage of first stimulation) denotes the rise in [Ca2+]i induced by the second K+ (45 mM) stimulation after 5-min pretreatment with the respective agent, expressed as a percentage of the first K+ (45 mM)-induced Ca2+ response in the same cell. Values are the mean ± SEM of 4–16 experiments.

 


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  		Figure 3. Effect of W-7 (100 µM) on Ca2+ currents through VSCC of mouse ß-cells. This recording was performed in the perforated patch configuration with nystatin in the pipette. Currents were elicited every 15 sec by 50-msec voltage steps from the holding potential of -70 to 0 mV. W-7 was applied at the time indicated by the horizontal bar. The lower panel shows the currents at a, b, and c on an extended time scale. This recording is representative of four experiments with similar results.

 
CaM antagonists inhibit internal Ca2+mobilization by PLC-linked agonists
IP3-mediated mobilization of internal Ca2+ is central to the generation of PLC-linked Ca2+ signals. In Ca2+-free medium, AVP or bombesin caused one or two Ca2+ transients, demonstrating mobilization of internal Ca2+ in HIT-T15 cells. As depicted for AVP in Fig. 5AGo, pretreatment (5 min) with W-7 (30 µM) inhibited the AVP- or bombesin-induced peak Ca2+ by 75 ± 6% (n = 25; P < 0.0001) and the amount of Ca2+ released, as calculated by the area under the curve, by 67 ± 13% (n = 25; P < 0.005) compared with control values (n = 48). W-5 (30 µM), however, neither changed peak Ca2+ nor the area under the curve of the PLC-linked Ca2+ signal in Ca2+-free medium compared with control values (n = 20; not shown). To investigate whether inhibition of PLC-linked mobilization of internal Ca2+ is secondary to inhibition of agonist-induced IP3 formation, the effects of W-7 and W-5 on AVP-stimulated IP3 production were determined. As shown in Fig. 5CGo pretreatment with W-5 (30 µM), but not W-7 (30 µM), caused a small, but significant, decrease in the basal IP3 concentration. However, neither W-7 (30 µM) nor W-5 (30 µM) affected the AVP (100 nM)-induced increase in IP3 formation (Fig. 5CGo). This demonstrates that W-7 interacts with the mobilization of internal Ca2+ distal to the formation of IP3, which is a prerequisite for the generation of AVP- or bombesin-induced Ca2+ transients.



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  		Figure 5. Effect of W-7 on AVP-induced mobilization of internal Ca2+ (A) and effect of W-7 and W-5 on thapsigargin-induced Ca2+-influx in the presence of verapamil (B) and on AVP-induced IP3 production (C). A, Effect of W-7 (30 µM) on AVP (1 nM)-induced mobilization of internal Ca2+ in Ca2+-free medium. Ca2+-free medium was added 7 min before the stimulation with AVP (1 nM). Control, Representative tracing of a control cell (n = 24). W-7 30 µM, W-7 (30 µM) was added 5 min before the stimulation with AVP. For average values, see text. B, Effects of W-7 and W-5 on thapsigargin (2 µM)-induced rises in [Ca2+]i in the presence of verapamil (50 µM). Verapamil (50 µM) was added 7 min and W-7 (30 µM) or W-5 (30 µM) was added 5 min before thapsigargin (2 µM) stimulation, and they were present throughout the experiment. For each group the mean from 8–12 cells of the thapsigargin (2 µM)-induced increase in [Ca2+]i above basal ({Delta} [Ca2+]i) is depicted. C, Effects of W-7 (30 µM) and W-5 (30 µM) on the AVP (100 nM)-stimulated formation of IP3. Solvent (control), W-7 (30 µM), or W-5 (30 µM) was added 5 min before treatment with or without (co) AVP (100 nM). The reaction was stopped after 15 sec, and IP3 was determined as described in Materials and Methods. Values are the mean ± SEM of three independent experiments determined in triplicate. *, P < 0.05.

 
CaM antagonists inhibit capacitative Ca2+influx through VICC
Depletion of internal Ca2+ stores activates Ca2+ influx across the plasma membrane to the cytosol, a mechanism termed capacitative Ca2+ entry (29). The endoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor thapsigargin, which depletes internal Ca2+ stores without the formation of IP3, is a major tool to study this type of Ca2+ entry (30). Thapsigargin (2 µM) caused a biphasic increase 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 VSCC and VICC (9, 10). To assess whether W-7 could interact with capacitative Ca2+ entry through VICC, cells were stimulated with thapsigargin (2 µM) in the presence of verapamil (50 µM). As shown in Fig. 5BGo, pretreatment (5 min) with W-7 (30 µM), but not W-5 (30 µM), reduced the plateau phase of the thapsigargin (2 µM)-induced Ca2+ signal, demonstrating inhibition of Ca2+ influx through VICC.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Frequency-modulated Ca2+ transients, rather than amplitude-modulated sustained increases in [Ca2+]i, constitute the intracellular signal activated by PLC-linked hormones in many tissues, including ß-cells (13, 14, 17). The mechanisms underlying the phenomenon of PLC-linked Ca2+ transients, however, are only partly understood. Here we demonstrate that the CaM antagonists, W-7 and W-13, reversibly inhibited the frequency and amplitude of the Ca2+ transients or stopped them completely. By contrast, the dechlorinated control compounds, W-5 and W-12, had no effect at even higher concentrations. As W-7, but not W-5, inhibited the carbachol-induced biphasic rise in [Ca2+]i in primary mouse ß-cells, this indicates that inhibition of PLC-linked Ca2+ signals in insulin-secreting cells by the CaM antagonists is not restricted to the generation of PLC-linked Ca2+ transients in HIT-T15 cells. The naphthalenesulfonamide compounds were specifically synthesized in analog pairs to better control for nonspecific effects unrelated to their interaction with the Ca2+/CaM complex (31). Within each pair, the difference in structure, one chlorine atom, increased the affinity for the Ca2+/CaM complex. Thus, at any given concentration, these analog pairs should exhibit differential effects, reflecting their differing affinities for the Ca2+/CaM complex, if the involvement of CaM is to be deduced. The facts that W-7 and W-12 inhibited the Ca2+ transients at concentrations associated with their reported anticalmodulin activity in other systems (31, 32), and that the respective control compounds, W-5 and W-13, were ineffective suggest, but does not definitely prove, that Ca2+ via activation of CaM may provide positive feedback involved in the generation of the PLC-linked Ca2+ transients in HIT-T15 cells.

W-7 and W-13 inhibited high K+-induced Ca2+ influx through VSCC, whereas W-5 (100 µM) and W-12 (300 µM) were ineffective. Furthermore, W-7 (100 µM) reversibly caused a 60% reduction, whereas an equimolar concentration of W-5 resulted in a 28% reduction of whole cell Ca2+ currents in mouse ß-cells. As the W compounds are hydrophobic, nonspecific interactions with VSCC are conceivable, thereby inhibiting voltage-sensitive Ca2+ influx. This, however, appears to be unlikely, as W-13, which is markedly more hydrophobic than W-7 (33), was 2–3 times less potent in inhibiting the K+-induced [Ca2+]i rise. As the difference in action of the W compounds on the K+-induced [Ca2+]i rise parallels their difference in anticalmodulin activity, a role for Ca2+/CaM could be assumed in the modulation of VSCC function in HIT-T15 cells, as has been previously suggested for RINm5F and rat pancreatic islet cells (34, 35). This view is supported by findings from mouse ß-cells overexpressing an inactive CaM that show impaired VSCC function (36). The mechanisms by which Ca2+/CaM may modulate VSCC function are as yet unclear. The fact that the time constant for the W-7-induced inhibition of the plateau phase of the K+ (45 mM)-induced [Ca2+]i was 5–6 times longer than the time constants for the VSCC blocker nifedipine and verapamil is consistent with, but does not prove, an indirect action of Ca2+/CaM modulating VSCC function, e.g. by phosphorylation of VSCC by CaM-dependent enzymes (20, 21, 22). Recently, it was shown that CaM can directly interact with VSCC, thereby modulating their activity (37). Thus, the time delay in the action of CaM antagonists could alternatively reflect the time required to access and interact with CaM already associated with the VSCC in the activated state. Although Ca2+-dependent inactivation of VSCC is well documented (38), Ca2+-dependent facilitation of voltage-sensitive Ca2+ influx has been demonstrated in several cell types in recent years (39). As CaM plays a pivotal role in both Ca2+-dependent inactivation and facilitation of Ca2+ influx through VSCC of the L type, this might be important during repetitive cellular activities (37). Thus, a rise in [Ca2+]i could provide positive feedback on voltage-sensitive Ca2+ influx by Ca2+/CaM-dependent enhancement of VSCC function, which, in turn, is necessary for the sustained generation of PLC-linked Ca2+ transients in HIT-T15 cells. However, additional Ca2+/CaM-dependent feedback mechanisms appear to exist. First, W-7 or W-13 inhibited PLC-linked Ca2+ transients at concentrations that only partly suppressed voltage-sensitive Ca2+ influx. Second, W-7 caused further inhibition of Ca2+ transients occurring in the presence of verapamil (50 µM) that completely blocked the K+-induced [Ca2+]i rise.

Mobilization of Ca2+ from internal Ca2+ stores is a prerequisite for PLC-linked Ca2+ transients. We found that W-7, but not W-5, inhibited AVP- or bombesin-induced Ca2+ mobilization from internal Ca2+ stores in Ca2+-free medium. As Ca2+ influx is abolished under these conditions, this effect of W-7 is independent of its inhibitory effect on voltage-sensitive Ca2+ influx. There are several ways that W-7 could interact with PLC-linked Ca2+ mobilization from internal Ca2+ stores. W-7 may influence agonist-induced formation of IP3 and/or IP3-dependent Ca2+ mobilization due to modulation of IP3 receptor function or due to altering the amount of Ca2+ stored within intracellular Ca2+ pools. The first possibility could be ruled out, as AVP-induced formation of IP3 was unaffected by W-7. In permeabilized rat islets, W-7 caused Ca2+ release from the endoplasmic reticulum and potentiated IP3-induced Ca2+ mobilization (40). These results, however, cannot be reconciled with our studies of W-7 in HIT-T15 cells. No evidence could be found for W-7-induced mobilization of internal Ca2+ either directly or by enhanced IP3 formation. Although W-7 caused a small rise in [Ca2+]i in a subset of HIT-T15 cells in the presence of external Ca2+, no such effect could be observed in Ca2+-free medium. Therefore, partial emptying of internal Ca2+ stores by W-7, either directly or indirectly, cannot account for the inhibition of AVP- or bombesin-induced internal Ca2+ mobilization in HIT-T15 cells. Activation of IP3-mediated Ca2+ release appears to require CaM-dependent IP3 receptor phosphorylation, most likely at the CaM kinase II phosphorylation site described for the purified receptor (24, 25). Thus, W-7 could inhibit agonist-induced mobilization of internal Ca2+ by inhibiting IP3-mediated Ca2+ mobilization via prevention of CaM-dependent IP3 receptor phosphorylation as in intact and permeabilized rat hepatocytes (23). IP3-dependent mobilization of internal Ca2+, therefore, appears to be an additional site where Ca2+, through activation of Ca2+/CaM-dependent processes, could exert positive feedback on the generation of AVP- or bombesin-induced Ca2+ transients in HIT-T15 cells.

Depletion of internal Ca2+ stores as a consequence of mobilization of internal Ca2+ by IP3 activates Ca2+ influx across the plasma membrane to the cytosol, termed capacitative Ca2+ entry (29). In HIT-T15 cells capacitative Ca2+ entry occurs through VSCC and VICC (9, 10). Ca2+ store depletion by the endoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor thapsigargin in the presence of verapamil (50 µM) resulted in an initial Ca2+ peak showing mobilization of internal Ca2+ followed by a sustained plateau rise in [Ca2+]i, reflecting capacitative Ca2+ entry through VICC. W-7 significantly reduced the thapsigargin-induced sustained plateau rise in [Ca2+]i under these conditions, suggesting that W-7 inhibits capacitative Ca2+ influx through VICC, which is independent from inhibition of VSCC. As W-5 was ineffective, this suggests that the action of W-7 could involve CaM-dependent processes. This is in agreement with findings from fibroblasts and thyroid FRTL-5 cells, where a role of CaM-dependent processes in capacitative Ca2+ influx through VICC has been suggested (41, 42). Further studies, however, are required to determine whether CaM-dependent mechanisms control voltage-insensitive Ca2+ influx either directly by modulation of VICC function or indirectly by interfering with the activation process of capacitative Ca2+ entry that is still poorly understood.

In summary, we could demonstrate that the W compounds differentially inhibited PLC-linked Ca2+ transients in single HIT-T15 cells. This appears to be caused by inhibition of mobilization of internal Ca2+ and of Ca2+ influx through VSCC and VICC, which are both necessary for the generation of PLC-linked Ca2+ transients. As the W compound effects corresponded well to their reported anticalmodulin activity, the involvement of CaM-dependent mechanisms is suggested. If CaM was involved, these data indicate that Ca2+ via activation of CaM-dependent processes provide positive feedback on the generation of PLC-linked Ca2+ transients in HIT-T15 cells. This appears to involve CaM-dependent regulation of both mobilization of internal Ca2+ and Ca2+ influx through VSCC and VICC.


   Acknowledgments
 
We thank Prof. Irene Schulz, Department of Physiology, University of the Saarland (Homburg/Saar, Germany), for the measurement of IP3.


   Footnotes
 
1 This work was supported by Deutsche Forschungsgemeinschaft Grant Scho 466/1–3. Back

Received April 16, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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