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Endocrinology Vol. 140, No. 3 1200-1204
Copyright © 1999 by The Endocrine Society

ARTICLES

A Low Voltage-Activated Ca2+ Current Mediates Cytokine-Induced Pancreatic ß-Cell Death1

Lin Wang, Arin Bhattacharjee, Zhuang Zuo, Fuquan Hu, Richard E. Honkanen, Per-Olof Berggren and Ming Li

Departments of Pharmacology (L.W., A.B., F.H., M.L.) and Biochemistry (Z.Z., R.E.H.), University of South Alabama College of Medicine, Mobile, Alabama 36688; and The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute (P.-O.B.), S-171 76 Stockholm, Sweden

Address all correspondence and requests for reprints to: Ming Li, Ph.D., Department of Pharmacology, University of South Alabama College of Medicine, Mobile, Alabama 36688. E-mail: mli{at}jaguar1.usouthal.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-dependent diabetes mellitus is characterized by the selective destruction of pancreatic ß-cells. Chronic treatment with cytokines induced a low voltage-activated (LVA) Ca2+ current in mouse ß-cells. The concomitant increase in the basal cytoplasmic free Ca2+ concentration ([Ca2+]i) was associated with DNA fragmentation and cell death. Antagonists of LVA Ca2+ channels prevented this elevation of basal [Ca2+]i and DNA fragmentation and reduced the percentage of cell death. Exposure to cytokines did not affect the profile of Ca2+ currents or basal [Ca2+]i in glucagon-secreting {alpha}-cells. An increased Ca2+ signal through LVA Ca2+ channels may thus be a key feature in cytokine-induced ß-cell destruction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HALLMARK of insulin-dependent diabetes mellitus (IDDM) is an almost complete destruction of pancreatic ß-cells, with maintenance of the {alpha}- (glucagon-secreting) and {delta}- (somatostatin-secreting) cells within the islets of Langerhans (1). Accumulating evidence has implicated cytokines as key mediators of ß-cell killing in rodent models of IDDM (2, 3, 4, 5) and in human islet preparation (6). The mechanisms that determine cytokine-mediated ß-cell selective death have not been clarified.

Previous studies have shown that the basal cytoplasmic free Ca2+ concentration ([Ca2+]i) in nonobese diabetic (NOD; a model system of IDDM) mouse islet cells is abnormally elevated and that these cells express a low voltage-activated (LVA) Ca2+ current (7). As enhanced excitability and/or [Ca2+]i overload, resulting from an overexpression of LVA Ca2+ currents, play crucial roles in the pathogenesis of several diseases (8, 9, 10), we were interested in determining the role of LVA Ca2+ channels in the process of cytokine-induced ß-cell death. In this report, we demonstrate that an increased Ca2+ signal through LVA Ca2+ channels is a key feature in cytokine-induced ß-cell destruction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and islet cell preparation
The ß-TC3 cell line was provided by G. G. Holz (New York University, New York, NY) and grown at 37 C in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 11 mM glucose, 10% FBS, penicillin (25 U/ml), and streptomycin (25 µg/ml).

The pancreases of Swiss-Webster mice (Charles River Laboratories, Inc., Wilmington, MA) were removed after intrapancreatic perfusion with 2 ml Hanks’ solution (Life Technologies) containing collagenase (4 mg/ml; Boehringer Mannheim, Indianapolis, IN), deoxyribonuclease I (10 µg/ml; Sigma Chemical Co., St. Louis, MO), CaCl2 (1.28 mM), and BSA (1 mg/ml; Life Technologies). The pancreatic tissue was incubated at 37 C for 20 min and then washed five times with enzyme-free Hanks’ solution. Islets were isolated and treated with 0.1% pancreatin (Sigma Chemical Co.) for 5 min at 37 C. Single cells were obtained by triturating the islets with plastic pipette tips; they were then transferred into 35-mm culture dishes. Cells were cultured in RPMI 1640 medium (Life Technologies) containing 5 mM glucose, 10% FBS, 25 U/ml penicillin, and 25 µg/ml streptomycin at 37 C, 5% CO2 for 2–5 days before the experiments. It has been shown that more than 80% of dispersed mouse islet cells cultured in this way are insulin-secreting ß-cells (11).

Patch-clamp electrophysiology and data analysis
Whole cell recordings were carried out by the standard "giga-seal" patch-clamp technique. The whole cell recording pipettes were made of hemocapillaries (Warner Instrument Corp., Hamden, CT), pulled by a two-stage puller (Sutter Instrument, Novato, CA), and heat polished before use. The currents were recorded using an EPC-9 patch-clamp amplifier (HEKA, Lambrecht/Pfalz, Germany). All experiments were performed in extracellular solution containing 10 mM CaCl2, 110 mM tetra-ethylammonium-Cl, 10 mM CsCl, 10 mM HEPES, 40 mM sucrose, and 0.5 mM 3,4-diaminopyridine, pH 7.3. The intracellular solution contained 130 mM N-methyl-D-glucamine, 20 mM EGTA (free acid), 5 mM bis(2-aminophenoxy)-ethane-N',N',N'N'-tetraacetate, 10 mM HEPES, 6 mM MgCl2, and 4 mM Ca(OH)2, with pH adjusted to 7.4 with methanesulfonate. The pipette solution contained 2 mM Mg ATP in all experiments to minimize run-down of Ca2+ currents. Data were acquired with Pulse/PulseFit software (HEKA) and filtered at 2.5 kHz. The recordings were performed at room temperature (20–23 C).

Cytokines
Interferon-{gamma} (IFN{gamma}) is murine recombinant with specific activity of 4 x 106 U/mg (Life Technologies). Interleukin-1ß (IL-1ß) and tumor necrosis factor-{alpha} (TNF{alpha}) are mouse recombinants with specific activities of 1.1 x 106 and 2.7 x 105 units/µg, respectively (Sigma Chemical Co.).

Measurements of [Ca2+]i
Indo-1/AM (Molecular Probes, Inc., Eugene, OR) was used for [Ca2+]i determination on an ACAS 570 Interactive Laser Cytometer (Meridian Instruments, Inc., Okemos, MI). This fluorescent indicator was excited with the 363-nm laser line. The calculated ratio of fluorescence emission (485 and 405 nm) of indo-1 was compared with that of a standard curve to determine [Ca2+]i. Cells were cultured in RPMI 1640 medium (Life Technologies) on poly-D-lysine (50 µg/ml; Sigma Chemical Co.)-coated glass coverslips (no. 1 grade, Fisher, Pittsburgh, PA) for 2–5 days at 37 C in 5% CO2. Before measurements, cells were treated with either cytokines or cytokines plus Ca2+ channel antagonists for a period of 6 h. Cells were then loaded in an extracellular solution (150 mM NaCl, 4.7 mM KCl, 3 mM glucose, 2 mM CaCl2, 2 mM MgCl2, and 5 mM HEPES, pH 7.2) and 2.5 µM indo-1/AM (Molecular Probes, Inc.) for 20 min at 37 C. Indo-1/AM was washed out before conducting the experiments. [Ca2+]i was measured in the line scan mode.

DNA fragmentation
The ß-TC3 cell DNA fragmentation was assayed by agarose gel electrophoresis with ethidium bromide staining as previously described (12). In all experiments, DNA was extracted from 2 x 106 cells after 22- to 24-h incubation in control medium or medium containing cytokines and Ca2+ channel antagonists.

Cell death
Cell death was analyzed by trypan blue staining. All cells after treatment were resuspended and then mixed with trypan blue in a 1:1 ratio (volume). A random sample of cells were transferred to coverslips and visually scored under a light microscope. More than 400 cells were counted from multiple fields under each experimental condition.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We examined the effects of chronic cytokine treatment on the voltage-sensitive Ca2+ currents in primary cultured mouse islet cells. After treatment with IL-1ß (25 U/ml) and IFN{gamma} (300 U/ml) for 6 h, an LVA Ca2+ current was induced in these cells (Fig. 1AGo). This current was present in 48% of cytokine-treated mouse islet cells. No such current was observed when the cells were treated with either IL-1ß or IFN{gamma} alone. We have conducted experiments at different times recording LVA Ca2+ currents induced by cytokines, and the results indicate that no further increase in current density occurs even after treatment for 48 h. This LVA current has not been observed in nontreated cells (7, 12, 13, 14, 15, 16). The steady state inactivation curve of the cytokine-induced LVA Ca2+ currents displayed a low voltage property (Fig. 1EGo) similar to the inactivation curve of the LVA currents in NOD mouse islets cells (7). This current was also blocked by NiCl2 (10 µM; n = 4; Fig. 1FGo). It has been reported that low concentrations of NiCl2 selectively block LVA Ca2+ channels in various cell types (17, 18, 19, 20), including pancreatic ß-cells (21). In addition to the cytokine-induced LVA current, a profound increase in Ca2+ current density was observed over the voltages between -20 and 20 mV. These high voltage-activated Ca2+ currents are nifedipine-sensitive currents (completely blocked by 10 µM nifedipine; data not shown), and the increase in this current density is similar to the increased L-type Ca2+ current density observed after treatment of ß-cells with serum from IDDM patients (22).



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  		Figure 1. LVA Ca2+ currents were induced by cytokine treatment (IL-1ß, 25 U/ml; IFN[{gamma}, 300 U/ml) for 6 h in primary cultured mouse islet cells, but not in {alpha}-TC1 cells. An LVA current was elicited by a -40 mV test pulse in an islet cell (A), but the same current was not detected in {alpha}-TC1 cells (C). The Ca2+ current density-voltage relationships obtained from islet cells (B) and {alpha}-TC1 cells (D) with and without cytokine treatment are shown. The open circles represent the current densities of untreated cells (n = 10 for islet cells; n = 20 for {alpha}-TC1 cells), and the filled circles represent the current densities of cells treated by cytokines (n = 21 for islet cells; n = 21 for {alpha}-TC1 cells). The recordings were elicited by voltages ranging from -50 to +20 mV for 100 msec. All experiments were performed at the holding potential of -80 mV. E, Steady state inactivation of LVA tail currents elicited by a 10-msec depolarizing (-10 mV) pulse followed by a 50-msec hyperpolarizing pulse (-100 mV), with a holding potential of -80 mV. The data (n = 4; error bars represent the SE) were fit to Boltzman equation (V1/2 = -50.7 mV; k = 9.25). F, NiCl2 (10 µM) blocked the cytokine-induced LVA Ca2+ current elicited at a -30 mV step pulse.

 
As {alpha}-cells are more resistant to the toxic effects of cytokines than ß-cells (23), we also examined the effects of cytokines on the Ca2+ currents in a glucagon-secreting cell line, {alpha}-TC1 (24). This cell line, like {alpha}-cells, is more resistant to the cytotoxic effect of cytokines (4, 25). Treatment of {alpha}-TC1 cells with IL-1ß and IFN{gamma} failed to induce LVA Ca2+ currents and did not alter the current density (Fig. 1Go, C and D). Therefore, the induction of LVA Ca2+ currents and increased Ca2+ current density observed after chronic treatment with cytokines showed specificity for ß-cells.

LVA Ca2+ channels are activated at low membrane potentials. This unique feature may allow them to regulate [Ca2+]i under nonstimulatory conditions. Indeed, basal [Ca2+]i in cytokine-treated cells was approximately 3-fold higher than that in nontreated cells (Fig. 2AGo). This increase in basal [Ca2+]i was blocked by NiCl2 (10 µM), but not by the L-type Ca2+ channel antagonist, nifedipine (10 µM). Cytokines failed to increase basal [Ca2+]i in {alpha}-TC1 cells (Fig. 2BGo). These results suggest that Ca2+ influx through LVA Ca2+ channels is responsible for the cytokine-induced elevation in basal [Ca2+]i in ß-cells.



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  		Figure 2. Effects of cytokines on [Ca2+]i in mouse islet cells and {alpha}-TC1 cells. A, Basal [Ca2+]i of primary cultured mouse islet cells was approximately 3-fold higher after cytokine treatment. NiCl2 (10 µM), but not nifedipine (10 µM), prevented the increase in [Ca2+]i. B, Basal [Ca2+]i in {alpha}-TC1 cells was unaffected by cytokine treatment. Cytokine treatment consisted of 25 U/ml IL-1ß and 300 U/ml IFN[{gamma} for 6 h. Numbers above bars represent the numbers (n) of experiments. *, P < 0.01, by one-way ANOVA in conjunction with Dunnett’s multiple comparison test.

 
It has been shown that cytokines induce apoptosis in human pancreatic islet cells (6). Apoptosis is also the mode of cell death in the development of IDDM in the NOD mouse (26) and in multiple low dose streptozotocin-induced IDDM in the mouse (27), and is involved in ß-cell destruction induced by serum of patients with IDDM (22). As a marker of apoptosis, DNA fragmentation has been reported to precede ß-cell lysis (3, 28, 29).

We have used ß-TC3 cells, a mouse ß-cell line (30), to demonstrate the role of LVA Ca2+ channels in cytokine-mediated DNA fragmentation. We first examined the LVA Ca2+ current density before and after cytokine treatment. The LVA Ca2+ current (at Vm = -30 mV) in ß-TC3 cells was increased from 1.86 ± 0.33 (pA/pF; n = 30) to 3.45 ± 0.47 (pA/pF; n = 10) after treatment with cytokines (25 U/ml IL-1ß, 100 U/ml IFN{gamma}, and 100 U/ml TNF{alpha}) for 24 h. This indicates that the LVA Ca2+ current in ß-TC3 cells is regulated by cytokines, as seen in mouse islet cells. As shown in Fig. 3Go, cytokine-induced DNA fragmentation displayed a ladder pattern of oligonucleosomal fragments. The three LVA Ca2+ channel blockers, NiCl2, amiloride (7, 31, 32, 33), and mibefradil (10, 34, 35), all independently prevented cytokine-induced DNA fragmentation. In contrast, nifedipine had no inhibitory effect on DNA fragmentation induced by cytokines. This experiment has been repeated in ß-TC3 cells (n = 2) as well as in NIT-1 cells (n = 3), a ß-cell line derived from NOD mice, and the same results were obtained.



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  		Figure 3. Effects of LVA Ca2+ channel antagonists on cytokine-induced DNA fragmentation. Cytokine-induced DNA fragmentation revealed by a ladder pattern of oligonucleosomal fragments. The fragmentation was prevented by NiCl2 (10 µM), amiloride (100 µM), or mibefradil (1 µM). Nifedipine (10 µM) did not prevent DNA fragmentation induced by cytokines. These agents alone did not cause DNA fragmentation.

 
We next examined the function of LVA Ca2+ channels in cytokine-mediated cell death in ß-TC3 cells. Many cells died when the medium contained IL-1ß, IFN{gamma}, and TNF{alpha}; however, NiCl2 (20 µM) effectively reduced the ß-cell killing potency of cytokines in both a time- and dose-dependent manner (Fig. 4Go, A and B, respectively). In contrast, nifedipine did not exhibit a protective effect. Similar results were obtained from an experiment conducted in NIT-1 cells with mibefradil, which also reduced ß-cell death induced by cytokines. These results demonstrate that LVA Ca2+ channels enhance the vulnerability of ß-cells to the cytotoxic effects of cytokines.



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  		Figure 4. Effects NiCl2 on cytokine-induced ß-TC3 cell death. NiCl2 (20 µM) significantly reduced cell death induced by cytokines in both a time- (A) and dose-dependent (B) manner (n = 3). Cytokine treatment consisted of IL-1ß (25 U/ml), IFN[{gamma} (100 U/ml), and TNF{alpha} (100 U/ml) in A and of IL-1ß (25 U/ml), TNF{alpha} (100 U/ml), and various concentrations of IFN[{gamma} as indicated in B. The first dose, 0, represents zero concentrations for all three cytokines (IL-1ß, IFN[{gamma}, and TNF{alpha}). The concentration of nifedipine was 10 µM in both A and B. Error bars represent the SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Some previous studies suggest that cytokine-induced apoptosis in human islets is slower than that in murine islets. This may be due to a relatively high expression of protective enzymes (36). However, patients with newly diagnosed IDDM have high concentrations of macrophage-derived cytokines (37), suggesting that cytokines play a critical role in IDDM pathogenesis. Our results demonstrate that cytokines are able to both induce the expression of LVA Ca2+ currents and increase HVA Ca2+ currents in ß-cells. However, LVA Ca2+ currents are responsible for elevated basal [Ca2+]i and are also involved in cytokine-induced ß-cell apoptosis.

The mechanisms underlying cytokine-mediated selective ß-cell apoptosis remain the central question in IDDM pathogenesis. It was found that IL-1 selectively induces ß-cell lysis in rat islets via the induction of endogenous inducible nitric oxide (NO) synthesis (38, 39, 40), but studies in human islets did not show a major contribution of inducible NO synthase to cytokine toxicity in ß-cells (41, 42). Thus, the NO theory may not explain human ß-cell destruction.

It has also been suggested that cytokines may damage ß-cells by inducing oxygen free radical production and lipid peroxidation (43). However, the toxicity of oxidants and their protection by nicotinamide, which scavenges hydroxyl free radicals, is not ß-cell specific (44, 45). Our hypothesis suggests that the key element for selectivity is basal [Ca2+]i, which is regulated differently in ß-cells and non-ß-cells (e.g. {alpha}-cells).

The mechanisms of cytokine-induced expression of LVA Ca2+ channels also remain to be clarified. This expression may be regulated at the transcriptional level, as LVA Ca2+ channel expression has been linked to cell cycle progression and proliferation (46, 47). Alternatively, it may involve the recruitment of channels into the plasma membrane or to alterations in intracellular signaling pathways that regulate channel function, such as protein phosphorylation. These regulatory mechanisms may partially account for the observation that only half of the tested islet cells expressed LVA Ca2+ currents at certain times. Recently, a rat neuronal T-type Ca2+ channel was identified (48). However, the Northern blot analysis failed to show the expression of this channel in pancreatic tissue. It is possible that the ß-cell LVA channel is an isoform of the neuronal T-type Ca2+ channel. The molecular identification of the ß-cell LVA Ca2+ channel is, therefore, crucial for understanding the manner in which these channels are regulated by cytokines.

It has been shown that LVA Ca2+ channels mediate sustained increases in [Ca2+]i induced by angiotensin II (49), endothelin (50), and platelet-derived growth factor (51). The role of Ca2+ influx in apoptosis is crucial in multiple cell types (52), A recent study in a mouse ß-cell line (MIN6) showed that treatment of these cells with Ca2+ ionophores did not result in cell death (53), suggesting that high [Ca2+]i alone is insufficient to kill the ß-cell. In addition to the elevated [Ca2+]i requirement for diabetic serum-induced apoptosis (22), our data suggest that elevated [Ca2+]i is also required for cytokine-induced ß-cell death. Enhanced basal [Ca2+]i may make ß-cells more vulnerable to further cytokine toxicity. Indeed, as cytokines had no effect on the Ca2+ current density or on basal [Ca2+]i in glucagon-secreting {alpha}-cells, these differences may explain the preferential destruction of ß-cells occurring during insulitis.


   Acknowledgments
 
We thank Dr. J. P. Clozel for kindly providing mibefradil, and Dr. R. M. Whitehurst, Jr., for constructive discussions.


   Footnotes
 
1 This work was supported by the Pharmaceutical Research and Manufacturers of America Foundation, Inc., the American Diabetes Association, the American Heart Association, the Juvenile Diabetes Foundation International, the NIH (Grant DK-50151; to M.L.), the Swedish Medical Research Council (Grants 03X-09890, 03XS-12708, and 19X-00034; to P.-O.B.), the Swedish Diabetes Association (to P.-O.B.), Funds of the Karolinska Institute (to P.-O.B.), and the Juvenile Diabetes Foundation International (to P.-O.B.). Back

Received July 9, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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