Thapsigargin-sensitive cationic current leads to membrane depolarization, calcium entry, and insulin secretion in rat pancreatic {beta}-cells

R. Cruz-Cruz,1 A. Salgado,2 C. Sánchez-Soto,1 L. Vaca,2 and M. Hiriart1

Departments of 1Biophysics and 2Cell Biology, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, Mexico City, Mexico

Submitted 23 February 2005 ; accepted in final form 2 April 2005


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
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Glucose-induced insulin secretion by pancreatic {beta}-cells depends on membrane depolarization and [Ca2+]i increase. We correlated voltage- and current-clamp recordings, [Ca2+]i measurements, and insulin reverse hemolytic plaque assay to analyze the activity of a thapsigargin-sensitive cationic channel that can be important for membrane depolarization in single rat pancreatic {beta}-cells. We demonstrate the presence of a thapsigargin-sensitive cationic current, which is mainly carried by Na+. Moreover, in basal glucose concentration (5.6 mM), thapsigargin depolarizes the plasma membrane, producing electrical activity and increasing [Ca2+]i. The latter is prevented by nifedipine, indicating that Ca2+ enters the cell through L-type Ca2+ channels, which are activated by membrane depolarization. Thapsigargin also increased insulin secretion by increasing the percentage of cells secreting insulin and amplifying hormone secretion by individual {beta}-cells. Nifedipine blocked the increase completely in 5.6 mM glucose and partially in 15.6 mM glucose. We conclude that thapsigargin potentiates a cationic current that depolarizes the cell membrane. This, in turn, increases Ca2+ entry through L-type Ca2+ channels promoting insulin secretion.

nonselective cationic channels; calcium channels; stimulus-secretion coupling


GLUCOSE-INDUCED INSULIN SECRETION by pancreatic {beta}-cells depends on the rise of intracellular Ca2+ concentration ([Ca2+]i). In nonstimulating glucose concentrations (<6.0 mM), the membrane potential is at resting level. When glucose concentration increases, {beta}-cell membrane exhibits an oscillating electrical activity. First, a slow membrane depolarization is observed; this is followed by a fast depolarization phase to a plateau level, on which bursts of action potentials are superimposed, and finally the membrane repolarizes. The time that the membrane spends firing in the depolarized plateau state is determined by extracellular glucose concentration (17).

It is well accepted that, when the glucose concentration rises, the ATP-to-ADP ratio increases as a result of glucose metabolism, leading to the closure of ATP-sensitive potassium (KATP) channels (1). This leads to a slow membrane depolarization via nonselective cationic channels, resulting in the subsequent activation of voltage-sensitive Na+ and Ca2+ channels (22). Ca2+ entry through L-type channels is a determining factor for insulin secretion (11).

The slow depolarization that follows KATP channel closure is an important event that is not fully understood. It has been suggested that it is due to sodium entrance through a yet unidentified voltage-independent cationic channel, since a study shows that this depolarization is eliminated by the removal of extracellular Na+ (3). Alternatively, it has been shown that maneuvers that deplete intracellular calcium stores, such as the application of the microsomal Ca2+-ATPase inhibitor thapsigargin (TG) or the activation of G protein-coupled receptors associated with the inositol triphosphate (IP3) cascade, also result in the activation of nonselective cationic currents in a wide variety of cells, including the murine insulinoma {beta}TC3-neo cells (18). This current, referred to as Ca2+-release-activated nonselective current (ICRAN) is activated also by the nonselective cationic current activator maitotoxin (MTX).

It has been shown that MTX induces cell membrane depolarization as a result of increased Na+ permeability in mouse {beta}-cells, resembling the effects of high glucose concentrations on cell membrane potential (23).

To explore cationic channels that could be important for membrane depolarization in rat pancreatic {beta}-cells, we analyzed the early effects of TG on 1) cationic currents, 2) electrical activity, 3) intracellular calcium concentration, and 4) insulin secretion.

We demonstrate a direct correlation between the activation of a TG-sensitive cationic channel, cell depolarization, changes in [Ca2+]i, and insulin secretion in pancreatic {beta}-cells.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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Materials. Reagents were obtained from the following sources: collagenase type IV from Worthington (Freehold, NJ); guinea pig insulin antiserum from Biogenesis (Sandown, NH); bovine serum albumin, Hanks' balanced salt solution (HBSS), chromium chloride, staphylococcal protein A, HEPES, trypsin, trypan blue, nystatin, and poly-L-lysine from Sigma (St. Louis, MO); tissue culture dishes (Corning); Spinner-Eagles' salt solution from Microlab (Mexico City, Mexico); fetal bovine serum from Equitech-Bio (Ingram, TX); guinea pig complement, RPMI 1640 salts, and penicillin-streptomycin-amphotericin B solution from Life Technologies (Grand Island, NY); TG from Alomone Labs (Jerusalem, Israel); fluo 4-AM from Molecular Probes (Eugene, OR).

Pancreatic {beta}-cell culture. All methods used in this study were approved by the Internal Council and the Animal Care Committee of the Instituto de Fisiologia Celular, Universidad Nacional Autónoma de México. Animal care was performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH No. 85-23, reviewed 1985).

Young adult male Wistar rats (250–280 g) were obtained from the local animal facility, maintained in a 14-h light (0600–2000)/10-h dark cycle, and allowed free access to a standard laboratory rat diet and tap water. Animals were anesthetized with pentobarbital sodium (40 mg/kg) and after pancreas dissection were killed by cervical dislocation.

Single pancreatic {beta}-cells were obtained by collagenase digestion and islet dissociation by mechanical disruption in calcium-free medium, as described previously (15). Single {beta}-cells were plated at low density (10,000 cells/cm2) on glass coverslips previously coated with poly-L-lysine and cultured for 24–72 h in RPMI 1640 supplemented with 200 U/ml penicillin G, 200 mg/ml streptomycin, 0.5 mg/ml amphotericin B, and 10% fetal bovine serum (FBS).

Electrophysiological recordings and analysis. Whole cell voltage-clamp recordings (2) were performed at 20–22°C using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Patch electrodes were pulled from Kimax-51 capillary tubes (Kimble Glass, Vineland, NJ) and had a tip resistance of 1.5–3 M{Omega}. Electrode tips were coated with Sylgard (Dow Corning, Midland, MI).

The pulse protocol used for the analysis of the currents consisted of applying depolarizing test pulses from –120 to +60 mV in 20 mV steps for 250 ms from a holding potential of –80 mV. After recording of control currents, TG (200 nM) was added with the aid of a Picospritzer (General Valve, Fairfield, NJ).

The whole cell conductance of the TG-activated current was calculated using Eq. 1:

where I2 is the current measured at –100 mV (V2) and I1 is the current measured at the holding potential at –80 mV (V1).

Current-clamp experiments. The perforated mode of the patch clamp technique was utilized in all measurements. An EPC9 amplifier was used. Data acquisition was controlled by the pulse connected to the bath solution via a 100 mM KCl-agar bridge. Nystatin was dissolved in methanol and added to the pipette solution at a final concentration of 100 µg/ml.

Current-clamp experiments were performed as previously described (20). Briefly, the patch pipette was filled with nystatin solution, and once a G{Omega} seal was formed, cell capacitative artifacts were monitored in response to a 1-mV voltage step until electrical access was gained. After this, the amplifier mode was switched from voltage-clamp to current-clamp, and the experiments were conducted as described in the legend of Fig. 3.



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Fig. 3. TG depolarizes {beta}-cells. A: current-clamp representative experiment of a single {beta}-cell measured with the perforated patch-clamp technique (MATERIALS AND METHODS). At time 0, (indicated by vertical arrow) 200 nM TG was applied, resulting in a slow depolarization that produced cell action potential firing. B: average firing frequency before and after TG addition. Values measured from 14 independent cells obtained from 3 different experiments.

 
Recording solutions. The ionic composition of the external solutions is summarized in Table 1.


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Table 1. External solutions

 
Internal Solution. The internal solution consisted of (in mM) 115 CsAsp, 10 CsCl, 5 CsF, 2.5 EGTA HEPES, 10, pH 7.2, 290–295 mosM. For perforated patch experiments the CsAsp was replaced by 115 KAsp.

Confocal calcium measurements. Changes in cytosolic calcium in pancreatic {beta}-cells were monitored using the fluorescent calcium indicator fluo 4-AM, as previously described (21). Briefly, cells were placed on glass coverslips and allowed to attach to the glass surface for 24 h in RPMI with 10% FBS. The cells were then loaded with 5 µM fluo 4-AM diluted in RPMI medium and incubated for 30 min at 37°C with 5% CO2. After incubation, cells were washed once with low-glucose physiological solution (containing in mM: 120 NaCl, 1.2 KH2PO4, 1.2 Mg2SO4, 4.75 KCl, 5.6 glucose, 20 HEPES, and 0.05% BSA). Calcium measurements were carried out using a Bio-Rad MRC 1024 confocal microscope. The excitation wavelength was 488 nm and emission was collected at 535 nm. An increment in the intensity of fluorescence indicated an increase in cytosolic calcium. The average of three images was recorded every 3 s. All drugs were applied to the dish with a micropipette. In some experiments, cells were incubated for 5 min at room temperature with nifedipine (5 µM) before the addition of TG. Cell fluorescence was subtracted from background and autofluorescence.

Reverse hemolytic plaque assay. To identify insulin-secreting cells and measure insulin secretion by single cells, we used a reverse hemolytic plaque assay (RHPA) (16), as described previously (15). Briefly, after 48 h in culture, cells were challenged for 1 h in HBSS containing 5.6 or 15.6 mM glucose, in control conditions or with TG (200 nM), in the presence of an insulin antiserum (1:20 in HBSS), and further incubated for 30 min with guinea pig complement. Insulin released during the incubation time was revealed by the presence of hemolytic plaques surrounding insulin-secreting cells. The size of the plaques was measured by projecting the image on a monitor attached to a video camera and Nikon Axiophot inverted microscope with the aid of the JAVA video analysis software (version 1.40; Jandel Scientific, Corte Madera, CA).

Plaque size was expressed as area; cells that formed plaques were counted, and the results were expressed as the percentage of insulin-secreting cells. All experiments were performed in duplicate, and ≥100 cells were counted per experimental condition. The overall secretory activity of {beta}-cells under a given experimental condition was expressed as a secretion index, calculated by multiplying the average plaque area by the percentage of plaque-forming cells.

Statistical analysis. Significant differences between data were evaluated by analysis of variance, followed by Fisher's multiple range test, using the Number Cruncher Statistical System (NCSS 4.2; Dr. Jerry L. Hintze, Kaysville, UT). All results are expressed as means ± SE.


    RESULTS
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TG increases a cationic current in {beta}-cells. We used whole cell configuration of the patch-clamp technique to measure the effects of TG on membrane currents in single rat {beta}-cells. Figure 1A illustrates representative families of current traces recorded in response to different voltage pulses from a holding potential of –80 mV before (1) and after exposure to 200 nM TG (2). The effects of TG developed immediately after application of the reagent and were not reversible after its withdrawal (data not shown). Figure 1B shows the means ± SE current-to-voltage (I-V) relationships obtained in four different experiments. The reversal potential of the current in control conditions is ~0 mV and does not significantly change with TG application.



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Fig. 1. Thapsigargin (TG) activates an inwardly rectifying current in {beta}-cells. A: representative current families elicited by voltage steps from a holding potential of –80 mV (MATERIALS AND METHODS).Cells under control conditions (1) and immediately after 200 nM TG (2). B: mean current-to-voltage (I-V) relationships obtained from 9 cells from 4 different experiments, control conditions ({bullet}) and after 200 nM TG ({circ}).

 
The slope conductances were calculated according to Eq. 1 (MATERIALS AND METHODS). Assuming a capacitance of 8.4 pF, slope conductance was 20 pS/pF for control cells and 33 pS/pF for TG-treated {beta}-cells. These results demonstrate that TG increases a small cationic current (Icat).

We then studied the ionic selectivity of Icat by replacing Na+ with N-methyl-D-glucamine (NMDG) or removing Ca2+ from the external solution. I-V curves obtained in each condition are illustrated in Fig. 2. As shown in Fig. 2A, Icat decreased when Na+ was replaced with NMDG in the external solution (Table 1, row 2), compared with control conditions (Fig. 1B). The current measured at –100 mV (Fig. 2D) decreased by nearly 65% compared with control conditions (Table 1, row 1).



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Fig. 2. TG-induced current is carried mainly by sodium ions. A: mean I-V relationships obtained in Na+-free solution (Table 1, row 2) under control conditions ({bullet}) and after 200 nM TG ({circ}). B: mean I-V relationships obtained with Ca2+-free solution (Table 1, row 3). C: mean I-V relationships obtained with extracellular solution Na+ and Ca2+ free (Table 1, row 4). D: mean peak current obtained at –100 mV for the conditions explained above. Note the small current obtained with 10 mM Ca2+ in the absence of extracellular Na+. In all cases, measurements were obtained from ≥20 different cells from 4 different experiments.

 
By contrast, in Ca2+-free solution (Table 1, row 3) the current magnitude decreased, but changes were not statistically significant (Fig. 2, B and D) compared with control conditions (Fig. 1B and Table 1, row 1).

When both cations were omitted from the external medium (Table 1, row 4), the currents were minimized (Fig. 2, C and D). Under these conditions, TG-sensitive Icat was reduced by 82% compared with the current recorded in the external solution with Na+ and Ca2+ (Fig. 1B and Table 1, row 1). These results indicate that the principal charge carrier of Icat is Na+, with a minimal contribution of Ca2+ ions.

TG increases electrical activity in {beta}-cells. We studied the effects of TG on single {beta}-cell electrical activity in 5.6 mM glucose with the current-clamp perforated patch-clamp technique. Figure 3A shows that, in control conditions, cells were polarized around –70 mV and electrically silent. A few seconds after TG application, the membrane slowly depolarized to a plateau level where action potentials were superimposed. Figure 3B shows the mean firing frequency of 14 different cells in the presence of 5.6 mM glucose, indicating that TG induced depolarization and electrical activity even at this low glucose concentration, when the KATP is presumably active.

TG increases [Ca2+]i in {beta}-cells. To evaluate the effect of TG on [Ca2+]i in 5.6 mM glucose, we measured single {beta}-cells' fluorescence with confocal microscopy. Figure 4A shows that TG increased [Ca2+]i in the cells, which remained elevated throughout the recording period. In contrast, in the presence of nifedipine, calcium signals were transient (Fig. 4B). This result indicates that the sustained phase of the [Ca2+]i increment is the result of calcium influx through L-type Ca2+ channels. Moreover, when [Ca2+]i was measured in a Ca2+-free solution, the sustained phase of the calcium signal induced by TG decreased by nearly 92% (Fig. 4C), consistent with the notion that the sustained phase is the result of L-type Ca2+ channels activity.



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Fig. 4. Effect of nifedipine (NIF) on TG-induced calcium increment. Single {beta}-cells cultured for 48 h were plated on glass coverslips and loaded with 5 µM fluo 4-AM, (MATERIALS AND METHODS). Each panel illustrates a representative single cell (A and B, top) obtained at time points indicated by vertical arrows. A: cells exposed to 200 nM TG under control conditions. B: cells exposed to 200 nM TG in the continuous presence of 5 µM nifedipine. Each solid line shown in A and B, bottom, illustrates the fluorescence increment ({Delta}F/{Delta}F0) in response to TG from a single cell. C: means ± SD of the fluorescence increment measured at the time point illustrated by the rightmost arrow in A and B. Fluorescence increments induced by addition of 200 nM TG under control conditions (+Ca2+), in the presence of 5 mM EGTA in the bath (Ca2+ free), and in the presence of normal extracellular Ca2+ and 5 µM nifedipine. Measurements obtained from ≥20 different cells from 4 independent experiments.

 
TG increases insulin secretion in different glucose concentrations. In control experiments, when single {beta}-cells were incubated for 1 h in the presence of an insulin antiserum with a stimulatory glucose concentration of 15.6 mM, they secreted 2.5-fold more insulin than in the basal glucose in 5.6 mM glucose (Fig. 5). In these conditions, TG increased individual insulin secretion by 100% in both glucose concentrations (Table 2). TG also increased the percentage of {beta}-cells that secreted insulin (plaque-forming cells) by 18 and 34% in basal and stimulatory glucose concentrations, respectively. Consequently, TG increases total insulin secretion, given by the insulin secretion index (seeMATERIALS AND METHODS), 2.5-fold in both glucose concentrations with respect to controls (Fig. 5 and Table 2). The cellular mechanism that explains this increment involves the amplification of hormone secretion by individual cells and an increase in the percentage of insulin-secreting cells.



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Fig. 5. Effect of TG on insulin secretion by a single {beta}-cell. Insulin secretion by single {beta}-cells was measured with reverse hemolytic plaque assay (MATERIALS AND METHODS). Overall secretory activity of {beta}-cells under a given experimental condition is expressed as a secretion index. C, control cells; TG, cells exposed to TG (200 nM); NIF, cells exposed to nifedipine (5 µM). Data are means ± SE of 4 different experiments in duplicate. Symbols denote statistically significant differences: *control cells in 5.6 mM glucose; {dagger}TG-treated cells in each glucose concentration; {ddagger}control cells in 15.6 mM glucose; §cells exposed to nifedipine to 15.6 mM glucose; P < 0.05.

 

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Table 2. Insulin secretion by single {beta}-cells in response to a 1-h incubation with TG in different extracellular glucose concentrations

 
We have previously shown that the L-type channel blocker nifedipine does not affect basal insulin secretion (19). In this study, no difference was found when cells were incubated simultaneously with TG and nifedipine in 5.6 mM glucose (Fig. 5 and Table 2).

We (19) have previously observed that, in 15.6 mM glucose, nifedipine decreases the insulin secretion index by nearly 60%. In the present study, we have reproduced this result and observed that TG and nifedipine together decreased the insulin secretion index by 41% compared with control cells in 15.6 mM glucose.

To estimate the percentage of insulin secretion that is not dependent on L-type channel activation, we subtracted the insulin secretion index in the presence of nifedipine from the value obtained with TG and nifedipine together, and this result was expressed as a percentage of the control index in 15.6 mM glucose. When TG is present, nearly 23% of the secretion in this high glucose concentration is not dependent on L-type calcium channel activation.

Finally, we found in three different experiments that the insulin secretion index in the complete absence of glucose is 176 ± 64 and 398 ± 100 in control conditions and with TG, respectively (P < 0.05). In the absence of glucose in the external solution, only a small percentage of cells secreted enough insulin to be detected by the RHPA, 21 and 26% in control and TG, respectively. Compared with the basal glucose concentration (5.6 mM), individual cells secreted nearly 0.5 times less insulin in zero glucose. However, in the latter condition, TG increased insulin secretion by 126%, indicating that the TG-induced increase is not dependent on the presence of glucose in the extracellular medium.


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 MATERIALS AND METHODS
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In this study, we demonstrate that TG potentiates an Icat already present in single {beta}-cells under resting conditions. Icat is carried mainly by Na+, with a small contribution of Ca2+. This current depolarizes the membrane, which leads to Ca2+ entry through L-type Ca2+ channels and increased insulin secretion by single rat {beta}-cells.

Several studies have shown the involvement of KATP and voltage-dependent channels in the electrical activity of {beta}-cells; however, few studies account for the importance of cationic channels in this process. These channels play an important role because they participate in the slow depolarization phase that precedes the fast depolarization and the plateau potential, where action potential firing is observed. Without the presence of these cationic channels, blockade of KATP channels would not result in membrane depolarization.

In the first experiments, we used TG as a tool to deplete [Ca2+]i stores and observed that TG depolarized the membrane. In this study, we focused on determining the immediate effects of TG on {beta}-cells. We found that TG increases the magnitude of an Icat already present in {beta}-cells. Icat increment induces membrane depolarization and the concomitant activation of voltage-gated Na+ and Ca2+ channels, resulting in action potential firing and calcium influx through nifedipine-sensitive L-type channels. The final consequence of the activation of Icat is a marked increment in insulin secretion even at basal glucose concentrations (where KATP channels are presumably active).

Other studies have shown the presence of similar cationic currents, mainly permeable to Na+, that are modulated by the muscarinic agonist carbachol, in mouse pancreatic {beta}-cells, and in the insulinoma cell line HIT-T15 (13). Moreover, a similar Icat activated by MTX has been described in mouse pancreatic {beta}-cells (23).

On the other hand, other Na+ currents activated by MTX and by depletion of intracellular Ca2+ stores, referred to as ICRAN, have been described in mouse {beta}-cells (18). The depletion of Ca2+ stores may also induce a sustained, voltage-independent Ca2+ entry (10, 14), which enhances glucose-induced electrical activity (23, 12) in {beta}-cells.

We also demonstrate that TG induces a slow depolarization and increases electrical activity, maintaining high [Ca2+]i. Moreover, when nifedipine was added, Ca2+ signals decreased even in the continuous presence of TG (Fig. 4B), indicating the importance of L-type Ca2+ channel activity for the sustained influx of calcium evoked by TG. It is unlikely that TG's effects on electrical activity and insulin secretion would be produced by the direct activation of L-type Ca2+ channels, because it has been shown that TG does not directly affect L-type Ca2+ currents in {beta}-cells (9).

It has been previously shown that exposure of islets to TG in 10 mM glucose resulted in increased action potential firing of {beta}-cells (6). However, in this stimulating glucose concentration, TG's effect was considered unlikely to be due to the activation of a depolarizing cationic conductance.

A Ca2+-dependent, nonselective cation current, activated by glucagon-like peptide-1 (GLP-1), has been observed in HIT and mouse and human {beta}-cells (7). The GLP1-activated channel is active in the presence of low glucose, which suggests that it could play a role in membrane depolarization (8). Like ICRAN, this current is activated by MTX and blocked by SKF96365 but whether or not these currents are identical remains to be determined.

We have previously described that insulin secretion by single {beta}-cells in 15.6 mM glucose is nearly 2.5-fold higher than in 5.6 mM glucose (4). In this study, we observed that acute TG treatment increases insulin secretion in both glucose concentrations. This effect is reflected in two measured parameters: 1) amplification of insulin secretion by individual cells, because TG increases the plaque area, which is proportional to the amount of hormone secreted by the cell, by nearly 100% in both glucose concentrations; and 2) the recruitment of previously silent cells, which in control conditions do not secrete a detectable amount of insulin, because in the presence of TG the percentage of insulin-secreting cells increases by 18 and 34% in 5.6 mM and 15.6 mM glucose, respectively.

TG also increases insulin secretion in the absence of glucose in the extracellular medium by 126% compared with the control. This increment is very similar to that previously observed in single {beta}-cells treated with carbachol in zero glucose (5).

TG may use different mechanisms to increase insulin secretion. For example, TG could transiently increase [Ca2+]i by depleting intracellular Ca2+ stores. However, our results suggest that the principal mechanism involved is an increase in the cationic current that leads to membrane depolarization and Ca2+ entry through L-type channels. In fact, the effect of TG on insulin secretion in 5.6 mM glucose is completely abolished by nifedipine, whereas in 15.6 mM glucose it is only partially inhibited.

We conclude that, in basal glucose concentration (5.6 mM), depletion of intracellular Ca2+ stores by TG application increases the activity of an Icat. The mechanism leading to Icat activation after depletion of internal Ca2+ stores remains to be identified.

Icat carried mostly by Na+ depolarizes the plasma membrane, which results in the subsequent activation of voltage-sensitive Na+ and Ca2+ channels, Ca2+ entry, and, as a direct consequence of these actions, insulin secretion (Fig. 6).



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Fig. 6. Proposed model for TG's effect on membrane depolarization and insulin secretion. In basal glucose concentration (5.6 mM), depletion of intracellular Ca2+ stores by TG application increases activity of cationic current (Icat). The mechanism leading to Icat activation after depletion of internal Ca2+ stores remains to be identified (?). This current depolarizes the plasma membrane, which results in subsequent activation of voltage-sensitive Na+ and Ca2+ channels, Ca2+ entry, and insulin secretion by pancreatic {beta}-cells.

 
The mechanism leading to cationic channel modulation in normal {beta}-cells remains to be established. However, the properties of the cationic current described here resemble those previously found in the literature for nonselective cationic channels activated upon depletion of intracellular Ca2+ stores (18, 10, 14) and, more interestingly, to carbachol-induced cationic currents (13). Because carbachol application would result in IP3 production and the release of Ca2+ from internal stores, it is probable that TG could promote similar effects on [Ca2+]i, mimicking the effects of carbachol. This could be a plausible mechanism for the modulation of Icat to control insulin secretion.


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This work was supported by Grant no. D39822 [GenBank] from Consejo Nacional de Ciencia y Tecnología.


    ACKNOWLEDGMENTS
 
We thank Tamara Rosenbaum, Elvira Arellanes, and Victor Navarro for proofreading and discussion of the manuscript, and Ana M. Escalante and Francisco Pérez for computing assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Hiriart, Dept. of Biophysics, Instituto de Fisiología Celular, UNAM; Ciudad Universitaria, AP 70-253 Coyoacán, México D.F. 04510, Mexico (e-mail: mhiriart{at}ifc.unam.mx)

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


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 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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 REFERENCES
 

  1. Aguilar-Bryan L and Bryan J. Molecular biology of adenosine triphosphate-sensitive potassium channels. Endocr Rev 20: 101–135, 1999.[Abstract/Free Full Text]
  2. Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85–100, 1981.[CrossRef][ISI][Medline]
  3. Henquin JC, Garcia MC, Bozem M, Hermans MP, and Nenquin M. Muscarinic control of pancreatic B cell function involves sodium-dependent depolarization and calcium influx. Endocrinology 122: 2134–2142, 1988.[Abstract]
  4. Hiriart M and Ramirez-Medeles MC. Functional subpopulations of individual pancreatic B-cells in culture. Endocrinology 128: 3193–3198, 1991.[Abstract]
  5. Hiriart M and Ramirez-Medeles MC. Muscarinic modulation of insulin secretion by single pancreatic beta-cells. Mol Cell Endocrinol 93: 63–69, 1993.[CrossRef][ISI][Medline]
  6. Kanno T, Rorsman P, and Gopel SO. Glucose-dependent regulation of rhythmic action potential firing in pancreatic beta-cells by K(ATP)-channel modulation. J Physiol 545: 501–507, 2002.[Abstract/Free Full Text]
  7. Leech CA and Habener JF. Insulinotropic glucagon-like peptide-1-mediated activation of nonselective cation currents in insulinoma cells is mimicked by maitotoxin. J Biol Chem 272: 17987–17993, 1997.[Abstract/Free Full Text]
  8. Leech CA and Habener JF. A role for Ca2+-sensitive nonselective cation channels in regulating the membrane potential of pancreatic beta-cells. Diabetes 47: 1066–1073, 1998.[Abstract]
  9. Lemmens R, Larsson O, Berggren PO, and Islam MS. Ca2+-induced Ca2+ release from the endoplasmic reticulum amplifies the Ca2+ signal mediated by activation of voltage-gated L-type Ca2+ channels in pancreatic beta-cells. J Biol Chem 276: 9971–9977, 2001.[Abstract/Free Full Text]
  10. Liu YJ and Gylfe E. Store-operated Ca2+ entry in insulin-releasing pancreatic beta-cells. Cell Calcium 22: 277–86, 1997.[CrossRef][ISI][Medline]
  11. Mears D. Regulation of insulin secretion in islets of Langerhans by Ca(2+)channels. J Membr Biol 200: 57–66, 2004.[CrossRef][ISI][Medline]
  12. Mears D, Sheppard NF Jr, Atwater I, Rojas E, Bertram R, and Sherman A. Evidence that calcium release-activated current mediates the biphasic electrical activity of mouse pancreatic beta-cells. J Membr Biol 155: 47–59, 1997.[CrossRef][ISI][Medline]
  13. Mears D and Zimliki CL. Muscarinic agonists activate Ca2+ store-operated and -independent ionic currents in insulin-secreting HIT-T15 cells and mouse pancreatic beta-cells. J Membr Biol 197: 59–70, 2004.[CrossRef][ISI][Medline]
  14. Miura Y, Henquin JC, and Gilon P. Emptying of intracellular Ca2+ stores stimulates Ca2+ entry in mouse pancreatic beta-cells by both direct and indirect mechanisms. J Physiol 503: 387–398, 1997.[Abstract]
  15. Navarro-Tableros V, Sanchez-Soto MC, Garcia S, and Hiriart M. Autocrine regulation of single pancreatic beta-cell survival. Diabetes 53: 2018–2023, 2004.[Abstract/Free Full Text]
  16. Neill JD and Frawley LS. Detection of hormone release from individual cells in mixed populations using a reverse haemolytic plaque assay. Endocrinology 112: 1135–1137, 1983.[Abstract]
  17. Ozawa S and Sand O. Electrophysiology of excitable endocrine cells. Physiol Rev 66: 887–952, 1986.[Free Full Text]
  18. Roe MW, Worley JF III, Qian F, Tamarina N, Mittal AA, Dralyuk F, Blair NT, Mertz RJ, Philipson LH, and Dukes ID. Characterization of a Ca2+ release-activated nonselective cation current regulating membrane potential and [Ca2+]i oscillations in transgenically derived beta-cells. J Biol Chem 273: 10402–10410, 1998.[Abstract/Free Full Text]
  19. Rosenbaum T, Sanchez-Soto MC, and Hiriart M. Nerve growth factor increases insulin secretion and barium current in pancreatic beta-cells. Diabetes 50: 1755–1762, 2001.[Abstract/Free Full Text]
  20. Vaca L. Calmodulin inhibits calcium influx current in vascular endothelium. FEBS Lett 390: 289–293, 1996.[CrossRef][ISI][Medline]
  21. Vaca L and Sampieri A. Calmodulin modulates the delay period between release of calcium from internal stores and activation of calcium influx via endogenous TRP1 channels. J Biol Chem 277: 42178- 42187, 2002.[Abstract/Free Full Text]
  22. Vidaltamayo R, Sanchez-Soto MC, and Hiriart M. Nerve growth factor increases sodium channel expression in pancreatic beta cells: implications for insulin secretion. FASEB J 16: 891–892, 2002.[Abstract/Free Full Text]
  23. Worley JF III, McIntyre MS, Spencer B, and Dukes ID. Depletion of intracellular Ca2+ stores activates a maitotoxin-sensitive nonselective cationic current in beta-cells. J Biol Chem 269: 32055–32058, 1994.[Abstract/Free Full Text]




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