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
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ABSTRACT |
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nonselective cationic channels; calcium channels; stimulus-secretion coupling
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 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 -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 -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 -cells.
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MATERIALS AND METHODS |
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Pancreatic -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 (250280 g) were obtained from the local animal facility, maintained in a 14-h light (06002000)/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 -cells were obtained by collagenase digestion and islet dissociation by mechanical disruption in calcium-free medium, as described previously (15). Single
-cells were plated at low density (10,000 cells/cm2) on glass coverslips previously coated with poly-L-lysine and cultured for 2472 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 2022°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 1.53 M. 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:
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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 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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Confocal calcium measurements.
Changes in cytosolic calcium in pancreatic -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
-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.
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RESULTS |
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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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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 -cells.
We studied the effects of TG on single
-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 -cells.
To evaluate the effect of TG on [Ca2+]i in 5.6 mM glucose, we measured single
-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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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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DISCUSSION |
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Several studies have shown the involvement of KATP and voltage-dependent channels in the electrical activity of -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 -cells. We found that TG increases the magnitude of an Icat already present in
-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 -cells, and in the insulinoma cell line HIT-T15 (13). Moreover, a similar Icat activated by MTX has been described in mouse pancreatic
-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 -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
-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 -cells (9).
It has been previously shown that exposure of islets to TG in 10 mM glucose resulted in increased action potential firing of -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 -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 -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 -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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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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