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RECEPTORS AND SIGNAL TRANSDUCTION
-cells
Department of Cellular and Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka City, Shizuoka, Japan
Submitted 17 October 2005 ; accepted in final form 22 June 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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-cells, hypotonic stimulation elicited an increase in cytosolic Ca2+ concentration ([Ca2+]c) at 2.8 mM glucose. The hypotonically induced [Ca2+]c elevation was significantly suppressed by nicardipine, a voltage-dependent Ca2+ channel blocker, and by Gd3+, amiloride, 2-aminoethoxydiphenylborate, and ruthenium red, all cation channel blockers. In contrast, the [Ca2+]c elevation was not inhibited by suramin, a P2 purinoceptor antagonist. Whole cell patch-clamp analyses showed that hypotonic stimulation induced membrane depolarization of -cells and produced outwardly rectifying cation currents; Gd3+ inhibited both responses. Hypotonic stimulation also increased insulin secretion from isolated rat islets, and Gd3+ significantly suppressed this secretion. Together, these results suggest that osmotic cell swelling activates cation channels in rat pancreatic -cells, thereby causing membrane depolarization and subsequent activation of voltage-dependent Ca2+ channels and thus elevating insulin secretion. calcium ion; swelling; patch-clamp; gadolinium
-cells, osmotic cell swelling also induces insulin secretion (2, 4, 6, 12, 22). Because high concentrations of glucose have been shown to increase -cell volume (18, 20), cell swelling may be involved in the glucose-induced insulin secretion. Several studies have suggested that cell swelling releases insulin by activating volume-sensitive Cl– channels, thus depolarizing the -cell membrane and increasing the cytosolic Ca2+ concentration ([Ca2+]c) via the activation of voltage-dependent Ca2+ channels (VDCC) (2, 3, 6, 13, 21). However, several reports have argued against this proposition by showing that hypotonically induced insulin secretion persists even in the presence of Cl– channel blockers such as niflumic acid and DIDS (12, 22). Thus the possibility remains that a mechanism independent of Cl– channels is involved in hypotonically induced insulin secretion.
Osmotic cell swelling mechanically stretches the plasma membrane. It is thus possible that stretch-activated cation channels participate in the responses to hypotonic stimulation in -cells. However, little information exists about stretch-activated cation channels in -cells. In the present study, therefore, we investigated the possible involvement of stretch-activated cation channels in the responses to hypotonic stimulation in rat pancreatic -cells. The data shown here provide direct evidence for a contribution of cation channels to -cell swelling-induced responses.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Measurement of [Ca2+]c.
Pancreatic -cells were isolated from male Wistar rats (9–13 wk old, 200–300 g; SLC, Hamamatsu, Japan) with a previously described collagenase digestion technique (11). Rats were treated as approved by the Institutional Animal Care and Use Committee and according to the Guidelines for Animal Experiments established by the Japanese Pharmacological Society. The isolated -cells were plated on coverslips and cultured for 1 day in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin.
[Ca2+]c was measured in fura PE3-loaded cells by dual-wavelength fluorometry as described previously (11). The cells were loaded with 4 µM fura PE3-AM for 3 h at 37°C and then mounted in a chamber on the stage of an inverted microscope (Diaphot TMD 300, Nikon, Tokyo, Japan). The cells were continuously superfused with isotonic solution (in mM: 85 NaCl, 5 KCl, 1.2 MgCl2, 1.2 CaCl2, 10 HEPES, 2.8 glucose, and 100 mannitol; 290 mosmol/l, pH 7.4) maintained at 37°C with the use of a peristaltic pump at a flow rate of 2 ml/min. [Ca2+]c was measured by an Argus-50/CA system (Hamamatsu Photonics, Hamamatsu, Japan), with alternating excitation of cells at 340 ± 10 and 380 ± 10 nm. The resultant emission was monitored at 510 ± 20 nm. Pairs of 340- and 380-nm fluorescence images were captured every 6 s and were converted to 340-to-380 ratio images. The 340-to-380 ratio was used to indicate the relative [Ca2+]c. Drugs were applied in the superfusing solution. Hypotonic stimulation was performed by exchanging the superfusing isotonic solution for 35% hypotonic solution (in mM: 85 NaCl, 5 KCl, 1.2 MgCl2, 1.2 CaCl2, 10 HEPES, and 2.8 glucose; 190 mosmol/l, pH 7.4).
Measurements of cell volume, membrane potential, and ion currents.
The isolated -cells were mounted in a chamber on the stage of an inverted microscope (Diaphot TMD 300, Nikon) and were continuously superfused with the isotonic solution at a flow rate of 1 ml/min at room temperature. Hypotonic stimulation was performed by exchanging the superfusing solution for the hypotonic solution.
For the measurement of cell volume, images of -cells were recorded with a digital camera (Coolpix 990, Nikon) connected to the microscope. The digital images were analyzed with graphic software (Canvas 7, Deneba Software, Miami, FL). Cell volume was calculated under the assumption that the cells were spherical and was expressed relative to volume with respect to the volume measured during exposure to the isotonic solution according to the method of Miley et al. (18).
The membrane potential of isolated -cells was measured with the whole cell patch-clamp technique. Patch pipettes, which were pulled from borosilicate glass capillaries and fire polished before use, had a resistance of 2–5 M
when filled with pipette solution containing (in mM) 140 KCl, 5 K2-ATP, 0.1 EGTA, and 10 HEPES (pH 7.2). The measurement of membrane potential was made in the I = 0 mode of a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Foster City, CA).
Ion currents were also measured with the whole cell patch-clamp technique. Patch pipettes had a resistance of 2–5 M when filled with pipette solution containing (in mM) 130 KCl, 10 tetraethylammonium chloride, 5 K2-ATP, 1 BAPTA, and 10 HEPES (pH 7.2). The ion currents were recorded in the voltage-clamp mode of the patch-clamp amplifier. pCLAMP software (version 8.1.0, Axon Instruments) was used for generating command pulses and recording data. Current records were low-pass filtered at 1 kHz (–3 dB), digitized at 5 kHz by an analog-to-digital converter (DigiData 1200A, Axon Instruments), and stored on a computer (Endeavor Pro-500L, Epson, Nagano, Japan). Membrane voltages were corrected for the liquid junction potential between the pipette and bath solutions. Leak subtraction was not performed.
Measurement of insulin secretion. Rat pancreatic islets isolated with the collagenase digestion technique were cultured for 1 day in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin. Insulin secretion was analyzed by a perifusion method as described previously (10). Fifty islets were placed in the chamber (1 ml) and perifused with isotonic solution containing 5.5 mM glucose at 37°C at a rate of 1 ml/min. After preincubation for 30 min, the isotonic solution was exchanged for the hypotonic solution for 10 min. The hypotonic stimulation was repeated in the absence of Gd3+ and then, after a 30-min interval, in the presence of Gd3+. The perifusate was collected every 2 min and kept at –70°C for later assay. Insulin released into the perifusate was assayed with a radioimmunoassay (Insulin Radioimmunoassay Kit, Eiken Chemical, Tokyo, Japan).
Statistics. Data are expressed as means ± SE. The effect of each treatment was analyzed with the paired Student’s t-test. A probability of P < 0.05 was accepted as the level of statistical significance.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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-cell volume induced by hypotonic stimulation.
Exposure of -cells to 35% hypotonic solution resulted in an increase in cell volume (Fig. 1). Swelling began at 2–4 min and reached a maximum within 8 min after exposure to the hypotonic solution. The increase in cell volume was followed by a small decrease in it, i.e., RVD. Gd3+ (1 µM) did not affect the hypotonically induced volume increase (data not shown).
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-cells induced by hypotonic stimulation.
In isolated rat pancreatic -cells, [Ca2+]c was stable in isotonic solution at 2.8 mM glucose. Exposing -cells to 35% hypotonic solution caused an increase in [Ca2+]c (Fig. 2). [Ca2+]c was rapidly returned to basal level when the superfusing solution was returned to the isotonic solution. The [Ca2+]c response was reproducible (Fig. 2A), and was largely suppressed by nicardipine (1 µM), a VDCC blocker (Fig. 2B; see 
Fig. 4). Although a similar [Ca2+]c elevation was produced by exposure to 15% or 25% hypotonic solution, the response was not reproducible when stimulated repetitively (data not shown). Therefore, the following analyses were performed with 35% hypotonic stimulation.
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-cells (21), Gd3+ at 1 µM had no significant effect on 30 mM K+-induced [Ca2+]c elevation (Fig. 3B). Similarly, the hypotonically induced [Ca2+]c elevation was significantly reduced by three other cation channel blockers, amiloride (3 µM), 2-APB (100 µM), and ruthenium red (3 µM) (Fig. 4). We also confirmed that each cation channel blocker at the concentration used in the present study had no effects on the 30 mM K+-induced [Ca2+]c elevation (data not shown). We further investigated the contribution of ATP to the hypotonically induced [Ca2+]c elevation. However, the [Ca2+]c elevation was not inhibited by suramin (50 µM), a P2 purinoceptor antagonist (Fig. 4). The same concentration of suramin completely abolished the [Ca2+]c elevation induced by ATP (30 µM; data not shown).
Changes in membrane potential of -cells induced by hypotonic stimulation.
The results of the [Ca2+]c measurements suggest that hypotonic stimulation activates cation channels, thereby depolarizing the -cell membrane and opening VDCC. To confirm this hypothesis, the effect of hypotonic stimulation on membrane potential was investigated with the patch-clamp technique. When the patch membrane was ruptured, the membrane potential of -cells exhibited oscillations, but they ceased within 10 min. At that time, the membrane potential was –53.9 ± 2.8 mV (n = 7). Exposure of -cells to 35% hypotonic solution depolarized the membrane significantly, by 
20 mV (Fig. 5A). In most cases, the gigaohm seal could not be maintained for a long time because of cell swelling. Therefore, the effect of Gd3+ on hypotonically induced depolarization was examined in a separate series of experiments, in which the gigaohm seal was formed in hypotonic solution within 10 min after the superfusing solution was exchanged for the hypotonic solution. As shown in Fig. 5B, the membrane potential in the hypotonic solution measured in this procedure was somewhat more depolarized than that shown in Fig. 5A, but the difference was not significant. Under this condition, the membrane potential was significantly polarized by Gd3+ (1 µM; Fig. 5B). These results suggest that hypotonic stimulation leads to membrane depolarization of -cells by opening Gd3+-sensitive cation channels.
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-cells to 35% hypotonic solution increased both inward and outward currents. Figure 7B represents the difference current, which shows the current elicited by the hypotonic stimulation. The difference current exhibited small outward rectification, and the reversal potential of the current was –6.7 ± 2.4 mV (n = 4). When NaCl concentration in the bath solution was reduced from 85 to 35 mM by the substitution of NaCl with mannitol, the reversal potential of the difference current was shifted toward a more negative potential, i.e., –32.5 ± 2.3 mV (n = 4; Fig. 7, C and D).
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Changes in insulin secretion induced by hypotonic stimulation. Finally, we investigated the contribution of cation channels to hypotonically induced insulin secretion from isolated rat pancreatic islets. Exposing islets to 35% hypotonic solution caused an increase in insulin secretion at 5.5 mM glucose (Fig. 8). The insulin secretion increased gradually until it peaked 6–8 min after the start of hypotonic stimulation. The insulin secretion induced by the hypotonic stimulation was reproduced at least twice. Gd3+ (1 µM) significantly inhibited the hypotonically induced insulin secretion (Fig. 8). The Cl– channel blocker DIDS (100 µM) also inhibited the hypotonically induced insulin secretion, although the inhibition by DIDS was smaller than that by Gd3+.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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-cells. The cation channel blocker Gd3+ at 1 µM suppressed hypotonically induced [Ca2+]c elevation, membrane depolarization, and insulin secretion. The involvement of cation channels was also supported by experiments with other cation channel blockers, such as amiloride, 2-APB, and ruthenium red. Moreover, hypotonic stimulation elicited cation currents sensitive to Gd3+. These results suggest a novel mechanism involving cation channels for swelling-induced insulin secretion from rat pancreatic -cells.
We show here that isolated rat pancreatic -cells respond to hypotonic stimulation with an increase in [Ca2+]c. Similar [Ca2+]c elevation induced by hypotonic stimulation has been reported in isolated mouse -cells (6) and the RINm5F -cell line (21). Swelling may increase [Ca2+]c by activation of Ca2+-permeable channels and/or Ca2+ release from intracellular stores (15, 24). The hypotonically induced [Ca2+]c elevation in rat pancreatic -cells was nearly abolished by the VDCC blocker nicardipine, in accordance with earlier studies (6, 21), indicating that the [Ca2+]c elevation results from the Ca2+ influx through VDCC. The results of the present study also confirm that hypotonic stimulation induces membrane depolarization of -cells (2, 6). It is thus likely that hypotonic stimulation causes -cell depolarization, thereby activating VDCC. Several studies have suggested that activation of volume-sensitive Cl– channels is responsible for the membrane depolarization of -cells induced by hypotonic stimulation (2, 3, 6, 13). However, a recent study in HIT clonal -cells clearly demonstrated that Cl– channel blockers such as niflumic acid and DIDS potentiate cell swelling and block RVD induced by hypotonic stimulation, but the insulin secretion persists even in the presence of the Cl– channel blockers (12). It has also been shown that hypotonically induced insulin secretion from the HC9 -cell line is inhibited by the VDCC blockers nitrendipine and calciseptine, but not by the Cl– channel blocker DIDS (22). Thus there should be another pathway involved in hypotonically induced insulin secretion. The present study proposes a novel mechanism involving cation channels, which are probably activated by the stretching of the membrane resulting from cell swelling.
In several cell types, such as epithelial and endothelial cells, osmotic cell swelling causes increases in [Ca2+]c via the activation of cation channels (5, 16). We have also demonstrated (25) the contribution of cation channels to hypotonically induced contraction of basilar artery myocytes. Moreover, some members of the transient receptor potential (TRP) family of nonselective cation channels have recently been shown to be sensitive to mechanical stresses, including hyposmolarity (1, 17). However, little information is available about the cation channels sensitive to hyposmolarity in pancreatic -cells. Sheader et al. (21) reported that 20 µM Gd3+ inhibited swelling-induced [Ca2+]c elevation in RINm5F cells; however, the same concentration of Gd3+ also blocked high K+-induced Ca2+ entry. Therefore, those authors did not conclude as to whether or not Gd3+-sensitive cation channels contribute to the swelling-induced response. The present study clearly shows that hypotonically induced [Ca2+]c elevation was partly suppressed by a lower concentration of Gd3+ (1 µM). In addition, this concentration of Gd3+ did not inhibit 30 mM K+-induced [Ca2+]c elevation, indicating that 1 µM Gd3+ has no direct inhibitory effect on VDCC of -cells. Furthermore, the [Ca2+]c elevation in response to hypotonic stimulation was also inhibited by other cation channel blockers, amiloride, 2-APB, and ruthenium red. These results support the hypothesis that cation channels participate in hypotonically induced [Ca2+]c elevation in rat pancreatic -cells. Our patch-clamp data also show that cation channels primarily mediate the outwardly rectifying currents elicited by hypotonic stimulation, because the reversal potential of the currents shifted toward negative potentials when external Na+ and Cl– concentrations were reduced. If Cl– is a main carrier of the current, then the reversal potential ought to be shifted toward positive potentials according to the reduction of the external Cl– concentration. Therefore, under our experimental conditions, hypotonic stimulation seems to activate cation channels, thereby depolarizing the membrane of rat pancreatic -cells.
However, these results are not compatible with those of earlier studies that suggest that the activation by cell swelling of volume-sensitive Cl– channels is responsible for hypotonically induced responses in -cells (2, 3, 6, 13). The discrepancy may be due to the differences in experimental conditions. For example, the glucose concentrations in external solutions differed: 2.8 mM in the present study and 4, 11.1, and 15 mM in previous studies (2, 3, 6, 13). In a study in which the Cl– channel blocker DIDS did not inhibit hypotonically induced insulin secretion, an external solution containing 0.1 mM glucose was used for the insulin secretion assay (22). Because volume-sensitive Cl– channels of -cells have been shown to be sensitive to intracellular cAMP and ATP (13, 18), metabolic conditions, which are drastically changed by glucose in -cells, may affect the activity of the Cl– channels. Alternatively, the discrepancy may be due to the method for hypotonic stimulation. In most studies on hypotonically induced responses in -cells, hypotonic stimulation was achieved by reducing the NaCl concentration in external solutions (2, 6, 12, 21, 22). The reduction of the external NaCl concentration would decrease the concentration gradients of Na+ and Cl–, which would in turn lead to a decrease in inward Na+ currents and an increase in inward Cl– currents. Thus this method would lead to underestimation and overestimation of the responses induced by cation and Cl– currents, respectively. In the present study, a hypotonic solution was prepared by removing mannitol from an isotonic solution; thus there were no changes in the concentrations of Na+ and Cl– from before to after hypotonic stimulation. Although there remains the possibility that the resultant low-Na+ external solution affected the reactivity, we confirmed that the hypotonic stimulation achieved by reducing NaCl also caused an increase in [Ca2+]c in rat -cells, which 1 µM Gd3+ inhibited (data not shown). Although the present study suggests that cation channels primarily contribute to -cell swelling-induced responses, it does not rule out the possible involvement of Cl– channels in the responses: The insulin secretion induced by hypotonic stimulation was partly inhibited by the Cl– channel blocker DIDS, and the hypotonically induced [Ca2+]c elevation was only partly inhibited by the cation channel inhibitors.
Osmotic cell swelling has been shown to induce ATP release from a variety of cell types, such as hepatoma (23), epithelial (8, 19), and endothelial (14) cells. It is known that ATP stimulates insulin secretion by acting on P2Y receptors (9). Moreover, -cells may secrete ATP in response to glucose stimulation, thereby increasing the ATP concentration close to the cell surface sufficiently high to enhance insulin secretion from -cells (7). Therefore, it is attractive to speculate that hypotonic stimulation releases ATP, which causes membrane depolarization and an increase in [Ca2+]c via the activation of P2Y receptors. However, the hypotonically induced [Ca2+]c elevation was not affected by the P2 receptor antagonist suramin, which, in contrast, completely abolished the [Ca2+]c elevation induced by ATP. Thus ATP is unlikely to be involved in hypotonically induced responses in -cells.
The mechanisms underlying glucose-induced insulin secretion are divided into at least two pathways: the ATP-sensitive K+ (KATP) channel-dependent pathway and the KATP channel-independent pathway. The former is the consensus model for glucose-induced insulin secretion: An increase in ATP resulting from the glucose metabolism closes plasma membrane KATP channels, leading to membrane depolarization, the opening of VDCC, and [Ca2+]c elevation. The latter pathway is estimated under conditions in which the contribution of KATP channels is eliminated by either the KATP channel opener or the blocker. Because high concentrations of glucose have been shown to increase -cell volume (18, 20), it is possible that the mechanism for cell swelling-induced insulin secretion is involved in the KATP channel-independent pathway. Thus stretch-activated cation channels may be an important factor in glucose-induced insulin secretion. Further experiments will be required to verify this hypothesis.
In conclusion, we have demonstrated that hypotonic stimulation causes an increase in [Ca2+]c in -cells by the following pathway: Osmotic cell swelling activates Gd3+-sensitive cation channels, which are most likely to be stretch-activated cation channels, thereby causing membrane depolarization, which activates VDCC and increases [Ca2+]c, finally leading to insulin secretion.
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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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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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) and presence (
) of 1 µM Gd3+ (A) or 100 µM DIDS (B). Each point represents the mean ± SE of 4 experiments. *P < 0.05, **P < 0.01.