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Endocrinology Vol. 143, No. 2 569-576
Copyright © 2002 by The Endocrine Society

INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS

Intracellular Ca2+ Modulation of ATP-Sensitive K+ Channel Activity in Acetylcholine-Induced Activation of Rat Pancreatic ß-Cells

Kyoko Nakano, Sechiko Suga, Teruko Takeo, Yoshiji Ogawa, Toshihiro Suda, Takahiro Kanno and Makoto Wakui

Department of Physiology (K.N., S.S., T.T., T.K., M.W.) and The Third Department of Internal Medicine (Y.O., T.S.), Hirosaki University School of Medicine, Hirosaki 036-8562, Japan

Address all correspondence and requests for reprints to: M. Wakui, M.D., Department of Physiology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan. E-mail: mw1224{at}cc.hirosaki-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the mechanism by which acetylcholine (ACh) regulates insulin secretion from rat pancreatic ß-cells. In an extracellular solution with 5.5 mM glucose, ACh increased the rate of insulin secretion from rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, ACh still increased insulin secretion, but the increment was lower than that without BIM. In the presence of nifedipine, an L-type Ca2+ channel blocker, on the other hand, ACh did not increase insulin secretion. In isolated rat pancreatic ß-cells, ACh caused depolarization followed by action potentials. This ACh effect was observed even in cells treated with BIM. In the presence of nifedipine, ACh caused only depolarization. These ACh effects were prevented by atropine. In the perforated whole-cell configuration, ramp pulses from -90 to -50 mV induced membrane currents mostly through ATP-sensitive K+ channels (KATP). These currents were reduced in size by ACh in cells either treated or untreated with BIM; whereas the loading of cells with U-73122 (a phospholipase C inhibitor) or BAPTA/AM (a Ca2+ chelator) abolished the ACh effect. In the standard whole-cell configuration, ACh reduced the currents through KATP with 0.5 mM EGTA, but not with 10 mM EGTA, in the pipette solution. Intracellular application of GDPßS or heparin also inhibited the ACh effect. In the inside-out single-channel recordings, elevation of the Ca2+ concentration inside the membrane from 10 nM–10 µM decreased KATP activity only in the presence of ATP. The affinity of ATP to KATP became 4.5 times higher with the higher concentration of Ca2+. These results suggest that Ca2+ from ACh receptor signaling modulates the sensitivity of KATP to ATP. A positive-feedback mechanism of intracellular Ca2+-dependent Ca2+ influx was also demonstrated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PARASYMPATHETIC NERVOUS ACTIVITY, as well as glucose stimulation, plays an important role in insulin secretion from pancreatic ß-cells (1, 2, 3). The muscarinic receptor activation by acetylcholine (ACh) leads to activation of phospholipase C, which, in turn, hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce IP3 and diacylglycerol (4, 5). In pancreatic ß-cells, IP3 mobilizes Ca2+ from intracellular stores, resulting in an elevation of the intracellular concentration of Ca2+ ([Ca2+]i) (6, 7, 8, 9, 10) and allowing activation of Ca2+/calmodulin. On the other hand, diacylglycerol activates PKC (11). PKC (12, 13, 14), like Ca2+/calmodulin (15, 16), accelerates exocytosis of insulin granules.

ACh is also known to depolarize ß-cells with bursts of Ca2+ action potentials (17, 18, 19, 20). The Ca2+ influx through voltage-dependent Ca2+ channels (L-type) results in an increase in the levels of [Ca2+]i (21). A study of mouse pancreatic islets revealed that an increase in the plasma membrane Na+ permeability participates in the ACh-induced depolarization (19).

On the other hand, the resting membrane potentials of pancreatic ß-cells are regulated largely by the ATP-sensitive K+ channels (KATP) (22), and a decrease in KATP activity caused by an increase of the ATP concentration and/or an increase in the ATP/ADP ratio (23) result in depolarization. In fact, the glucose stimulation of ß-cells increases the level of ATP, which results in a depolarization that opens L-type Ca2+ channels, allowing the Ca2+ influx responsible for insulin secretion (22). Furthermore, a variety of factors, including phosphorylation processes and phospholipid molecules constituting the cell membrane, are known to change KATP activity. For example, at least in cardiac myocytes, activation of PKC was shown to increase the open-time probability of KATP, leading to hyperpolarization, by reducing the ATP sensitivity to KATP (24, 25, 26, 27). However, the effect of ACh or PKC on KATP activity in ß-cells is not well understood, although the intervention of PKC in ACh-induced depolarization has been excluded, at least in mouse pancreatic ß-cells (19).

To elucidate the mechanism of ACh regulation of insulin secretion from rat islet ß-cells, focusing particularly on the mechanism of ACh-induced depolarization, in this study, we examined the effect of ACh on insulin secretion, membrane potentials, and membrane currents through KATP. The results of the present study indicate that, contrary to the case of cardiac myocytes, ACh inhibits KATP activity of rat pancreatic ß-cells, resulting in depolarization, by elevating ATP sensitivity to KATP. The depolarization induced by ACh is responsible for opening L-type Ca2+ channels, which allows Ca2+ influx. This ACh-induced depolarization is not mediated by activation of PKC but rather by a novel positive-feedback mechanism of [Ca2+]i-dependent Ca2+ influx.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
This study was carried out in accordance with the Guidelines for Animal Experimentation, Hirosaki University, Japan.

Islet preparation and ß-cell isolation
Isolation of rat islets and separation of islets into single cells were performed as previously described (28, 29). In short, the animals were anesthetized with diethyl ether, and 10 ml HBSS containing collagenase (200 U/ml; Wako Pure Chemical Industries Ltd., Osaka, Japan) was injected into the common bile duct. The pancreas swollen with the digestion solution was quickly excised and incubated in a plastic culture bottle for 20 min at 37 C. The suspension obtained by shaking the bottle was filtered through 0.5-mm metal mesh and washed with HBSS including 2% BSA. With the Histopaque (specific gravity, 1.077; Sigma, St. Louis, MO) gradient method, about 200 islets were obtained from one rat. After being washed with HBSS containing 2% BSA, islets were cultured for 24 h with 5% CO2 in the tissue culture medium. Separation of islets was carried out using dispase (1000 U/ml; Godo Shusei, Tokyo, Japan). Separated cells were again cultured for 1–2 d. Only single ß-cells were chosen for electrophysiological experiments. Identification of ß-cells was carried out by detecting the excitatory responses to 15 mM glucose or 0.5 mM tolbutamide (Sigma). Islets were used for measurements of insulin secretion.

Measurement of insulin secretion
Twenty islets were hand-picked under microscopy and placed in a polypropylene syringe filter (0.45-mm filter; Corning, Inc. Laboratory, Corning, NY) and continuously perifused with a control solution of HBSS containing 5.5 mM glucose, 10 mM HEPES, and 2% BSA, at a rate of 1 ml/min, for 30 min, before stimulation of islets with ACh. To stimulate the islets with ACh, the perifusion solution was changed to HBSS containing 1 µM ACh. The perifusate through the syringe filter was collected, and the concentration of insulin in the collected perifusate was measured by ELISA using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA). Insulin measuring kits were purchased from Morinaga Seikagaku Corp. Institute (Yokohama, Japan).

Electrical recordings
Isolated cells were kept in a 35-mm Petri dish, and the dish was placed on an inverted microscope (IMT-2; Olympus Corp., Tokyo, Japan). The membrane potential and the whole-cell membrane currents were recorded with a patch-clamp amplifier (EPC-7; List Electronic, Darmstadt, Germany) (30). The patch pipettes, made of borosilicate microcapillary tubes (Drummond Co., Broomall, PA), had a tip resistance of 2–4 megohms when they were filled with the pipette solution. After the whole-cell configuration was established by the amphotericin B or nystatin perforation method (31), the membrane potential was recorded in the current-clamp mode. In the voltage-clamp mode under the perforated whole-cell condition, voltage ramp pulses from -90 to -50 mV were applied to elicit the membrane currents through KATP. To apply various drugs to the cell through the pipette, the standard whole-cell method was also used (30), and only experiments with a series resistance below 20 megohms were accepted for the data analysis. The membrane capacitances of single ß-cells were 8–12 picofarads (pF). Single-channel current recordings were carried out in the inside-out configuration. Data of single-channel currents were low-pass-filtered at 1 KHz, digitized at 10 KHz, and analyzed using a single-channel current analysis program (QP-120J; Nihon Kohden, Tokyo, Japan). The open-time probability (Po) of the single channel was calculated according to the equation Po = 1 - Pc1/N, where Pc is the total closed time probability and N is the total number of channels. The Po of KATP was shown as a function of the concentration of ATP. The theoretical curves for the ATP inhibition of KATP activity were obtained by using the Hill equation (P/P(0)) = 1/{1+ ([ATP]/ki)h}, where P is the open time probability with ATP at each concentration, P(0) is the open time probability without ATP inside the membrane, [ATP] is the concentration of ATP, ki is the ATP concentration at which the inhibition is half-maximal, and h is the Hill coefficient. The mean values of ki and h were used for obtaining the curves, taken from several series of experiments in which various concentrations of ATP were examined in the same patch. All electrophysiological experiments were carried out at room temperature (24 C).

Solutions and drugs
For electrophysiological studies, the extracellular solution contained 135 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 1 mM CaCl2, 5.5 mM glucose, and 10 mM HEPES. The pH of the solution was 7.3. The pipette solution for recording the membrane potential and whole-cell current, with the perforation method, contained 100 mM K-gluconate, 35 mM KCl, 0.5 mM EGTA, 10 mM HEPES, 5.5 mM glucose, and 240 µg/ml amphotericin B (Sigma) or 200 µg/ml nystatin (Sigma). The pH of this solution was 7.2. The pipette solution for recording the whole-cell current with the standard method contained 100 mM K-gluconate, 35 mM KCl, 1.2 mM MgCl2, 2 mM ATP, 1 mM GTP, 0.5 mM EGTA, 10 mM HEPES, and 5.5 mM glucose. The pH of this solution was 7.2. When the concentration of EGTA was elevated to 10 mM, the concentration of K-gluconate was reduced to 90 mM. Cells in the experimental bath were continuously exposed to a stream of the extracellular solution throughout the experiment. For the inside-out single channel recordings, the pipette solution contained 135 mM KCl, 1.2 mM MgCl2, 5.5 mM glucose, 0.5 mM EGTA, and 10 mM HEPES. The pH of this solution was 7.3. The ionic composition of the solution inside the membrane was the same as that in the pipette solution for the standard whole-cell current recordings, but the concentration of ATP was varied, and the pH was 7.2. In the inside-out recordings, ATP was added to the bath solution at various concentrations. Using a program for calculating metal ion/ligand binding (S. Oiki and Y. Okada, Okazaki National Institute for Physiology, Okazaki, Japan), the concentrations of ATP, Mg2+, Ca2+, and EGTA in the solution were adjusted. For example, to make a solution containing 10 µM free ATP, 10 nM Ca2+, and 1.2 mM Mg2+, we used 5 mM EGTA, 0.189 mM CaCl2, and 1.297 mM MgCl2. To make 10 µM free ATP, 10 µM Ca2+, and 1.2 mM Mg2+, we used 5 mM EGTA, 4.886 mM CaCl2, and 1.211 mM MgCl2. ACh, nifedipine, atropine, heparin, U-73122, and bisindolylmaleimide (BIM) were purchased from Sigma. GDPßS was a purchase from Boehringer Ingelheim GmbH (Mannheim, Germany). BAPTA/AM was purchased from Calbiochem (La Jolla, CA). Nifedipine was dissolved in methanol, and the final concentration of methanol in the experimental solution was 0.01%. BAPTA/AM, U-73122, and BIM were dissolved in dimethylsulfoxide (DMSO), and the final concentration of DMSO in the experimental solution was less than 0.2%. For the treatment of cells with BAPTA/AM, U-73122, or BIM, cells were preincubated in a solution containing one of BAPTA/AM (100 µM), U-73122 (2 µM), or BIM (1 µM) for 10–30 min; and, further, U-73122 or BIM was present in the extracellular solution throughout the experiments. The effect of BIM on the activity of PKC in islets was evaluated using a MESACAP Protein Kinase Assay Kit (MBL, Nagoya, Japan) based on ELISA. Phosphorylated PS peptide (coated on microwells) by PKC was detected with biotinylated monoclonal antibody and streptavidin conjugated to peroxidase. After adding peroxidase substrate, the intensity of the color was measured photometrically at 492 nm. The net PKC activity was shown by subtracting the intensity of null PKC activity (no Ca2+, no phosphatidylserine, 1 mM EGTA) from the values measured in the presence of 2 mM Ca2+ and 50 µg/ml phosphatidylserine. The reaction buffer contained 25 mM Tris-HCl (pH 7.0), 3 mM MgCl2, 0.1 mM ATP, 0.5 mM EDTA, and 5 mM 2-mercaptoethanol. The PKC activities of islets with 5.5 mM glucose were 0.06 ± 0.03 (n = 6) in the resting state and 0.17 ± 0.01 (n = 6) during application of 80 nM PMA (P < 0.01). The PKC activity of islets treated with 1 µM BIM was 0.01 ± 0.01 (n = 6). However, this value was not significantly different from that of islets not treated with BIM. The value of PKC activity of BIM-treated islets did not increase during the PMA stimulation [0.01 ± 0.01 (n = 5)]. This value was significantly lower than that of BIM-untreated islets (P < 0.01).

Statistics
Data were expressed as mean ± SEM of several experiments, and statistical significance was evaluated by using the two-tail paired or unpaired t test. A value of P < 0.05 was accepted as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ACh increases insulin secretion from rat islets
Figure 1Go shows the time course of the rate of insulin secretion from islets, in response to 1 µM ACh, measured in perifusate collected every 2 min. In the presence of 5.5 mM glucose in the perifusion (extracellular) solution, insulin was secreted at the basal level from the islets. When the perifusion solution was changed to a solution containing 1 µM ACh, the rate of insulin secretion increased, and this increment lasted for at least 24 min, during which ACh was present. After removal of the ACh stimulation, the rate of insulin secretion gradually decreased to the basal level (data not shown). Treatment of islets with BIM (1 µM) did not alter the basal secretion of insulin, and ACh still increased the rate of insulin secretion. Treatment of islets with nifedipine (1 µM) decreased the basal insulin secretion. ACh, applied to islets in the presence of nifedipine, showed little effect on insulin secretion. To analyze the data quantitatively, the perifusate passing through the syringe filter was collected every 12 min, and the concentrations of insulin in the samples were measured. The rates of the basal insulin secretion measured in samples of the first and the second 12-min perifusions were the same (Fig. 2AGo). Application of ACh (1 µM) produced a 2.6-fold increase in the rate of insulin secretion in the samples of the third 12-min perifusion. The treatment of islets with 1 µM BIM, a PKC inhibitor (32 , see also Materials and Methods), by itself did not change the rate of insulin secretion. Application of ACh on top of BIM produced a 1.6-fold increase of insulin secretion (Fig. 2BGo). Nifedipine (1 µM) decreased the rate of insulin secretion, and further application of ACh failed to increase the insulin secretion (Fig. 2CGo). Because BIM was dissolved in ethanol (0.01%) and nifedipine in DMSO (0.1%), the effects of these solvents on insulin secretion were also examined. Neither solvent at these concentrations affected insulin secretion (data not shown).



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  		Figure 1. The time course of insulin secretion from rat islets, in response to ACh. The rates of insulin secretion from rat islets, obtained by the perifusion method, are shown. Insulin secretion, in response to 1 µM ACh, was measured in three different series of experiments: without any treatment (none), treatment with BIM (1 µM), or nifedipine (1 µM). Periods of the drug treatment were shown by a dotted line (BIM) and an interrupted line (nifedipine), respectively. The perifusion (extracellular) solution contained 5.5 mM glucose throughout the experiment. Mean values of three experiments in each protocol are shown. Vertical bars represent SEM. Before sampling the solution passing through the syringe filter, islets in the syringe filter were perifused with the control solution for 30 min. Successive aliquots of each 2-min perifusion solution were collected in chronological order.

 


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  		Figure 2. The effect of BIM or nifedipine on insulin secretion from rat islets by ACh. The solution passing through the syringe filter with islets was collected in 12-min aliquots in chronological order. The extracellular solution contained 5.5 mM glucose throughout the experiment. A, The insulin secretion without any stimulation (basal) was measured twice, in order. ACh (1 µM) increased the rate of insulin secretion. B, The treatment of islets with BIM (1 µM) did not change the rate of insulin secretion. ACh (1 µM), applied on top of BIM, increased insulin secretion. C, Nifedipine (1 µM) decreased insulin secretion. In the presence of nifedipine, ACh (1 µM) had little effect on insulin secretion. Mean values are shown, and vertical bars represent SEM. *, P < 0.05; **, P < 0.01.

 
ACh causes depolarization of ß-cells through activation of muscarinic receptors
In isolated rat ß-cells, the membrane potential with 5.5 mM glucose (resting potential) subtly changed (fluctuated); and in some cells, action potentials spontaneously developed. Measured in a period without an action potential, the average membrane potential varied in different cells from -43 to -68 mV. Application of ACh (1 µM) caused depolarization, followed by action potentials (Fig. 3AGo). In the presence of nifedipine (1 µM), ACh caused only depolarization (Fig. 3BGo). The magnitude of depolarization was different under various conditions. In the presence of atropine (1 µM), no potential change was observed in response to 1 µM ACh (Fig. 3CGo). In cells treated with 1 µM BIM, ACh still caused depolarization associated with action potentials (Fig. 3DGo). Examined in the presence of nifedipine (1 µM), the magnitude of ACh-induced depolarization depended on the concentration of ACh (Fig. 3EGo).



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  		Figure 3. Depolarization of rat pancreatic ß-cells induced by ACh through activation of muscarinic receptors. The membrane potential of isolated rat pancreatic ß-cells, recorded by the perforation method, is shown. A, In a control condition with 5.5 mM glucose, ACh (1 µM) caused depolarization, followed by action potentials. The dotted line indicates the original potential level before application of ACh. B, In the presence of nifedipine (1 µM), ACh caused only depolarization. C, In the presence of atropine (1 µM), ACh (1 µM) had little effect on the membrane potential. D, In cells treated with BIM (1 µM), ACh still induced depolarization, followed by action potentials. In A–D, representative tracings from at least five experiments are shown. E, Concentration-response relationships of ACh-induced depolarization from records obtained in the presence of 1 µM nifedipine. The depolarization was observed in the presence or absence of atropine (1 µM), and the magnitude of depolarization was measured as the mean value around the peak. Mean values are shown, taken from 5–7 experiments at each concentration of ACh. Vertical bars represent SEM. *, P < 0.05; **, P < 0.01, both compared with the value with ACh at 0.01 µM in the absence of atropine.

 
ACh reduces KATP activity through PLC activation
In the perforated whole-cell configuration, membrane currents in response to voltage ramp pulses from -90 to -50 mV were recorded, and these currents were strongly inhibited by application of tolbutamide (0.5 mM), a selective inhibitor of KATP (21), to the bath solution (Fig. 4AGo), suggesting that most of the currents recorded were passing through KATP. Application of ACh to the bath solution at 1 µM reduced the magnitude of the currents (Fig. 4BGo). In the presence of atropine (1 µM), ACh (1 µM) failed to reduce the currents (Fig. 4CGo). In cells treated with BIM (1 µM), ACh (1 µM) still reduced the magnitude of the currents (Fig. 4DGo). On the other hand, treatment of the cells with BAPTA/AM (100 µM) abolished the ACh effect on the currents (Fig. 4EGo). The inhibition of the currents by ACh depended on the concentration of ACh in cell groups both treated and untreated with BIM (1 µM) (Fig. 4FGo).



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  		Figure 4. PKC-independent reduction of ß-cell membrane currents through KATP, by ACh. Whole-cell currents, in response to voltage ramps from -90 to -50 mV, recorded by the perforated whole-cell method, are shown. A, Application of tolbutamide (TOL, 0.5 mM) to the bath solution strongly inhibited the currents. The dotted line indicates zero current. B, Application of ACh reduced the currents. C, In the presence of atropine (1 µM), ACh failed to affect the currents. D, In cells treated with BIM (1 µM), ACh still reduced the magnitude of the currents. E, In cells treated with BAPTA/AM, ACh had little effect on the currents. In A–E, representative tracings from at least six experiments are shown. F, Concentration-response relationships of the ACh-induced reduction of currents in cells treated and untreated with BIM (1 µM). Mean values are taken from six experiments at each concentration of ACh. *, P < 0.05; **, P < 0.01, both compared with the value with ACh at 0.01 µM in each group of cells (treated or untreated with BIM).

 
ACh inhibition of KATP activity is mediated by elevation of [Ca2+]i
Figure 5AGo shows the whole-cell currents induced by voltage ramps from -90 to -50 mV, recorded by the standard whole-cell method with a pipette solution containing 2 mM ATP and 0.5 mM EGTA. Under this condition, application of ACh (1 µM) reduced the magnitude of the currents. When GDPßS (400 µM), a G protein inhibitor, was present in the pipette solution, which allowed GDPßS to enter the cell, ACh showed little effect on the currents (Fig. 5BGo). Recorded in the perforated whole-cell configuration, the treatment of cells with U-73122, a PLC inhibitor (33), abolished the ACh effect on the current (Fig. 5CGo). When heparin (200 mg/ml), an IP3 antagonist (34), was present in the pipette solution, ACh had no effect on the currents, recorded in the standard whole-cell configuration (Fig. 5DGo). When the pipette solution contained 10 mM EGTA, ACh failed to reduce the currents (Fig. 5EGo). The inhibition by ACh of the currents, recorded with 0.5 mM EGTA in the pipette solution, depended on the concentration of ACh (Fig. 5FGo).



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  		Figure 5. Involvement of Ca2+ signal pathway in ACh inhibition of KATP activity. A, Whole-cell currents, in response to voltage ramps from -90 to -50 mV, recorded by the standard whole-cell method, are shown. The pipette solution contained 2 mM ATP and 0.5 mM EGTA. ACh reduced the magnitude of the currents. The dotted line indicates zero current. B, In the presence of GDPßS (400 µM) in the pipette solution, ACh (1 µM) did not affect the currents. C, Whole-cell currents, recorded by the perforated whole-cell method. In cells pretreated with U-73122 (2 µM), ACh (1 µM) showed little effect on the currents. D, Whole-cell currents, by the standard whole-cell method. In the presence of heparin (200 mg/ml) in the pipette solution, ACh (1 µM) did not change the currents. E, When the pipette solution contained 2 mM ATP and 10 mM EGTA, ACh had little effect on the currents. In A–E, representative tracings from at least five experiments are shown. F, The concentration-response relationships of ACh-induced decrease in the currents, from experiments shown in A and E, recorded by the standard whole-cell method. Values of the ACh effect were shown by comparing the magnitudes of currents before application of ACh (I0) and currents (I) showing the maximum effect of ACh during approximately 1-min application of ACh. Mean values are taken from five to eight experiments at each concentration of ACh. **, P < 0.01, compared with each pair with 10 mM EGTA.

 
Intracellular Ca2+ modulates ATP sensitivity to KATP
Single-channel currents were recorded in the inside-out mode. Application of ATP at 1 mM in the bath solution (inside the membrane) markedly inhibited the channel activity (Fig. 6AGo), suggesting that KATP activity was observed. In the absence of ATP inside the membrane, elevation of the Ca2+ concentration from 10 nM to 10 µM in the solution inside the membrane had little effect on KATP activity (Fig. 6BGo). In the presence of ATP at 10 µM and Ca2+ at 10 nM, KATP activity was lower than that without ATP, and elevation of the Ca2+ concentration to 10 µM, keeping the free-ATP concentration constant, further decreased KATP activity (Fig. 6CGo). In Fig. 6DGo, the effects of elevation of the Ca2+ concentration on the open-time probabilities of KATP, with and without 10 µM ATP, were summarized. Furthermore, from relationships between KATP activity and the concentration of ATP (Fig. 6EGo), ki decreased with elevation of the Ca2+ concentration. The value of h was also increased by elevation of the Ca2+ concentration.



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  		Figure 6. Inhibition of KATP activity, by elevation of Ca2+ concentration inside the membrane. Single-channel current recordings in the inside-out configuration. All tracings shown were recorded with the transmembrane potential at -60 mV, inside negative. A, The solution inside the membrane contained 10 nM Ca2+, throughout. Application of 1 mM ATP strongly inhibited the channel activity, suggesting that KATP activity was recorded. The dotted line indicates the current level with channels closed. B, Without ATP in the solution inside the membrane, elevation of Ca2+ concentration to 10 µM had little effect on KATP activity. C, In the presence of 10 µM ATP and 10 nM Ca2+ in the solution inside the membrane, KATP activity was lower than that without ATP. Elevation of Ca2+ to 10 µM inhibited KATP activity. Lower tracings are 1-sec sections of tracings with a higher sweep, starting from the points indicated. In A–C, representative tracings from at least five experiments are shown. D, The mean values of open-time probabilities (Po) of KATP are taken from six to eight experiments. In the absence of ATP, Po values of KATP were 0.19 ± 0.01 (n = 8) with Ca2+ at 10 nM and 0.18 ± 0.02 (n = 7) with Ca2+ at 10 µM (P > 0.05). In the presence of 10 µM ATP, the values of Po were 0.10 ± 0.01 (n = 8) with Ca2+ at 10 nM and 0.04 ± 0.01 (n = 7) with Ca2+ at 10 µM (**, P < 0.01). E, Relationship of KATP activity to the concentration of ATP. The curves for the ATP inhibition of KATP activity were obtained by the Hill equation, with the values of ki being 12.3 ± 1.0 µM (n = 4) with Ca2+ at 10 nM (a solid line) and 2.7 ± 0.5 µM (n = 7) with Ca2+ at 10 µM (a dotted line) in the solution inside the membrane (P < 0.05). The values of h were 0.88 ± 0.04 (n = 4) with Ca2+ at 10 nM and 1.05 ± 0.04 (n = 7) with Ca2+ at 10 µM (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Insulin secretion from rat ß-cells caused by ACh is mediated largely by Ca2+ influx
In the present study, application of 1 µM ACh increased insulin secretion with glucose at 5.5 mM in the extracellular solution (Figs. 1Go and 2AGo). It is already known that ACh and carbachol directly stimulate pancreatic ß-cells to secrete insulin via activation of muscarinic receptors (1, 2, 3, 4, 5). Activation of PKC, one of the signaling pathways distal to the muscarinic receptors in ß-cells, is known to potentiate insulin secretion (12, 13, 14). In the present study, BIM, a PKC inhibitor, at the maximum concentration (Ref. 32 , see also Materials and Methods), partly inhibited the increment of insulin secretion by ACh (Fig. 2BGo). On the other hand, treatment of the islets with nifedipine reduced the rate of basal insulin secretion (Fig. 2CGo), indicating that the basal insulin secretion is attributable, in part, to Ca2+ influx through L-type Ca2+ channels, based on the finding in the present study that some ß-cells developed spontaneous action potentials, as already described. Furthermore, the treatment of islets with nifedipine completely inhibited the ACh effect on insulin secretion (Fig. 2CGo). Together, these results suggest that insulin secretion in response to ACh is mediated by both PKC and Ca2+ mechanisms, and both mechanisms are attributable largely to Ca2+ influx through L-type Ca2+ channels. Ca2+ signaling is known to be another important pathway distal to muscarinic receptors for ACh-induced insulin secretion (6, 7, 8, 9, 10). Dependency of ACh-induced insulin secretion on Ca2+ uptake in rat (35) and mouse (19) pancreatic islets was already known. ACh, through activation of muscarinic receptors, produces IP3, which liberates Ca2+ from intracellular stores. The Ca2+ release from intracellular stores is transient, and Ca2+ influx is required to maintain the elevation of [Ca2+]i induced by ACh (21). The results shown in Fig. 2CGo indicate that in the Ca2+-dependent insulin secretion of rat ß-cells induced by ACh, the major route for the Ca2+ supply is Ca2+ influx through L-type Ca2+ channels.

Depolarization of ß-cells by ACh is caused by a decrease in activity of KATP
The ACh-induced Ca2+ influx through L-type Ca2+ channels in rat ß-cells is mediated by depolarization of the cell. Electrophysiological studies have revealed that ACh causes depolarization with bursts of action potentials (17, 18, 19, 20). The results of the present study also show that ACh (0.01–10 µM) causes depolarization of rat pancreatic ß-cells in a concentration-dependent manner through activation of muscarinic receptors and the subsequent action potentials attributable to the opening of L-type Ca2+ channels (Fig. 3Go).

The ionic mechanism of ACh-induced depolarization has been studied in mouse pancreatic ß-cells. Henquin et al. (19) found that 22Na+ influx, 45Ca2+ influx, and 86Rb+ efflux were increased in response to ACh, and they concluded that an increase in plasma membrane Na+ permeability is responsible for ACh-induced depolarization, at least in mouse pancreatic ß-cells. In the present study on rat ß-cells, however, ACh reduced whole-cell membrane currents through KATP (Figs. 4Go and 5Go), suggesting that in rat ß-cells, a decrease of KATP activity is responsible, at least in part, for the depolarization induced by ACh.

With respect to the mechanism of ACh inhibition of KATP activity, the possibility of a change (elevation) of ATP concentration in the cell is not excluded. However, the present finding that in a condition where the ATP concentration in the cell was fixed, by using the standard whole-cell method, ACh reduced KATP activity (Fig. 4AGo), suggests that the ACh action on KATP activity is independent of its putative effect on the concentration of intracellular ATP. Because the treatment of cells with GDPßS (Fig. 4BGo) or U-73122 (Fig. 4CGo) prevented the ACh inhibition of KATP activity, the activity of G proteins and the subsequent activation of PLC distal to muscarinic receptors seem to be involved in the ACh inhibition of KATP activity in rat ß-cells. Furthermore, heparin, loaded in the cell, prevented the ACh inhibition of the currents through KATP (Fig. 4DGo), suggesting that IP3 is involved in the signal transduction pathway of the ACh inhibition of KATP activity, resulting in depolarization. On the other hand, the treatment of ß-cells with BIM did not prevent ACh-induced electrical response (Fig. 2Go, D and E) or ACh-induced inhibition of KATP activity (Fig. 3DGo), suggesting that PKC is not involved in these electrical effects of ACh. In cardiac muscles, on the other hand, activation of PKC is known to increase KATP activity (24, 25, 26, 27). A difference between cardiac myocytes and ß-cells in their major isotypes of PKC [{delta} and {epsilon} in cardiac myocytes (36), {alpha} and {epsilon} in ß-cells (37)] or a difference in subtypes of KATP (Kir6.1 in cardiac myocytes, Kir6.2 in ß-cells) may produce different sensitivities of KATP to PKC.

Intracellular Ca2+ modulates ATP affinity to KATP
The finding that ACh failed to inhibit KATP activity when [Ca2+]i was strongly chelated by a high concentration of EGTA (Fig. 4EGo) or in cells loaded with BAPTA/AM (Fig. 4FGo) indicates that an increase in the level of [Ca2+]i is responsible for ACh inhibition of KATP activity. IP3 was also shown to be the molecule responsible for the ACh inhibition of KATP (see above). Together, these findings suggest that the elevation of [Ca2+]i of rat ß-cells by ACh, first caused by IP3-induced Ca2+ release from intracellular stores, leads to inhibition of KATP activity.

Divalent cations, including Mg2+, Sr2+, and Ca2+, are known to regulate KATP activity; and the effect of Mg2+, in particular, has been extensively studied (38, 39, 40). Findley ( 38) showed that Mg2+ reduced channel openings without affecting single-channel conductance of a rat insulin-secreting cell line, RINm5F. He also found that Mg2+ is required for KATP activity to be maintained by a phosphorylation process (38). A Mg2+-free solution was shown to potentiate the run-down of KATP in CRI-G1 cells (40). Ashcroft and Kakei ( 39) also found that elevation of Mg2+ in the solution inside the membrane inhibited KATP activity by increasing ATP efficacy on KATP. However, the relationships between KATP activity and free ATP concentration with Mg2+-free and with 2 mM Mg2+ solutions were the same, suggesting that Mg2+ changed the concentration of free ATP, actually regulating KATP activity (39). Findley ( 38) found also that Sr2+ (1 mM) and Ca2+ (1 mM), but not Na+ (10 mM), inhibited KATP activity of RINm5F cells. However, the mechanism of inhibition of KATP by divalent cations has remained unclear.

In the present study, we found that in the inside-out single channel recordings, elevation of the Ca2+ concentration inside the membrane in a physiological range decreased KATP activity in the presence of ATP (Fig. 5Go, C and D). In these experiments, Ca2+ concentration changes did not alter KATP activity when ATP was absent from the solution inside the membrane (Fig. 5BGo). These results suggest that Ca2+ inside the membrane affects the efficacy of ATP on KATP. In fact, the value of ki for ATP inhibition of KATP activity decreased with increasing Ca2+ concentration inside the membrane (Fig. 5EGo). The value of h was also increased by elevation of the Ca2+ concentration (Fig. 5EGo), indicating that Ca2+ may also increase the ATP binding site of KATP.

The mechanism by which Ca2+ increases ATP affinity to KATP is not clear at present. Recently, the membrane PIP2 was shown to reduce the ATP sensitivity of KATP (41, 42, 43). Further experiments exploring phospholipid actions revealed that molecules with minus charges have the ability to reduce the ATP efficacy on KATP (44), leading to a hypothesis that electrostatic binding of the molecule to a portion of the channel pore, Kir, interferes with ATP binding to its binding site, which is also in Kir (45, 46). Accordingly, Ca2+, with its positive charges, may produce a reverse action of potentiating ATP binding affinity to Kir.

In summary, the present study reveals the presence of a novel ACh receptor signaling in rat pancreatic ß-cells. In this cell type, ACh, through activation of muscarinic receptors, releases Ca2+ from intracellular stores, and Ca2+ from the signaling increases the sensitivity of KATP to ATP. This Ca2+ action results in depolarization, to open the voltage-dependent Ca2+ channels, allowing Ca2+ influx. Thus, islet ß-cells possess a positive-feedback mechanism of Ca2+-dependent Ca2+ influx.


   Footnotes
 
This work was supported by Grant-in-Aid No. 12470005 from the Japan Ministry of Education, Science, Sports, and Culture (to M.W.) for scientific research and by a grant for Scientific Research Work from the Committee of the 50th Anniversary of Establishment of Hirosaki University, Hirosaki, Japan.

Abbreviations: ACh, Acetylcholine; BIM, bisindolylmaleimide; [Ca2+]i, intracellular concentration of Ca2+; DMSO, dimethylsulfoxide; h, the Hill coefficient; KATP, ATP-sensitive K+ channels; ki, the ATP concentration at which the inhibition is half-maximal; Kir, channel pore of KATP; pF, picofarads; PIP2, phosphatidylinositol 4,5-bisphosphate; Po, open-time probability.

Received February 8, 2001.

Accepted for publication October 10, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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