Insulinotropic Glucagon-like Peptide-1-mediated Activation of Non-selective Cation Currents in Insulinoma Cells Is Mimicked by Maitotoxin*

(Received for publication, June 4, 1996, and in revised form, May 15, 1997)

Colin A. Leech Dagger and Joel F. Habener

From the Laboratory of Molecular Endocrinology, Howard Hughes Medical Institute, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

Maitotoxin (MTX) activates a Ca2+-dependent non-selective cation current (ICa-NS) in insulinoma cells whose time course is identical to non-selective cation currents activated by incretin hormones such as glucagon-like peptide-1 (GLP-1), which stimulate glucose-dependent insulin secretion by activating cAMP signaling pathways. We investigated the mechanism of activation of ICa-NS in insulinoma cells using specific pharmacological reagents, and these studies further support an identity between MTX- and GLP-1-activated currents. ICa-NS is inhibited by extracellular application of genistein, econazole, and SKF 96365. This inhibition by genistein suggests that tyrosine phophorylation may play a role in the activation of ICa-NS. ICa-NS is not inhibited by incubation of cells in glucose-free solution, by extracellular tetrodotoxin, nimodipine, or tetraethylammonium, or by intracellular dialysis with 4-aminopyridine, ATP, ryanodine, or heparin. ICa-NS is also not significantly inhibited by staurosporine, which does, however, partially inhibit the MTX-induced rise of intracellular Ca2+ concentration. These effects of staurosporine suggest that protein kinase C may not be involved in the activation of ICa-NS but that it may regulate intracellular Ca2+ release. Alternatively, ICa-NS may have a small component that is carried through separate divalent cation-selective channels that are inhibited by staurosporine. ICa-NS is neither activated nor inhibited by dialysis with KF, KF + AlF3 or GTPgamma S (guanosine 5'-O-(3-thiotriphosphate)), suggesting that GTP-binding proteins do not play a major role in the activation of this current.


INTRODUCTION

The consensus model of glucose-stimulated insulin secretion is that closure of ATP-sensitive K+ channels (K+ATP)1 permits membrane depolarization, activation of voltage-dependent Ca2+ channels (VDCCs) and the influx of Ca2+ (1). Individual beta -cells, however, are often unresponsive to glucose alone, but can become responsive by combined stimulation with glucose and hormones, such as insulinotropic hormone glucagon-like peptide-1 (GLP-1; Ref. 2), that elevate intracellular cAMP levels (3-5). One mechanism underlying this increased responsiveness is the enhanced closure of K+ATP channels (2). A second mechanism by which GLP-1, pituitary adenylate cyclase-activating polypeptide (PACAP), and cAMP can induce beta -cell depolarization is through the activation of voltage-independent, non-selective cation currents (6, 7). Similar cation currents are also activated by MTX, a polyether toxin isolated from dinoflagellates that in beta -cells has been shown to stimulate insulin secretion and inositol trisphosphate (Ins(1,4,5)P3) production (8) and to enhance the influx of monovalent cations (9).

MTX-sensitive currents are activated by depletion of intracellular Ca2+ stores (10), and GLP-1 enhances intracellular Ca2+ mobilization through the potentiation of ryanodine-sensitive Ca2+-induced Ca2+ release in beta TC3 cells (11, 12). Increased cAMP levels stimulate Ca2+ release from secretory granules and reduce mitochondrial Ca2+ uptake in beta -cells (13, 14). These observations raise the possibility that the activation of non-selective cation currents by PACAP and GLP-1 may be a secondary consequence of glucose- and cAMP-dependent intracellular Ca2+ release. The physiological role of Ca2+ release-activated currents in beta -cells remains controversial, but such currents have been suggested to play a role in the cholinergic modulation of electrical bursting activity (15), and may control the membrane potential and intracellular Ca2+ ([Ca2+]i) oscillations in response to nutrient stimulation (10).

Both PACAP (16) and GLP-1 (17) are potent insulin secretagogues in the presence of slightly elevated glucose levels. The activation of a voltage-independent, non-selective cation current by these hormones under conditions that stimulate insulin secretion suggests that this current may play an important role in depolarizing beta -cells to initiate insulin secretion. The aim of this study is to examine the mechanism of activation of the MTX-sensitive current and to compare the properties of ICa-NS with the current activated by GLP-1, PACAP, and cAMP to determine whether these currents are likely to be carried through the same channels.


MATERIALS AND METHODS

Preparation of Cell Cultures

HIT-T15 cells were obtained from the American Type Culture Collection. beta TC6 cells were obtained from Dr. Shimon Efrat (Albert Einstein College of Medicine, New York, NY). HIT-T15 cells (passages 67-75) were maintained in Ham's F-12 medium containing 10 mM glucose, 10% heat-inactivated horse serum, and 2.5% fetal bovine serum. beta TC6 cells (passages 24-59) were maintained in Dulbecco's modified Eagle's medium containing 25 mM glucose, 15% horse serum, and 2.5% fetal bovine serum. Culture media also contained 100 units/ml penicillin G and 100 µg/ml streptomycin. Cells were plated onto glass coverslips coated with 1 mg/ml of type V concanavalin A (Sigma), which facilitates their adherence to glass. Cultures were maintained at 37 °C in a 5% CO2 atmosphere incubator, and experiments were conducted 1-5 days post-plating.

Test Solutions

Cells were bathed in a standard extracellular solution (SES) containing: 138 mM NaCl, 5.6 mM KCl, 2.6 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES (295 mosM; pH adjusted to 7.4 with NaOH, approximately 4 mM), and 0.8 mM D-glucose unless indicated as being different in the text. Na+-free, N-methyl-D-glucamine (NMG) solutions were prepared using 138 mM NMG substituted for NaCl and adjusted to pH 7.4 with HCl. A Na+-free, 100 Ca solution was also prepared containing: 100 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES (282 mosM; pH adjusted to 7.4 with KOH, approximately 6 mM) Other test solutions were prepared by substitution of NaCl with 140 mM KCl, CsCl, LiCl, or choline chloride. Ca2+-free solutions were prepared by substituting MgCl2 or MnCl2 for CaCl2. Low [Cl-]o solution was prepared by substituting NaCl with Na-aspartate.

Test solutions containing MTX were applied to individual cells by focal application from micropipettes using a PicoSpritzer II pressure ejection system (General Valve, Fairfield, NJ). A gravity-fed bath superfusion system was used to exchange and refresh bath solutions. Maitotoxin, econazole, staurosporine, nimodipine, and glyburide were obtained from Sigma. Tetrodotoxin, SKF 96365, genistein, GTPgamma S, ryanodine, and heparin were obtained from Calbiochem. Tetraethylammonium chloride (TEA) and 4-aminopyridine (4AP) were obtained from Aldrich.

Measurement of Intracellular Calcium

Cells were prepared for measurement of [Ca2+]i by incubation in fura-2 acetoxymethyl ester (fura-2 AM; Molecular Probes, Inc., Eugene, OR). Cells were loaded in SES supplemented with 2% fetal bovine serum, 0.03% pluronic F-127, and 1 µM fura-2 AM for 15 min at room temperature (20-22 °C). Coverslips with fura-loaded adherent cells formed the base of a recording chamber mounted on a temperature-controlled stage (Micro Devices, Jenkintown, PA). Cells were visualized using a Zeiss IM35 microscope equipped with a Nikon UVF100 100× objective. Measurements of [Ca2+]i were performed at 1-s intervals from the average of 10 video frames using a dual excitation wavelength video imaging system (IonOptix Corp., Milton, MA). Experiments were conducted at 32 °C. [Ca2+]i was estimated from the ratio of 510 nm emission fluorescences due to excitation by 350 nm and 380 nm wavelength light from the following equation (18).
[<UP>Ca</UP>]<SUB>i</SUB>=K<SUB>d</SUB> &bgr;[(R−R<SUB><UP>min</UP></SUB>)/(R<SUB><UP>max</UP></SUB>−R)] (Eq. 1)
Kd is the dissociation constant of fura-2 (225 nM), beta  is the ratio of 380 nm induced fluorescences of free/bound fura-2, R is the measured ratio of 350 nm/380 nm fluorescences and Rmin and Rmax are 350 nm/380 nm fluorescence ratios in zero [Ca2+] and saturating [Ca2+], respectively. Values of beta , Rmin, and Rmax were determined using fura-2 pentapotassium salt and calibration solutions from Molecular Probes, Inc.

Patch Clamp Recording Techniques

Cell resting potentials and membrane currents were measured under current clamp or voltage clamp using either whole-cell or perforated patch configurations (19, 20). Patch pipettes pulled from borosilicate glass (Kimax-51, tip resistance 2-4 megohms) were fire polished and tip-dipped in K- or Cs-pipette solution containing: 95 mM K2SO4 (or Cs2SO4), 7 mM MgCl2, 5 mM HEPES (pH adjusted to 7.4 with NaOH; final concentration of Na+ approximately 2 mM). Pipettes were then back-filled with the same solution, to which nystatin (240 µg/ml) was added for perforated patch recording.

The patch pipette was connected to an Heka Electronik EPC-9 patch clamp amplifier (Instrutech Corp., Mineola, NY) interfaced with a Macintosh Quadra 840AV computer running Pulse version 8.0 software (Instrutech Corp.). The series resistance (Rs) and cell capacitance (Cm) were monitored following seal formation, and experiments were conducted when Rs declined to <35 megohms. In voltage clamp experiments, Rs was compensated for by 60%.

Values are given as mean ± S.E. Statistical significance was determined using Student's t test computed using Lotus 1-2-3 spreadsheets.


RESULTS

MTX activates a non-selective cation current in beta TC6 cells (Fig. 1) that has a reversal potential of -7.8 ± 1.0 mV (n = 72) in SES (142 mM [Na+]o). This current was observed in all cells tested, and its reversal potential becomes more negative as [Na+]o is reduced (Fig. 1, Table I), although the shift is less than predicted from the Nernst potential for Na+. ICa-NS is also observed in solutions where extracellular Na+ is replaced by Cs+, K+, Li+, choline+, or NMG+ and in cells dialyzed with K+, Cs+, Na+, choline+, or NMG+ with Na+ in the bathing solution (Table II), confirming the non-selective nature of this current. Changing from normal [Cl-]o to low [Cl-]o extracellular solution did not affect the amplitude or reversal potential of the current (data not shown), confirming the cation selectivity of ICa-NS.


Fig. 1. MTX activates a non-selective cation current mainly carried by Na+. Panel A shows the whole cell current from a perforated patch (Cs-pipette solution) voltage-clamped beta TC6 cell (Cm 11.3 pF) initially bathed in SES (142 mM Na+). All bath solutions contained 5 mM TEA, 2 µM TTX, and 100 µM Cd2+ (to inhibit Ca2+-activated K+ channels, voltage-dependent Na+ channels, and VDCCs respectively). Breaks in the trace (labeled 1-6) mark where series of four voltage ramps from -70 mV to +30 mV (1 V/s) were applied. Currents were filtered at 1 kHz. A 2-s pulse of 10 pM MTX was applied (arrow) and the bath solution subsequently changed to 14 mM Na+ (Tris-HCl-substituted), returned to SES, exchanged with 14 mM Na+ (NMG-substituted), and again returned to SES as indicated. Panel B shows the averaged currents from series of voltage ramps (as indicated in A) before MTX (1), after MTX stimulation (2), and after changing the bath to 14 mM Na+ (3). Panel C shows the currents from the ramp series subtracted as indicated. The reversal potential of the MTX-activated current in this cell was 0 mV in 142 mM Na+ and reversibly shifted to -31 mV in 14 mM Na+ (Table I). The currents during ramp series (5) were too small to allow accurate determination of the reversal potential.
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Table I. Sodium dependence of ICa-NS reversal potential (mV)

Table shows the effect of changing [Na+]o on the reversal potential (mV) of the MTX-activated current (ICa-NS) in beta TC6 cells. The reversal potential shifted by less than would be predicted from the Nernst potential for Na+ suggesting that the channels are non-selective. Other values given in this table are control values obtained from cells subsequently transferred to different [Na+]o solutions. The number of cells recorded is indicated in parentheses.

142 Na 70 Na/70 NMG 70 Na/50 Ca 14 Na/NMG Na-free/NMG Na-free/100 Ca

 -7.8  ± 1.0  (72)
 -6.0  ± 3.7  (5)  -21.6  ± 2.1
 -4.6  ± 2.1  (5)  -30.6  ± 2.9
 -3.4  ± 1.2  (5)  -38.4  ± 2.5
 -13.6  ± 3.3  (6)  -39.7  ± 1.8
 -1.4  ± 0.5  (9)  -25.0  ± 1.4

Table II. Ionic dependence of reversal potentials (mV) for MTX-induced currents

Table shows the effect of extracellular (bath) and intracellular (pipette) ion substitutions on the reversal potential of ICa-NS. The reversal potential with SES (Na+) bath solution and Cs+-pipette solution is taken to be the reference value and ion substitutions that produced a significant change in the reversal potential are indicated. These data confirm the non-selective nature of the MTX-sensitive channels. *, p < 0.001; **, p = 0.005; ***, p = 0.014. 

Cytosol Bath
Na K Cs Li Choline

Cs  -7.8  ± 1.0 (72)  -2.6  ± 0.7 (5) +2.7  ± 1.7 (6)**  -3.9  ± 0.6 (7)  -22.8  ± 1.0 (6)*
Na  -2.7  ± 1.2 (6)
Choline +1.8  ± 3.3 (7)***
NMG +10.0  ± 4.9 (9)*

It is reported that Ca2+ is impermeant through MTX-activated channels in mouse beta -cells (10) in contrast with a report that Ca2+ is permeant through such channels in mouse L-cells (21). We therefore decided to re-examine the Ca2+ permeability of ICa-NS. Ca2+ influx through MTX-sensitive channels in HIT-T15 cells is suggested by an increase of [Ca2+]i when hyperpolarizing voltage steps are applied following activation of the MTX-sensitive current and by the rapid and reversible fall of [Ca2+]i when extracellular Ca2+ is removed (Figs. 2 and 3A). Fig. 2 shows that a hyperpolarizing voltage clamp step from -70 mV to -100 mV had no effect on [Ca2+]i in a HIT-T15 cell before stimulation with MTX, but that similar voltage steps applied after activation of ICa-NS resulted in a pronounced increase of [Ca2+]i that was reversibly abolished by removal of extracellular Ca2+. Hyperpolarizing pulses applied following activation of non-selective cation currents by GLP-1 (22) or 8-bromo-cAMP (7) also increased [Ca2+]i.


Fig. 2. Hyperpolarizing voltage steps increase [Ca2+]i following MTX stimulation. Fig. 2 shows simultaneous records of membrane current (top trace) and [Ca2+]i (lower trace) from a HIT-T15 cell (Cm 14.0 pF) held initially at -70 mV in perforated patch voltage clamp. Membrane potential is shown as the inset. The cell was bathed in 0.8 mM glucose SES plus 1 µM TTX and was dialyzed with Cs-pipette solution. A 15-s hyperpolarizing step to -100 mV before stimulation with MTX produces a small increase in the holding current and no detectable increase of [Ca2+]i. The cell was then stimulated with 25 pM MTX (indicated by bar), and a step from -70 mV to -100 mV following MTX produces an increase in the holding current and a reversible rise of [Ca2+]i. The bath solution was then changed to a Ca2+-free (Mg2+-substituted + 50 µM EGTA + 1 µM TTX) solution resulting in a decrease of [Ca2+]i and inhibition of the hyperpolarization-induced increase of [Ca2+]i. On return to SES (2.6 mM Ca2+), [Ca2+]i increases and hyperpolarization again produces a further rise of [Ca2+]i.
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Fig. 3. Effects of removing extracellular Ca2+ on membrane currents and changes in [Ca2+]i. Panel A shows simultaneous recordings of membrane current (top trace) and [Ca2+]i (lower trace) from a beta TC6 cell (Cm 23.5 pF) bathed in SES and held at -70 mV in perforated patch voltage clamp using K-pipette solution. Bath perfusion with Ca2+-free solution (50 µM EGTA, indicated by bar) produces a small reduction of [Ca2+]i from 138 nM to 118 nM and a small increase in the holding current, these effects are reversed by perfusion with SES. A 10-s pulse of 50 pM MTX induces an inward current and rise of [Ca2+]i. The rise of [Ca2+]i was rapidly and reversibly reduced by bath perfusion with Ca2+-free solution (bar). ICa-NS was slightly reduced by the Ca2+-free solution in this cell. The beta TC6 cell shown in B (Cm 7.7 pF) was bathed in SES plus 5 mM TEA, 1 µM TTX, and 1 µM nimodipine and was held at -70 mV using Cs-pipette solution. An inward current developed, and [Ca2+]i fell from 52 nM to 41 nM during bath perfusion with Ca2+-free solution (50 µM EGTA plus TEA, TTX, and nimodipine, indicated by bar). The reversal potential of the current was estimated from voltage ramps (r, data not shown) to be -13 mV. The current rapidly inactivates when SES is reintroduced into the bath and [Ca2+]i increases to 95 nM. A subsequent pulse of 50 pM MTX (30 s, starting at arrow) activates an inward current (reversal potential -12 mV) and a rise in [Ca2+]i. Panel C shows that activation of ICa-NS increases Mn2+ quenching of intracellular fura-2 fluorescence. This HIT-T15 cell (Cm 38.8 pF) was bathed in SES plus 5 mM TEA, 1 µM TTX, and 1 µM nimodipine and was held at -70 mV using Cs-pipette solution. Application of a 60-s pulse of Ca2+-free, Mn2+-substituted solution before MTX produces a gradual quenching of fura-2 fluorescences (i and ii), a small increase in the holding current (iii) similar to that seen in panel A, i, with little change in the fluorescence ratio (iv). Stimulation with MTX (1-s pulse, arrow) activates the inward current and rise in [Ca2+]i, and a subsequent 60-s pulse of the Ca2+-free, Mn2+-substituted solution produces rapid quenching of fura-2 fluorescence (i and ii) and a fall in [Ca2+]i (iv) with little effect on membrane current (iii).
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We further tested the effects of removal of extracellular Ca2+ to confirm the activation of MTX-sensitive currents by Ca2+-free solutions (10) and to determine the effects of activation of the current on [Ca2+]i in the absence of extracellular Ca2+. In 15/18 cells tested, bath perfusion with a Ca2+-free solution containing 50 µM EGTA (the Ca2+ concentration in this solution was measured (using K5 fura-2) to be 150 nM) before stimulation with MTX caused a small, reversible increase of the membrane current and a fall of [Ca2+]i (Fig. 3A). Holding currents at -70 mV increased from -0.96 ± 0.16 pA/pF in SES to -1.64 ± 0.30 pA/pF (p = 0.005) and [Ca2+]i decreased from 117 ± 15 nM to 99 ± 15 nM on removal of extracellular Ca2+ (not statistically significant, p = 0.4). In 3/18 cells the holding current showed a much larger increase on transfer to Ca2+-free solution (Fig. 3B). The holding current in these cells increased by -21.8 ± 4.3 pA/pF and [Ca2+]i reduced from 75 ± 15 nM to 37 ± 4 nM (p = 0.1). This current inactivates rapidly, and [Ca2+]i is transiently elevated when SES (2.6 mM Ca2+) is reintroduced to the bath (Fig. 3B). Elevation of [Ca2+]i and activation of a non-selective cation current were also observed in a similar subset of cells following stimulation with thapsigargin (2-10 µM, data not shown), as reported previously (10).

Bath perfusion with Ca2+-free solution after the activation of ICa-NS results in a rapid, reversible fall of [Ca2+]i but has only a small effect on the current amplitude (Figs. 2 and 3A). The reversal potential of the MTX-activated current in Ca2+-free solution is -10.1 ± 2.8 mV (n = 12), not significantly different (p = 0.43) from the reversal potential measured in SES. These data suggest that Ca2+-influx carries only a small proportion of the current through MTX-sensitive channels in the presence of Na+. However, the rapid and reversible effects of removing extracellular Ca2+ on [Ca2+]i suggests that ICa-NS does permit Ca2+ entry.

Further evidence for the influx of divalent cations through MTX-sensitive channels is suggested by increased Mn2+ quenching of intracellular fura-2 fluorescence following MTX-stimulation (Fig. 3C). Fig. 3C (i and ii) shows the raw fura-2 fluorescence emission values, application of a 60-s pulse of Ca2+-free, Mn2+-substituted solution before stimulation with MTX produces a gradual quenching of the fluorescence signals, a small increase in the holding current (Fig. 3C, iii), and little or no change in the 350 nm/380 nm fluorescence ratio (Fig. 3C, iv). Following activation of the inward current and rise in [Ca2+]i, a pulse of Ca2+-free, Mn2+-substituted solution produces rapid quenching of fura-2 fluorescence (Fig. 3C, i and ii) with little or no effect on membrane current (Fig. 3C, iii) and reduces the fluorescence ratio (Fig. 3C, iv), consistent with a decrease in [Ca2+]i. Similar activation of Mn2+ quenching has been observed following stimulation of insulinoma cells with PACAP (6), cAMP (22), and thapsigargin (23, 24).

A role for Ca2+ influx through MTX-sensitive channels is further supported by the effects of applying high [Ca2+]o solutions. A Na+-free test solution containing 100 mM Ca2+ was applied to a beta TC6 cell prior to stimulation with MTX and produces a small increase of [Ca2+]i (Fig. 4), similar to the effects of applying elevated [Ca2+]o solutions to mouse islets (25). Application of 100 mM [Ca2+]o solution following stimulation with MTX produces a pronounced inhibition of ICa-NS (Fig. 4) and a negative shift in the reversal potential of the current (Table I). This inhibition of ICa-NS by the 100 mM Ca2+ solution is accompanied by a rise of [Ca2+]i (Fig. 4). These data could be explained by Ca2+ influx through a single class of non-selective cation channel with a lower permeability to Ca2+ than to Na+ under these experimental conditions. Alternatively, ICa-NS may have two (or more) components, one component being monovalent cation selective, and the other, smaller, component being selective for divalent cations.


Fig. 4. Na+-free, 100 mM Ca2+ solutions decrease ICa-NS and raise [Ca2+]i. A beta TC6 cell (Cm 28.7 pF) was held at -70 mV in perforated patch voltage clamp with Cs-pipette solution and SES plus 5 mM TEA, 1 µM TTX, and 10 µM nimodipine. The bath solution was then exchanged with Na+-free, 100 mM Ca2+, and this solution caused a gradual increase of [Ca2+]i (lower trace) with no apparent effect on the membrane current (upper trace). The bath was then returned to SES and [Ca2+]i recovered. A 1-s pulse of MTX (arrow) elicited an inward current and a rise of [Ca2+]i. The bath solution was then exchanged with Na+-free, 100 mM Ca2+ and the current was inhibited, but [Ca2+]i increased further. Bath perfusion with SES again led to recovery of the current and a fall of [Ca2+]i, indicating that the MTX-sensitive, non-selective cation channels are permeant to Ca2+. The reversal potential of the current shifted in the hyperpolarizing direction in Na+-free, 100 mM Ca2+ solution (Table I).
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The non-selective cation current activated by 8-bromo-cAMP in HIT-T15 cells is inhibited by whole cell dialysis with Ca2+-free, EGTA-buffered solutions or by loading the cells with BAPTA (1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid), a Ca2+ chelator (7) leading us to test the dependence of ICa-NS activation on [Ca2+]i (Fig. 5). Whole cell recordings from HIT-T15 cells were performed with either normal K-pipette solution (nominally Ca2+-free) or the same solution with 5 mM EGTA added (Ca2+-free). Whole cell dialysis with Ca2+-free intracellular solution inhibited activation of ICa-NS compared with control cells from the same platings dialyzed with nominally Ca2+-free solution or compared with cells in perforated patch voltage clamp (Fig. 5). These observations suggest that physiological [Ca2+]i levels are required for activation of the current. It is notable that dialysis of the cells with Ca2+-free solution does not induce activation of the current alone, whereas dialysis with this solution might be expected to deplete intracellular Ca2+ stores and thus activate store-operated currents.


Fig. 5. Activation of the MTX-sensitive current is dependent upon intracellular Ca2+. Bar graph of MTX-induced current amplitudes (inverted scale) in HIT cells held at -70 mV in perforated patch (perf., n = 5) or whole cell recording dialyzed with nominally Ca2+-free (WCR, n = 5) and Ca2+-free (+ 5 mM EGTA, n = 6) K-pipette solutions. Whole cell recording currents are not significantly different from perforated patch current amplitudes (p = 0.39), whereas calcium-free currents (EGTA) are significantly inhibited compared with whole cell recording currents (p = 0.003).
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Ca2+-activated non-selective cation (Ca-NS) channels are expressed in beta -cells that are activated at cytosolic [Ca2+] > 10-4 M and are blocked by 1 mM ATP and also by 10 mM 4AP when applied to the cytosolic face of isolated, inside-out patches (26, 27). Activation of ICa-NS is observed with physiological [Ca2+]i levels but inhibition of the current by dialysis of cells with Ca2+-free solutions suggests that it may be carried through Ca-NS channels. To test this possibility, beta TC6 cells were bathed in glucose-free SES with 5 mM TEA and 10 nM glyburide added (to block Ca2+-activated K+ channels and K+ATP channels) and dialyzed in the whole cell recording mode with K-pipette solution and 10 mM 4AP either with or without 2 mM ATP. MTX-induced currents in cells dialyzed without ATP had a mean peak amplitude of -23.6 ± 7.9 pA/pF (n = 6), and cells from the same platings dialyzed with 2 mM ATP had peak amplitudes of -22.5 ± 7.5 pA/pF (n = 6, not significantly different, p = 0.9). These data indicate that activation of ICa-NS is not glucose-dependent, consistent with previous reports of MTX-stimulated, glucose-independent insulin secretion (28). These differences in the sensitivity of ICa-NS to [Ca2+]i and to block by ATP and 4AP in whole cell recordings compared with inside-out patches may indicate that ICa-NS is not carried through the Ca-NS channels reported previously (26, 27) or may reflect the different recording configurations.

The non-selective cation current activated by PACAP in beta TC6 cells is inhibited by SKF 96365 (22), a blocker of depletion-activated currents (29) that inhibits MTX-induced Ca2+ influx and insulin secretion (30). Fig. 6 shows that application of 50 µM SKF 96365 reversibly inhibits ICa-NS in beta TC6 cells, similar to its effect on the PACAP-induced current in these cells (22), further supporting the suggestion that these currents are carried by the same channel type.


Fig. 6. Inhibition of ICa-NS by SKF 96365. A beta TC6 cell (Cm 8.0 pF) was held at -70 mV in perforated patch voltage clamp with Cs-pipette solution and bathed in SES. A 1-s pulse of 50 pM MTX was applied (arrow) and elicited an inward current. 50 µM SKF 96365 was then applied (indicated by bar) by pressure ejection from a pipette positioned close to the cell. SKF 96365 caused a reversible inhibition of the MTX-activated current in 8 cells tested.
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The activation of Mn2+ quenching of intracellular fura-2 fluorescence by MTX (Fig. 3C) is similar to that observed following thapsigargin treatment of insulinoma cells (23, 24). Thapsigargin-sensitive Ca2+ pools can also be depleted by econazole, which inhibits Ca2+-ATPases (31) and thereby elevates [Ca2+]i and also inhibits Mn2+ quenching in HIT cells (23). We therefore tested the effect of econazole on the activation of ICa-NS. Fig. 7A shows that 10 µM econazole significantly reduces the amplitude of ICa-NS compared with control cells from the same platings. The basal (pre-MTX) [Ca2+]i rose from 52 ± 7 nM to 182 ± 41 nM (n = 5, p = 0.01) in the presence of econazole and the peak amplitude of ICa-NS decreased from -12.6 ± 2.3 pA/pF to -3.6 ± 1.1 pA/pF (n = 5, p = 0.01). The rise of [Ca2+]i above basal during the MTX response was reduced from 449 ± 206 nM to 49 ± 14 nM (p = 0.09) by econazole.


Fig. 7. Effects of inhibitors on ICa-NS and the associated rise of [Ca2+]i. Panel A shows normalized ICa-NS amplitudes (left panel, inverted scale) in beta TC6 cells held at -70 mV in perforated patch voltage clamp (K-pipette solution). Control currents (Cont., n = 5) were obtained from cells bathed in SES, 10 µM econazole was then added to the bath solution (Econ., n = 5), and currents from the same platings of cells recorded in this solution. Econazole produced a significant reduction in the current amplitude (p = 0.01), and the rise of [Ca2+]i was also reduced (p = 0.09). Panel B shows control ICa-NS amplitudes (left panel, inverted scale, n = 6) and ICa-NS after addition of 100 µM genistein (genis., n = 8) to the bath solution. Genistein produces a significant reduction in current amplitude (p = 0.01). The equivalent reduction of the change of [Ca2+]i is shown in the right panel (p = 0.06). Panel C (left panel) shows control ICa-NS amplitudes (inverted scale, n = 5) and ICa-NS amplitudes following addition of 1 µM staurosporine (Stauro.) to the bath solution. Staurosporine had no significant effect on ICa-NS current amplitudes (p = 0.9) but significantly reduced the rise of [Ca2+]i (p < 0.001, right panel).
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The effect of genistein, a tyrosine kinase inhibitor, on the activation of ICa-NS was tested as capacitative Ca2+ influx activated by thapsigargin can also be inhibited by genistein (Ref. 32, Fig. 7B). The peak amplitude of ICa-NS reduces from -20.2 ± 3.3 pA/pF (n = 6) in control cells to -8.2 ± 2.6 pA/pF (n = 8, p = 0.01) following exposure of cells from the same platings to 100 µM genistein. Basal [Ca2+]i (pre-MTX) is not significantly affected by genistein (88 ± 12 nM in control cells, 118 ± 19 nM in genistein, p = 0.2), while the peak rise of [Ca2+]i during MTX responses reduced from 1026 ± 388 nM (control) to 313 ± 92 nM (genistein, p = 0.06).

Protein kinase C (PKC) activates capacitative Ca2+ entry in rat insulinoma (RINm5F) cells, and this activation of Ca2+ entry can be blocked by 1 µM staurosporine (24). We therefore tested the effects of 1 µM staurosporine on membrane currents and on the rise of [Ca2+]i following MTX stimulation of beta TC6 cells (Fig. 7C). Staurosporine had no significant effect on ICa-NS; the mean peak amplitude of control currents was -54.2 ± 19.5 pA/pF (n = 5) compared with -51.1 ± 7.7 pA/pF (n = 6, p = 0.9) in cells from the same platings after addition of 1 µM staurosporine to the bathing solution. The pre-MTX basal [Ca2+]i in control cells was 61 ± 11 nM and 96 ± 15 nM in cells bathed in staurosporine (p = 0.1), whereas the peak rise (increase above basal levels) of [Ca2+]i was reduced from 1653 ± 252 nM to 142 ± 28 nM (p < 0.001) by staurosporine.

Activation of some types of non-selective cation current has been shown to be mediated by GTP-binding proteins (33). We therefore examined the potential role of GTP-binding proteins in the activation of ICa-NS by dialysis of beta TC6 cells with Cs-pipette solution supplemented with 10 mM 4AP, 2 mM Na2ATP, and either 10 mM KF, 10 mM KF + 100 µM AlF3, or 100 µM GTPgamma S. The bath contained SES with 5 mM TEA and 1 µM TTX. Whole cell dialysis of cells for 15-20 min with KF (n = 4), KF + AlF3 (n = 5), or GTPgamma S (n = 5) failed to activate inward currents in cells that all subsequently responded to stimulation with MTX (data not shown).

MTX stimulates an increase in Ins(1,4,5)P3 levels in beta -cell lines (8), and this increase might activate ICa-NS through an intracellular Ca2+ release mechanism. Heparin is a specific blocker of Ins(1,4,5)P3 receptors that should inhibit activation of ICa-NS if Ins(1,4,5)P3-gated Ca2+ stores play an important role. beta TC6 cells were dialyzed in whole cell recordings with Cs-pipette solution plus 10 mM 4AP, 2 mM Na2ATP, and 0.5 mg/ml heparin for 3-4 min before stimulation with MTX. Activation of ICa-NS was not inhibited in cells dialyzed with heparin. The amplitude of the currents was not significantly different from that in control cells from the same platings, and the reversal potential of the currents was -7.0 ± 2.0 mV (n = 9), not significantly different from control values (p = 0.96).

GLP-1 enhances intracellular Ca2+ mobilization from ryanodine-sensitive stores in beta TC3 cells, and ryanodine reduces the amplitude of [Ca2+]i spikes produced by depolarizing voltage clamp steps within 1 or 2 min (11). We introduced 100 µM ryanodine into beta TC6 cells by whole cell dialysis in the Cs/4AP/ATP-pipette solution (as above) and allowed 3-4 min dialysis before stimulation with MTX. Ryanodine failed to prevent activation of ICa-NS under these conditions, and the current amplitude and reversal potential (-8.0 ± 1.9 mV, n = 5) are not significantly different from control cells (p = 0.97).


DISCUSSION

We propose that MTX activates the same non-selective cation current as stimulation with the peptide hormones GLP-1 and PACAP (6, 7, 22). These hormones couple through GTP-binding proteins (G-proteins) to activate adenylyl cyclase and elevate intracellular cAMP in beta -cells (3-5), and cAMP analogs can also activate these non-selective cation currents. However, activation of G-proteins, by dialysis of cells with KF, KF + AlF3, or GTPgamma S (compounds that stimulate G-protein mediated activation of non-selective cation currents in epithelial cells (33) and activate K+ATP channels in RINm5F and HIT-T15 insulinoma cells (34, 35)), neither activated nor inhibited the MTX-sensitive current.

Depletion of intracellular Ca2+ stores activates a MTX-sensitive current in mouse beta -cells (10), and the stimulation of Ins(1,4,5)P3 production by MTX (8) raises the possibility that Ins(1,4,5)P3-gated stores may play a role in the activation of this current. Parasympathetic, cholinergic stimulation of beta -cells stimulates Ins(1,4,5)P3 production, potentiates glucose-induced insulin secretion (36), and also activates a TTX-insensitive Na+-dependent depolarizing current (37) that may also be carried through Ca2+ release-activated non-selective cation channels (15). The role of intracellular Ca2+ release in the activation of this current has, however, been disputed, and cholinergic activation of this Na+ current is reported to be mediated by M3-type muscarinic receptors being coupled to Na+ channels (38). We observed that dialysis with heparin, a blocker of Ins(1,4,5)P3 receptors, failed to inhibit activation of ICa-NS, suggesting that Ca2+ release from these stores may not be critical.

The presence of a Ca2+ store depletion-activated current in pancreatic beta -cells was proposed from studies showing: 1) that the state of filling of endoplasmic reticulum stores could regulate the membrane potential in mouse beta -cells (39), 2) that thapsigargin can activate Mn2+ quenching of intracellular fura-2 in RINm5F cells (24), and 3) that the Mn2+ quenching pathway is inhibited by econazole in HIT-T15 cells (23). Activation of ICa-NS by thapsigargin and its inhibition by both econazole and SKF 96365 are consistent with the suggestion that ICa-NS may represent a Ca2+ release-activated current (10, 31).

A role for PKC in the activation of store-operated Ca2+ entry in RINm5F cells was suggested from observations that the sustained [Ca2+]i rise in response to combined stimulation with PKC-activating phorbol esters and thapsigargin is inhibited by staurosporine (24). We observed that staurosporine reduced the MTX-induced [Ca2+]i rise but did not significantly inhibit the amplitude of ICa-NS, suggesting that PKC may not play a direct role in the activation of ICa-NS but does regulate [Ca2+]i responses. Such effects could be mediated through inhibition of Ca2+ release from intracellular Ca2+ stores, or could reflect the inhibition of a small, divalent cation selective component of ICa-NS carried through a distinct set of channels other than the non-selective cation channels. The high concentration of staurosporine (1 µM) used in these experiments would also be expected to inhibit cAMP-dependent protein kinase, and further studies are required to elucidate the role(s) of PKC and cAMP-dependent protein kinase in the pathway(s) leading to the activation of ICa-NS and regulation of [Ca2+]i changes.

Inhibition of both ICa-NS and the MTX-induced [Ca2+]i rise by genistein, an inhibitor of tyrosine kinases (40) that blocks thapsigargin- and carbachol-induced Ca2+ entry (32, 41), suggests a role for tyrosine phosphorylation in the activation pathway of ICa-NS. However, the role of tyrosine phosphorylation remains ambiguous as only certain tyrosine kinase inhibitors (including genistein) effectively block capacitative Ca2+ entry (42). Thapsigargin-induced Ca2+ entry can also occur in the absence of detectable tyrosine phosphorylation but is still inhibited by tyrosine kinase inhibitors (43), and, therefore, the role of tyrosine kinases in activation of ICa-NS remains to be clarified.

Ca-NS channels are expressed in beta -cells that are activated at cytosolic [Ca2+] > 10-4 M (26, 27). Activation of ICa-NS is observed at physiological [Ca2+]i (approximately 100 nM) and is inhibited by dialysis of cells with Ca2+-free solution, similar to the inhibition of cAMP-activated currents (7) and suggesting a Ca2+-dependent step in the activation pathway of these channels. Ca-NS channels are also expressed in pancreatic acinar cells, and these channels are activated at much lower cytosolic Ca2+ concentrations in whole cell records than in isolated patches (44); a similar difference in Ca2+ sensitivity seems likely to occur for Ca-NS channels in beta -cells. Ca-NS channels are blocked by 1 mM ATP and by 10 mM 4AP when applied to the intracellular face of isolated membrane patches (27); however, these two compounds did not inhibit ICa-NS when introduced into the cytosol by whole cell dialysis (at 2 mM and 10 mM, respectively). It remains to be determined whether these differences in sensitivity to [Ca2+]i, 4AP, and ATP are a consequence of the different recording conditions or suggest that Ca-NS channels do not carry ICa-NS.

Reducing extracellular [Cl-] has no effect on ICa-NS amplitude or on its reversal potential, confirming the cation selectivity of the channel. Depletion of intracellular ATP levels through bathing cells in glucose-free media and dialyzing with ATP-free solutions or dialysis of cells with 2 mM ATP has no effect on ICa-NS amplitudes. This further distinguishes the MTX-induced current from the non-selective anion current described in insulin-secreting cells that increases in amplitude upon dialysis with 2 mM ATP (45).

Elevation of [Ca2+]i is observed following the activation of non-selective cation currents by MTX, GLP-1, or PACAP in voltage clamped cells where activation of voltage-dependent Ca2+ channels is prevented (6, 7, 22). This rise of [Ca2+]i is reversed by removal of extracellular Ca2+, suggesting that Ca2+ influx is associated with the non-selective current, although it remains to be determined whether a single class of channel is permeant to both monovalent and divalent cations, or if two (or more) distinct conductances are involved. The physiological role of Ca2+ influx associated with ICa-NS in the stimulation of insulin secretion remains to be determined. It has been reported that sustained Ca2+ influx through L-type VDCCs is strongly coupled to insulin secretion from HIT-T15 cells, whereas more transient Ca2+ influx through N-type Ca2+ channels is only weakly coupled (46). The Ca2+ influx associated with ICa-NS is prolonged and may, therefore, be able to contribute to the sustained [Ca2+]i elevation that triggers secretion. However, the magnitude of this Ca2+ influx is likely to be small compared with that through L-type channels as, under physiological conditions, the cells will depolarize to a value close to that for the reversal potential of ICa-NS, and also influx through L-type VDCCs raises [Ca2+]i very rapidly, whereas the [Ca2+]i increase associated with ICa-NS develops much more slowly. This slow time course of the rise in [Ca2+]i is consistent with a small amplitude Ca2+ influx and would explain why it is difficult to resolve a decrease in ICa-NS amplitude on changing to Ca2+-free solution with normal extracellular Na+ concentrations. It therefore seems that the main physiological role for these non-selective cation currents in the control of insulin secretion will be to depolarize the membrane potential and activate VDCCs.

The currents activated by GLP-1, PACAP, cAMP analogs, and MTX are Ca2+-dependent non-selective cation currents that activate over tens of seconds and persist for extended periods following removal of the stimulus. These currents are all insensitive to TTX, L-type Ca2+ channel blockers, TEA, and ryanodine but are inhibited by NMG, SKF96365, and La3+. Based upon these similarities between the temporal properties of the currents, and their associated [Ca2+]i changes, and the pharmacology of the currents, we propose that these agents activate the same non-selective cation channels. The precise mechanism(s) controlling the activation of these non-selective cation channels remains to be determined, but a role for tyrosine kinase-induced phosphorylation is suggested by the effects of genistein. Activation of ICa-NS may also be controlled by the state of filling of intracellular Ca2+ stores, or may be partly due to Ca2+ release from intracellular stores raising cytosolic Ca2+ levels. We propose that activation of MTX-sensitive non-selective cation channels may play an important role in depolarizing beta -cells in response to stimulation by GLP-1 and PACAP during feeding to initiate insulin secretion without large elevations of blood glucose. We also suggest that Ca2+ is permeant through the MTX-sensitive channels and suggest that spontaneous activity of these channels may form the depolarizing, non-selective background conductance that permits Ca2+ influx (25) and opposes the activity of ATP-sensitive K+ channels in regulating the resting potential of beta -cells under both basal conditions and in response to hormonal stimulation.


FOOTNOTES

*   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.
Dagger    To whom correspondence should be addressed. Tel.: 617-726-5191; Fax: 617-726-6954; E-mail: leech{at}helix.mgh.harvard.edu.
1   The abbreviations used are: K+ATP, ATP-sensitive K+ channel; MTX, maitotoxin; ICa-NS, Ca2+-dependent non-selective cation current activated by MTX and GLP-1; VDCCs, voltage-dependent Ca2+ channels; GLP-1, glucagon-like peptide-1; PACAP, pituitary adenylyl cyclase-activating polypeptide; TTX, tetrodotoxin; NMG, N-methyl-D-glucamine; [X]i, intracellular concentration of ion X (where X is any ion); [X]o, extracellular concentration of ion X; Ca-NS, Ca2+-activated non-selective cation channel; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; GTPgamma S, guanosine-5'-O-(3-thiotriphosphate) tetralithium salt; SES, standard extracellular solution; TEA, tetraethylammonium chloride; 4AP, 4-aminopyridine; pA, picoamps; pF, picofarads.

ACKNOWLEDGEMENT

We thank Maurice Castonguay for maintenance of cell cultures.


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