Muscarinic modulation of voltage-dependent Ca2+ channels in insulin-secreting HIT-T15 cells

Jeffrey A. Love, Neil W. Richards, Chung Owyang, and David C. Dawson

Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi 39216; and Departments of Internal Medicine and Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Potentiation of insulin secretion from pancreatic beta -cells by acetylcholine requires ongoing cyclic electrical activity initiated by other depolarizing secretagogues. Patch-clamp recordings in glucose-free solutions were made from the clonal beta -cell line HIT-T15 to determine whether the muscarinic agonist bethanechol (BCh) modulated voltage-dependent Ca2+ channels independent of effects on membrane potential. Only high-threshold, dihydropyridine-sensitive (L-type) Ca2+ channels with a mean conductance of 26 pS were observed in cell-attached patches. BCh (100 µM) caused a two- to threefold increase in both fractional open time and mean current of single Ca2+ channels. These changes resulted from a 44% decrease in the longer of two apparent mean closed times and a 25% increase in the mean open time. Similar BCh-stimulated increases in macroscopic Ca2+ currents were recorded in whole cell, perforated-patch recordings. The role of protein kinase C (PKC) in the muscarinic activation of Ca2+ channels was tested using a variety of PKC activators and inhibitors. Acute application of either the active phorbol ester phorbol 12-myristate 13-acetate (PMA) or the membrane-permeable diacylglycerol analog 1,2-didecanoyl-rac-glycerol mimicked the effects of BCh, whereas an inactive phorbol (4alpha ) had no effect. Depletion of PKC activity by chronic exposure to PMA or acute application of the PKC inhibitor staurosporine greatly reduced or abolished muscarinic activation of Ca2+ channels. These results are consistent with muscarinic activation of L-type, voltage-dependent Ca2+ channels mediated in large part by PKC.

pancreatic beta -cells; bethanechol; diacylglycerol; protein kinase C; L-type calcium channels

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

PANCREATIC beta -cells respond to increasing concentrations of extracellular glucose with gradual membrane depolarization until, at 6-8 mM glucose, a threshold is reached for the initiation of cyclic electrical activity. This activity consists of prolonged, voltage-dependent action potentials (plateau potentials) with superimposed spikes, both of which are Ca2+ dependent, interposed between repolarized, silent phases. Both the duration of the plateau potentials and the fraction of a given cycle spent at this potential are strongly glucose dependent and show a close correlation with Ca2+ influx and insulin secretion (20). Subsequent patch-clamp studies have revealed that glucose metabolism results in the inhibition of ATP-sensitive K+ channels, leading to membrane depolarization, activation of high-threshold voltage-dependent Ca2+ channels, and Ca2+ influx essential for stimulus-secretion coupling (2, 3, 36).

Acetylcholine (ACh) alone has little effect on insulin secretion and beta -cell electrical activity yet potentiates insulin secretion in the presence ongoing cyclic electrical activity initiated by glucose or other depolarizing secretagogues. The secretory effects are paralleled by both a depolarization of the plateau potentials and an increased frequency of these Ca2+-dependent potentials as well as the superimposed Ca2+ spikes (6, 9, 14). Because glucose-stimulated beta -cell electrical activity, insulin secretion, and the effects of ACh on them persist in tetrodotoxin- and Na+-free solutions, a significant part of the ACh effect is likely to be due to an increase in Ca2+ channel activity (11-13, 15, 19, 20, 26, 27). Although muscarinic depolarization of beta -cells resulting from a decrease in resting K+ conductance has been reported (27) and could explain the increased Ca2+ influx and insulin secretion, the possibility of a membrane potential-independent activation of Ca2+ channels by muscarinic agonists has not been studied in detail. Such a direct activation of beta -cell Ca2+ currents has been demonstrated for agents that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels (1).

The mechanism of cholinergic modulation of beta -cell electrical activity and its relationship to insulin secretion are poorly understood. However, it is well established that ACh, acting via G protein-coupled muscarinic receptors, stimulates phospholipid hydrolysis, yielding inositol trisphosphate (IP3) and diacylglycerol (DAG) (34). The activation of protein kinase C (PKC) by DAG results in the phosphorylation of endogenous protein substrates (8, 10) and appears to be required for the sustained, Ca2+-dependent cholinergic potentiation of glucose-stimulated insulin secretion (16, 22, 23). In contrast, IP3 production causes only a transient stimulation of insulin secretion, and only at high concentrations of cholinergic agonist (34). Protein kinase A, which is not stimulated by ACh, potentiates glucose-stimulated electrical activity and insulin secretion in a similar manner. These effects have been attributed to both an increased Ca2+ channel activity at a given membrane potential and a sensitization of the secretory process to Ca2+ (1, 24).

These observations led us to hypothesize that ACh, acting via PKC, could alter the gating of voltage-dependent Ca2+ channels activated by membrane depolarization. To test this hypothesis, we used the clonal beta -cell line HIT-T15. This continuous cell line provided a homogeneous population of insulin-secreting cells that exhibited all the salient electrophysiological and biochemical features of pancreatic beta -cells (15-17, 25). Cell-attached patch recordings of single Ca2+ channel currents and permeabilized-patch recordings of whole cell currents were used to study the effects of a muscarinic agonist under conditions that preserved the cytoplasmic integrity of the cells and allowed the control of membrane potential.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture. HIT-T15 cells were obtained from the American Type Culture Collection and grown in RPMI-1640 containing 11.1 mM glucose and supplemented with 10% fetal bovine serum, glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml). Cultures were maintained in a warm (37°C), humidified atmosphere containing 95% air-5% CO2. Cells (passages 59-75) were grown on glass coverslips for patch-clamp recordings. At least 30 min before patch-clamp recordings, coverslips were removed from the culture medium and placed in a glucose-free bath solution containing (in mM) 135 NaCl, 5 KCl, 2 MgCl2, 5 CaCl2, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-NaOH (pH 7.4). This ensured that any residual effects of glucose on Ca2+ channel activity (3, 31) were avoided.

Patch-clamp recordings. Single Ca2+ channel currents were recorded from cell-attached patches in a glucose-free bath solution containing (in mM) 115 KCl, 28 KOH, 1 MgCl2, 1 CaCl2, 10 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 10 HEPES (pH 7.4). This solution closely approximates the cytoplasmic K+ concentration and depolarizes the cells to a membrane potential of ~0 mV. Under these conditions, any depolarization resulting from application of BCh was prevented, and changes in channel activity were ascribed to a membrane potential-independent change in channel kinetics. Gigaohm seals were made using polystyrene-coated glass (Corning 7052) pipettes containing a solution of (in mM) 100 BaCl2, 10 tetraethylammonium chloride (TEA · Cl), and 10 HEPES-Ba(OH)2 (pH 7.4). This solution blocked all K+ currents in the patches while allowing Ba2+ to serve as the carrier of inward current and minimizing the Ca2+-dependent inactivation of channel activity reported in HIT cells (17). Membrane patches were voltage clamped using an Axopatch-1B amplifier (Axon Instruments, Foster City, CA), and the records were stored on computer hard disk. Currents were filtered at 1-3 kHz using an eight-pole Bessel filter and a digitization rate of 5-15 kHz. Capacity and leakage currents associated with voltage steps were removed by subtracting the composite null sweep. As has been observed in primary cultures of beta -cells (31), the brief nature of the Ca2+ channel openings combined with their low open probability prevented us from reliably establishing the number of channels in a patch. Because calculation of open-state probability presumes recordings from a single channel, we instead used fractional open time (Fo) as an index of Ca2+ channel activation. Single-channel currents were analyzed using pCLAMP software (Axon Instruments) with Fo calculated from the total open time of the channels divided by the total time of the record and the mean current (Iavg) calculated from the sum of single-channel currents divided by the total time of the record. Mean open and closed times were calculated from the time constants of the exponential functions fitted to the frequency distributions of open and closed times. All values were expressed as means ± SE, and statistical comparisons were made using two-tailed, paired Student's t-tests.

Macroscopic Ca2+ currents were recorded using the perforated-patch variation of whole cell recording. Amphotericin was dissolved in dimethyl sulfoxide (DMSO, 6 mg/100 µl) and then diluted in the pipette solution to a final concentration of 240 µg/ml. The pipette solution contained (in mM) 55 CsCl, 70 Cs2SO4, 7 MgCl2, and 10 HEPES (pH 7.4), and the bath solution contained (in mM) 136 NaCl, 10 BaCl2, or CaCl2 (pH 7.4). After formation of gigaohm seals, perforation began within 5 min and stabilized within 15-30 min. Only patches with stable access resistances of 8-25 MOmega were studied further. Whole cell currents were studied at membrane potentials from -100 to +50 mV using a sequence of voltage steps (10 mV; 200-500 ms) from a holding potential of -70 mV. Subtraction of linear leak currents extrapolated from currents measured at potentials negative to the holding potential was used to correct the observed values of macroscopic Ca2+ currents.

Chemicals. All pharmacological agents were ultimately dissolved in the bath solution described above. Concentrated stocks of lipophilic compounds were made using DMSO and diluted to yield a final concentration 0.01-0.1% DMSO. Chemical reagents included bethanechol (BCh), phorbol 12-myristate 13-acetate (PMA), 1,2-didecanoyl-rac-glycerol (DC10), 4alpha -phorbol, pertussis toxin (PTX), and TEA · Cl, all purchased from Sigma Chemical (St. Louis, MO). The dihydropyridine compounds BAY-K-8644 and nifedipine were obtained from Research Biochemicals (Natick, MA).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Single Ca2+ channel currents in cell-attached patches. Cell-attached patches exhibited single Ca2+ channel (Ba2+) currents in over 85% of the cells tested. After seal formation, the patches were hyperpolarized to pipette potentials of -60 to -100 mV, and channels were activated by 50- to 250-ms depolarizing voltage steps to pipette potentials up to +10 mV. Brief, low-frequency channel openings with a mean single-channel conductance of 26 ± 1 pS (n = 11) were observed only at pipette potentials positive to -40 mV (Fig. 1). Kinetic analysis of the channel openings revealed a single apparent mean open time (tau o) of 0.5 ± 0.1 ms and two apparent mean closed times (tau c1tau c2) of 1.3 ± 0.2 and 32.7 ± 6.5 ms, respectively (n = 11). Marked increases in Fo occurred only at pipette potentials positive to -20 mV (Fig. 2), and the threshold for these channel openings was unaffected by increasing the holding potential to values up to -100 mV. In addition, these channels exhibited little inactivation during voltage steps lasting several hundred milliseconds to several seconds. Excision of the patches to form inside-out patches resulted in a complete loss of activity within several minutes.


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Fig. 1.   Activation of single Ca2+ channel currents in a cell-attached patch from an HIT-T15 cell. Single-channel currents were recorded in a high (143 mM)-K+ bath solution, using pipettes filled with a solution that contained 100 mM BaCl2 and 10 mM TEA · Cl. Pipette potential was stepped from a holding potential of -90 mV to potentials indicated at right. In all patches tested, threshold for channel activation was positive to -40 mV, with most channel activation occurring positive to -20 mV. Channel openings were observed throughout duration of voltage steps, and simultaneous opening of at least 2 channels occurred at a step potential of 0 mV.


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Fig. 2.   HIT-T15 cells exhibited only high-threshold, voltage-dependent Ca2+ channels. In cell-attached patches, channel openings were recorded during a series of 10-mV depolarizing voltage steps from a holding potential of -100 mV, using solutions described in Fig. 1 legend. Threshold for channel activation occurred positive to -40 mV, with most of channel activity occurring at pipette potentials positive to -20 mV. Fractional open time (Fo) was fraction of total trial time (20 67-ms sweeps at each test potential) during which at least 1 channel was open. Each point represents mean ± SE of Fo recorded from 6 patches.

We also tested the dihydropyridine sensitivity of these high-threshold Ca2+ channels. Bath application of the dihydropyridine agonist BAY-K-8644 (0.1-1 µM) caused a dramatic change in the Ca2+ channel gating pattern to one of frequent, prolonged openings (Fig. 3). This resulted in a significant increase in Iavg from 0.04 ± 0.01 to 0.56 ± 0.19 pA (P <=  0.05; n = 5). In contrast, the dihydropyridine antagonist nifedipine (1-5 µM) significantly reduced Iavg from 0.05 ± 0.01 to 0.01 ± 0.01 pA (P <=  0.05; n = 6), and the remaining channel currents exhibited the same thresholds and single-channel conductances recorded in the absence of the antagonist (Fig. 4).


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Fig. 3.   Dihydropyridine agonist BAY-K-8644 increased high-threshold Ca2+ channel currents of HIT-T15 cells. Top: channel openings recorded from a cell-attached patch were elicited by stepping from a holding potential of -90 mV to a test potential of -30 mV. Brief, infrequent channel openings occurred throughout 200-ms steps. Bottom: 5 min after bath solution was changed to one containing BAY-K-8644 (1 µM), channel gating was characterized by prolonged openings that revealed at least 2 distinct channels within the patch. Mean current (Iavg), calculated from 100 sweeps, increased from 0.03 to 0.78 pA/ms in presence of agonist.


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Fig. 4.   Dihydropyridine antagonist nifedipine inhibited high-threshold Ca2+ channel currents of HIT-T15 cells. Top: channel openings recorded from a cell-attached patch were elicited by stepping from a holding potential of -70 mV to a test potential of -20 mV. Bottom: 10 min after switch to a solution that contained nifedipine (5 µM), Iavg, calculated from 100 sweeps, decreased from 0.06 to 0.01 pA/ms.

Macroscopic Ca2+ channel currents in permeabilized patches. Whole cell recordings in the permeabilized-patch configuration were used to confirm that the properties of the single Ca2+ channel currents accurately reflected the properties of macroscopic Ca2+ channel currents. Like the single-channel currents, whole cell currents exhibited a threshold positive to -40 mV, with over 80% of the maximal current recorded at potentials positive to -20 mV (Fig. 6, A and B). The threshold was not changed by increasing the holding potential from -70 to -100 mV. The mean peak current of 105 ± 13 pA (n = 6) occurred at +10 mV and reversed at +50 mV. Application of nifedipine (5 µM) significantly decreased mean peak current by 84% (from 91 ± 12 to 15 ± 4 pA) in all three cells tested.

Effects of BCh on Ca2+ channel currents. Single Ca2+ channel currents were activated by depolarizing voltage steps (40-80 mV; 0.1-0.5 Hz) from a holding potential of -70 mV. Under control conditions, single Ca2+ channel openings of HIT-T15 cells occurred as low-frequency bursts described by tau o, tau c1, and tau c2. Exposure of HIT-T15 cells to the muscarinic agonist BCh (100 µM) caused a large increase in the frequency of channel openings (Fig. 5) in 27 of 31 patches tested. Statistically significant (P <=  0.05) increases in tau o, Fo, and Iavg, as well as significant decreases in the longer of two apparent mean closed times, tau c2, accompanied the increased activity (Table 1). These effects occurred within 5 min of changing the bath solution and persisted throughout the duration of exposure (tested up to 45 min after completion of bath change). In contrast, all cells exposed to BCh after pretreatment with atropine (10 µM; n = 6) exhibited no significant changes in Fo (0.01 vs. 0.01) or Iavg (0.01 vs. 0.01 pA/ms), whereas, in the same passage of cells, two of three cells responded to BCh in the absence of atropine with significant increases in Fo (0.02 vs. 0.06) and Iavg (0.02 vs. 0.06 pA/ms). Pretreatment with PTX (100 ng/ml) for 12-48 h before recording had no effect on the BCh response. In four of five cells tested, BCh (100 µM) caused significant increases in Fo (0.03 vs. 0.07) and Iavg (0.04 vs. 0.08 pA/ms). In recordings from cells of the same passage not treated with PTX, BCh significantly increased Fo (0.03 vs. 0.08) and Iavg (0.04 vs. 0.08 pA/ms) in three of four cells tested.


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Fig. 5.   Muscarinic agonist bethanechol (BCh) increased high-threshold, single Ca2+ channel currents independent of a change in membrane potential. Top: single-channel currents were recorded from a cell-attached patch in a high-K+ bath solution that chemically clamped membrane potential at or close to 0 mV. Channel openings were elicited by depolarizing voltage steps from a holding potential of -70 mV to a test potential of -15 mV. Under these conditions, channel openings were brief and infrequent. Bottom: 10-min exposure to a bath solution that contained 100 µM BCh markedly increased Ca2+ channel activity, and Iavg, calculated from 100 sweeps, increased from 0.03 to 0.06 pA/ms.

                              
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Table 1.   Effects of bethanechol on single Ca2+ channel kinetics in HIT-T15 cells

Similar effects of BCh on whole cell Ca2+ channel currents were measured using permeabilized-patch recordings (Fig. 6). In all five cells tested, BCh (100 µM) significantly increased the mean peak current from 100 ± 14 to 197 ± 27 pA. As shown in Fig. 6, the effect of BCh was observed only at test potentials positive to -30 mV, with the greatest increases in current occurring at test potentials positive to -20 mV.


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Fig. 6.   BCh increased whole cell Ca2+ currents in HIT-T15 cells. A: whole cell Ca2+ currents evoked by 200-ms voltage steps (holding potential -70 mV) in absence (top) and presence (bottom) of BCh (100 µM; 10 min). Test potentials ranged from -30 to +40 mV in 10-mV increments. B: whole cell current-voltage relationship in same cell determined using voltage steps from -120 to +50 mV (holding potential -70 mV). Exposure to 100 µM BCh for 10 min increased peak Ba2+ current from 122 to 322 pA.

Activation of PKC mimics BCh effects. Because PKC has been implicated in muscarinic potentiation of glucose-stimulated insulin secretion (16, 22, 23), we compared the effects of exogenous activators of the enzyme with those of BCh. Acute application of the active phorbol ester PMA (10-100 nM) resulted in a marked increase in the frequency of Ca2+ channel openings (Fig. 7). Similar to the response to BCh, the increased channel activity was accompanied by significant (P <= 0.05) increases in Fo (0.03 ± 0.01 vs. 0.14 ± 0.02) and Iavg (0.03 ± 0.01 vs. 0.14 ± 0.03 pA/ms) and a significant decrease in tau c2 (35.5 ± 1.1 vs. 11.1 ± 1.9 ms) in four of five patches tested. Unlike the response to BCh, no significant increase in tau o was observed. In contrast, acute application of 4alpha -phorbol (100 nM), which does not activate PKC, had no effect on Fo (0.05 ± 0.02 vs. 0.04 ± 0.01), Iavg (0.04 ± 0.03 vs. 0.04 ± 0.01 pA/ms), tau c2 (20.8 ± 6.0 vs. 20.6 ± 6.0 ms), tau o (0.5 ± 0.1 vs. 0.6 ± 0.1 ms), or tau c1 (1.4 ± 0.2 vs. 1.2 ± 0.2 ms) in all five patches tested. The effects of the membrane-permeable DAG analog DC10 on single Ca2+ channel currents were also tested. Acute application of this compound also caused a significant increase in Ca2+ channel activity (Fig. 8) accompanied by significant (P <=  0.05) increases in Fo (0.04 ± 0.01 vs. 0.09 ± 0.01) and Iavg (0.03 ± 0.01 vs. 0.10 ± 0.02 pA/ms) and a significant decrease in tau c2 (27.1 ± 3.8 vs. 12.8 ± 1.6 ms), but tau o (0.4 ± 0.1 vs. 0.5 ± 0.1 ms) and tau c1 (2.7 ± 0.7 vs. 1.7 ± 0.2 ms) were unchanged in six of eight patches tested.


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Fig. 7.   Phorbol ester phorbol 12-myristate 13-acetate (PMA) increased high-threshold Ca2+ channel currents in HIT-T15 cells. Top: single-channel openings were evoked by voltage steps from a holding potential of -70 mV to a test potential of -10 mV. High-K+ bath solution contained 0.01% DMSO, which served as a vehicle control. Bottom: 5 min after bath solution was changed to one that contained PMA (100 nM; 0.01% DMSO), Iavg, calculated from 100 sweeps, increased from 0.02 to 0.06 pA/ms.


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Fig. 8.   Diacylglycerol analog 1,2-didecanoyl-rac-glycerol (DC10) increased high-threshold single Ca2+ channel currents in HIT-T15 cells. Top: single-channel currents were evoked by steps from a holding potential of -70 mV to a test potential of -10 mV in a high (143 mM)-K+ bath solution containing 0.01% DMSO as a vehicle control. Bottom: 5 min after bath solution was changed to one containing DC10 (5 µg/ml; 0.01% DMSO), Iavg, calculated from 100 sweeps, increased from 0.04 to 0.11 pA/ms.

Depletion or inhibition of PKC activity antagonizes effects of BCh. To further test the role of PKC in muscarinic activation, we examined the effects of depleting or inhibiting PKC activity. Depletion of PKC activity was accomplished by chronic (20-32 h) exposure to PMA (200 nM), a treatment known to deplete enzyme activity by >75% in beta -cells (25). After such treatment, BCh caused only a small (20%) but significant increase in Fo and Iavg and no change in the mean open or closed times in 9 of 12 cells tested. In contrast, seven of nine cells chronically exposed to 4alpha -phorbol (200 nM) responded to BCh with large increases in Fo (180%) and Iavg (225%) and a decrease (54%) in tau c2 (Table 2). The nature and magnitude of these effects were comparable to those observed previously in untreated cells (Table 1).

                              
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Table 2.   Effects of chronic exposure to phorbol esters on bethanechol activation of single Ca2+ channel currents

We also examined the effects of BCh in the presence of the protein kinase inhibitor staurosporine (100 nM). This concentration of staurosporine has been reported to inhibit islet PKC activity by >80% (12). Ca2+ channel activity was initially recorded in the presence of staurosporine alone before switching to a solution containing both staurosporine and BCh (100 µM). Under these conditions, BCh evoked small but significant increases in Fo (0.03 ± 0.01 vs. 0.04 ± 0.01) and Iavg (0.04 ± 0.01 vs. 0.05 ± 0.01 pA/ms), but no changes in tau o (0.3 ± 0.1 vs. 0.3 ± 0.1 ms), tau c1 (2.1 ± 0.3 vs. 1.9 ± 0.5 ms), or tau c2 (24.6 ± 4.2 vs. 23.3 ± 5.9 ms) were observed in seven of nine cells tested. In parallel control cells from the same passage, BCh, in the presence of the vehicle (0.01% DMSO) alone, caused significant increases in Fo (0.04 ± 0.01 vs. 0.10 ± 0.02) and Iavg (0.05 ± 0.01 vs. 0.10 ± 0.02 pA/ms) accompanied by a significant decrease in tau c2 (15.9 ± 1.6 vs. 8.4 ± 2.2 ms), but tau o (0.3 ± 0.1 vs. 0.4 ± 0.1 ms) and tau c1 (1.9 ± 0.6 vs. 1.2 ± 0.2 ms) were unchanged.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Ca2+ channels in HIT-T15 cells. HIT-T15 cells exhibited exclusively high-threshold voltage-dependent Ca2+ channel currents. Both single-channel and whole cell currents had thresholds of activation positive to -40 mV that were unaltered when the holding potential was increased from -60 to -100 mV. These high-threshold currents were predominantly, if not exclusively, L-type currents. Consistent with this conclusion were blockade of >80% of single-channel and whole cell currents by nifedipine, a mean single-channel conductance of 26 pS, little current inactivation during prolonged depolarizations, and rapid, complete loss of single-channel currents after excision of cell-attached patches. Using the solutions and recording protocols that revealed low-threshold, T-type channels in rat beta -cells (3), we observed no such channels in cell-attached patches from HIT cells. Our results are the first to characterize single Ca2+ channel currents in HIT-T15 cells and to correlate them with whole cell currents recorded under conditions that preserved the normal cytosolic components required for Ca2+ channel activity.

Using standard whole cell recordings of HIT cell Ca2+ currents, Keahey et al. (17) reported thresholds between -50 and -40 mV from a holding potential of -80 mV. These currents were reduced by ~85% in the presence of nimodipine, and the residual currents retained the kinetics of L-type Ca2+ currents. Parallel studies of the dihydropyridine sensitivity of K+-stimulated insulin secretion and intracellular Ca2+ accumulation revealed a strong correlation with that of the Ca2+ currents. These authors concluded that HIT cells contain almost exclusively high-threshold, L-type Ca2+ channels and that, as reported in islets and primary cultures of beta -cells, the entry of extracellular Ca2+ essential for insulin secretion occurs through these channels. Simultaneous measurements of Ca2+ transients and whole cell Ca2+ currents in HIT cells also indicated that voltage-dependent influx was carried exclusively by L-type channels (7). Our results are the first to confirm these conclusions at the single-channel level.

Fast, Ca2+-dependent and slow, voltage-dependent components of Ca2+ current inactivation have been described in HIT cells (28). Currents elicited at more negative test potentials (-50 to -30 mV) were either unaffected or increased by cell dialysis, whereas currents observed at more positive potentials were subject to rundown, suggesting the presence of high-threshold L- and N-type channels. An omega -conotoxin (CTX)-sensitive, high-threshold (N-type) Ca2+ current has also been recorded in the rat insulinoma cell line RINm5F (30). This high-threshold current comprised 15-25% of the total Ca2+ current, and its blockade with CTX inhibited stimulated insulin secretion by up to 51%. Although our experiments cannot prove that such a current does not exist in HIT cells, our results indicate that it could contribute only a small fraction (<20%) of the total current, whereas in HIT cells over 90% of insulin secretion is dihydropyridine sensitive (17). Thus the effects of BCh and PKC activators on Ca2+ currents in HIT cells can be ascribed to actions on high-threshold, L-type channels.

Muscarinic agonist increased voltage-activated Ca2+ channel currents in HIT-T15 cells. Stimulation of muscarinic receptors increased single-channel and whole cell L-type Ca2+ channel currents two- to threefold in HIT-T15 cells under recording conditions that chemically or electrically clamped resting membrane potential. This is the first direct evidence for a muscarinic receptor-mediated modulation of single Ca2+ channel currents in pancreatic beta -cells that is independent of effects on resting membrane potential. Antagonism of the BCh effects by atropine confirmed that this was a muscarinic receptor-mediated event, and the PTX-resistant nature of the effects suggests coupling of the receptor to G proteins other than Gi or Go, which are associated with PTX-sensitive inhibition of L-type Ca2+ channels in beta -cells (29). These observations are consistent with previous reports that cholinergic potentiation of glucose-stimulated insulin secretion is mediated by PTX-resistant G proteins (30, 34). Recent work indicates that muscarinic M3 receptors mediate cholinergic potentiation of glucose-stimulated insulin secretion (5), and in other tissues, these receptors appear preferentially coupled to Gq and G11 and lead to the activation of phospholipase Cbeta 1 (18). The specific stimulatory G proteins mediating cholinergic activation of phospholipase C in islets remain to be determined; however, one recent study has implicated Go (33).

Muscarinic stimulation of Ca2+ currents required PKC activation. Cholinergic agonists activate phospholipase C with the production of IP3 and DAG in both pancreatic islet cells and HIT cells. These two second messengers respectively cause the release of intracellular Ca2+ and the activation of PKC (8, 15, 16, 20, 22, 23, 34, 36). In HIT-T15 cells activation of PKC, using either a phorbol ester or a DAG analog, qualitatively and quantitatively mimicked the effects of BCh on single and whole cell Ca2+ channel currents. Conversely, both the PKC inhibitor staurosporine and depletion of PKC activity by chronic phorbol treatment markedly inhibited the effects of BCh. Biochemical studies of islet and HIT cells have demonstrated that staurosporine, at the concentrations used in our experiments, inhibited PKC activity by 70-80% (37), whereas chronic exposure to phorbol esters reduced PKC activity by 75%. These effects were accompanied by a comparable inhibition of the sustained cholinergic potentiation of glucose-stimulated insulin secretion that was dependent on Ca2+ influx (16, 22, 23, 32). We observed a comparable inhibition of BCh-stimulated increases in Ca2+ currents. Thus intact PKC activity appears essential for the muscarinic effects that we observed.

These results do not preclude the possibility that PKC might activate another second-messenger system, which in turn modulates Ca2+ channels. cAMP has been shown to increase Ca2+ influx through voltage-dependent channels in beta -cells (1, 24), and we have also recorded significant (P <=  0.05) increases in Fo (0.04 ± 0.01 vs. 0.10 ± 0.01; 5 of 6 cells) of single Ca2+ channel currents in HIT-T15 cells exposed to forskolin (5-10 µM) in the presence of 3-isobutyl-1-methylxanthine (100 µM). The membrane-permeable analog 8-bromo-cAMP (2 mM) also increased Fo (0.05 ± 0.01 vs. 0.09 ± 0.03; 3 of 3 cells) in HIT-T15 cells (unpublished observations). However, numerous biochemical studies on islet cells and clonal beta -cell lines have demonstrated muscarinic potentiation of glucose-stimulated insulin secretion without any effect on cAMP levels (9, 20, 36). The fact that our experiments were conducted with the cells depolarized to a membrane potential at or near 0 mV in the presence of extracellular Ca2+ argues against the activation of another protein kinase by BCh due to increased Ca2+ influx. Our experiments also cannot rule out some direct modulation of Ca2+ channel activity by interaction with G protein subunits.

Potential importance in insulin secretion. Numerous studies have confirmed that cholinergic agonists produce a biphasic potentiation of glucose-stimulated insulin secretion. An initial rapid, brief increase in secretion, mediated by IP3 generation and release of intracellular Ca2+, is followed by a larger, prolonged PKC-mediated increase in secretion that is dependent on extracellular Ca2+ (11, 15, 16, 20, 22, 23, 25, 32, 34, 36). Previous work indicated that cholinergic agonists, in the presence of substimulatory concentrations of glucose, depolarized the membrane potential of beta -cells (6, 9, 20, 27) and increased Na+ influx (12) but potentiated cyclic electrical activity, Ca2+ influx, or insulin secretion only in the presence of ongoing electrical activity initiated by glucose or some other depolarizing secretagogue (9, 11, 14, 16, 20, 26, 36). To date, two mechanisms have been postulated for the PKC-mediated effects: an increase in Ca2+ influx secondary to membrane depolarization, and a sensitization of the secretory process to Ca2+ (6, 9, 11-16, 22, 23, 26, 27). Our results suggest a third, PKC-mediated, cholinergic mechanism, an increase in Ca2+ channel activity at any given membrane potential above the threshold for channel opening. Given the close correlation of intracellular Ca2+ concentration and insulin secretion (20, 36), an increase in the Ca2+ currents would be expected to contribute to increased insulin secretion. It is likely that all three mechanisms, membrane depolarization, direct Ca2+ channel activation, and sensitization of exocytosis, act in concert during cholinergic potentiation of glucose-stimulated insulin secretion.

    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-44169, the University of Michigan Diabetes Research and Training Center, and the University of Michigan Gastrointestinal Peptide Research Center.

    FOOTNOTES

Address for reprint requests: J. A. Love, Dept. of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 North State St., Jackson, MS 39216-4505.

Received 26 March 1997; accepted in final form 28 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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