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Am J Physiol Endocrinol Metab 286: E463-E471, 2004. First published November 12, 2003; doi:10.1152/ajpendo.00365.2003
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Epinephrine-induced hyperpolarization of islet cells without KATP channels

Andrea Sieg,1 Jiping Su,1 Alvaro Muñoz,2 Michael Buchenau,1 Mitsuhiro Nakazaki,2 Lydia Aguilar-Bryan,3 Joseph Bryan,2 and Susanne Ullrich1,2

1Institut für Neurophysiologie, Universität zu Köln, 50931 Cologne, Germany; and 2Department of Molecular and Cellular Biology and 3Department of Medicine/Endocrinology, Baylor College of Medicine, Houston, Texas 77030

Submitted 14 August 2003 ; accepted in final form 28 October 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study examines the effect of epinephrine, a known physiological inhibitor of insulin secretion, on the membrane potential of pancreatic islet cells from sulfonylurea receptor-1 (ABCC8)-null mice (Sur1KO), which lack functional ATP-sensitive K+ (KATP) channels. These channels have been argued to be activated by catecholamines, but epinephrine effectively inhibits insulin secretion in both Sur1KO and wild-type islets and in mice. Isolated Sur1KO {beta}-cells are depolarized in both low (2.8 mmol/l) and high (16.7 mmol/l) glucose and exhibit Ca2+-dependent action potentials. Epinephrine hyperpolarizes Sur1KO {beta}-cells, inhibiting their spontaneous action potentials. This effect, observed in standard whole cell patches, is abolished by pertussis toxin and blocked by BaCl2. The epinephrine effect is mimicked by clonidine, a selective {alpha}2-adrenoceptor agonist and inhibited by {alpha}-yohimbine, an {alpha}2-antagonist. A selection of K+ channel inhibitors, tetraethylammonium, apamin, dendrotoxin, iberiotoxin, E-4130, chromanol 293B, and tertiapin did not block the epinephrine-induced hyperpolarization. Analysis of whole cell currents revealed an inward conductance of 0.11 ± 0.04 nS/pF (n = 7) and a TEA-sensitive outward conductance of 0.55 ± 0.08 nS/pF (n = 7) at -60 and 0 mV, respectively. Guanosine 5'-O-(3-thiotriphosphate) (100 µM) in the patch pipette did not significantly alter these currents or activate novel inward-rectifying K+ currents. We conclude that epinephrine can hyperpolarize {beta}-cells in the absence of KATP channels via activation of low-conductance BaCl2-sensitive K+ channels that are regulated by pertussis toxin-sensitive G proteins.

insulin secretion; ABCC8; sulfonylurea receptor-1 knockout mice; pertussis toxin; membrane potential; adenosine triphosphate-sensitive potassium channels

THE FIRST PHASE OF INSULIN SECRETION from pancreatic {beta}-cells is regulated by an ATP-sensitive potassium (KATP) channel that sets the resting membrane potential and thus modulates voltage-dependent Ca2+ influx (4, 12, 39). The increased open probability of KATP channels in a resting {beta}-cell, reflecting reduced rates of glucose metabolism and decreased ATP-to-ADP ratio (ATP/ADP), gives a membrane potential, approximately -65 mV, approaching the K+ equilibrium potential. Upon feeding, glucose metabolism increases ATP/ADP, closing KATP channels and depolarizing {beta}-cells to approximately -40 mV, which activates voltage-dependent Ca2+ channels and initiates oscillations of [Ca2+]i (8). Sulfonylureas used in the treatment of type 2 diabetes selectively inhibit {beta}-cell KATP channels and initiate the same sequence, thus stimulating insulin secretion (2, 5, 41, 49).

Mutations in the subunits, Kir6.2 and sulfonylurea receptor-1 (SUR1), of {beta}-cell KATP channels that result in loss or alteration of channel activity account for ~50% of the dominant, recessive, and sporadic cases of hyperinsulinemic hypoglycemia (3, 22, 28). Mutations that affect subunit trafficking result in loss of functional KATP channels and sustained severe hypoglycemia secondary to unregulated insulin release (10, 44). Several missense mutations produce less severe although sustained hypoglycemia and have implicated ADP as a physiological regulator (27, 34, 51). SUR1 knockout (Sur1KO) mice also lack functional {beta}-cell KATP channels (43, 47). At low glucose concentrations, isolated Sur1KO islet cells are depolarized, display Ca2+-dependent action potential, and exhibit elevated, oscillating cytosolic Ca2+ concentrations ([Ca2+]i) (6, 47). Surprisingly, although the electrophysiological phenotypes of hyperinsulinemic hypoglycemia and Sur1KO {beta}-cells are similar, Sur1KO mice are normoglycemic unless stressed, and their isolated islets display attenuated insulin secretion in response to a glucose challenge (33, 43).

The present study was undertaken to examine the potential contribution of inhibitory pathways to the control of insulin release in Sur1KO mice, specifically whether the inhibitory action of epinephrine is abrogated. Activation of the sympathetic nervous system is known to inhibit nutrient-induced insulin secretion (46). Catecholamines are reported to inhibit secretion via multiple mechanisms including: activation of {alpha}2-adrenoceptors that inhibit adenylyl cyclase activity (14, 53), inhibition of Ca2+ influx (15, 42), direct inhibition of exocytosis at a distal step (31, 35, 52), inhibition of Ca2+ currents mediated by G proteins [reported for RINm5F insulinoma cells (42) but not confirmed in mouse {beta}-cells (9)], and activation of an unidentified, hyperpolarizing K+ current (38). Pertussis toxin (PTX)-sensitive, G protein-mediated activation of K+ currents by epinephrine and other inhibitors of insulin secretion, somatostatin and galanin, for example, has been reported by several groups (15, 18, 38, 40). A specific role for KATP channels in hyperpolarization induced by epinephrine and these hormones has been suggested repeatedly (16, 21, 36, 40). In contrast, others have reported that the channels closed by glucose and sulfonylureas are insensitive to G protein activation (36) and that clonidine does not activate KATP channels (38). Here we show that epinephrine can hyperpolarize Sur1KO {beta}-cells lacking KATP channels, inhibiting Ca2+-dependent action potentials and insulin release. We use K+ channel inhibitors to characterize the underlying mechanism.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. The generation of Sur1KO mice has been described previously (43). The Sur1-null gene is on a C57Bl/6 background; C57Bl/6 age-matched animals were used as wild-type (WT) controls.

Male C56BL/6 and Sur1KO mice had free access to food and water. Experiments were started between 9:00 and 10:00 AM. Approximately 20 µl of blood were withdrawn from a tail vein and used to measure blood glucose with a commercial blood glucose monitoring system (FreeStyle; TheraSense, Alameda, CA) and to determine serum insulin by ELISA (Ultra Sensitive Rat Insulin ELISA Kit; Crystal Chem, Downers Grove, IL). Animals were challenged with a subcutaneous injection of a mixture of glucose plus carbachol (2 and 0.2 mg/kg, respectively) in the presence or absence of DL-epinephrine (2 mg/kg; ICN Biomedicals, Aurora, OH, prepared freshly at 1 mg/ml in phosphate-buffered saline). Blood samples were taken at the indicated times and analyzed as described above.

Animal protocols were approved by the Animal Research Committees of the respective institutions.

Isolation and handling of islets. Islets were isolated from pancreatic tissue following injection of 3 ml of collagenase solution (1 mg/ml; Serva, Heidelberg, Germany) into the pancreatic duct followed by digestion for 10 min at 37°C. Isolated islets were preincubated for 1 h at 37°C in buffer containing (in mmol/l) 140 NaCl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 2.8 glucose, 10 HEPES, pH 7.4, and 5 g/l bovine serum albumin (fraction V; Sigma, Deisenhofen, Germany). Batches of 5 islets/0.5 ml were incubated for 30 min at 37°C in the presence of test substances as indicated. Released insulin and islet insulin content, after acid-ethanol [1.5%:75% (vol/vol)] extraction, was measured by radioimmunoassay as described (52). The results are expressed as a percentage of islet insulin content.

For patch clamp experiments, islets were dissociated into single cells with trypsin (1 mg/ml), seeded onto glass coverslips coated with poly-L-ornithine (10 mg/l; Sigma, Munich, Germany) and cultured for 2-4 days in RPMI 1640 medium (GIBCO, Eggenstein, Germany) containing 11 mmol/l glucose supplemented with 50 µmol/l 2-mercaptoethanol, 1 mmol/l sodium pyruvate, 10 mmol/l HEPES, 100,000 IU/l penicillin, 100 mg/l streptomycin, and 10% fetal calf serum (Biochrom, Berlin, Germany) before the experiment. Pretreatment with pertussis toxin (100 ng/ml; Sigma, Munich, Germany) was done overnight (18-24 h) in culture medium. Epinephrine bitartrate and tetraethylammonium (TEA) were purchased from Sigma (Munich, Germany); the channel inhibitors were from Alomone Labs (Jerusalem, Israel).

Patch clamp experiments. Coverslips were mounted in a bath chamber on the stage of an inverted microscope (IM; Zeiss, Jena, Germany) and superfused (2-5 ml/min) with a solution containing (in mmol/l) 140 NaCl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 2.8 glucose, and 10 HEPES, pH 7.4 (adjusted at ~20-22°C). Patch clamp pipettes (Clark-Medical, Reading, UK) were pulled with a DMZ-Universal Puller (Zeitz, Augsburg, Germany) and had a resistance of 4-6 M{Omega} when filled with pipette solution containing (in mmol/l) 30 KCl, 95 K-gluconate, 1 MgCl2, 1.2 NaH2PO4, 4.8 Na2HPO4, 5 Na2ATP, and 1 Na3GTP, pH 7.2. For perforated patch clamp experiments, the pipette solution contained no ATP or GTP, and amphotericin B was added to a concentration of 1 mg/ml; the pipette resistance was 6-8 M{Omega}. The membrane potential was measured continuously using the current-clamp mode of the patch clamp amplifier (U. Fröbe and R. Busche, Freiburg, Germany) and was displayed on a pen recorder.

Statistics. Data are presented as means ± SE. Student's paired t-test was used to compare patch clamp experiments. ANOVA was used to compare blood glucose and serum insulin concentrations at different time points. Differences were judged significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Epinephrine induces a PTX-sensitive hyperpolarization via activation of {alpha}2-adrenoceptors. The perforated patch clamp method was used to compare membrane potential changes in intact, metabolically active WT and Sur1KO islet cells in response to glucose and {alpha}2-adrenoceptor activation. In low glucose (2.8 mmol/l), the membrane potential in WT cells was -60 ± 1.3 mV (n = 7; Fig. 1A). Increasing the glucose concentration to 16.7 mmol/l resulted in depolarization to -42 ± 1.4 mV (n = 6) and generation of continuous action potentials (Fig. 1A and Table 1). The addition of epinephrine (1 µmol/l) to WT cells, in high glucose, produced rapid hyperpolarization (to -58 ± 2.3 mV, n = 4; Fig. 1B and Table 1) and inhibition of action potentials. In low glucose, Sur1KO islet cells were depolarized to -37 ± 0.7 mV (n = 66) and exhibited action potentials (Fig. 1, C and D, and Table 1). Raising the glucose concentration to 16.7 mmol/l caused a transient hyperpolarization (Fig. 1C, n = 3). In both low and high glucose, epinephrine (1 µmmol/l) repolarized Sur1KO cells by 18.0 ± 0.9 mV (n = 66; Fig. 1, D and E, and Table 1) and by -17.0 ± 9.6 (n = 4; Table 1), respectively. The hyperpolarizing effect of epinephrine was blocked by {alpha}-yohimbine (10 µmol/l, n = 5), an {alpha}2-adrenoceptor antagonist, and mimicked by clonidine (10 µmol/l, n = 5), an {alpha}2-adrenoceptor agonist (Fig. 1, E and F). Pretreatment with 100 ng/ml PTX overnight completely blocked the epinephrine-induced hyperpolarization in either high or low glucose (n = 3; Fig. 1G). These observations confirm the sustained depolarization of Sur1KO islets in low glucose secondary to the loss of KATP channels (43, 47) and demonstrate that epinephrine can counteract this sustained depolarization, reversibly hyperpolarizing islet cells in the absence of KATP channels.



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  		Fig. 1. Epinephrine-induced hyperpolarization of sulfonylurea receptor-1 knockout mouse (Sur1KO) islet cells is due to {alpha}2-adrenoceptor activation. Membrane potential (Vm) of cultured mouse islet cells was measured using the perforated patch clamp technique as described in MATERIALS AND METHODS. Representative traces of membrane potential from wild-type cells are shown in A and B and from Sur1KO islet cells in C-G. In A and C, the glucose concentration was 2.8 mmol/l and then increased to 16.7 mmol/l, as indicated. Epinephrine and other agents were applied when indicated by bars. Concentrations were epinephrine (Epi), 1 µmol/l; clonidine, 10 µmmol/l; {alpha}-yohimbine (Yoh), 10 µmol/l; and pertussis toxin (PTX), 100 ng/ml.

 

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  		Table 1. Effect of epinephrine on Vm of WT and Sur1KO mouse islet cells in the absence and presence of KCl, nifedipine, and various K+ channel inhibitors

 

Epinephrine induces hyperpolarization by opening K+ channels, not by closing Ca2+ channels. The epinephrine-induced hyperpolarization persisted when membrane potential was assessed using the standard whole cell configuration with GTP (1 mmol/l) and ATP (5 mmol/l) in the pipette solution. All subsequent experiments were performed using this standard whole cell configuration. To determine whether inhibition of Ca2+ channels was responsible for repolarization, nifedipine (500 nmol/l) was applied. Nifedipine inhibited the Ca2+-dependent action potentials but did not prevent membrane hyperpolarization by epinephrine, indicating that the underlying mechanism does not depend solely on Ca2+ channel inhibition (Fig. 2A and Table 1). Increasing the extracellular K+ concentration raised the baseline membrane potential by ~9 mV (from -35 mV to -26 mV; Fig. 2B and Table 1). Under these conditions, application of epinephrine could not hyperpolarize {beta}-cells but did reduce the action potential firing rate, consistent with a modulatory effect on Ca2+ influx (15, 42). The application of BaCl2 (0.5 mmol/l), a nonspecific K+ channel inhibitor, abolished the hyperpolarizing effect of epinephrine without affecting action potentials (Fig. 2C and Table 1).



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  		Fig. 2. Contribution of Ca2+ and K+ currents to epinephrine-induced hyperpolarization of Sur1KO islet cells. Cells were prepared as described in MATERIALS AND METHODS. Representative membrane potential measurements, performed in the standard whole cell configuration, are shown. Nifedipine was applied at 500 nmol/l (A), epinephrine at 1 µmol/l (B), and BaCl2 at 0.5 mmol/l (C). Statistics of all experiments are given in Table 1.

 

Effect of epinephrine and guanosine 5'-O-(3-thiotriphosphate) on whole cell currents from Sur1KO islet cells. In the whole cell configuration, Sur1KO islet cells display a large, outward, TEA-sensitive current (0.55 ± 0.08 nS/pF; n = 7) and a smaller inward current (0.11 ± 0.04 nS/pF; n = 7; Fig. 3A). With 5 mmol/l ATP in the pipette, the outward and inward currents for WT cells were 1.42 ± 0.2 nS/pF (n = 11) and 0.13 ± 0.03 nS/pF (n = 11), respectively. As can be seen in Fig. 3, A and B, epinephrine had no significant effect on either of these two currents. The sensitivity of epinephrine-induced hyperpolarization to PTX (Fig. 1) implied activation via G proteins. Incorporation of guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S; 400 nmol/l) in the pipette solution did activate a small inward current in 2 of 10 cells under conditions of symmetric KCl (125 mmol/l; Fig. 3C). The majority of cells did not exhibit this behavior, implying that these currents are not in {beta}-cells, the major cell type, that the currents are small and hard to detect, or that dialysis has removed a required factor.



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  		Fig. 3. Whole cell currents of Sur1KO islet cells are not activated by guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S) or epinephrine. Cells were prepared as described in MATERIALS AND METHODS. A: current measurements (peak current) were done using 10-mV steps from -60 to +20 mV in the presence ({bullet}) and absence ({circ}) of 1 µmol/l epinephrine. Currents are superimposable, and the current-voltage relationship (mean ± SE, n = 10) is not affected by epinephrine. B: ramp currents from -140 to +10 mV, 300-ms duration, are identical in the presence (dashed line) and absence (control) of epinephrine (1 µmmol/l). C: at extra- and intracellular K+ concentrations ([K+]o = [K+]i) = 125 mmol/l, a small increase of inward current was measured after addition of 400 nmol/l GTP{gamma}S in 2 of 10 cells.

 

Effects of specific K+ channel inhibitors on epinephrine-induced hyperpolarization. The effects of K+ channel inhibitors on epinephrine-induced hyperpolarization are summarized in Figs. 4 and 5 and Table 1. TEA had a major effect on the amplitude of membrane potential oscillations (26, 37) but did not block hyperpolarization by epinephrine (Fig. 4A). The TEA effect on oscillations has been attributed to blockade of both the Kv channel (Kv2.1) and BKCa, the high-conductance, Ca2+-activated, voltage-dependent K+ channel (32). Dendrotoxin, a Kv channel blocker, slightly depolarized Sur1KO {beta}-cells and increased the amplitude of membrane potential oscillations but did not block the effect of epinephrine (Fig. 4B). Apamin had no significant effect on the amplitude of oscillations or epinephrine-induced hyperpolarization (Fig. 4C). Iberiotoxin, a BKCa channel blocker, had a similar effect on amplitude, without affecting the epinephrine response (Fig. 4D). Similarly, the human ether-a-go-go-related gene (HERG) channel inhibitor E-4130 had no effect on epinephrine-induced hyperpolarization (Fig. 5A). Although we have evidences on the expression of KvLQT1 in insulin-secreting cells, chromanol 293B did not alter epinephrine-induced hyperpolarization (Fig. 5B). In agreement with the lack of an activating effect of GTP{gamma}S on inward currents in the majority of cells, tertiapin, a G protein-activated inward-rectifying K+ (GIRK) channel inhibitor, had no effect on epinephrine-induced hyperpolarization (Fig. 5C). 293B, E-4130, and tertiapin did not show significant effects on the amplitude of membrane potential oscillations. The results imply that epinephrine-induced hyperpolarization does not involve activation of KATP channels, Kv channels, KCa channels, HERG channels, chromanol 293B-sensitive KvLQT1 channels, or tertiapin-sensitive GIRK channels.



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  		Fig. 4. Voltage- and Ca2+-activated K+ channels are not involved in epinephrine-induced hyperpolarization of Sur1KO islet cells. Cells were prepared as described in MATERIALS AND METHODS. Representative membrane potential measurements, performed in the standard whole cell configuration at 2.8 mmol/l glucose, are shown. Epinephrine (1 µmol/l) and K+ channel inhibitors were added as indicated. A: 10 mmol/l tetraethylammonium (TEA); B: 400 nmol/l {alpha}-dendrotoxin; C: 200 nmol/l apamin; D: 50 nmol/l iberiotoxin. Statistics of all experiments are given in Table 1.

 


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  		Fig. 5. K+ channel inhibitors do not block epinephrine-induced hyperpolarization of Sur1KO islet cells. Experiments were performed as described in Fig. 4. Substances were added when indicated. The concentrations applied were (A) epinephrine, 1 µmol/l, E-4130, 400 nmol/l; (B) chromanol 293B, 100 µmol/l; (C) tertiapin, 10 nmol/l. Statistics of all experiments are given in Table 1.

 

Epinephrine inhibits insulin secretion from Sur1KO {beta}-cells. To examine whether epinephrine contributes to glucose homeostasis in Sur1KO mice, we determined its effects on insulin secretion from isolated islets and on blood sugar and insulin levels in control and knockout animals. Consistent with the observed hyperpolarization, epinephrine (0.1 or 1 µmol/l) abolished insulin secretion, stimulated by glucose and elevated cAMP, in both WT and Sur1KO islets (Fig. 6A). It is worth noting that forskolin and IBMX were used to increase cAMP to supraphysiological levels to obtain comparable insulin release from control and knockout islets. High glucose alone is not sufficient to stimulate insulin release from Sur1KO islets (43, 47). To test whether epinephrine blocks insulin release in Sur1KO mice, we made use of the observation that the cholinergic stimulatory pathway is intact in Sur1KO islets whose glucose-stimulated insulin release is potentiated by carbachol and PKC activators (33). Challenging control and Sur1KO animals with glucose plus carbachol produced only a small increase in blood glucose, secondary to a large increase in insulin secretion (Fig. 6, B and C). The addition of epinephrine effectively inhibited glucose/carbachol-stimulated insulin secretion in both control and Sur1KO animals, resulting in a significant increase in their blood glucose levels (Fig. 6, B and C). These results indicate that, irrespective of the presence of {beta}-cell KATP channels, epinephrine can inhibit insulin secretion from either isolated islets or intact animals.



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  		Fig. 6. Epinephrine inhibits insulin secretion from isolated islets and lowers serum insulin levels in Sur1KO mice. A: islets were prepared as described in MATERIALS AND METHODS. Test substances were added as indicated. Results are expressed as %insulin content for indicated no. of observations from 3 independent experiments. Blood glucose (B) and serum insulin (C) concentrations were measured in wild-type (WT; squares) and Sur1KO (circles) mice challenged with a mixture of glucose plus carbachol (carb; 2 and 0.2 mg/kg, respectively) in the presence (filled symbols) or absence (open symbols) of epinephrine (2 mg/kg). Data are expressed as means + SD; n = 3 or 4. *P < 0.001 for WT vs. Sur1KO at 5 min.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study shows that {alpha}2-adrenoceptor activation hyperpolarizes insulin-secreting cells in the absence of functional KATP channels. This confirms our earlier report (1) that epinephrine and somatostatin can hyperpolarize {beta}-cells when KATP channels are closed by high glucose and tolbutamide, suggesting that distinct channels are involved. Rorsman et al. (38) also described a clonidine-induced K+ current independent of KATP channels in mouse islets. Neither study addressed directly the possibility that epinephrine or clonidine could activate KATP channels inhibited by tolbutamide or by increased glucose metabolism. Although the molecular identity of the underlying channels remains unknown, the present findings show conclusively that KATP channel openings are not required for the epinephrine-induced hyperpolarization of insulin-secreting cells. Other inhibitors of insulin secretion, i.e., galanin and somatostatin, have been reported to increase the open probability of KATP channels in insulin-secreting cell lines (16, 17), and G{alpha}i/o proteins are reported to activate KATP channels (36). Somatostatin is reported to increase the open-state probability of KATP channels in the absence but not presence of glucose, implying that it may hyperpolarize via a different channel(s) (36). Somatostatin has been reported to activate both KATP and GIRK channels in Min6 cells (48). Preliminary results testing the ability of galanin to hyperpolarize Sur1KO islets suggest that this neurotransmitter does not act through KATP channels (20), contrary to what has been reported using the pancreatic {beta}-cell line RINm5F (21).

Epinephrine-induced hyperpolarization persisted during dialysis of the cell in the whole cell configuration, suggesting a direct action on ion channels rather than regulation through soluble second messengers, such as [Ca2+]i or cAMP, both of which are lowered by the activation of {beta}-cell {alpha}2-adrenoceptors (45). Occasionally, the epinephrine-induced hyperpolarization was transient. This phenomenon has been described in WT islets and is dependent on the stimuli and the concentration of the catecholamine used (15). Consistent with earlier studies of rodent islet cells, the hyperpolarizing effect of epinephrine in Sur1KO islet cells is mediated by a PTX-sensitive G protein, since the effect depends on having GTP in the pipette, and is abolished by PTX pretreatment of the cells (Fig. 1G). GIRK channels activated at negative potentials and by GTP{gamma}S have been described in a variety of systems (13). Gi protein-activated GIRK channel subunits have also been identified in human {beta}-cells by using RT-PCR (24). Unexpectedly, tertiapin, a GIRK channel inhibitor (19), did not affect epinephrine-induced hyperpolarization, and GTP{gamma}S in the pipette solution activated inward currents in only a small number of cells. A possible explanation for these contradictory results is that {alpha}-, {delta}-, or PP cells express GIRK channels, whereas {beta}-cells do not have tertiapin-sensitive channels.

We were unable to detect changes in whole cell currents in the majority of WT or SUR1KO islet cells following application of epinephrine (Fig. 3). This is unexpected, because the effect of epinephrine on membrane potential was reproducible and the lack of KATP currents in Sur1KO islet cells makes them ideal candidates to examine other K+ currents. Pancreatic {beta}-cells have a high electrical resistance, and small currents are sufficient to hyperpolarize them. We conclude that the current change underlying the hyperpolarization that we observe is too small to be detected using our standard patch clamp technique.

A transient, glucose-induced hyperpolarization of Sur1KO islet cells was observed in the perforated patch configuration where cytoplasmic factors are preserved but not in the standard whole cell configuration where factors are dialysed out. A comparable effect of glucose has been observed in normal {beta}-cells when KATP channels are inhibited by tolbutamide (7) and observed in Sur1KO islet cells during measurements of [Ca2+]i (47). The mechanism responsible for the transient, glucose-induced hyperpolarization is not understood.

The failure of pharmacological inhibitors to block hyperpolarization indicates that Kv, KCa, HERG, and KCNQ1 channels do not play a major role in the epinephrine effect. On the basis of the observations that high extracellular KCl and a low concentration (0.5 mmol/l) of BaCl2 blocked epinephrine-induced hyperpolarization, we conclude that K+ channel openings are involved. The sensitivity to BaCl2 also excludes the possibility that activation of the electrogenic Na+-K+-ATPase may account for the hyperpolarization induced by epinephrine (11). This conclusion is supported by the finding that ouabain was unable to reverse the inhibition of insulin secretion by epinephrine (50). The nature of the underlying K+ channel remains to be elucidated, but its regulation is independent of the pathway(s) that modulates KATP channels, implying that it is an important regulator in {beta}-cells. PTX, also known as islet-activating protein, can increase glucose-induced insulin secretion by blocking G protein function (30).

Epinephrine abolished stimulated insulin secretion from isolated islets of WT and Sur1KO mice. Because glucose alone does not stimulate insulin secretion in the knockout animals (43, 47), challenging with high glucose, in the presence and absence of catecholamine, was not sufficient to establish that epinephrine could inhibit insulin release in Sur1KO mice. Thus we made use of our earlier observation (33) that the cholinergic stimulatory pathway is intact in KO islets. Epinephrine effectively inhibited the large stimula-tory effect of glucose plus carbachol on plasma insulin levels in both control and Sur1KO animals despite the large increase in blood glucose, presumably as a consequence of {beta}-adrenergic activation of hepatic glucose output. The observation that epinephrine can hyperpolarize Sur1KO {beta}-cells and inhibit insulin secretion, stimulated by several strategies, in both isolated islets and animals implies that the sympathetic branch of the counterregulatory reaction is intact in the Sur1KO animals and participates in glucose homeostasis. Similarly, the marked stimulatory effect of carbachol on insulin release from both isolated islets (33) and Sur1KO mice implies that the cholinergic stimulatory pathways (reviewed in Ref. 25) are intact and may play a major role in glucose homeostasis in these animals.

The channel activated by epinephrine remains to be identified. The present results suggest that it is a small-conductance K+ channel regulated by a G protein mechanism. We suggest that this is the same K+ current activated in {beta}-cells by galanin and somatostatin. The efficacy of octreotide, a somatostatin receptor agonist, as a means to counteract hypoglycemia in hyperinsulinemic hypoglycemic patients is consistent with this interpretation (29).


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Cologne Fortune program, Deutsche Forschungsgemeinschaft (DFG) Grant UL 140/6-1, a grant from the German Diabetes society (S. Ullrich), and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52771 (J. Bryan) and DK-57671 (L. Aguilar-Bryan). S. Ullrich was a recipient of a Heisenberg fellowship of the DFG (UL 140/2-1) and a DFG travel grant. A. Muñoz was supported by Juvenile Diabetes Research Foundation International (JDRFI) Grant 5-2003-508.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Ullrich, University of Tübingen, Institut für Physiologie, Gmelinstrasse 5, D-72076 Tübingen, Germany (E-mail: Susanne.Ullrich{at}uni-tuebingen.de).

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abel KB, Lehr S, and Ullrich S. Adrenaline-, not somatostatin-induced hyperpolarization is accompanied by a sustained inhibition of insulin secretion in INS-1 cells. Activation of sulphonylurea KATP channels is not involved. Pflügers Arch 432: 89-96, 1996.[CrossRef][Web of Science][Medline]
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