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• W2018904710 abstract "Antagonism of voltage-dependent K+ (Kv) currents in pancreatic β-cells may contribute to the ability of glucagon-like peptide-1 (GLP-1) to stimulate insulin secretion. The mechanism and signaling pathway regulating these currents in rat β-cells were investigated using the GLP-1 receptor agonist exendin 4. Inhibition of Kv currents resulted from a 20-mV leftward shift in the voltage dependence of steady-state inactivation. Blocking cAMP or protein kinase A (PKA) signaling (Rp-cAMP and H-89, respectively) prevented the inhibition of currents by exendin 4. However, direct activation of this pathway alone by intracellular dialysis of cAMP or the PKA catalytic subunit (cPKA) could not inhibit currents, implicating a role for alternative signaling pathways. A number of phosphorylation sites associated with phosphatidylinositol 3 (PI3)-kinase activation were up-regulated in GLP-1-treated MIN6 insulinoma cells, and the PI3 kinase inhibitor wortmannin could prevent antagonism of β-cell currents by exendin 4. Antagonists of Src family kinases (PP1) and the epidermal growth factor (EGF) receptor (AG1478) also prevented current inhibition by exendin 4, demonstrating a role for Src kinase-mediated trans-activation of the EGF tyrosine kinase receptor. Accordingly, the EGF receptor agonist betacellulin could replicate the effects of exendin 4 in the presence of elevated intracellular cAMP. Downstream, the PKCζ pseudosubstrate inhibitor could prevent current inhibition by exendin 4. Therefore, antagonism of β-cell Kv currents by GLP-1 receptor activation requires both cAMP/PKA and PI3 kinase/PKCζ signaling via trans-activation of the EGF receptor. This represents a novel dual pathway for the control of Kv currents by G protein-coupled receptors. Antagonism of voltage-dependent K+ (Kv) currents in pancreatic β-cells may contribute to the ability of glucagon-like peptide-1 (GLP-1) to stimulate insulin secretion. The mechanism and signaling pathway regulating these currents in rat β-cells were investigated using the GLP-1 receptor agonist exendin 4. Inhibition of Kv currents resulted from a 20-mV leftward shift in the voltage dependence of steady-state inactivation. Blocking cAMP or protein kinase A (PKA) signaling (Rp-cAMP and H-89, respectively) prevented the inhibition of currents by exendin 4. However, direct activation of this pathway alone by intracellular dialysis of cAMP or the PKA catalytic subunit (cPKA) could not inhibit currents, implicating a role for alternative signaling pathways. A number of phosphorylation sites associated with phosphatidylinositol 3 (PI3)-kinase activation were up-regulated in GLP-1-treated MIN6 insulinoma cells, and the PI3 kinase inhibitor wortmannin could prevent antagonism of β-cell currents by exendin 4. Antagonists of Src family kinases (PP1) and the epidermal growth factor (EGF) receptor (AG1478) also prevented current inhibition by exendin 4, demonstrating a role for Src kinase-mediated trans-activation of the EGF tyrosine kinase receptor. Accordingly, the EGF receptor agonist betacellulin could replicate the effects of exendin 4 in the presence of elevated intracellular cAMP. Downstream, the PKCζ pseudosubstrate inhibitor could prevent current inhibition by exendin 4. Therefore, antagonism of β-cell Kv currents by GLP-1 receptor activation requires both cAMP/PKA and PI3 kinase/PKCζ signaling via trans-activation of the EGF receptor. This represents a novel dual pathway for the control of Kv currents by G protein-coupled receptors. Voltage-dependent K+ (Kv) 1The abbreviations used are: Kvvoltage-dependent K+GLP-1glucagon-like peptide-1PKAprotein kinase API3phosphatidylinositol 3PKCprotein kinase CEGFepidermal growth factorTPA12-O-tetradecanoylphorbol-13-acetateMOPS4-morpholinepropanesulfonic acidMAPmitogen-activated proteinMEKMAP kinase/extracellular signal-regulated kinase kinase. channels are important regulators of membrane potential in excitable tissues where they generally mediate action potential repolarization (1Edwards G. Weston A.H. Diabetes Res. Clin. Pract. 1995; 28: 57-66Abstract Full Text PDF PubMed Scopus (34) Google Scholar). In pancreatic islet β-cells, Kv channels repolarize glucose-stimulated action potentials, limit entry of Ca2+ through voltage-dependent Ca2+ channels, and therefore act as negative regulators of insulin secretion (2MacDonald P.E. Wheeler M.B. Diabetologia. 2003; 46: 1046-1062Crossref PubMed Scopus (202) Google Scholar). Recent work by us and others (3Roe M.W. Worley III, J.F. Mittal A.A. Kuznetsov A. DasGupta S. Mertz R.J. Witherspoon III, S.M. Blair N. Lancaster M.E. McIntyre M.S. Shehee W.R. Dukes I.D. Philipson L.H. J. Biol. Chem. 1996; 271: 32241-32246Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 4MacDonald P.E. Sewing S. Wang J. Joseph J.W. Smukler S.R. Sakellaropoulos G. Wang J. Saleh M.C. Chan C.B. Tsushima R.G. Salapatek A.M. Wheeler M.B. J. Biol. Chem. 2002; 277: 44938-44945Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 5MacDonald P.E. Ha X.F. Wang J. Smukler S.R. Sun A.M. Gaisano H.Y. Salapatek A.M. Backx P.H. Wheeler M.B. Mol. Endocrinol. 2001; 15: 1423-1435Crossref PubMed Scopus (163) Google Scholar) demonstrates the importance of Kv channels, particularly Kv2.1, in the regulation of β-cell excitability and insulin secretion. Importantly, because β-cell Kv channels are closed under resting conditions, the excitatory and insulinotropic effects of Kv channel antagonists are glucose-dependent (6Henquin J.C. Biochem. Biophys. Res. Commun. 1977; 77: 551-556Crossref PubMed Scopus (47) Google Scholar, 7Atwater I. Ribalet B. Rojas E. J. Physiol. 1979; 288: 561-574PubMed Google Scholar). voltage-dependent K+ glucagon-like peptide-1 protein kinase A phosphatidylinositol 3 protein kinase C epidermal growth factor 12-O-tetradecanoylphorbol-13-acetate 4-morpholinepropanesulfonic acid mitogen-activated protein MAP kinase/extracellular signal-regulated kinase kinase. Glucagon-like peptide-1 (GLP-1) is secreted by intestinal l-cells in response to nutrient ingestion (8Drucker D.J. Endocrinology. 2001; 142: 521-527Crossref PubMed Scopus (310) Google Scholar). Although known to exert effects on cell growth and proliferation, satiety, and intestinal motility, the most well recognized action of GLP-1 is to enhance insulin secretion from pancreatic islet β-cells (9MacDonald P.E. El Kholy W. Riedel M.J. Salapatek A.M. Light P.E. Wheeler M.B. Diabetes. 2002; 51: 434-442Crossref PubMed Google Scholar). GLP-1 and its analogues are under intense investigation as potential treatments for type-2 diabetes, because their insulinotropic effect is dependent upon elevated glucose, avoiding the potentially dangerous complication of hypoglycemia (10Drucker D.J. Gastroenterology. 2002; 122: 531-544Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). The actions of GLP-1 are mediated by the G protein-coupled GLP-1 receptor and result from effects on many targets within the β-cell, the most well characterized being the cAMP and PKA-dependent inhibition of KATP channels (9MacDonald P.E. El Kholy W. Riedel M.J. Salapatek A.M. Light P.E. Wheeler M.B. Diabetes. 2002; 51: 434-442Crossref PubMed Google Scholar). Recent evidence also demonstrates a cAMP-dependent and PKA-independent component to the insulinotropic effect of GLP-1, mediated by enhanced Ca2+-induced Ca2+ release from the endoplasmic reticulum via activation of cAMP guanine nucleotide exchange factor II (Epac2) (11Kashima Y. Miki T. Shibasaki T. Ozaki N. Miyazaki M. Yano H. Seino S. J. Biol. Chem. 2001; 276: 46046-46053Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 12Kang G. Chepurny O.G. Holz G.G. J. Physiol. 2001; 536: 375-385Crossref PubMed Scopus (172) Google Scholar). Recently (13MacDonald P.E. Salapatek A.M. Wheeler M.B. Diabetes. 2002; 51: 443-447Crossref PubMed Google Scholar), we reported that GLP-1 and the GLP-1 receptor agonist exendin 4 antagonizes Kv currents in rat β-cells (13MacDonald P.E. Salapatek A.M. Wheeler M.B. Diabetes. 2002; 51: 443-447Crossref PubMed Google Scholar). Because this likely contributes to the insulinotropic effect of GLP-1, particularly the glucose dependence, we investigated the mechanism of Kv current reduction and the signal transduction pathway(s) involved. We show that exendin 4 antagonizes Kv currents in rat β-cells by causing a hyperpolarizing shift in the voltage dependence of steady-state inactivation. We further demonstrate that antagonism of Kv channels by exendin 4 depends on activation of both the cAMP/PKA and phosphatidylinositol 3 (PI3)-kinase/PKCζ signaling pathways. Activation of PI3 kinase can result from an Src kinase-mediated trans-activation of the EGF receptor. This study provides a novel dual-signal pathway mechanism for the regulation of Kv currents. Additionally, the present work suggests a potential mechanism contributing to the glucose dependence of the insulinotropic effect of GLP-1. Reagents—Exendin 4 and GLP-1 were from Bachem (Torrance, CA). AG1428 and PP1 were from Biomol (Plymouth Meeting, PA). Betacellulin, cAMP, GMP-PNP, H-89, insulin, MgATP, PD98059, Rp-cAMPs, and tetraethylammonium were from Sigma-Aldrich. Bisindolylmaleimide, calphostin C, rapamycin, TPA, wortmannin, the constitutively active PKA catalytic subunit, and the PKCζ pseudosubstrate were from Calbiochem. 8-pCPT-2′-O-Me-cAMP was from BIOLOG Life Science Institute (Bremen, Germany). When necessary, reagents were dissolved in dimethyl sulfoxide (Sigma-Aldrich). Final solutions contained no greater than 0.01% dimethyl sulfoxide, and control solutions contained the same when appropriate. Anti-V5 antibody (1:10000) was from Sigma-Aldrich, anti-Kv2.1 antibody (1:1000) was from Upstate (Charlottesville, VA), and anti-EGF receptor antibody (1:1000) was from Cell Signaling Technology (Beverly, MA). EGF receptor (erbB-1)-specific primers were as follows: forward, 5′-ACCTGTGTGAAGAAGTGCCC-3′; and reverse, 5′-ACTGGCAGGATGTGAAGGTC-3′. Islet Isolation and Cell Culture—Male Wistar rats (250–300 g), p110γ -/- mice (from J. M. Penninger and P. H. Backx, University of Toronto), and GLP-1 receptor -/- mice (from D. J. Drucker, University of Toronto) were anesthetized by intraperitoneal injection of ketamine hydrochloride and xylazine (100 and 20 mg/kg) and sacrificed by exsanguination according to University of Toronto guidelines. Islets of Langerhans were isolated by collagenase digestion and dispersed to single cells as described previously (5MacDonald P.E. Ha X.F. Wang J. Smukler S.R. Sun A.M. Gaisano H.Y. Salapatek A.M. Backx P.H. Wheeler M.B. Mol. Endocrinol. 2001; 15: 1423-1435Crossref PubMed Scopus (163) Google Scholar, 14Zhang C.Y. Baffy G. Perret P. Krauss S. Peroni O. Grujic D. Hagen T. Vidal-Puig A.J. Boss O. Kim Y.B. Zheng X.X. Wheeler M.B. Shulman G.I. Chan C.B. Lowell B.B. Cell. 2001; 105: 745-755Abstract Full Text Full Text PDF PubMed Scopus (825) Google Scholar). Cells were plated on glass coverslips in 35-mm dishes in RMPI 1640 medium with 2.5 mm glucose, 0.25% HEPES, 7.5% fetal bovine serum, 100 units/ml penicillin G sodium, 100 μg/ml streptomycin sulfate (penicillin-streptomycin from Invitrogen) and cultured for 1–3 days prior to electrophysiological recordings. MIN6 insulinoma cells (passage 35–40), from S. Seino (Chiba University, Chuo-ku, Japan), were cultured in high glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, and β-mercaptoethanol (2 μl/500 ml) at 37 °C and 5% CO2. HIT-T15 cells (passage 80–95), from R. P. Robertson (Pacific NW Research Institute, Seattle, WA), were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% l-glutamine, 100 units/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate (penicillin-streptomycin from Invitrogen) at 37 °C and 5% CO2. Electrophysiology—Cells were patch-clamped in the whole-cell configuration using an EPC-9 amplifier and PULSE software (HEKA Electronik, Lambrecht, Germany). Patch pipettes were prepared from 1.5-mm thin walled borosilicate glass tubes using a two-stage micropipette puller (Narishige, Tokyo, Japan) and had typical resistances of 3–6 megaohms when fire polished and filled with intracellular solution containing the following (in mm): KCl, 140; MgCl2·6H20, 1; EGTA, 1; HEPES, 10; MgATP, 5; pH 7.25 with KOH. Extracellular solutions contained the following (in mm): NaCl, 140; CaCl2, 2; KCl, 4; MgCl2·6H2O, 1; HEPES, 10; pH 7.30 with NaOH. β-Cells were positively identified by the lack of an inward voltage-dependent Na+ current from a holding potential of -70 mV (15Gopel S. Kanno T. Barg S. Galvanovskis J. Rorsman P. J. Physiol. 1999; 521: 717-728Crossref PubMed Scopus (199) Google Scholar, 16Lou X.L. Yu X. Chen X.K. Duan K.L. He L.M. Qu A.L. Xu T. Zhou Z. J. Physiol. 2003; 548: 191-202Crossref PubMed Scopus (33) Google Scholar). Outward currents were elicited from a holding potential of -70 mV by a series of 500-ms depolarizing pulses in 20-mV increments to +70 mV or by a single depolarizing pulse to +30 mV for 500 ms or 8 s. Sustained outward current was taken as the mean current during the final 25 ms of the depolarization. The voltage dependence of steady-state inactivation was investigated by holding the cells at potentials from -80 to +30 mV for 15 s followed by a 5-ms pre-pulse to -70 mV and a 500-ms depolarization to +30 mV to elicit outward currents. Electrophysiological recordings were obtained at 32–35 °C and normalized to cell capacitance. The bath (∼0.5 ml) was perfused continuously at 1 ml/min (Minipuls peristaltic pump; Gilson Inc., Middleton, WI). Currents were filtered upon acquisition at 10 kHz, and capacitive transients were cancelled with the automatic compensation functions of the Pulse software (HEKA Electronik). Whole-cell conductance was calculated as G = I/(V - Veq), where G is whole-cell conductance, I is current, V is the test potential, and Veq is the equilibrium potential of K+ (-94.1 mV under the present conditions). Current-voltage relationships were compared by repeated measures analysis of variance followed by a Tukey post-test to compare currents elicited at various voltages. Voltage dependence of activation and steady-state inactivation curves were fit with a Boltzman function: G/Gmax = 1/[1 + exp([V -V50]/s)], where G is whole-cell conductance, V is test potential, V50 is the voltage at which half the channels are inactivated, and s is the slope of the curve, using Prism 3.03 (Graphpad Software, San Diego, CA). Time constants of activation or inactivation were derived by fitting the waveform with either a single or double exponential function using PulseFit software (HEKA Electronik) and compared using the Student's t test (two groups). Immunoprecipitation and Western Blotting—HIT-T15 cells, plated in 10-cm culture dishes, were transfected with a construct expressing a V5-His-tagged GLP-1 receptor in the pcDNA3.1-V5-His vector (Invitrogen) using LipofectAMINE 2000 (Invitrogen). Thirty h after transfection, cells were washed with ice-cold phosphate-buffered saline and lysed with 0.3 ml of lysis buffer (50 mm NaH2PO4, pH 8.0, 150 mm NaCl, 0.5% Triton X-100, 5 mm imidazole). Fifty μl of lysate was saved as the non-immunoprecipitated sample, whereas 25 μl of a 50% slurry of nickel-nitrilotriacetic acid resin (Qiagen) was added to the remainder and mixed gently for 2 h at 4 °C. The mixture was centrifuged for 10 s at 15000 × g, and the pellet was washed twice with washing buffer (50 mm NaH2PO4, pH 8.0, 150 mm NaCl, 0.5% Triton X-100, 10 mm imidazole). Protein was eluted with 3 × 35 μl of elution buffer (50 mm NaH2PO4, pH 8.0, 150 mm NaCl, 0.5% Triton X-100, 250 mm imidazole). Immunoblotting was performed as described previously (5MacDonald P.E. Ha X.F. Wang J. Smukler S.R. Sun A.M. Gaisano H.Y. Salapatek A.M. Backx P.H. Wheeler M.B. Mol. Endocrinol. 2001; 15: 1423-1435Crossref PubMed Scopus (163) Google Scholar). Twenty-five μg of protein from each sample was separated on a 10% polyacrylamide gel and transferred to PVDF-Plus™ membrane (Fisher). Primary antibodies (see above) were detected with appropriate secondary antibodies (sheep anti-mouse, 1:3000 and donkey anti-rabbit, 1:7500; Amersham Biosciences) for 1 h at room temperature. Visualization was by chemiluminescence (ECL; Amersham Biosciences) and exposure to Kodak film (Eastman Kodak Co.) for 5 s to 10 min. Phospho-site Screen—MIN6 cells were grown in monolayer to 70–80% confluence in 10-cm dishes, and the cells were washed twice with phosphate-buffered saline and pre-incubated for 30 min with a Krebs-Ringer bicarbonate buffer containing the following (in mm): NaCl, 115; KCl, 5; NaHCO3, 24; CaCl2, 2.5; MgCl2, 1; HEPES, 10; glucose, 5; and 0.1% (w/v) bovine serum albumin at 37 °C and 5% CO2. The cells were washed twice again with Krebs-Ringer bicarbonate buffer and incubated in the same with or without 10-7m GLP-1 for 10 min. Whole cell proteins were extracted from MIN6 cells treated with GLP-1 using lysis buffer containing the following (in mm): MOPS, 20; EGTA, 2; EDTA, 5; sodium fluoride, 30; β-glycerophosphate, 40; sodium pyrophosphate, 10; sodium orthovanadate, 2; phenylmethylsulfonyl fluoride, 1; benzamidine, 3; pepstatin A, 0.005; leupeptin, 0.01; and 0.5% Nonidet P-40 and 0.5% Triton X-100 at pH 7.0. The phosphorylation levels of 40 sites were screened by Kinetworks™ KPPS 1.1 (Kinexus, Vancouver, Canada). Antagonism of β-Cell Kv Currents by GLP-1 and Exendin 4—We have shown recently (4MacDonald P.E. Sewing S. Wang J. Joseph J.W. Smukler S.R. Sakellaropoulos G. Wang J. Saleh M.C. Chan C.B. Tsushima R.G. Salapatek A.M. Wheeler M.B. J. Biol. Chem. 2002; 277: 44938-44945Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 5MacDonald P.E. Ha X.F. Wang J. Smukler S.R. Sun A.M. Gaisano H.Y. Salapatek A.M. Backx P.H. Wheeler M.B. Mol. Endocrinol. 2001; 15: 1423-1435Crossref PubMed Scopus (163) Google Scholar) that Kv channels are important glucose-dependent regulators of insulin secretion and can be regulated by GLP-1 receptor activation in rat β-cells (13MacDonald P.E. Salapatek A.M. Wheeler M.B. Diabetes. 2002; 51: 443-447Crossref PubMed Google Scholar). Because GLP-1 antagonizes Kv currents in β-cells, we investigated whether the GLP-1 receptor could physically associate with Kv2.1, a prominent β-cell Kv channel (3Roe M.W. Worley III, J.F. Mittal A.A. Kuznetsov A. DasGupta S. Mertz R.J. Witherspoon III, S.M. Blair N. Lancaster M.E. McIntyre M.S. Shehee W.R. Dukes I.D. Philipson L.H. J. Biol. Chem. 1996; 271: 32241-32246Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 4MacDonald P.E. Sewing S. Wang J. Joseph J.W. Smukler S.R. Sakellaropoulos G. Wang J. Saleh M.C. Chan C.B. Tsushima R.G. Salapatek A.M. Wheeler M.B. J. Biol. Chem. 2002; 277: 44938-44945Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 5MacDonald P.E. Ha X.F. Wang J. Smukler S.R. Sun A.M. Gaisano H.Y. Salapatek A.M. Backx P.H. Wheeler M.B. Mol. Endocrinol. 2001; 15: 1423-1435Crossref PubMed Scopus (163) Google Scholar). A V5-His-tagged GLP-1 receptor construct was expressed in HIT-T15 insulinoma cells and immunoprecipitated by binding to a nickel-nitrilotriacetic acid resin. HIT-T15 cells were used, because these express low levels of endogenous GLP-1 receptor (17Salapatek A.M. MacDonald P.E. Gaisano H.Y. Wheeler M.B. Mol. Endocrinol. 1999; 13: 1305-1317Crossref PubMed Scopus (36) Google Scholar) but abundant Kv2.1 protein (5MacDonald P.E. Ha X.F. Wang J. Smukler S.R. Sun A.M. Gaisano H.Y. Salapatek A.M. Backx P.H. Wheeler M.B. Mol. Endocrinol. 2001; 15: 1423-1435Crossref PubMed Scopus (163) Google Scholar). Western blotting for Kv2.1 and V5-His-tagged GLP-1 receptor demonstrated the abundant expression of Kv2.1 and no expression of the GLP-1 receptor construct in whole lysates from untransfected HIT-T15 cells (Fig. 1A, lane 1). Whole lysates from HIT-T15 cells transfected with the GLP-1 receptor construct showed abundant expression of both Kv2.1 and the GLP-1 receptor (Fig. 1A, lane 2). Immunoprecipitation of lysates from untransfected HIT-T15 cells did not pull down Kv2.1 protein (Fig. 1A, lane 3), whereas Kv2.1 could be pulled down from lysates of cells expressing the tagged GLP-1 receptor (Fig. 1A, lane 4). Rat β-cells were voltage-clamped in the whole-cell configuration at near-physiological temperatures (32–35 °C). Outward voltage-dependent K+ currents were similar to those we have reported previously (4MacDonald P.E. Sewing S. Wang J. Joseph J.W. Smukler S.R. Sakellaropoulos G. Wang J. Saleh M.C. Chan C.B. Tsushima R.G. Salapatek A.M. Wheeler M.B. J. Biol. Chem. 2002; 277: 44938-44945Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 13MacDonald P.E. Salapatek A.M. Wheeler M.B. Diabetes. 2002; 51: 443-447Crossref PubMed Google Scholar, 18MacDonald P.E. Salapatek A.M. Wheeler M.B. J. Physiol. 2003; 546: 647-653Crossref PubMed Scopus (33) Google Scholar) (Fig. 1B) and could be blocked with the general Kv channel antagonist tetraethylammonium (15 mm) (Fig. 1B). Maximum steady-state outward currents were blocked by 40.8 ± 4.1% (n = 9, p < 0.001) upon perfusion of GLP-1 (10-8m) (Fig. 1B). To ensure that currents were stable, control currents were recorded every min for ∼5 min prior to addition of GLP-1. Kv currents were maximally inhibited at ∼6 min after exposure to GLP-1 (Fig. 1C). Partial washout of this effect was observed (not shown). However, because of technical difficulties in recording from cells at 32–35 °C for prolonged periods (>20 min), experiments were not of sufficient duration to observe complete washout. Subsequent experiments were performed using the GLP-1 receptor agonist exendin 4, which has a recently demonstrated therapeutic relevance in clinical trials (19Egan J.M. Meneilly G.S. Elahi D. Am. J. Physiol. Endocrinol. Metab. 2003; 284: E1072-E1079Crossref PubMed Scopus (90) Google Scholar). Confirming our previous studies (13MacDonald P.E. Salapatek A.M. Wheeler M.B. Diabetes. 2002; 51: 443-447Crossref PubMed Google Scholar), exendin 4 (10-8m) inhibited voltage-dependent outward K+ currents from rat β-cells by 43.4 ± 6.3% (n = 7, p < 0.001) (Fig. 2A). At a concentration two orders of magnitude lower (10-10m), exendin 4 antagonized rat β-cell voltage-dependent K+ currents by 20.8 ± 3.9% (n = 4, p < 0.05). The current activation time constant in the presence of 10-8m exendin 4 (τa = 3.14 ± 0.27 ms, n = 7) was not significantly different from controls (τa = 2.69 ± 0.22 ms, n = 7) (Fig. 2B). We reported previously (18MacDonald P.E. Salapatek A.M. Wheeler M.B. J. Physiol. 2003; 546: 647-653Crossref PubMed Scopus (33) Google Scholar) that rat β-cell Kv currents inactivate with both fast (τf) and slow (τs) time constants at 32–35 °C (18MacDonald P.E. Salapatek A.M. Wheeler M.B. J. Physiol. 2003; 546: 647-653Crossref PubMed Scopus (33) Google Scholar). Neither τf nor τs in the presence of 10-8m exendin 4 (τf = 303.4 ± 45.1 ms and τs = 2.00 ± 0.20 s, n = 7) were different from controls (τf = 258.5 ± 54.5 ms and τs = 2.52 ± 0.42 s, n = 7) (Fig. 2B). The contribution of fast inactivation to total inactivation was also not changed (41.2 ± 5.9 versus 36.7 ± 2.9%, n = 7). Also, the voltage dependence of current activation under control conditions (V50 = -7.57 ± 4.31 mV, n = 7) was not significantly different after treatment with 10-8m exendin 4 (V50 = -9.77 ± 5.15 mV, n = 7) (Fig. 2C). Interestingly, the reduction in Kv currents results from an ∼20-mV hyperpolarizing shift in the voltage dependence of steady-state inactivation from -43.4 ± 3.2 to -66.6 ± 5.1 mV (n = 4 and 3, p < 0.01) (Fig. 2C). cAMP/PKA Signaling Is Necessary but Not Sufficient for Antagonism of Kv Currents by Exendin 4 —The importance of cAMP/PKA signaling for the insulinotropic effect of GLP-1 receptor activation is well known (9MacDonald P.E. El Kholy W. Riedel M.J. Salapatek A.M. Light P.E. Wheeler M.B. Diabetes. 2002; 51: 434-442Crossref PubMed Google Scholar). To investigate the involvement of cAMP and PKA signaling in the inhibitory effect of exendin 4 on Kv currents, β-cells were pre-treated (30 min) with either the cAMP antagonist Rp-cAMPs (100 μm, n = 5) or the PKA antagonist H-89 (1 μm, n = 6). Voltage-dependent outward K+ currents in cells pre-treated with either antagonist were not different from untreated cells and could not be antagonized significantly by treatment with exendin 4 (10-8m) (Fig. 3A). These results demonstrate the necessity of the cAMP/PKA signaling pathway for Kv current inhibition by exendin 4. Because activation of the cAMP/PKA pathway by GLP-1 is mediated by G-protein activation of adenylyl cyclase (9MacDonald P.E. El Kholy W. Riedel M.J. Salapatek A.M. Light P.E. Wheeler M.B. Diabetes. 2002; 51: 434-442Crossref PubMed Google Scholar), we investigated whether general G-protein activation was sufficient to reproduce the inhibitory effect of GLP-1 receptor activation on β-cell Kv channels. Inclusion of the non-hydrolysable GTP analogue GMP-PNP (10 nm) in the pipette solution decreased outward K+ current (by 55.7 ± 6.0%, n = 8, p < 0.001) similar to exendin 4 (Fig. 3B). We next investigated whether direct activation of the cAMP/PKA pathway could replicate this inhibitory effect. Addition of cAMP (100 μm) to the pipette solution was unable to decrease voltage-dependent outward K+ currents in rat β-cells (n = 7) (Fig. 3B). Also, intracellular dialysis of the constitutively active PKA catalytic subunit (200 units/ml) had no effect on currents compared with dimethyl sulfoxide controls (n = 6), which were themselves not different from untreated cells (Fig. 3B). Forskolin (5 μm) and isobutylmethylxanthine (100 μm) together blocked currents by 35.6 ± 7.5% (n = 5, p < 0.05) (Fig. 3B). However, forskolin (and its inactive analogs) directly inhibit Kv currents in insulin secreting cells (20Zunkler B.J. Trube G. Ohno-Shosaku T. Pflugers Arch. 1988; 411: 613-619Crossref PubMed Scopus (40) Google Scholar, 21Su J. Yu H. Lenka N. Hescheler J. Ullrich S. Pflugers Arch. 2001; 442: 49-56Crossref PubMed Scopus (57) Google Scholar). Additionally, β-cell Kv currents could not be antagonized by treatment with the Epac-selective cAMP analog 8-pCPT-2′-O-Me-cAMP (50 μm, n = 7) (Fig. 3B). Our inability to antagonize voltage-dependent K+ channels in β-cells by activating the cAMP/PKA pathway is in agreement with previous reports (20Zunkler B.J. Trube G. Ohno-Shosaku T. Pflugers Arch. 1988; 411: 613-619Crossref PubMed Scopus (40) Google Scholar, 21Su J. Yu H. Lenka N. Hescheler J. Ullrich S. Pflugers Arch. 2001; 442: 49-56Crossref PubMed Scopus (57) Google Scholar) and suggests that additional pathway(s) are also required for the inhibitory action of GLP-1. Phospho-site Screening Identifies PI3 Kinase as an Additional Candidate Pathway—To identify candidate signaling pathways that may be involved in the GLP-1 receptor-mediated reduction of voltage-dependent K+ currents, MIN6 insulinoma cells were treated with GLP-1 (10-7m) for 10 min, and protein lysates were screened with multiple phosphosite-specific antibodies using the Kinetworks™ KPPS 1.1 screen. MIN6 insulinoma cells were used in this case, because they allow for preparation of the necessary amount of protein lysates for the phospho-screen, and in our hands they exhibit good insulin secretory responses to GLP-1 (∼1.5-fold). Two separate experiments were conducted, and the -fold increases in phosphorylation of 40 sites on 30 different proteins are shown in Table I.Table IFold increase in phosphorylation of signal transduction proteins following GLP-1 treatmentProtein (abbreviation)Epitope-Fold changeAdducin-α (120)Ser-7241.01Adducin-γ (80)Ser-6620.61cAMP response element-binding protein (CREB)Ser-133N.D.aN.D., not detectedCyclin-dependent kinase 1 (CDK1)Tyr-151.12dsRNA-dependent protein kinase (PKR)Thr-451N.D.Extracellular regulated kinase 1 (ERK1)Thr-202/Tyr-2040.96Extracellular regulated kinase 2 (ERK2)Thr-185/Tyr-187N.D.Glycogen synthase kinase 3α (GSK3α)Tyr-2791.29Glycogen synthase kinase 3α (GSK3α)Ser-21N.D.Glycogen synthase kinase 3β (GSK3β)Tyr-2161.38Glycogen synthase kinase 3β (GSK3β)Ser-9N.D.MAP kinase kinase 1/2 (MEK1/2)Ser-221/Ser-2252.95MAP kinase kinase 3 (MEK3)Ser-189/Thr-1930.54MAP kinase kinase 6 (MEK6)Ser-207/Thr-211N.D.79-kDa mitogen- and stress-activated protein kinase 1/2 (MSK1/2)Ser-376N.D.80-kDa mitogen- and stress-activated protein kinase 1/2 (MSK1/2)Ser-376N.D.N-methyl-d-aspartate glutamate receptor subunit 1 (NR1)Ser-8960.72Oncogene JUN (JUN)Ser-73N.D.63-kDa oncogene Raf 1 (RAF1)Ser-2591.1871-kDa oncogene Raf 1 (RAF1)Ser-2592.78Oncogene Src (Src)Tyr-529N.D.Oncogene Src (Src)Tyr-4180.38p38 MAP kinase (p38 MAPK)Thr-180/Tyr-182N.D.Protein kinase Bα (Akt1)Ser-4731.37Protein kinase Bα (Akt1)Thr-3082.17Protein kinase Cα (PKCα)Ser-6570.90Protein kinase Cα/β (PKCα/β)Thr-638/Thr-6411.22Protein kinase Cδ (PKCδ)Thr-5051.71Protein kinase Cϵ (PKCϵ)Ser-7190.97Retinoblastoma 1 (RB)Ser-7804.80Retinoblastoma 1 (RB)Ser-807/Ser-811N.D.Ribosomal S6 kinase 1 (RSK1)Thr-360/Ser-364N.D.70-kDa S6 kinase p70 (P70 S6K)Thr-389N.D.77-kDa S6 kinase p70 (P70 S6K)Thr-3891.11Signal transducer and activator of transcription 1 (STAT1)Ser-701N.D.Signal transducer and activator of transcription 3 (STAT3)Ser-7271.11Signal transducer and activator of transcription 5 (STAT5)Tyr-694N.D.SMA- and MAD-related protein 1 (SMAD1)Ser-463/Ser-465N.D.40-kDa stress-activated protein kinase (JNK)Thr-183/Tyr-185N.D.44-kDa stress-activated protein kinase (JNK)Thr-183/Tyr-185N.D.a N.D., not detected Open table in a new tab Interestingly, a number of known PDK1 phosphorylation sites are up-regulated. These include Akt1 Ser-473 and Thr-308, PKCα/β Thr-638/Thr-641, and PKCδ Thr-505 (Table I). Because PDK1 is a direct effector of PI3 kinase signaling, these results may be" @default.
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• W2018904710 title "Antagonism of Rat β-Cell Voltage-dependent K+ Currents by Exendin 4 Requires Dual Activation of the cAMP/Protein Kinase A and Phosphatidylinositol 3-Kinase Signaling Pathways" @default.
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