ABSTRACT
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels mediate the pacemaker current (Ih or If) observed in electrically rhythmic cardiac and neuronal cells. Here we describe a hyperpolarization-activated time-dependent cationic current, beta-Ih, in pancreatic beta-cells. Transcripts for HCN1-4 were detected by RT-PCR and quantitative PCR in rat islets and MIN6 mouse insulinoma cells. beta-Ih in rat beta-cells and MIN6 cells displayed biophysical and pharmacological properties similar to those of HCN currents in cardiac and neuronal cells. Stimulation of cAMP production with forskolin/3-isobutyl-1-methylxanthine (50 microM) or dibutyryl-cAMP (1 mM) caused a significant rightward shift in the midpoint activation potential of beta-Ih, whereas expression of either specific small interfering (si)RNA against HCN2 (siHCN2b) or a dominant-negative HCN channel (HCN1-AAA) caused a near-complete inhibition of time-dependent beta-Ih. However, expression of siHCN2b in MIN6 cells had no affect on glucose-stimulated insulin secretion under normal or cAMP-stimulated conditions. Blocking beta-Ih in intact rat islets also did not affect membrane potential behavior at basal glucose concentrations. Taken together, our experiments provide the first evidence for functional expression of HCN channels in the pancreatic beta-cell.
Subject(s)
Insulin-Secreting Cells/metabolism , Potassium Channel Blockers/metabolism , Potassium Channels/metabolism , Animals , Benzazepines/pharmacology , Cells, Cultured , Cyclic AMP/physiology , Cyclic Nucleotide-Gated Cation Channels , Electrophysiology , Exocytosis/drug effects , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/physiology , Insulinoma/pathology , Membrane Potentials/drug effects , Mice , Piperidines/pharmacology , Pyrimidines/pharmacology , RNA, Small Interfering/pharmacology , RatsABSTRACT
Antagonism of voltage-dependent K+ (Kv) currents in pancreatic beta-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 beta-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 beta-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 PKCzeta pseudosubstrate inhibitor could prevent current inhibition by exendin 4. Therefore, antagonism of beta-cell Kv currents by GLP-1 receptor activation requires both cAMP/PKA and PI3 kinase/PKCzeta 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.
Subject(s)
Peptides/physiology , Phosphatidylinositol 3-Kinases/metabolism , Potassium/chemistry , Androstadienes/pharmacology , Animals , Blotting, Western , Catalysis , Cell Line , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Electrophysiology , Enzyme Activation , Enzyme Inhibitors/pharmacology , ErbB Receptors/metabolism , Exenatide , Glucagon/chemistry , Glucagon-Like Peptide 1 , Glucose/metabolism , Insulin/metabolism , Insulin Secretion , Kinetics , Male , Mice , Peptide Fragments/chemistry , Peptides/chemistry , Phosphorylation , Potassium/metabolism , Precipitin Tests , Protein Precursors/chemistry , Quinazolines , Rats , Rats, Wistar , Signal Transduction , Time Factors , Transcriptional Activation , Tyrphostins/pharmacology , Up-Regulation , Venoms/chemistry , WortmanninABSTRACT
Voltage-dependent K+ (Kv) channels negatively regulate Ca2+ entry into pancreatic beta-cells by repolarizing glucose-stimulated action potentials. A role for phosphatidylinositol 3-kinase (PI3K) modulation of Kv channel function was investigated using the PI3K inhibitors wortmannin and LY294002, and LY303511, a negative control compound with respect to PI3K activity. In MIN6 insulinoma cells, wortmannin (100 nM) had no effect on whole-cell outward K+ currents, but LY294002 and LY303511 reversibly blocked currents in a dose-dependent manner (IC50=9.0+/-0.7 microM and 64.6+/-9.1 microM, respectively). Western blotting confirmed the specific inhibitory effects of LY294002 and wortmannin on insulin-stimulated PI3K activity. Kv currents in rat beta-cells at near physiological temperatures were inhibited 92% by 25 microM LY294002. Kv2.1 and Kv1.4 are highly expressed in beta-cells, and in Kv2.1-transfected tsA201 cells, 50 microM LY294002 and 100 microM LY303511 reversibly inhibited currents by 99% and 41%, respectively. In Kv1.4-transfected tsA201 cells, 50 microM LY294002 reduced the inactivation time constant from 73 to 18 ms. The insulinotropic properties of LY294002 and its effects in other excitable cells may be caused by inhibition of Kv currents rather than PI3K antagonism. Furthermore, LY294002 may represent a novel structure from which future Kv channel blockers may be developed.
Subject(s)
Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Potassium Channels, Voltage-Gated/physiology , Androstadienes/pharmacology , Animals , Cell Line , Cells, Cultured , Dichlororibofuranosylbenzimidazole/pharmacology , Dose-Response Relationship, Drug , Glucose/pharmacology , Humans , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Islets of Langerhans/physiology , Membrane Potentials/drug effects , Patch-Clamp Techniques , Piperazines/pharmacology , Potassium Channels, Voltage-Gated/genetics , Rats , Signal Transduction/drug effects , Transfection , WortmanninABSTRACT
The physiological effects of glucagon-like peptide-1 (GLP-1) are of immense interest because of the potential clinical relevance of this peptide. Produced in intestinal L-cells through posttranslational processing of the proglucagon gene, GLP-1 is released from the gut in response to nutrient ingestion. Peripherally, GLP-1 is known to affect gut motility, inhibit gastric acid secretion, and inhibit glucagon secretion. In the central nervous system, GLP-1 induces satiety, leading to reduced weight gain. In the pancreas, GLP-1 is now known to induce expansion of insulin-secreting beta-cell mass, in addition to its most well-characterized effect: the augmentation of glucose-stimulated insulin secretion. GLP-1 is believed to enhance insulin secretion through mechanisms involving the regulation of ion channels (including ATP-sensitive K(+) channels, voltage-dependent Ca(2+) channels, voltage-dependent K(+) channels, and nonselective cation channels) and by the regulation of intracellular energy homeostasis and exocytosis. The present article will focus principally on the mechanisms proposed to underlie the glucose dependence of GLP-1's insulinotropic effect.
