RESUMEN
Cell-to-cell interactions play an important role in insulin secretion. Compared with intact islets, dispersed pancreatic beta-cells show increased basal and decreased glucose-stimulated insulin secretion. In this study, we used mouse MIN6B1 cells to investigate the mechanisms that control insulin secretion when cells are in contact with each other or not. RNAi-mediated silencing of the adhesion molecule E-cadherin in confluent cells reduced glucose-stimulated secretion to the levels observed in isolated cells but had no impact on basal secretion. Dispersed cells presented high cytosolic Ca(2+) activity, depolymerized cytoskeleton and ERK1/2 activation in low glucose conditions. Both the increased basal secretion and the spontaneous Ca(2+) activity were corrected by transient removal of Ca(2+) or prolonged incubation of cells in low glucose, a procedure that restored the ability of dispersed cells to respond to glucose (11-fold stimulation). In conclusion, we show that dispersed pancreatic beta-cells can respond robustly to glucose once their elevated basal secretion has been corrected. The increased basal insulin secretion of dispersed cells is due to spontaneous Ca(2+) transients that activate downstream Ca(2+) effectors, whereas engagement of cell adhesion molecules including E-cadherin contributes to the greater secretory response to glucose seen in cells with normal intercellular contacts.
Asunto(s)
Calcio/metabolismo , Comunicación Celular/fisiología , Inhibición de Contacto/fisiología , Citosol/metabolismo , Insulina/metabolismo , Animales , Cadherinas/antagonistas & inhibidores , Cadherinas/metabolismo , Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Glucosa/farmacología , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Proinsulina/metabolismo , Transporte de Proteínas , ARN Interferente Pequeño/farmacologíaRESUMEN
The sulfonylurea receptor SUR is an ATP binding cassette (ABC) protein of the ABCC/MRP family. Unlike other ABC proteins, it has no intrinsic transport function, neither active nor passive, but associates with the potassium channel proteins Kir6.1 or Kir6.2 to form the ATP-sensitive potassium (K(ATP)) channel. Within the channel complex SUR serves as a regulatory subunit which fine-tunes the gating of Kir6.x in response to alterations in cellular metabolism. It constitutes a major pharmaceutical target as it binds numerous drugs, K(ATP) channel openers and blockers, capable of up- or down-regulating channel activity. We here review current knowledge on the molecular basis of the interaction of classical K(ATP) channel openers (cromakalim, pinacidil, diazoxide) with SUR.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/fisiología , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/fisiología , Canales de Potasio/química , Receptores de Droga/química , Receptores de Droga/fisiología , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/química , Animales , Cromakalim/farmacología , Citoplasma/metabolismo , Diazóxido/farmacología , Regulación hacia Abajo , Humanos , Canales KATP , Modelos Biológicos , Pinacidilo/farmacología , Canales de Potasio/fisiología , Canales de Potasio de Rectificación Interna/metabolismo , Unión Proteica , Isoformas de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Sulfonilureas , Regulación hacia ArribaRESUMEN
Extracellular Zn(2+) has been identified as an activator of pancreatic K(ATP) channels. We further examined the action of Zn(2+) on recombinant K(ATP) channels formed with the inward rectifier K(+) channel subunit Kir6.2 associated with either the pancreatic/neuronal sulphonylurea receptor 1 (SUR1) subunit or the cardiac SUR2A subunit. Zn(2+), applied at either the extracellular or intracellular side of the membrane appeared as a potent, reversible activator of K(ATP) channels. External Zn(2+), at micromolar concentrations, activated SUR1/Kir6.2 but induced a small inhibition of SUR2A/Kir6.2 channels. Cytosolic Zn(2+) dose-dependently stimulated both SUR1/Kir6.2 and SUR2A/Kir6.2 channels, with half-maximal effects at 1.8 and 60 microm, respectively, but it did not affect the Kir6.2 subunit expressed alone. These observations point to an action of both external and internal Zn(2+) on the SUR subunit. Effects of internal Zn(2+) were not due to Zn(2+) leaking out, since they were unaffected by the presence of a Zn(2+) chelator on the external side. Similarly, internal chelators did not affect activation by external Zn(2+). Therefore, Zn(2+) is an endogenous K(ATP) channel opener being active on both sides of the membrane, with potentially distinct sites of action located on the SUR subunit. These findings uncover a novel regulatory pathway targeting K(ATP) channels, and suggest a new role for Zn(2+) as an intracellular signalling molecule.