RESUMO
BACKGROUND/AIM: Pancreatic acinar cells are involved in the secretion of digestive enzymes. Digestive enzymes in pancreatic acinar cells are stored in membrane-bound secretory vesicles called zymogen granules (ZGs). The swelling of ZGs is implicated in the regulation of the expulsion of intravesicular contents during secretion. The molecular mechanism of ZG swelling has been previously elucidated. It has been further demonstrated that the water channel aquaporin-1, the potassium channel IRK-8, and the chloride channel CLC-2, are present in the ZG membrane and involved in ZG swelling. However, a direct measurement of these ion channels at the ZG membrane in intact ZGs had not been performed. The aim of this study was to investigate the electrical activity of single ZGs and verify the types of channels found within their membrane. METHODS: ZGs from pancreatic acinar cells were isolated from the pancreas of Sprague-Dawley rats. Direct measurements of whole vesicle currents, in the presence and absence of ion channel blockers (quinine, glyburide and DIDS), were recorded following successful patching of single ZGs. CONCLUSION: In this study, we were able, for the first time, to patch single ZGs and study ion channels in their membrane. We were able to record currents across the ZG membrane and, utilizing ion channel blockers, confirm the presence of the chloride channels CLC-2 and the potassium channel IRK-8 (Kir6.1), and additionally demonstrate the presence of a second chloride channel CLC-3.
Assuntos
Precursores Enzimáticos/metabolismo , Canais Iônicos/metabolismo , Pâncreas/enzimologia , Técnicas de Patch-Clamp/métodos , Vesículas Secretórias/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aquaporina 1 , Aquaporinas/metabolismo , Canais de Cloro CLC-2 , Canais de Cloreto/metabolismo , Canais Iônicos/antagonistas & inibidores , Canais KATP , Masculino , Microscopia de Força Atômica , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos , Ratos Sprague-Dawley , Vesículas Secretórias/efeitos dos fármacosRESUMO
Opioid-like peptides mediate analgesia and induce behavioral effects such as tolerance and dependence by ligand-receptor-mediated mechanisms. The classical opioid prohormones can generate several bioactive peptides, and these divergent families of prohormones share a common well conserved ancestral opioid motif (Tyr-Gly-Gly-Phe). Evidence from pharmacological and molecular cloning studies indicates the presence of multiple isoforms of opioid ligands and receptors that are as yet uncharacterized. To identify potential new members we used the opioid motif as an anchor sequence and isolated two distinct isoforms (Xen-dorphins A and B) of an opioid prohormone from Xenopus laevis brain cDNA library. Xen-dorphin prohormones can generate multiple novel opioid ligands distinct from the known members of this family. Both isoforms are present in a wide variety of tissues including the brain. Two potential bioactive peptides, Xen-dorphin-1A and -1B, that were chemically synthesized showed opioid agonist activity in frog and rat brain membranes using a [35S]GTPgammaS assay. Initial radioligand binding experiments demonstrated that Xen-dorphin-1B binds with high affinity to opioid receptor(s) and with potential preference to the kappa-opioid receptor subtype. Cloning of the Xen-dorphin prohormone provides new evidence for the potential presence of other members in the opioid peptide superfamily.
Assuntos
Encefalinas/genética , Hormônios Peptídicos/química , Hormônios Peptídicos/genética , Precursores de Proteínas/química , Precursores de Proteínas/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Membrana Celular/metabolismo , Clonagem Molecular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Encefalinas/química , Biblioteca Gênica , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Hormônios/metabolismo , Cinética , Ligantes , Dados de Sequência Molecular , Isoformas de Proteínas , Ratos , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Distribuição TecidualRESUMO
Aquaporins (AQP) are involved in rapid and active gating of water across biological membranes. The molecular regulation of AQP is unknown. Here we report the isolation, identification and reconstitution of the regulatory complex of AQP-1. AQP-1 and Galphai3 have been implicated in GTP-induced gating of water in zymogen granules (ZG), the secretory vesicles in exocrine pancreas. In the present study, detergent-solubilized ZGs immunoprecipitated with monoclonal AQP-1 antibody, co-isolates AQP-1, PLA2, Galphai3, potassium channel IRK-8, and the chloride channel ClC-2. Exposure of ZGs to either the potassium channel blocker glyburide, or the PLA2 inhibitor ONO-RS-082, blocked GTP-induced ZG swelling. RBC known to possess AQP-1 at the plasma membrane, swell on exposure to the Galphai-agonist mastoparan, and respond similarly to ONO-RS-082 and glyburide, as ZGs. Liposomes reconstituted with the AQP-1 immunoisolated complex from solubilized ZG, also swell in response to GTP. Glyburide or ONO-RS-082 abolished the GTP effect. Immunoisolate-reconstituted planar lipid bilayers demonstrate conductance, which is sensitive to glyburide and an AQP-1 specific antibody. Our results demonstrate a Galphai3-PLA2 mediated pathway and potassium channel involvement in AQP-1 regulation.