Glucagon induces the plasma membrane insertion of functional aquaporin-8 water channels in isolated rat hepatocytes

Authors

  • Sergio A. Gradilone,

    1. Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina
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  • Fabiana García,

    1. Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina
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  • Robert C. Huebert,

    1. Center for Basic Research in Digestive Diseases, Departments of Internal Medicine and Biochemistry and Molecular Biology, Mayo Medical School, Clinic, and Foundation, Rochester, MN
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  • Pamela S. Tietz,

    1. Center for Basic Research in Digestive Diseases, Departments of Internal Medicine and Biochemistry and Molecular Biology, Mayo Medical School, Clinic, and Foundation, Rochester, MN
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  • M. Cecilia Larocca,

    1. Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina
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  • Arlinet Kierbel,

    1. Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina
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  • Flavia I. Carreras,

    1. Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina
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  • Nicholas F. LaRusso,

    1. Center for Basic Research in Digestive Diseases, Departments of Internal Medicine and Biochemistry and Molecular Biology, Mayo Medical School, Clinic, and Foundation, Rochester, MN
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  • Raúl A. Marinelli Ph.D.

    Corresponding author
    1. Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina
    • Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 570, 2000 Rosario, Santa Fe, Argentina; fax: (54) 341-4399473
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Abstract

Although glucagon is known to stimulate the cyclic adenosine monophosphate (cAMP)-mediated hepatocyte bile secretion, the precise mechanisms accounting for this choleretic effect are unknown. We recently reported that hepatocytes express the water channel aquaporin-8 (AQP8), which is located primarily in intracellular vesicles, and its relocalization to plasma membranes can be induced with dibutyryl cAMP. In this study, we tested the hypothesis that glucagon induces the trafficking of AQP8 to the hepatocyte plasma membrane and thus increases membrane water permeability. Immunoblotting analysis in subcellular fractions from isolated rat hepatocytes indicated that glucagon caused a significant, dose-dependent increase in the amount of AQP8 in plasma membranes (e.g., 102% with 1 μmol/L glucagon) and a simultaneous decrease in intracellular membranes (e.g., 38% with 1 μmol/L glucagon). Confocal immunofluorescence microscopy in cultured hepatocytes confirmed the glucagon-induced redistribution of AQP8 from intracellular vesicles to plasma membrane. Polarized hepatocyte couplets showed that this redistribution was specifically to the canalicular domain. Glucagon also significantly increased hepatocyte membrane water permeability by about 70%, which was inhibited by the water channel blocker dimethyl sulfoxide (DMSO). The inhibitors of protein kinase A, H-89, and PKI, as well as the microtubule blocker colchicine, prevented the glucagon effect on both AQP8 redistribution to hepatocyte surface and cell membrane water permeability. In conclusion, our data suggest that glucagon induces the protein kinase A and microtubule-dependent translocation of AQP8 water channels to the hepatocyte canalicular plasma membrane, which in turn leads to an increase in membrane water permeability. These findings provide evidence supporting the molecular mechanisms of glucagon-induced hepatocyte bile secretion.

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