Membrane microdomains in hepatocytes: Potential target areas for proteins involved in canalicular bile secretion

P. Tietz, J. Jefferson, R. Pagano, and N. F. LaRusso

Department of Internal Medicine, Mayo Clinic College of Medicine, Rochester, MN 55905

Corresponding Author: larusso.nicholas{at}mayo.edu

Formation of bile requires that large volumes of water be rapidly transported across liver epithelia, including hepatocytes. Previous work from our laboratory supports important roles for aquaporins (AQPs) in bile secretion. We recently reported that rat hepatocytes express three AQPs: AQP8, AQP9, and AQP0 (1). Upon exposure of hepatocyte couplets to a choleretic agonist (dibutyryl cyclic AMP), AQP8, the most abundant AQP in hepatocytes, redistributed to the canalicular plasma membrane, while the subcellular distributions of AQP0 and 9 were unaffected. Recognizing that cholesterol and sphingolipids are thought to promote assembly of proteins into specialized membrane microdomains, we hypothesized that canalicular bile secretion involves the regulated trafficking of vesicles to and from localized lipid-enriched microdomains within the canalicular plasma membrane. We isolated membrane microdomains from rat hepatocytes total plasma membranes. Fractions were sonicated in Triton, centrifuged overnight on a sucrose gradient to yield a Triton-soluble, pellet and a Triton-insoluble, sphingolipid-enriched microdomain fraction at the 5/30% sucrose interface. The detergent insoluble, portion of the hepatocyte plasma membrane was enriched in alkaline phosphatase (microdomain-positive marker) and devoid of amino peptidase-N (microdomain-negative marker). Immunoblotting showed this fraction was also enriched in caveolin (a cholesterol-binding protein frequently associated with membrane microdomains), both AQP8 and 9, but negative for clathrin. The microdomain fractions contained AE2 (chloride-bicarbonate anion exchanger isoform-2) and MRP2 (multidrug resistance associated protein 2). Exposure to glucagon, a choleretic agonist, significantly increased the expression of AQP8, but not AQP9 associated with the microdomain fractions. Sphingolipid analysis of the detergent insoluble fraction showed the most predominant species to be sphingomyelin. These data support the presence of sphingolipid-enriched microdomains enriched in cholesterol and sphingomyelin that represent potential localized target areas for clustering of AQPs and functionally related proteins involved in canalicular bile secretion.

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