Abstract
The formation of hepatic bile requires that water be transported across liver epithelia. Rat hepatocytes express three aquaporins (AQPs): AQP8, AQP9, and AQP0. Recognizing that cholesterol and sphingolipids are thought to promote the assembly of proteins into specialized membrane microdomains, we hypothesized that canalicular bile secretion involves the trafficking of vesicles to and from localized lipid-enriched microdomains in the canalicular plasma membrane. Hepatocyte plasma membranes were sonicated in Triton and 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 (a microdomain-positive marker) and devoid of amino-peptidase N (a microdomain-negative marker), enriched in caveolin, both AQP8 and AQP9, but negative for clathrin. The microdomain fractions contained chloride-bicarbonate anion exchanger isoform 2 and multidrug resistance-associated protein 2. Exposure of isolated hepatocytes to glucagon increased the expression of AQP8 but not AQP9 in the microdomain fractions. Sphingolipid analysis of the insoluble fraction showed the predominant species to be sphingomyelin. These data support the presence of sphingolipid-enriched microdomains of the hepatocyte membrane that represent potential localized target areas for the clustering of AQPs and functionally related proteins involved in canalicular bile secretion.
Highlights
The formation of hepatic bile requires that water be transported across liver epithelia
AQP8 is present in intracellular vesicles; in response to a choleretic agonist, AQP8 is redistributed to the canalicular plasma membrane, where it facilitates the transport of water across the hepatocyte epithelial barrier in concert with AQP9, which is constitutively expressed on the basolateral plasma membrane [2]
We used a variety of biochemical approaches and provide evidence that Triton-insoluble microdomains exist in hepatocyte plasma membranes, are enriched in caveolin, cholesterol, and sphingomyelin, and contain AQP8 and AQP9, multidrug resistance-associated protein 2 (MRP2), and anion exchanger isoform 2 (AE2)
Summary
The formation of hepatic bile requires that water be transported across liver epithelia. We used a variety of biochemical approaches and provide evidence that Triton-insoluble microdomains exist in hepatocyte plasma membranes, are enriched in caveolin, cholesterol, and sphingomyelin, and contain AQP8 and AQP9, multidrug resistance-associated protein 2 (MRP2), and anion exchanger isoform 2 (AE2).
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