Defective hepatocyte aquaporin-8 expression and reduced canalicular membrane water permeability in estrogen-induced cholestasis.
Our previous work supports a role for aquaporin-8 (AQP8) water channels in rat hepatocyte bile formation mainly by facilitating the osmotically driven canalicular secretion of water. In this study, we tested whether a condition with compromised canalicular bile secretion, i.e., the estrogen-induced intrahepatic cholestasis, displays defective hepatocyte AQP8 functional expression. After 17alpha-ethinylestradiol administration (5 mg x kg body wt(-1).day(-1) for 5 days) to rats, the bile flow was reduced by 58% (P < 0.05). By subcellular fractionation and immunoblotting analysis, we found that 34 kDa AQP8 was significantly decreased by approximately 70% in plasma (canalicular) and intracellular (vesicular) liver membranes. However, 17alpha-ethinylestradiol-induced cholestasis did not significantly affect the protein level or the subcellular localization of sinusoidal AQP9. Immunohistochemistry for liver AQPs confirmed these observations. Osmotic water permeability (P(f)) of canalicular membranes, measured by stopped-flow spectrophotometry, was significantly reduced (73 +/- 1 vs. 57 +/- 2 microm/s) in cholestasis, consistent with defective canalicular AQP8 functional expression. By Northern blotting, we found that AQP8 mRNA expression was increased by 115% in cholestasis, suggesting a posttranscriptional mechanism of protein level reduction. Accordingly, studies in primary cultured rat hepatocytes indicated that the lysosomal protease inhibitor leupeptin prevented the estrogen-induced AQP8 downregulation. In conclusion, hepatocyte AQP8 protein expression is downregulated in estrogen-induced intrahepatic cholestasis, presumably by lysosomal-mediated degradation. Reduced canalicular membrane AQP8 expression is associated with impaired osmotic membrane water permeability. Our data support the novel notion that a defective expression of canalicular AQP8 contributes as a mechanism for bile secretory dysfunction of cholestatic hepatocytes.
Carreras
Flavia I
FI
Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 570, 2000 Rosario, Santa Fe, Argentina.
Lehmann
Guillermo L
GL
Ferri
Domenico
D
Tioni
Mariana F
MF
Calamita
Giuseppe
G
Marinelli
Raúl A
RA
D000818:Animals; D020346:Aquaporins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000502:physiology*; D002779:Cholestasis / Q000139:chemically induced / Q000378:metabolism* / Q000503:physiopathology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D004967:Estrogens; D004997:Ethinyl Estradiol / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D014867:Water / Q000378:metabolism*
C491637:Aqp9 protein, rat; D020346:Aquaporins; D015853:Cysteine Proteinase Inhibitors; D004967:Estrogens; D007976:Leupeptins; C108330:aquaporin 8; D014867:Water; C025483:estradiol-17 beta-glucuronide; D004997:Ethinyl Estradiol; D004958:Estradiol; C032854:leupeptin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde