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1 Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Instituto de Fisiología Experimental. IFISE-CONICET, Rosario, Argentina
2 Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Instituto de Fisiología Experimental. IFISE-CONICET, Rosario, Santa Fe, Argentina
3 Università degli Studi di Bari, Dipartimento di Zoologia, Bari, Bari, Italy
4 Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Instituto de Biología Molecular y Celular de Rosario. IBR-CONICET, Rosario, Santa Fe, Argentina
5 Università degli Studi di Bari, Dipartimento di Fisiologia Generale ed Ambientale, Bari, Bari, Italy
6 Facultad de Cs. Bioquímicas y Farmacéuticas. Universidad Nacional de Rosario, Instituto de Fisiología Experimental. IFISE-CONICET, Rosario, Santa Fe, Argentina
* To whom correspondence should be addressed. E-mail: rmarinel{at}unr.edu.ar.
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 EE administration (5 mg/kg b.w./day 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 about 70% in plasma (canalicular) and intracellular (vesicular) liver membranes. However, EE-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 (Pf) of canalicular membranes, measured by stopped-flow spectrophotometry, was significantly reduced (73 ± 1 vs. 57 ± 2 µm.sec-1) 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 post-transcriptional mechanism of protein level reduction. Accordingly, studies in primary cultured rat hepatocytes indicated that the lysosomal protease inhibitor leupeptin prevented the estrogen-induced AQP8 down-regulation. In conclusion, hepatocyte AQP8 protein expression is down-regulated in estrogen-induced intrahepatic cholestasis presumably by lysosomal-mediated degradation. Reduced canalicular membrane AQP8 expression is associated to 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.
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