The sodium-taurocholate (TC) cotransporting polypeptide (NTCP) facilitates bile formation by mediating sinusoidal Na ϩ -TC cotransport. During sepsis-induced cholestasis, there is a decrease in NTCPdependent uptake of bile acids and an increase in nitric oxide (NO) levels in hepatocytes. In rat hepatocytes NO inhibits Na ϩ -dependent uptake of taurocholate. The aim of this study was to extend these findings to human NTCP and to further investigate the mechanism by which NO inhibits TC uptake. Using a human hepatoma cell line stably expressing NTCP (HuH-NTCP), we performed experiments with the NO donors sodium nitroprusside and S-nitrosocysteine and demonstrated that NO inhibits TC uptake in these cells. Kinetic analyses revealed that NO significantly decreased the V max but not the K m of TC uptake by NTCP, indicating noncompetitive inhibition. NO decreased the amount of NTCP in the plasma membrane, providing a molecular mechanism for the noncompetitive inhibition of TC uptake. One way that NO can modify protein function is through a posttranslational modification known as S-nitrosylation: the binding of NO to cysteine thiols. Using a biotin switch technique we observed that NTCP is S-nitrosylated under conditions in which NO inhibits TC uptake. Moreover, dithiothreitol reversed NO-mediated inhibition of TC uptake and S-nitrosylation of NTCP, indicating that NO inhibits TC uptake via modification of cysteine thiols. In addition, NO treatment led to a decrease in Ntcp phosphorylation. Taken together these results indicate that the inhibition of TC uptake by NO involves S-nitrosylation of NTCP. sodium-taurocholate cotransporting polypeptide TRANSPORT OF SOLUTES FROM the sinusoidal space to the canaliculus provides the driving force for bile formation (1). Bile formation, in turn, depends on the proper functioning of a number of hepatobiliary transporters present on the hepatic sinusoidal (basolateral) and canalicular (apical) membranes. NTCP, present at the basolateral membrane of hepatocytes, mediates the transport of conjugated bile salts such as taurocholate (TC) and taurochenodeoxycholate in a Na ϩ -dependent fashion using the sodium gradient produced by the Na , 13, 19, 39). Lipopolysaccharide (LPS)-induced cholestasis is associated with a decrease in TC uptake and an increase in proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin-6 (IL-6), and interleukin-1 (IL-1) (6,11,19). At the same time, LPS also initiates a burst of NO production by inducible nitric oxide synthase in all major cell types of the liver (11).The roles for NO in the liver are diverse and varied. NO plays important roles in regulating hepatic blood flow including portal hypertension (11). NO has both proapoptotic and antiapoptotic actions in the liver (31). Additionally, NO plays important roles in alcoholic liver disease and ischemia/reperfusion injury (31). NO has also been implicated in regulating bile formation. Whereas lower levels of NO have been linked to increased bile flow, higher levels of NO are associate...
The Na(+) taurocholate (TC) cotransporting polypeptide Ntcp/NTCP mediates TC uptake across the sinusoidal membrane of hepatocytes. Previously, we demonstrated that nitric oxide (NO) inhibits TC uptake through S-nitrosylation of a cysteine residue. Our current aim was to determine which of the eight cysteine residues of Ntcp is responsible for NO-mediated S-nitrosylation and inhibition of TC uptake. Thus, we tested the effect of NO on TC uptake in HuH-7 cells transiently transfected with cysteine-to-alanine mutant Ntcp constructs. Of the eight mutants tested, only C44A Ntcp displayed decreased total and plasma membrane (PM) levels that were also reflected in decreased TC uptake. C266A Ntcp showed a decrease in TC uptake that was not explained by a decrease in total expression or PM localization, indicating that C266 is required for optimal uptake. We speculated that NO would target C266 since a previous report had shown the thiol reactive compound [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET) inhibits TC uptake by wild-type NTCP but not by C266A NTCP. We confirmed that MTSET targets C266 of Ntcp, but, surprisingly, we found that C266 was not responsible for NO-mediated inhibition of TC uptake. Instead, we found that C96 was targeted by NO since C96A Ntcp was insensitive to NO-mediated inhibition of TC uptake. We also found that wild-type but not C96A Ntcp is S-nitrosylated by NO, suggesting that C96 is important in regulating Ntcp function in response to elevated levels of NO.
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