Characterization of cloned rat liver Na(+)-bile acid cotransporter using peptide and fusion protein antibodies

Ananthanarayanan, M.; Ng, Oi Cheng; Boyer, James L.; Suchy, Frederick J.

doi:10.1152/ajpgi.1994.267.4.g637

Cited by 65 publications

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“…Anti-fusion protein antibodies to the cloned Ntcp were prepared as described previously (5,6). Male Wistar rats (200 -300 g) obtained from Charles River Laboratories served as liver donors.…”

Section: Methodsmentioning

confidence: 99%

“…Two different proteins, namely Na ϩ /TC cotransport polypeptide (Ntcp) and epoxy hydrolase, have been shown to mediate Na ϩ /TC cotransport into hepatocytes (3, 4). The rat liver Ntcp is a ϳ51-kDa glycoprotein with seven transmembrane domains (3,5,6). Recent studies suggest that Na ϩ /TC cotransport and Ntcp are up-regulated by hormones and down-regulated by cholestatic agents.…”

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confidence: 99%

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Role of Protein Phosphatases in Cyclic AMP-mediated Stimulation of Hepatic Na+/Taurocholate Cotransport

Mukhopadhyay

Webster

Anwer

1998

Journal of Biological Chemistry

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Cyclic AMP has been proposed to stimulate Na ؉ /taurocholate (TC) cotransport in hepatocytes by translocating Na ؉ /TC cotransport polypeptide (Ntcp) to the plasma membrane and to induce Ntcp dephosphorylation. Whether protein phosphatases 1 and 2A (PP1/2A) are involved in the regulation of Na ؉ /TC cotransport by cAMP was investigated in the present study. Okadaic acid and tautomycin, inhibitors of PP1/2A, inhibited cAMP-mediated increases in TC uptake and cytosolic [Ca 2؉ ], and only tautomycin inhibited basal TC uptake. Removal of cAMP reversed cAMP-mediated increases in TC uptake and plasma membrane Ntcp mass. Okadaic acid alone increased Ntcp phosphorylation without affecting Ntcp mass in plasma membranes and homogenates. In the presence of okadaic acid, cAMP failed to increase plasma membrane Ntcp mass, induce Ntcp dephosphorylation, and decrease endosomal Ntcp mass. Phosphorylated Ntcp was detectable in endosomes isolated from okadaic acid-treated hepatocytes but not in endosomes from control and cAMP-treated hepatocytes. PP1 was found to be enriched in plasma membranes, whereas PP2A was mostly in the cytosol. Cyclic AMP did not activate either PP1 or PP2A, whereas okadaic acid inhibited primarily PP2A. These results suggest that 1) the effect of cAMP on Na ؉ /TC cotransport is not mediated via either PP1 or PP2A; rather, cAMP-mediated signaling pathway is maintained by PP2A and inhibition of PP2A overrides cAMP-mediated effects, and 2) okadaic acid, by inhibiting PP2A, inhibits cAMP-mediated increases in Na ؉ /TC cotransport by decreasing the ability of cAMP to increase cytosolic [Ca 2؉ ]. It is proposed that cAMP-mediated dephosphorylation of Ntcp leads to an increased retention of Ntcp in the plasma membrane, and okadaic acid, by inhibiting PP2A, inhibits cAMP-mediated stimulation of Na ؉ /TC cotransport by reversing the ability of cAMP to increase cytosolic [Ca 2؉ ] and to induce Ntcp dephosphorylation.Efficient hepatic uptake of conjugated bile acids, like taurocholate (TC), 1 is mediated predominantly via Na ϩ -coupled cotransport mechanism (1, 2). Two different proteins, namely Na ϩ /TC cotransport polypeptide (Ntcp) and epoxy hydrolase, have been shown to mediate Na ϩ /TC cotransport into hepatocytes (3, 4). The rat liver Ntcp is a ϳ51-kDa glycoprotein with seven transmembrane domains (3,5,6). Recent studies suggest that Na ϩ /TC cotransport and Ntcp are up-regulated by hormones and down-regulated by cholestatic agents. Prolactin increases Na ϩ /TC cotransport and Ntcp mRNA in hepatocytes (7). Endotoxin and estradiol decrease TC uptake, Ntcp content of plasma membranes, and Ntcp mRNA (8, 9). These results indicate that Ntcp undergoes transcriptional/translational regulation.Recent studies also suggest that Ntcp undergoes short term post-translational regulation, but the molecular mechanism underlying this regulation is unclear. In a previous study (10), we reported that cAMP, acting via protein kinase A, stimulates Na ϩ /TC cotransport by increasing its maximal transport rate; the effect of cA...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Role of Protein Phosphatases in Cyclic AMP-mediated Stimulation of Hepatic Na+/Taurocholate Cotransport

Mukhopadhyay

Webster

Anwer

1998

Journal of Biological Chemistry

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“…Immunofluorescence experiments localized Ntcp to the basolateral plasma membrane of rat [9,294] and human hepatocytes [177]. It displays an even expression throughout the liver acinus.…”

Section: Subcellular Expression and Tissue Distributionmentioning

confidence: 97%

The Role of the Sodium-Taurocholate Cotransporting Polypeptide (NTCP) and of the Bile Salt Export Pump (BSEP) in Physiology and Pathophysiology of Bile Formation

Stieger

2010

Handbook of Experimental Pharmacology

246

260

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“…17,26 We examined the cellular localization of ntcp in WIF-B cells grown for 10 days in the absence or presence of TC (50 µmol/L). Cells were fixed, permeabilized, and immunostained with a polyclonal antiserum directed against ntcp, as described in Materials and Methods.…”

Section: Indirect Immunofluorescent Examination Of the Localization Omentioning

confidence: 99%

Enhanced Na+-dependent bile salt uptake by WIF-B cells, a rat hepatoma hybrid cell line, following growth in the presence of a physiological bile salt

et al. 1998

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Western blots immunostained for the basolateral Na ؉ -dependent plasma membrane protein, ntcp, revealed the appropriate Ϸ50-kd band in control and TC-grown cells, and confocal immunofluorescence microscopy demonstrated staining along the basolateral plasma membrane. Northern blots hybridized with a cDNA probe directed against ntcp indicated a modest TC-induced increase in mRNA levels. Reverse-transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from WIF-B cells and oligonucleotide primers specific for rat ntcp or human NTCP transcripts revealed only the presence of the rat ntcp transcript. We conclude that bile salts, at concentrations normally found in mammalian portal blood, may be capable of promoting enhanced hepatocellular bile salt uptake via an increase in basolateral Na ؉ -dependent plasma membrane transport capacity. (HEPATOLOGY 1998;27:191-199.)The mammalian liver is continually exposed to low concentrations of bile salts present in portal and peripheral blood. Hepatocytes avidly take up bile salts across the basolateral plasma membrane and rapidly secrete them into bile; less than 5% of secreted bile salts are derived from de novo synthesis within the hepatocyte. 1 In rats, the postprandial bile salt concentration in the portal blood is Ϸ60 µmol/L, with taurocholate (TC) comprising up to half of the bile salt species. 2 In humans, the bile salt load reaching the liver fluctuates considerably over a 24-hour period, with portal vein plasma concentrations ranging from Ϸ14 µmol/L in the fasting state to Ϸ43 µmol/L in the postprandial state. 3 Hepatocytes are thus exposed routinely to changes in rates of bile salt delivery, and therefore must respond with variable rates of bile salt uptake across the basolateral plasma membrane. High plasma bile salt concentrations (up to 1 mmol/L) are associated with down-regulation of basolateral bile salt uptake into hepatocytes. 4 It is unclear, however, whether plasma bile salt concentrations within physiological ranges may promote bile salt uptake into hepatocytes.Difficulties are encountered when examining the physiological role of bile salts in intact animals, because many variables change simultaneously when conducting in vivo studies. A major experimental problem also is encountered when addressing normal bile salt regulation of hepatic physiology in vitro, because of the difficulty in maintaining liver preparations with stable rates of basolateral bile salt uptake. Isolated perfused rat livers maintain physiological rates of bile salt uptake and secretion for up to 3 hours, but bile flow decreases thereafter. 5 Isolated rat hepatocytes exhibit profound decreases in rates of bile salt uptake over the first 72 hours of primary culture. [6][7][8] Bile salt transport may be virtually absent in cultured hepatocyte tumor cell lines. 9 Recently, an in vitro polarized hepatocyte cell system, WIF-B cells, has been reported by Hubbard et al. 10,11 as a refinement of an existing rat hepatoma-human fibroblast cell line developed by Cassio et al. 12 WIF-B cell...

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Characterization of cloned rat liver Na(+)-bile acid cotransporter using peptide and fusion protein antibodies

Cited by 65 publications

References 0 publications

Role of Protein Phosphatases in Cyclic AMP-mediated Stimulation of Hepatic Na+/Taurocholate Cotransport

Role of Protein Phosphatases in Cyclic AMP-mediated Stimulation of Hepatic Na+/Taurocholate Cotransport

The Role of the Sodium-Taurocholate Cotransporting Polypeptide (NTCP) and of the Bile Salt Export Pump (BSEP) in Physiology and Pathophysiology of Bile Formation

Enhanced Na+-dependent bile salt uptake by WIF-B cells, a rat hepatoma hybrid cell line, following growth in the presence of a physiological bile salt

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