Abstract
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 [Ca2+], 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 [Ca2+]. 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 [Ca2+] and to induce Ntcp dephosphorylation.
Highlights
Efficient hepatic uptake of conjugated bile acids, like taurocholate (TC),1 is mediated predominantly via Naϩ-coupled cotransport mechanism [1, 2]
The present study showed that inhibitors of phosphatases 1 and 2A (PP1/2A) decreased the ability of cAMP to stimulate TC uptake in hepatocytes
The effect of okadaic acid and tautomycin on cAMP-stimulated TC uptake is most likely mediated via inhibition of PP2A in hepatocytes, both agents are known inhibitors of PP1 and 2A [16, 34]
Summary
Efficient hepatic uptake of conjugated bile acids, like taurocholate (TC), is mediated predominantly via Naϩ-coupled cotransport mechanism [1, 2]. Our recent study showed that Ntcp is a serine/threonine phosphoprotein, phosphorylated Ntcp is detectable in the plasma membrane, but not in microsomes and Golgi complex, and treatment of hepatocytes with cAMP results in Ntcp dephosphorylation [15]. These results raise the possibility that the regulation of Naϩ/TC cotransport by cAMP may involve phosphorylation/dephosphorylation of Ntcp. Values represent enrichment relative to respective homogenate and are expressed as means Ϯ S.D.; n ϭ 3– 6 membrane preparations
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