Abstract
Cyclic AMP stimulates taurocholate (TC) uptake and sodium taurocholate co-transporting polypeptide (Ntcp) translocation in hepatocytes via the phosphoinositide-3 kinase (PI3K) signaling pathway. The aim of the present study was to determine whether protein kinase (PK) Czeta, one of the downstream mediators of the PI3K signaling pathway, is involved in cAMP-mediated stimulation of TC uptake. Studies were conducted in isolated rat hepatocytes and in HuH-7 cells stably transfected with rat liver Ntcp (HuH-Ntcp cells). Studies in hepatocytes showed that cAMP activates PKCzeta in a PI3K-dependent manner without inducing translocation of PKCzeta to the plasma membrane. Inhibition of cAMP-induced PKCzeta activity by myristoylated PKC (zeta/lambda) pseudosubstrate, a specific inhibitor of PKCzeta, and Gö 6850, a PKC inhibitor, resulted in inhibition of cAMP-induced increases in TC uptake and Ntcp translocation. Studies in HuH-Ntcp cells showed that inhibition of cAMP-induced PKCzeta activation by dominant-negative (DN) PKCzeta resulted in inhibition of cAMP-induced increases in TC uptake and Ntcp translocation. DN PKCzeta also inhibited wild-type PKCzeta-induced increases in PKCzeta activity, TC uptake, and Ntcp translocation. Myristoylated PKC (zeta/lambda) pseudosubstrate and DN PKCzeta also inhibited cAMP-induced activation of PKB in hepatocytes and HuH-Ntcp cells, respectively. Neither DN PKB nor constitutively active PKB affected cAMP-induced activation of PKCzeta, and wild-type PKCzeta did not activate PKB. Taken together, these results suggest that cAMP-induced activation of PKB is dependent on cAMP-induced stimulation of PKCzeta. It is proposed that cAMP-induced Ntcp translocation involves the activation of the PI3K/PKCzeta signaling pathway followed by the activation of the PI3K/PKB signaling pathway.
Highlights
The phosphoinositide-3 kinase (PI3K) signaling pathway has been shown to be involved in the regulation of solute transport in various cell types, including hepatocytes [1,2,3,4,5]
CAMP did not increase plasma membrane content of PKC in hepatocytes (Fig. 1). These results indicated that cAMP activated PKC in a PI3Kdependent manner and that this activation was not associated with translocation to the plasma membrane in hepatocytes
Effect of PKC Inhibitors on Taurocholate Uptake, Ntcp Translocation, and PKC Activity in Hepatocytes—To determine whether the effect of cAMP on TC uptake and Ntcp translocation is mediated via PKC, we studied the effect of PKC inhibitors
Summary
The PI3K signaling pathway has been shown to be involved in the regulation of solute transport in various cell types, including hepatocytes [1,2,3,4,5]. Cyclic AMP and cell swelling rapidly increase Naϩ/TC cotransport and Ntcp translocation to the plasma membrane [25,26,27], and this effect is mediated via the PI3K/PKB signaling pathway [11]. Considering that the effect of insulin on GLUT4 translocation involves PKB and PKC, cAMP-mediated Ntcp translocation may involve PKC. It is unknown whether cAMP activates the PI3K/PKC signaling pathway in hepatocytes and whether this pathway is involved in the regulation of hepatic TC uptake. The aim of the present study was to determine whether PKC is involved in cAMP-mediated stimulation of hepatic TC uptake and Ntcp translocation. Results are consistent with a role of PKC in hepatic uptake of bile acids
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