Cross-talk between Protein Kinases C (cid:1) and B in Cyclic AMP-mediated Sodium Taurocholate Co-transporting Polypeptide Translocation in Hepatocytes*

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) C (cid:1) , 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 PKC (cid:1) in a PI3K-dependent manner without inducing translocation of PKC (cid:1) to the plasma membrane. Inhibition of cAMP-induced PKC (cid:1) activity by myristoylated PKC ( (cid:1) / (cid:2) ) pseudosubstrate, a specific inhibitor of PKC (cid:1) , and Go¨ 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 PKC (cid:1) activation by dominant-negative (DN) PKC (cid:1) resulted in inhibition of cAMP-induced increases in TC uptake and Ntcp translocation. DN PKC (cid:1)

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). Three classes of serine/threonine kinases, namely PKB 1 (also known as Akt), atypical PKC /, and p70 S6K , have been proposed to act downstream of PI3K (6). The PI3K/PKB signaling pathway has been reported to be involved in the regulation of Glut4 translocation (7)(8)(9)(10) in adipocytes and myocytes and Ntcp translocation in hepatocytes (11). The activation of PKC, like that of PKB, requires phosphoinositides (12) and phosphorylation by PDK1 (13,14). Recent studies indicate that insulin also stimulates PI3K-dependent PKC in various cell types (15,16), including hepatocytes (17), and the PI3K/PKC signaling pathway is involved in insulin-stimulated GLUT4 translocation (16,18) and NHE1 activity in human erythrocytes (19). In hepatocytes, PKC has been reported to have an antiapoptotic effect (20) and to mediate peroxovanadium-induced activation of glycogen synthase (17). The role of PKC in hepatic bile acid uptake has not been studied.
Conjugated bile acids, such as TC, are efficiently taken up by hepatocytes primarily via a Na ϩ /TC cotransport mechanism; Ntcp, a serine/threonine phosphoprotein (21), is believed to be the major protein involved in Na ϩ /TC cotransport (22)(23)(24). Cyclic AMP and cell swelling rapidly increase Na ϩ /TC cotransport and Ntcp translocation to the plasma membrane (25)(26)(27), and this effect is mediated via the PI3K/PKB signaling pathway (11). The PI3K/p70 S6K pathway is not involved in cell swelling-and cAMP-induced increases in TC uptake (25,27). Considering that the effect of insulin on GLUT4 translocation involves PKB and PKC, cAMP-mediated Ntcp translocation may also involve PKC. However, it is unknown whether cAMP also activates the PI3K/PKC signaling pathway in hepatocytes and whether this pathway is also 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. The role of PKC was evaluated by determining the effect of PKC inhibitors in hepatocytes and the effect of transient transfection with WT and/or DN PKC in HuH-Ntcp cells. Results are consistent with a role of PKC in hepatic uptake of bile acids.
Hepatocyte Preparation-Hepatocytes were isolated from rat livers using a previously described collagenase perfusion method (32). Freshly prepared hepatocytes suspended (100 mg/ml, wet weight) in a HEPES assay buffer, pH 7.4, containing 20 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgSO 4 , 1.0 mM CaCl 2 , 0.8 mM KH 2 PO 4 and 5 mM glucose were incubated for 30 min at 37°C under air before initiating studies. Hepatocytes were pretreated with PKC inhibitors and then with 10 M CPT-cAMP for 15 min followed by determination of TC uptake, Ntcp translocation, and PKC activity. Details of these experiments are given in the figure legends. All studies were repeated in at least three different cell preparations.
HuH-Ntcp Cell Culture and Transfections-HuH-Ntcp cells were cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 100,000 units/liter penicillin, 100 mg/liter streptomycin, 1.2 g/liter G418 at 37°C in a 5% CO 2 /95% O 2 air incubator. For transfection experiments, cells were cultured for 24 h in the culture medium and then transiently transfected with a PKC plasmid using LipofectAMINE as described previously (11). After 24 h incubation in the transfection medium, cells were cultured for additional 24 h in culture media. Cells were then incubated in the HEPES buffer (see above) for 3 h at 37°C before treatments.
TC Uptake-The initial uptake rate of TC in hepatocytes was determined as described previously (33). In brief, at various times after incubation of hepatocytes with different agents and/or CPT-cAMP, an aliquot of cell suspension (5-8 mg protein/ml) was withdrawn and used to determine the initial uptake rate of TC (20 M). Transport was initiated by adding cells to the incubation medium containing [ 14 C]TC and [ 3 H]inulin, with uptake determined at different time points. Initial uptake rates were calculated from the slope of the linear portion of time-dependent uptake curves and were expressed as nanomoles per minute per milligram of protein.
TC uptake in HuH-Ntcp cells was determined as described previously (11). In brief, after pretreatment with 100 M CPT-cAMP for 15 min, cells were incubated with 20 M TC containing [ 3 H]TC (100 dpm/ pmol) for 2 min. Cells were then washed twice with 0.5 ml of ice-cold HEPES buffer and lysed in 0.25 ml of a lysis buffer (20 mM Tris, 150 mM NaCl, 1% Triton, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 10 g/ml aprotinin, 10 g/ml leupeptin, 500 nM okadaic acid, and 1 mM orthovanadate, pH 7.5). Aliquots of cell lysate were counted for radioactivity and TC uptake rate was expressed in picomoles per minute per milligram of protein.
Ntcp Translocation-A cell surface protein biotinylation method was used to quantitate Ntcp translocation in hepatocytes (25,27) and HuH-Ntcp cells (11). In brief, after various treatments, cell surface proteins were biotinylated by exposing hepatocytes (or HuH-Ntcp cells) to sulfo-D-biotin-N-hydroxysuccinimide ester (0.5 mg/ml; Pierce) followed by preparation of cell lysate used to determine biotinylated and total Ntcp mass. Biotinylated proteins were isolated using streptavidin-agarose beads and then subjected to immunoblot analysis to determine plasma membrane Ntcp. For immunoblot analysis, proteins (10 -50 g) from whole-cell lysate and streptavidin-agarose-containing biotinylated proteins were subjected to 12% SDS-PAGE by the method of Laemmli (34) as described previously (35). Proteins were transferred electophoretically from SDS gels to nitrocellulose membranes (Transblot transfer membrane, 0.45 m; Bio-Rad) and probed with the Ntcp antibody (1:2000 dilution). Peroxidase conjugated anti-IgG was used as the secondary antibody. The immunoblots were developed with the Amersham Biosciences ECL kit according to the manufacturer's instructions.
Protein Kinase Assay-Cell lysates obtained after various treatments of hepatocytes and HuH-Ntcp cells were assayed for PKC using a previously described method (20). In brief, PKC was immunoprecipitated by incubating 25-50 g of total cellular protein with PKC antibody (Upstate), and the immunocomplexes were incubated for 15 min at 37°C with a 25-l reaction mixture (kinase buffer containing 200 M ATP, 2 Ci of [␥-32 P]ATP and 1 g of myelin basic protein). Reactions were stopped by adding Laemmli sample buffer and phosphorylated

FIG. 1. Effect cAMP on PKC activity (top) and distribution (bottom) in hepatocytes.
Top, hepatocytes were treated with 10 M CPT-cAMP (cAMP) or buffer (CON) for 15 min in the presence and absence of 100 nM wortmannin (Wort; 15-min pretreatment) followed by determination of PKC activity. Results of densitometric analysis (mean Ϯ S.E., n ϭ 7) and a representative blot of myelin basic protein phosphorylation (PKC activity) are shown. Bottom, hepatocytes were treated with 10 M CPT-cAMP for 15 min followed by isolation of plasma membrane (PM) and determination of PKC mass. Results of densitometric analysis (mean Ϯ S.E., n ϭ 3) and a representative PKC immunoblot are shown. *, significantly different from the control value.

FIG. 2. Gö 6850, but not Gö 6976, inhibits basal cAMP-induced increases in PKC activity (top) and TC uptake (bottom) in hepatocytes.
Hepatocytes were treated with 5 or 10 M Gö6850 or Gö6976 for 30 min before addition of 10 M CPT-cAMP. PKC activity and TC uptake were determined 15 min after the addition of buffer (Basal) or CPT-cAMP (cAMP). Values are mean Ϯ S.E. (n ϭ 6 for PKC activity; n ϭ 3 for TC uptake). *, significantly different from respective basal values.
myelin basic protein was resolved by SDS-PAGE followed by autoradiography. All samples were assayed in triplicate. PKB activity was determined from the ratio of phosphorylated PKB (active form) to total PKB as described previously (25,27).
Other Methods-The Lowry method was used to determine cell protein (36). The blots were scanned in gray scale using Adobe Photoshop (Adobe Systems, Mountain View, CA) and the relative band densities were quantitated using Sigma Gel (SPSS Inc., Chicago, IL). All values are expressed as mean Ϯ S.E. Paired t test was used to statistically analyze data with p Ͻ 0.05 considered significant.

Effect of cAMP on PKC in
Hepatocytes-PKC is considered to be downstream of PI3K, and its activation depends on PI3K products (12) and phosphorylation by PDK1 followed by autophosphorylation in some cells (37). We have shown previously that cAMP activates PI3K as well as PKB and p70 S6K , two downstream effectors of PI3K, in hepatocytes (25,38). Thus, the first series of studies were conducted to determine whether cAMP activates PKC in a PI3K-dependent manner in hepatocytes. PKC activity increased in hepatocytes treated with 10 M CPT-cAMP and wortmannin inhibited both basal and cAMP-induced increases in PKC activity (Fig. 1). It may be noted that the effect of cAMP was variable and ranged from no activation in some cell preparations to 2-3-fold activation in others. PKC activation is usually associated with its translocation to membranes (37). However, 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. Studies in Sf9 cells showed that Gö 6850, but not Gö 6976, inhibit PKC activity (39). Thus, studies were conducted with these two inhibitors first.
PKC PS (100 M, but not 10 M) inhibited basal as well as cAMP-stimulated PKC activity in hepatocytes (Fig. 4). PKC PS (80 M) has been shown to inhibit insulin-stimulated PKC activity in hepatocytes (17). PKC PS (100 M, but not 10 M) also inhibited cAMP-induced increases in TC uptake (Fig. 4) and plasma membrane Ntcp (Fig. 5); cAMP does not affect total Ntcp level in hepatocytes (25). Taken together, these results indicate that cAMP-induced translocation of Ntcp may be mediated via PKC.
DN PKC Inhibited cAMP-induced TC Uptake and Ntcp Translocation in HuH-Ntcp Cells-To determine the role of PKC more directly, HuH-Ntcp cells were transiently transfected with DN PKC followed by determination of PKC activity, TC uptake, and Ntcp translocation. We have shown previously that cAMP stimulates TC uptake and Ntcp translocation in HuH-Ntcp cells (11). Expression of DN PKC was confirmed by immunoblot analysis. As in hepatocytes, cAMP increased PKC activity and TC uptake in HuH-Ntcp cells (Fig.  6). DN PKC did not affect either basal TC uptake or PKC activity. However, cAMP-induced increases in TC uptake and PKC activity were inhibited in cells transfected with DN PKC (Fig. 6). Likewise, DN PKC also inhibited the ability of cAMP to increase plasma membrane Ntcp without affecting the basal level of Ntcp (Fig. 6). Total Ntcp level was not affected by either cAMP or DN PKC. These results further support that cAMP-induced increases in TC uptake and Ntcp translocation is dependent on PKC activity.
WT PKC Stimulates Uptake and Ntcp Translocation in HuH-Ntcp Cells-If the effect of cAMP is mediated via PKC, activation of PKC independent of cAMP should also stimulate TC uptake and Ntcp translocation. This hypothesis was tested by determining TC uptake and Ntcp translocation in HuH-Ntcp cells transiently transfected with WT PKC, DN PKC, or WT PKC ϩ DN PKC-. PKC activity in HuH-Ntcp cells transfected with WT PKC-was more than 2-fold higher than in cells transfected with the empty vector (Fig. 7). This increase in PKC-activity was abolished when the expression of WT PKC was inhibited by cotransfecting HuH-Ntcp cells with 10-fold excess of DN PKC; DN PKC alone did not affect basal PKC activity. Similar to the effect on PKC-activity, the expression of WT PKC also increased TC uptake and plasma membrane Ntcp content, and these effects were inhibited by cotransfection with 10-fold excess DN PKC (Fig. 7). These results would indicate that PKC-is involved in the regulation of TC uptake and Ntcp translocation.
Cross-talk between PKB and PKC-Recent studies suggest that PKC may be involved in PKB activation (37,40) and our previous studies suggested a role for the PI3K/PKB signaling pathway in cAMP-induced Ntcp translocation (11). Thus, the effect of PKC observed in the present study may involve PKB. To assess possible interactions between PKC and PKB in cAMP-induced Ntcp translocation, we determined the effect of PKC PS and DN PKC on cAMP-induced PKB activation. PKC PS (100 M but not 10 M) inhibited cAMP-induced activation of PKB in hepatocytes (Fig. 8). Likewise, cAMP failed to stimulate PKB in HuH-Ntcp cells transiently trans- fected with DN PKC (Fig. 9); DN PKC inhibited cAMP-induced increases in PKC by 80% as in Fig. 6. Both PKC PS and DN PKC decreased basal PKB activity by 20%, although this decrease did not reach statistical significance. These results raised the possibility that PKC may be a positive regulator of PKB in hepatocytes. However, when PKC activity was increased 2.8 Ϯ 0.35-fold (n ϭ 3) by transfecting HuH-Ntcp cells with WT PKC, there was no increase in PKB activity (1.1 Ϯ 0.13 relative to empty vector). Thus, it would seem that cAMPinduced activation of PKB is dependent on cAMP-induced activation of PKC, but PKC does not affect basal PKB activity.
To further define the possible mechanism of interaction between PKB and PKC, we determined the effect of DN PKB on cAMP-stimulated PKC activity. It is interesting that neither basal nor cAMP-stimulated PKC activity was affected in HuH-Ntcp cells transiently transfected with DN PKB (Fig. 10); DN PKB decreased cAMP-stimulated PKB activity as previously reported (11). Furthermore, stimulation of PKB activity in HuH-Ntcp cells by constitutively active PKB was not associated with a change in PKC activity (0.9 Ϯ 0.11, n ϭ 3); constitutively active PKB (1.0 g) increased PKB activity 7-fold in HuH-Ntcp cells as reported previously (11). Thus, PKB does not seem to be involved in cAMP-induced PKC activation.
In separate studies, we determined whether PKB and PKC co-immunoprecipitate with each other. For these studies, either PKC or PKB was immunoprecipitated from hepatocytes or HuH-Ntcp cell followed by immunodetection of PKB or PKC, respectively. PKB and PKC do not co-immunoprecipitate (data not shown). Thus, a direct interaction between PKB and PKC seems unlikely. DISCUSSION The present study was designed to determine the role of PKC-in cAMP-induced TC uptake and Ntcp translocation in hepatocytes. Our studies in hepatocytes showed that cAMP activates PKC in a PI3K-dependent manner and that inhibition of PKC-decreases cAMP-induced increases in TC uptake and Ntcp translocation. Studies in HuH-Ntcp cells showed that cAMP and overexpression of WT PKC increase PKC activity, TC uptake, and Ntcp translocation and that these effects were blocked by DN PKC. Inhibition of cAMP-induced activation of PKC also resulted in inhibition of cAMP-induced activation of PKB. These results would suggest that cAMP-induced in-creases in TC uptake and Ntcp translocation are mediated via the PI3K/PKC/PKB signaling pathway.
The present study showed that cAMP-induced activation of PKC was dependent on PI3K, as evidenced by the inhibitory effect of wortmannin (Fig. 1). This is an expected result considering that cAMP activates PI3K in hepatocytes (38) and the PI3K product, phosphatidylinositol 3,4,5-trisphosphate, activates PKC (12). The dependence of hepatocyte PKC activity on PI3K has also been reported for the activation of PKC by insulin (17) and bile acid (20). This result raises the possibility that other cellular effects of cAMP may be mediated via the PI3K/PKC signaling pathway.
Our study also showed that the activation of PKC by cAMP was not associated with translocation of PKC to the plasma membrane (Fig. 1). A recent study in HL-60 cells also showed that cAMP does not increase PKC in the postnuclear particulate fraction (41), and we have also observed similar results in hepatocytes. 2 Thus, activation of PKC may not be associated with its translocation to membranes. It is believed that generation of diacylglycerol and calcium recruits PKCs to the membrane by engaging C1 and C2 domains of PKCs to the membrane. PKC lacks the C2 domain, and the ligand binding pocket of its C1 domain (atypical C1) is impaired (37). Although this explains why PKC does not respond to either diacylglycerol or calcium, it may also be a reason for the lack of translocation to the membrane. It is of interest to note that cAMP induces translocation of PKC to the nuclear membrane in HL-60 cells (41), and activation of PKC by cAMP in PC12 cells is associated with a slight increase in cytosol (42). Thus, the membrane-targeting signal of PKC may reside on the target membrane rather than on PKC itself.
Our conclusion that the PI3K/PKC signaling pathway is involved in cAMP-mediated TC uptake and Ntcp translocation is based on complimentary results in hepatocytes and HuH-Ntcp cells. Studies in hepatocytes showed that inhibition of cAMP-induced PKC activity by Gö 6850 and PKC PS was associated with inhibition of cAMP-mediated increases in TC uptake and Ntcp translocation. These results are consistent with a role for PKC but do not rule out other possibilities. For example, the differential effects Gö 6850 and Gö 6976 may be caused by inhibition of PKC␦ or PKC⑀, because Gö 6850, but not Gö 6976, also inhibits PKC␦ and PKC⑀ in Sf9 cells (39). The effect of PKC PS may also be caused by inhibition of PKC. To rule out these possibilities, studies were conducted in HuH-Ntcp cells transfected with WT PKC and DN PKC. These studies showed that WT PKC increased TC uptake and Ntcp translocation, and the effects of cAMP and WT PKC were inhibited by DN PKC (Fig. 6 and 7). Because PKC is the common denominator in all cases, these results, taken together, strongly implicate the PI3K/PKC signaling pathway in cAMP-mediated TC uptake and Ntcp translocation in hepatocytes.
In the present study, DN PKC did not inhibit basal PKCzeta activity but did inhibit PKC activation by cAMP and WT PKC. This is not an unusual finding. For example, over-expression of kinase inactive PKC does not inhibit basal PKC activity in adipocytes but does inhibit insulin-stimulated PKC activity (18). Moreover, overexpression of DN PKB or kinaseinactive PKB does not inhibit basal PKB activity in hepatocytes (11) and myocytes (8). It should also be noted that DN kinase inhibits "basal" activity when the "basal" activity is caused by overexpression of wild-type kinase, as we have observed in the present study (Fig. 7). One likely explanation may be that because overexpression of DN kinase affects activation by competing with wild-type kinase, such overexpression may not affect the basal activity, which is caused by already activated kinase. Overexpression of DN kinase, however, affects activation of the kinase by a stimulant, because this requires activation of inactive kinase.
In a previous study, we reported that cAMP-induced Ntcp translocation is mediated via the cAMP/PI3K/PKB signaling pathway (11). Thus, it would seem that both PI3K/PKC and PI3K/PKB signaling pathways are involved in cAMP-induced Ntcp translocation. Insulin-mediated Glut4 translocation has also been shown to be dependent on both PKC/ and PKB (43), although atypical PKCs seem to play a prominent role in the Glut4 translocation induced by insulin and non-insulin agonists (44 -46). It should be noted that inhibition of either PKB activity (11) or PKC activity resulted in near complete inhibition of cAMP-induced Ntcp translocation and TC uptake. Such a result would be consistent with the following possibilities: either a common mediator downstream of PKC and PKB further mediates the effect of cAMP and/or there is a cross-talk between PKC and PKB. Because translocation of transporters involve vesicle trafficking and cAMP stimulates vesicle trafficking in hepatocytes (47), PKC and PKB may stimulate vesicle trafficking by affecting any of the many proteins (putative common mediator) involved in vesicle trafficking. However, the identity of such a common mediator(s) is unknown at this time.
Results of the present study are consistent, however, with cross-talks between PKB and PKC in cAMP-induced Ntcp translocation. PKC PS and DN PKC, which inhibit cAMPstimulated PKC activity ( Fig. 4 and 6), also inhibit cAMPinduced increases in PKB activity ( Fig. 8 and 9). DN PKB, which inhibits cAMP-induced activation of PKB (11), does not inhibit cAMP-induced activation of PKC (Fig. 10). These results suggest that cAMP-induced activation of PKB is dependent on cAMP-induced activation of PKC. In that case, cAMPinduced Ntcp translocation may involve the following signaling sequence: cAMP-induced activation of the PI3K/PKC signaling pathway leads to the activation of PKB, which in turn stimulates Ntcp translocation. Such a mechanism would be consistent with the finding that inhibition of either cAMPinduced increases in PKB (11) or PKC activity resulted in near-complete inhibition of cAMP-induced Ntcp translocation and TC uptake ( Fig. 5 and 6). A cross-talk between PKB and PKC is feasible based on our current understanding of the PI3K-dependent activation of PKB and PKC, which can also explain why WT PKC may not activate PKB, as discussed below.
PI3K is one of the phosphoinositide kinases that phosphorylate the inositol ring at the 3 position. The resulting phosphoinositide phosphates, acting in concert with PDKs, are involved in the activation of downstream kinases, such as PKC/, PKB/ Akt, and p70 S6K (6,22,48). Although the activation of PKC is a result of phosphorylation by PDK1 followed by autophosphorylation, the mechanism of activation of PKB remains controversial (6,37). PKB activation has been proposed to involve phosphoinositide phosphate-dependent phosphorylation by PDK1, followed by either autophosphorylation or phosphorylation by a second kinase, PDK2, that phosphorylates and activates PKB fully when associated with PKC (37,40). Thus, it is possible that activation of PKC by cAMP allows PDK2 to fully phosphorylate and activate PKB in hepatocytes. Activation of PKB requires generation of phosphoinositide phosphates to target PKB to the membrane before phosphorylation by PDK1 (37). Thus, activation of PI3K by cAMP (38) is a necessary step in the activation of PKB by PKC. Overexpression of WT PKC (a downstream kinase) is not expected to stimulate PI3K, which may explain why WT PKC does not stimulate PKB in HuH-Ntcp cells. It may be noted that PKC has been reported to be a negative regulator of PKB in breast cancer cells (49), vascular smooth muscle cells (50), and COS-7 cells (51). However, this is unlikely in hepatocytes because cAMP activates both PKB and PKC. It seems, then, that the nature of crosstalk between PKC and PKB is cell-specific.
Based on the above discussions, it can be proposed that cAMP-induced activation of PKC is necessary for cAMP-induced activation of PKB and PKB is the downstream mediator of cAMP-induced Ntcp translocation. Thus, the signaling pathway can be as follows: cAMP/PI3K/PKC/PKB. This signaling pathway however does not explain our result that overexpression of WT PKC stimulates Ntcp translocation (Fig. 7) without activating PKB. Thus, a PKC-dependent, but PKB-independent, mechanism may also be involved in Ntcp translocation.
In summary, the present study revealed that the PI3K/PKC signaling pathway activated by cAMP is involved in cAMPinduced stimulation of Ntcp translocation in hepatocytes. In addition, cAMP-induced activation of PKB is dependent on cAMP-induced stimulation of PKC. It is proposed that cAMPinduced Ntcp translocation involves the activation of the PI3K/ PKC signaling pathway followed by the activation of the PI3K/ PKB signaling pathway.