Role of the PI3K/PKB signaling pathway in cAMP-mediated translocation of rat liver Ntcp

Cynthia R. L. Webster and M. Sawkat Anwer

Departments of Biomedical Sciences and Clinical Sciences, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

cAMP stimulates Na+-taurocholate (TC) cotransport by translocating the Na+-TC-cotransporting peptide (Ntcp) to the plasma membrane. The present study was undertaken to determine whether the phosphatidylinositol-3-kinase (PI3K)-signaling pathway is involved in cAMP-mediated translocation of Ntcp. The ability of cAMP to stimulate TC uptake declined significantly when hepatocytes were pretreated with PI3K inhibitors wortmannin or LY-294002. Wortmannin inhibited cAMP-mediated translocation of Ntcp to the plasma membrane. cAMP stimulated protein kinase B (PKB) activity by twofold within 5 min, an effect inhibited by wortmannin. Neither basal mitogen-activated protein kinase (MAPK) activity nor cAMP-mediated inhibition of MAPK activity was affected by wortmannin. cAMP also stimulated p70S6K activity. However, rapamycin, an inhibitor of p70S6K, failed to inhibit cAMP-mediated stimulation of TC uptake, indicating that the effect of cAMP is not mediated via p70S6K. Cytochalasin D, an inhibitor of actin filament formation, inhibited the ability of cAMP to stimulate TC uptake and Ntcp translocation. Together, these results suggest that the stimulation of TC uptake and Ntcp translocation by cAMP may be mediated via the PI3K/PKB signaling pathway and requires intact actin filaments.

mitogen-activated protein kinase; p70S6K; rapamycin; cytochalsin D; cytosolic calcium concentration; protein kinase B; phosphatidylinositol-3-kinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

BILE ACIDS UNDERGO extensive enterohepatic circulation and are efficiently transported across hepatocytes after their absorption from the intestine (2, 38). Hepatic transport of conjugated bile acids like taurocholate (TC) involves sinusoidal uptake primarily via a Na+-TC cotransport mechanism and canalicular secretion via an ATP-dependent transport mechanism (3). Although two different proteins, namely Na+-TC-cotransporting peptide (Ntcp) (20) and epoxy hydrolase (51), have been proposed to mediate Na+-TC cotransport into hepatocytes, Ntcp appears to represent the major sinusoidal membrane Na+-TC cotransporter (31). The rat liver Ntcp is a ~51-kDa serine/threonine phosphorylated glycoprotein (36) with seven transmembrane domains (1, 20, 44). Na+-TC cotransport and Ntcp are upregulated by hormones and downregulated by cholestatic agents. For example, prolactin increases Na+-TC cotransport and Ntcp mRNA in hepatocytes (29). Endotoxin, ethinyl estradiol, and extrahepatic cholestasis decrease TC uptake, Ntcp content of plasma membranes, and Ntcp mRNA (18, 34, 42). Both Na+-TC cotransport and Ntcp mass, but not Ntcp mRNA, were reduced in livers of multidrug resistance-associated protein 2 P-glycoprotein-deficient mice (25). Thus Ntcp undergoes both transcriptional and posttranscriptional regulation.

Our recent studies suggest that Ntcp also undergoes short-term posttranslational regulation. We demonstrated that cAMP, acting via protein kinase A (PKA), rapidly enhances Na+-TC cotransport in hepatocytes by increasing transport maximum (19). This effect of cAMP is potentiated by Ca2+/calmodulin-dependent processes and downregulated by protein kinase C (19). In addition, cAMP does not increase transporter synthesis but stimulates translocation of Ntcp to the plasma membrane (35). The ability of cAMP to translocate Ntcp is dependent on cAMP-mediated increases in cytosolic Ca2+ concentration ([Ca2+]) (36) and protein phosphatase 2A (37).

Recent studies suggest that the phosphatidylinositol-3-kinase (PI3K)-signaling pathway plays an important regulatory role in the translocation of insulin-regulatable glucose transporter (9, 11, 48) and Na+/H+ exchanger (26). This enzyme catalyzes the phosphorylation of phosphatidylinositols at the D3 position, yielding phosphoinositides that have been implicated in vesicular transport, cell adhesion, actin rearrangements, cell growth, and cell survival (12, 47). Determination of a role for PI3K has been facilitated by the availability of wortmannin, a potent and relatively specific inhibitor of mammalian PI3K activity (54). In addition, some of the effects of wortmannin on membrane trafficking in the endocytic pathway can be mimicked by procedures that directly interfere with PI3K functions (22). In the liver, the PI3K signaling pathway has also been shown to regulate endocytotic and transcytotic transport of fluid phase marker (17), ATP-dependent bile canalicular transporters (32, 33), and cell volume (15).

The regulatory effect of PI3K in insulin-stimulated glucose transport is proposed to be mediated via protein kinase B (PKB), also known as RAC/Akt kinase (24, 50). The activation of PKB is dependent on the presence of PI3K products and requires sequential phosphorylation by two phosphoinositide-dependent kinases, PDK1 and PDK2 (13, 43, 45). These studies raise the possibility that the PI3K/PKB pathway may be involved in cAMP-mediated translocation of Ntcp to the plasma membrane. To test this hypothesis, we studied the effect of cAMP with and without wortmannin on Na+-TC cotransport, Ntcp translocation, and PKB activity in hepatocytes. In addition, we studied the effect of cytochalasin D, an inhibitor of actin filament formation, to determine the role of microfilaments in Ntcp translocation. Our results are consistent with a role for the PI3K/PKB signaling pathway and microfilaments in cAMP-mediated Ntcp translocation.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Materials. TC (sodium salt) and rapamycin were purchased from Calbiochem (San Diego, CA). 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP), aprotinin, leupeptin, and collagenase were obtained from Sigma (St. Louis, MO). [24-14C]taurocholic acid (56 mCi/mmol) and [methoxy-3H]inulin (80 Ci/mmol) were purchased from DuPont NEN (Boston, MA). Antifusion protein antibodies to the cloned Ntcp were prepared as previously described (1, 44) and were a generous gift from the laboratories of Drs. Suchy and Meier. Male Wistar rats (200-300 g) obtained from Charles River Laboratories served as liver donors.

Hepatocyte preparation. Hepatocytes were isolated from rat livers using a previously described collagenase perfusion method (6). Freshly prepared hepatocytes suspended (100 mg wet wt/ml) in a HEPES assay buffer (pH 7.4) containing (in mM) 20 HEPES, 140 NaCl, 5 KCl, 1 MgSO4, 1.0 CaCl2, 0.8 KH2PO4, and 5 glucose were incubated for 30 min at 37°C under air before initiating studies. Four different types of experiments were conducted with hepatocytes: 1) the effect of PI3K (wortmannin and LY-294002) and p70S6K (rapamycin) inhibitors on basal and cAMP-stimulated TC uptake; 2) the effect of wortmannin on cAMP-induced translocation of Ntcp; 3) the effect of wortmannin on cAMP-induced increases in cytosolic [Ca2+]; and 4) the effect of cytochalasin D, an inhibitor of actin filament formation (10), on cAMP-induced TC uptake and Ntcp translocation. In addition, the effect of wortmannin on PKB and mitogen-activated protein kinase (MAPK) activity and the effect of rapamycin on PKB and p70S6K activity in the presence and absence of cAMP were determined. Details of these experiments are given in the legend of each figure. All studies were repeated in at least three different cell preparations.

TC uptake in hepatocytes. The initial uptake rate of TC in hepatocytes was determined as previously described (5). Briefly, at various times after incubation of hepatocytes with various agents (wortmannin, LY-294002, rapamycin, or cytochalasin D) 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 µM). Transport was initiated by adding cells to the incubation medium containing [14C]TC and [3H]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 in nanomoles per minute per milligram of protein. Further details of each uptake study are provided in appropriate figure legends.

Ntcp translocation studies. To determine whether either wortmannin or cytochalasin D affects basal and cAMP-induced changes in Ntcp translocation, hepatocytes were pretreated with either 100 nM wortmannin or 5 µM cytochalasin D before incubation with 10 µM CPT-cAMP for an additional 15 min. Cell-surface proteins were then biotinylated (see below), followed by separation of biotinylated proteins and detection of Ntcp using immunoblot analysis. This method allowed determination of cell-surface Ntcp (biotinylated) as a fraction of total cellular Ntcp using the same cell lysate as described below.

Biotinylation of cell surface proteins. Cell-surface proteins were biotinylated using a method similar to that described by Janecki et al. (21). Hepatocytes (200 mg wet wt/ml) pretreated with various agents were washed twice in ice-cold PBS (pH 8.0) and then exposed to sulfo-NHS-LC-biotin (0.5 mg/ml; Pierce) in PBS for 1 h at 4°C, followed by washing three times with excess PBS. Cell pellets were resuspended in lysis buffer (20 mM Tris, 150 mM NaCl, 1% Triton, 1 mM phenylmethlysulfonyl fluoride, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta -glycerophosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 500 nM okadaic acid, and 1 mM orthovanadate, pH 7.5) and then incubated for 1 h at 4°C. The cell lysates obtained after centrifugation at 16,500 g for 5 min were used to determine biotinylated and total Ntcp mass. To assay for biotinylated Ntcp, the lysates were incubated with streptavidin-agarose beads for 1 h. The beads were separated by centrifugation, followed by washing with lysis buffer, and then were boiled in Laemmli sample buffer for 5 min, followed by centrifugation. The resulting supernatant containing biotinylated proteins was subjected to immunoblot analysis to determine plasma membrane Ntcp. The supernatant was also analyzed for the presence of actin to determine whether cytosolic proteins were also biotinylated by our procedure. Although actin can be easily detected in the whole cell lysate, no actin was biotinylated (data not shown), indicating that cytoplasmic proteins were not biotinylated.

Immunoblot analysis. Proteins (5-50 µg) from plasma membranes, whole cell lysate, and supernatant containing biotinylated proteins were subjected to 12% SDS-PAGE by the method of Laemmli (27) as previously described (35). Proteins were transferred electophoretically from SDS gels to nitrocellulose membranes (Transblot transfer membrane, 0.45 µm; Bio-Rad) and probed with the Ntcp antibody (1:2,000 dilution). Peroxidase-conjugated anti-IgG was used as the secondary antibody. The immunoblots were developed with the Amersham enchanced chemiluminescence kit according to the manufacturer's instructions.

Protein kinase assays. The activity of PKB and MAPK was determined using commercially available assay kits from New England Biolabs (Beverly, MA). Hepatocytes were treated with 10 µM CPT-cAMP with or without 100 nM wortmannin or 200 nM rapamycin followed by preparation of cell lysates as described in Biotinylation of cell surface proteins. For PKB assay, cell lysates were subjected to 10% SDS-PAGE cells. Separated proteins were transferred electophoretically from SDS gels to nitrocellulose membranes and probed with the phospho-PKB (Akt-Ser-473) antibody (1:1,000 dilution) to detect the activated form of PKB. The blot was then stripped and reprobed with PKB (Akt) antibody to detect total PKB. For MAPK assay, activated MAPK in cell lysates was immunoprecipitated with phospho-p44/42 MAPK (Thr-202/Tyr-204) monoclonal antibody, followed by incubation with MAPK substrate (fusion protein Elk-1), 10% SDS-PAGE, and detection of the product (phospho-Elk-1) using phospho-Elk-1 (Ser-383) polyclonal antibody. The activity of p70S6K in cell lysate was determined according to the procedure provided by Upstate Biotechnology (Lake Placid, NY) using a peptide substrate. The assay was conducted in the presence of three protein kinase inhibitors (PKA, protein kinase C, and calmodulin kinase), and the activity of p70S6K was obtained after subtracting endogenous substrate phosphorylation.

Other methods. The effect of cAMP in the presence and absence of wortmannin on cytosolic [Ca2+] was determined using a Ca2+-selective fluorescence indicator, quin 2, as previously described (4). The Lowry method was used to determine cell protein (30). The blots and autoradiograms were scanned in grayscale using Adobe Photoshop (Adobe Systems, San Jose, CA), and the relative band densities were quantitated using Sigmal Gel (Jandel Scientific Software, San Rafael, CA). All values are expressed as means ± SE. Paired or Student's t-test was used to statistically analyze data, with P < 0.05 considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of PI3K inhibitors on TC uptake. When hepatocytes were pretreated with wortmannin or LY-294002 (inhibitors of PI3K) for up to 30 min, basal TC uptake remained unaltered (Fig. 1A). However, both wortmannin and LY-294002 decreased the ability of cAMP to stimulate TC uptake (Fig. 1B). The effect of wortmannin was dose dependent, with 70% inhibition at 100 nM. Because cAMP-stimulated TC uptake was inhibited by two structurally different inhibitors of PI3K, this result suggests a regulatory role for PI3K in hepatic TC uptake.


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Fig. 1.   Effect of phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY-294002 on basal (A) and cAMP-stimulated (B) taurocholate (TC) uptake. Hepatocytes were treated with inhibitors for 15 min before addition of 10 µM 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) or buffer. TC (20 µM) uptake was determined immediately before and 15 min after addition of buffer or CPT-cAMP. Difference between uptake before and after addition of CPT-cAMP is shown in B. Values are means ± SE; n = 3-5 different cell preparations. Basal TC uptake (0.85 ± 0.08 nmol · min-1 · mg protein-1) was increased to 1.49 ± 0.14 nmol · min-1 · mg protein-1 by 10 µM CPT-cAMP. * P < 0.05 vs. respective control values.

To determine whether the inhibitory effect of PI3K inhibitors persists once TC uptake is stimulated by cAMP, TC uptake was determined in hepatocytes first treated with CPT-cAMP for 20 min and then with either 100 nM wortmannin or 80 µM LY-294002 for an additional 20 min (Fig. 2). When DMSO was added instead of a PI3K inhibitor, TC uptake remained elevated. However, when either wortmannin or LY-294002 was added, TC uptake rate decreased to a level 25-30% above the basal rate. Thus PI3K inhibitors decrease the ability of cAMP to stimulate and maintain stimulated TC uptake.


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Fig. 2.   Effect of wortmannin and LY-294002 on cAMP-stimulated TC uptake. After determination of basal TC uptake, hepatocytes were treated with 10 µM CPT-cAMP for 20 min, and TC uptake was determined. Hepatocytes were then treated with either DMSO, 100 nM wortmannin, or 80 µM LY-294002, and TC uptake was determined 20 min later. Values are means ± SE; n = 4 different cell preparations. * P < 0.05 vs. values after CPT-cAMP/DMSO treatment.

Effect of wortmannin on plasma membrane and total Ntcp. To determine whether cAMP-mediated translocation of Ntcp was affected by wortmannin, we used a cell-surface biotinylation technique to determine plasma membrane Ntcp mass. In selected studies, plasma membranes were also isolated, and Ntcp mass was determined. Because similar results were obtained by both methods, results from biotinylation studies are shown. As previously reported, cAMP treatment resulted in a 55% increase in plasma membrane Ntcp mass without a change in total Ntcp mass (Fig. 3). Wortmannin alone did not affect either plasma membrane or total Ntcp mass. However, pretreatment with wortmannin inhibited the ability of cAMP to increase plasma membrane Ntcp. Thus wortmannin inhibited cAMP-mediated translocation of Ntcp to the plasma membrane.


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Fig. 3.   Effect of wortmannin with and without cAMP on Na+-TC cotransporting peptide (Ntcp) mass in cell lysate (A) and biotinylated proteins (B). Hepatocytes were treated with DMSO or 100 nM wortmannin for 15 min, followed by addition of buffer [control (CON) or wortmannin (WORT)] or 10 µM CPT-cAMP (cAMP and cAMP + WORT). Bottom: after additional 15-min incubation, cell lysates were prepared after biotinylation of cell-surface proteins. Cell lysate (Total) and biotinylated proteins (Membrane) were subjected to Ntcp immunoblot analysis using 5-20 µg protein. Typical Ntcp immunoblots are shown in A and B, and results of densitometric analysis (means ± SE; n = 5) are shown at bottom. * P < 0.05 vs. control values.

Effect of wortmannin on cytosolic [Ca2+]. The stimulatory effect of cAMP on TC uptake is in part dependent on cAMP-mediated increases in cytosolic [Ca2+] (19). To determine whether the inhibitory effect of wortmannin was due to a decrease in cAMP-mediated increases in cytosolic [Ca2+], we determined the effect of wortmannin on cytosolic [Ca2+] in the presence and absence of 10 µM CPT-cAMP. The ability of cAMP to increase cytosolic [Ca2+] in hepatocytes was not affected by wortmannin (Fig. 4). Thus the effect of wortmannin on cAMP-induced increases in TC uptake is not due to an interference with the ability of cAMP to increase cytosolic [Ca2+].


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Fig. 4.   Effect of wortmannin on cytosolic Ca2+ concentration ([Ca2+]). Hepatocytes pretreated with DMSO (0 nM wortmannin) or wortmannin (10, 50, or 100 nM) for 15 min were transferred to a cuvette. After a 100-s recording to determine basal cytosolic [Ca2+], 10 µM CPT-cAMP was added, and recording continued for another 5 min. A: representative changes in cytosolic [Ca2+] averaged over 10 s are shown. B: effect of wortmannin on cAMP-induced increases in spike concentration was expressed as percent of control (0 nM okadaic acid) Values are means ± SE; n = 3-6.

Effect of cAMP/wortmannin on PKB. Results showing that wortmannin inhibits cAMP-mediated stimulation of TC uptake and Ntcp translocation would suggest that PI3K (and/or its products) is involved in this process. Our previous studies showed that cAMP does not stimulate PI3K in hepatocytes (53), raising the possibility that cAMP may activate an effector downstream of PI3K. One such downstream effector is PKB, which requires binding to PI3K products for activation (45). To test this possibility, we determined the effect of cAMP on PKB activity in hepatocytes. Treatment of hepatocytes with 10 µM CPT-cAMP resulted in a twofold increase in phosphorylated PKB (active form) within 5 min, without any effect on total PKB (Fig. 5). Pretreatment of hepatocytes with 100 nM wortmannin (or 80 µM LY-294002; data not shown) significantly decreased basal as well as cAMP-mediated increases in phosphorylated PKB. This result indicates that cAMP activates PKB in hepatocytes and that this activation is sensitive to wortmannin and LY-294002.


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Fig. 5.   Effect of cAMP with and without wortmannin on protein kinase B (PKB) activity. Cell lysates were prepared from hepatocytes treated with 10 µM CPT-cAMP for 0, 5, 10, and 15 min in presence and absence of 100 nM wortmannin (15-min pretreatment) and then subjected to immunoblot analysis for phosphorylated PKB (pPKB) and total PKB (tPKB). A: representative blot is shown. B: results of densitometric analysis are shown. Values are means ± SE; n = 4. * P < 0.05 vs. 0-min values in absence of wortmannin. # P < 0.05 vs. values in absence of wortmannin at same time points.

Effect of rapamycin on TC uptake and protein kinases. Another downstream effector of PI3K is p70S6K. To determine whether the effect of cAMP is mediated via p70S6K, we studied the effect of rapamycin, an inhibitor of p70S6K (8). Rapamycin (200 nM) did not affect either basal or cAMP-stimulated TC uptake (Fig. 6). The activity of p70S6K was increased threefold by cAMP, and rapamycin inhibited both basal and cAMP-stimulated p70S6K activity in hepatocytes (Fig. 6). Rapamycin did not affect the ability of cAMP to stimulate PKB activity (data not shown). These results would indicate that cAMP-induced stimulation of TC uptake is not mediated via p70S6K.


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Fig. 6.   Effect of rapamycin (RAPA) on TC uptake (A) and p70S6K activity (B). Hepatocytes were treated with DMSO or 200 nM rapamycin for 30 min before addition of 10 µM CPT-cAMP (cAMP and RAPA + cAMP) or buffer (CON and RAPA), followed by determination of TC (20 µM) uptake and p70S6K activity. Values are means ± SE; n = 3 different cell preparations. * P < 0.05 vs. respective control values. # P < 0.05 vs. cAMP values.

Effect of cAMP/wortmannin on MAPK. Wortmannin has been shown to partially inhibit MAPK activation by platelet-activating factor in neutrophils (16), and MAPK has been suggested to be involved in the stimulation of bile acid secretion induced by cell swelling (39). Thus it is possible that the effect of wortmannin in hepatocytes is mediated via inhibition of MAPK. To test this possibility, we determined the effect of cAMP on MAPK in the presence and absence of wortmannin. Treatment of hepatocytes with CPT-cAMP resulted in a decrease in MAPK activity by 50 and 75% within 5 and 10 min, respectively (Fig. 7). MAPK activity in the presence and absence of cAMP was not affected by pretreatment with wortmannin. Thus it is unlikely that the inhibitory effect of wortmannin on cAMP-stimulated Ntcp translocation is secondary to its effect on MAPK in hepatocytes.


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Fig. 7.   Effect of cAMP with and without wortmannin on mitogen-activated protein kinase (MAPK) activity. Cell lysates were prepared from hepatocytes treated with 10 µM CPT-cAMP for 0, 5, 10, and 15 min in presence and absence of 100 nM wortmannin (15-min pretreatment) and then assayed for MAPK activity. Values are means ± SE; n = 3 different cell preparations. * P < 0.05 vs. respective 0-min values.

Effect of cytochalasin D on TC uptake and Ntcp translocation. Cytochalasin D did not affect basal TC uptake, but it decreased cAMP-stimulated TC uptake by 58 and 83% at 2 and 5 µM, respectively (Fig. 8). Cytochalasin D alone did not affect either plasma membrane or total Ntcp mass (Fig. 9). However, pretreatment with cytochalasin D inhibited the ability of cAMP to increase plasma membrane Ntcp. Thus cytochalasin D inhibited cAMP-mediated translocation of Ntcp to the plasma membrane, indicating that Ntcp translocation is dependent on intact microfilaments.


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Fig. 8.   Effect of cytochalasin D on basal and cAMP-stimulated TC uptake. Hepatocytes were treated with DMSO or cytochalasin D (2 or 5 µM) for 60 min before addition of 10 µM CPT-cAMP (cAMP) or buffer (Basal), followed by determination of TC (20 µM) uptake. Values are means ± SE; n = 4 different cell preparations. * P < 0.05 vs. respective control values (0 µM cytochalasin D).



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Fig. 9.   Effect of cytochalasin D with and without cAMP on Ntcp mass in cell lysate (A) and biotinylated proteins (B). Hepatocytes were treated with DMSO or 5 µM cytochalasin D for 60 min, followed by addition of buffer (CON and Cyto D) or 10 µM CPT-cAMP (cAMP and Cyto D+cAMP). Bottom: after additional 15-min incubation, cell lysates were prepared after biotinylation of surface proteins. Cell lysate (Total) and biotinylated proteins (Membrane) were subjected to Ntcp immunoblot analysis using 5-20 µg protein. Typical Ntcp immunoblots are shown in A and B, and results of densitometric analysis (means ± SE; n = 3) are shown at bottom. * P < 0.05 vs. control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study showed that PI3K inhibitors and cytochalasin D decreased the ability of cAMP to stimulate TC uptake in hepatocytes and to translocate Ntcp to the plasma membrane. In addition, cAMP activated PKB, an effect inhibited by wortmannin. These results suggest a role for the PI3K/PKB signaling pathway and microfilaments in cAMP-induced stimulation of TC uptake and translocation of Ntcp to the plasma membrane, as discussed below.

cAMP has been proposed to stimulate TC uptake by translocating Ntcp from an endosomal pool to the plasma membrane (35). Insulin-induced translocation of glucose transporter GLUT4 in adipocytes and muscles has been shown to involve PI3K (40, 48). Results of the present study with PI3K inhibitors would suggest that PI3K is also involved in cAMP-mediated translocation of Ntcp. This is supported by our findings that cAMP failed to stimulate TC uptake and failed to translocate Ntcp to the plasma membrane in the presence of PI3K inhibitors (Figs. 1-3). There is, however, an important difference between the effects of insulin and cAMP on PI3K. Although insulin stimulates PI3K in adipocytes and muscles (11), cAMP does not activate PI3K in hepatocytes (53). This may suggest either that the effect of PI3K inhibitors is not mediated via inhibition of PI3K or that cAMP stimulates the PI3K signaling pathway by activating a downstream effector.

The possibility that the effect of the PI3K inhibitors is mediated via a PI3K-independent mechanism seems unlikely for the following reasons. Similar inhibitory effects were observed by wortmannin and LY-294002 (Figs. 1 and 2), two structurally unrelated inhibitors. The possibility that wortmannin may have inhibited cAMP-stimulated PKA activity also seems unlikely because wortmannin did not affect the ability of cAMP to increase cytosolic [Ca2+] (Fig. 4). We have previously shown that cAMP, acting via PKA, increases cytosolic [Ca2+] in hepatocytes (19). Wortmannin has been shown to inhibit Ca2+-independent activation of MAPK by platelet-activating factor in neutrophils by a PI3K-independent mechanism (16). Such a mechanism is also unlikely because MAPK activity in the presence and absence of cAMP was not affected by wortmannin (Fig. 7).

The possibility that cAMP stimulates PI3K pathway by activating a downstream effector is consistent with our results. Two known downstream effectors of PI3K are PKB and p70S6K (13). PI3K phosphorylates lipids to produce phosphatidylinositol phosphates, such as phosphatidylinositol 3,4,5-trisphosphate, which in turn affect cellular functions by allowing activation of PKB and p70S6K (13). The effect of cAMP does not appear to involve p70S6K, because rapamycin, an inhibitor of p70S6K, failed to inhibit the effect of cAMP on TC uptake (Fig. 6). A recent study suggests that insulin-stimulated amino acid transport in 3T3-L1 adipocytes is also not mediated via p70S6K (46). On the other hand, cAMP activated PKB in hepatocytes, an effect inhibited by wortmannin (Fig. 5). Because an inhibitor of PKB is not available, the effect of PKB could not be studied directly. Nevertheless, on the basis of the known PI3K inhibitory effect of wortmannin and the postulated role of PKB on glucose transporter translocation (23, 50), we propose that the PI3K/PKB signaling pathway may be involved in cAMP-mediated translocation of Ntcp. It should, however, be noted that the role of PKB in glucose transporter translocation is still controversial (11).

The mechanism by which cAMP activates PKB is speculative at this point. Although the activation of PKB is usually sensitive to wortmannin, wortmannin-insensitive activation of PKB has also been reported. For example, cAMP-mediated activation of PKB in human embryo kidney cells (293 EBNA) is wortmannin insensitive (41). Ca2+/calmodulin-dependent protein kinase kinase activates PKB directly in COS-7 cells, an effect insensitive to wortmannin (55). Because the activation of PKB by cAMP in hepatocytes is sensitive to wortmannin (Fig. 5) and wortmannin did not affect cAMP-mediated increases in cytosolic [Ca2+] (Fig. 4), it is likely that the wortmannin-sensitive activation of PKB by cAMP involves PI3K products and not Ca2+/calmodulin kinase kinase. The activation of PKB requires its binding to PI3K products, followed by sequential phosphorylation at Thr-308 by PDK1 and at Ser-473 by PDK2 (13). It is possible that cAMP, acting via PKA, enhances the interaction between PI3K products and PKB and thereby allows activation of PKB without activating PI3K. A recent study reported that PKA activates rat kidney inward-rectifier K+ channels by enhancing interactions between phosphatidylinositol bisphosphate and the channel (28). It is, however, also possible that PKA activates PKB either by directly phosphorylating PKB or indirectly by activating PDK1 or PDK2.

Results of our studies with cytochalasin D (Figs. 8 and 9) suggest that cAMP-mediated translocation of Ntcp is dependent on intact microfilaments. A recent study using green fluorescence protein conjugate of Ntcp showed that cytochalasin D inhibited cAMP-mediated insertion of Ntcp into the plasma membrane (14). Insulin-mediated translocation of glucose transporter GLUT4 in L6 muscle cells is also dependent on intact microfilaments (49). PI3K products have been implicated in vesicular trafficking (12, 17), and insulin has been shown to stimulate actin polymerization by a wortmannin-sensitive mechanism (52). Recently, PKB has been suggested to be involved in glucose transporter containing vesicle translocation (7). It is tempting to speculate, based on these observations, that cAMP-mediated Ntcp translocation may involve PI3K/PKB-mediated activation of vesicular transport along microfilaments.

In summary, the present study showed that cAMP-mediated stimulation of TC uptake and Ntcp translocation involves a wortmannin-sensitive mechanism and requires intact microfilaments. Because cAMP activates PKB in a wortmannin-sensitive manner, it is proposed that the effect of cAMP is mediated via the PI3K/PKB signaling pathway. Although wortmannin (100 nM) completely inhibited cAMP-mediated translocation of Ntcp (Fig. 3), it decreased cAMP-stimulated TC uptake by only 70% (Fig. 1). Thus it is likely that the stimulation of TC uptake by cAMP may be mediated via pathways in addition to the PI3K/PKB signaling pathway. In view of other recent studies on the role PI3K on canalicular transporters (17, 32, 33), it would appear that PI3K plays an important regulatory role in bile acid transport across hepatocytes.


    ACKNOWLEDGEMENTS

This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-33436.


    FOOTNOTES

We thank Holly Jameson for excellent technical assistance and Dr. I. M. Arias for helpful discussion. We also thank Drs. P. J. Meier, B. Stieger, M. Ananthanarayanan, and F. J. Suchy for kindly providing the Ntcp antibody.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. S. Anwer, Dept. of Biomedical Sciences, Tufts Univ. School of Veterinary Medicine, 200 Westboro Road, North Grafton, MA 01536 (E-mail: sanwer{at}infonet.tufts.edu).

Received 19 July 1999; accepted in final form 16 August 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 277(6):G1165-G1172
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