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Am J Physiol Gastrointest Liver Physiol 285: G316-G324, 2003. First published April 17, 2003; doi:10.1152/ajpgi.00048.2003
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LIVER AND BILIARY TRACT

Mechanisms by which cAMP increases bile acid secretion in rat liver and canalicular membrane vesicles

Suniti Misra, Lyuba Varticovski, and Irwin M. Arias

Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

Submitted 29 January 2003 ; accepted in final form 11 April 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane. Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus. We examined mechanisms involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent taurocholate in these vesicles. Addition of a decapeptide that activates PI3K to canalicular membrane vesicles increased ATP-dependent transport above baseline activity. In contrast to effects induced by taurocholate, cAMP-stimulated intracellular trafficking of the canalicular ABC transporters was unaffected by wortmannin, and recruitment of multidrug resistance protein 2, but not bile salt excretory protein (bsep), was partially decreased by colchicine. These studies indicate that trafficking of bsep and other canalicular ABC transporters to the canalicular membrane in response to cAMP is independent of PI3K activity. In addition, PI3K lipid products are required for activation of bsep in the canalicular membrane. These observations prompt revision of current concepts regarding the role of cAMP and PI3K in intracellular trafficking, regulation of canalicular bsep, and bile acid secretion.

ATP binding cassette transporters; bile acid secretion; bile salt export protein; multidrug resistance protein 2; phosphatidylinositol 3-kinase

THE ENTEROHEPATIC CIRCULATION of bile acids efficiently provides for their availability in response to requirements for emulsification of dietary fats and results in postprandial elevations of plasma bile acids (4). The hepatic response is rapid and includes enhanced bile acid uptake, transcellular transport, and subsequent secretion in to the bile. These processes and unresolved issues regarding bile acid secretion and intracellular transport have been reviewed elsewhere (4, 43, 51). Intracellular trafficking of canalicular ATP binding cassette transmembrane proteins (ABC transporters) requires complex motor-driven vesicular movement along microtubules, phosphatidylinositol 3-kinase (PI3K) lipid products, small GTPases, and other unidentified components (21, 37). Bile salt excretory protein (bsep), which is also termed abcb11, is an ABC transporter that is required for ATP-dependent secretion of bile acids across the canalicular membrane (12). Of importance to the present study is the observation that, on the basis of kinetic analysis of transport rates using isolated canalicular membrane vesicles (CMV), bsep should be saturated at basal bile acid concentrations (30). Obviously, this could not account for rapid postprandial increments in bile acid secretion or choleretic responses that immediately follow intravenous or oral administration of taurocholate (TC) and choleretic agents, including cAMP (4, 9, 13, 21). Recent studies demonstrate that bsep and other canalicular ABC transporters, such as multidrug resistance (mdr) 1 and 3, and multidrug resistance protein 2 (mrp2), traffic from Golgi directly to the canalicular membrane (20, 39). Recruitment of these transporters to the canalicular membrane in response to TC and dibutyryl cAMP (dBcAMP), a soluble analog of cAMP, is rapid, is reversible, occurs in the absence of new protein synthesis, and is additive, which suggests that different pathways may be involved (11, 22, 27). These responses do not involve enhanced transcription or translation, which have been elucidated as regulatory determinants of sterol synthesis, secretion, absorption, and transport (25). The effects of TC on intracellular trafficking and bile secretion are reduced by pretreatment with colchicine, a microtubular inhibitor, and by wortmannin (WM), an inhibitor of PI3K (11, 27). In addition, the activity of several ABC transporters recruited in response to TC to the canalicular membrane is regulated by PI3K lipid products and restored by incubation of CMV with 3'-phosphoinositides in the presence of WM (28). These observations indicate that TC increases microtubule-dependent trafficking of ATP-dependent transporters and that bile acid secretion requires activation of PI3K. In WIF-B9 cells, cAMP and TC stimulated PI3K activity through a phosphotyrosine-independent mechanism in association with increased bile acid secretion (19).

PI3K and its 3'-polyphosphoinositide lipid products regulate many biological responses, including receptor-initiated mitogenesis, vesicular trafficking, oxidative burst, membrane ruffling, glucose uptake, and activation of membrane ion channels (3, 7). Typically, mitogens that activate protein tyrosine kinases stimulate type I PI3K-{alpha} or -{beta}, whereas those that activate G protein-coupled receptors lead to activation of PI3K-{gamma} (3, 46); however, exceptions have been noted (29, 35). Less is known about mechanisms responsible for PI3K activation by cAMP. Although cAMP-stimulated PI3K and p70 S6 kinase are sufficient to stimulate cell cycle progression (6, 34), there are additional cell type-specific mechanisms by which cAMP stimulates PI3K activity, proliferation, and bile acid secretion (6, 19, 34, 48).

The present studies were performed to identify mechanisms by which cAMP increases bile acid secretion and intracellular trafficking of ABC transporters, particularly in relation to PI3K activity in CMV. Results were compared with those described after treatment with TC (11, 27).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Reagents. Tris base, EDTA, CaCl2, sucrose, HEPES, TC, WM, colchicine, protein A-Sepharose beads, L-leucine-p-nitroanilide, L-glutamyl-p-nitroanilide, dBcAMP, and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO) and were of highest purity. A synthetic rhodamine-linked membrane-permeable 10-mer peptide based on the phosphoinositide-binding sequence of gelsolin and designated as PI3K peptide was a gift of P. Janmey (University of Pennsylvania) and J. Hartwig (Brigham and Women's Hospital, Boston, MA) (17, 18). [3H]TC (2–5 Ci/mmol) and [{gamma}-32P]ATP (6,000 Ci/mmol) were from Perkin Elmer (Boston, MA). EAG15 (polyclonal anti-mrp2) was a gift from D. Keppler (University of Heidelberg, Heidelberg, Germany); K12 (polyclonal anti-bsep) was a gift from B. Stieger (University of Zurich, Zurich, Switzerland). Polyclonal anti-P110{gamma} antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-p110{alpha} and p110{beta} and monoclonal anti-phosphotyrosine antibodies were from Upstate Biotechnology (Lake Placid, NY).

In situ perfusion of the rat liver. The procedures using animals were approved by the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats weighing 250–300 g were purchased from Charles River Farms (Wilmington, MA). Rats were anesthetized with sodium pentobarbital (50 mg/kg), and nonrecirculating single-pass liver perfusion was performed in situ according to Hems et al. (14). Liver viability was sustained by maintaining portal pressure (average 10 cmH2O), O2 supply, temperature (37°C), and buffer pH (7.35–7.40) throughout the perfusion. Animals were perfused at 30 ml/min with 95% O2-5% CO2 oxygenated Krebs-Ringer-bicarbonate buffer containing 5.5 mM glucose and 10 µM TC for 10 min as described (11, 16). [3H]TC (2 x 107 cpm/ml) was added to the perfusate 10 min later. The effect of dBcAMP on TC secretion and bile volume was determined following addition of 100 µM dBcAMP in the perfusion buffer at indicated times. The effect of WM on TC- or dBcAMP-induced bile acid secretion was determined following addition of 100 nM WM to the buffer before or after perfusion with dBcAMP as indicated. Colchicine was administered intraperitoneally over 1 min at 2.5 mg/kg in 0.5 ml of PBS at 2.5 h before perfusion. No toxicity to colchicine was apparent, and respiration and body temperature were maintained. Bile was collected every 3 min, samples were weighed to determine volume, and TC secretion was quantified. Immediately following perfusion, the liver was removed for preparation of CMV.

In vivo treatment of rats. For these experiments, rats received a single injection of PBS (control), 100 µmol dBcAMP or TC into the tail vein in a 0.5 ml PBS over 1 min, or a single intraperitoneal injection of colchicine at 2.5 mg/kg in 1 ml of PBS at 45 min before preparation of CMV or liver extracts.

Preparation of CMV. After removal from control and experimental animals, the liver was perfused briefly at room temperature with 0.25 M sucrose, 10 mM HEPES-Tris with protease inhibitors (in µg/ml: 2 aprotinin, 2 leupeptin, 2 pepstatin, 100 phenylmethylsulfonyl fluoride, and 5 benz-amidine) to remove blood, minced, and homogenized in 5 volumes of buffer. CMV were isolated from liver homogenates following nitrogen cavitation and Ca2+ precipitation as described (16). Vesicle purity was determined by using leucine aminopeptidase (38) and {gamma}-glutamyl transpeptidase as markers (32). With respect to activity in CMV compared with homogenate, enrichment was 50- to 70-fold with leucine aminopeptidase and 20- to 30-fold with {gamma}-glutamyl transpeptidase. The yield of CMV was 1–1.2 mg protein/60 g rat liver. CMV were stored in aliquots in buffer A (10 mM HEPES-Tris, pH 7.4, 0.25 M sucrose, 0.2 mM CaCl2) at -70°C until use.

Bile acid transport studies. Transport of TC was measured by an optimized rapid filtration method (16, 30). The reaction mixture contained 1.2 mM ATP, an ATP-regeneration system (3 mM creatine phosphate, 100 µg/ml creatine kinase) in buffer B (10 mM HEPES-Tris, pH 7.4, 0.25 M sucrose, 10 mM MgCl2, 0.2 mM CaCl2), and 10 µM TC, which contained a trace amount of [3H]TC. For each time point, transport was initiated by adding 20 µl of reaction mixture, which had been prewarmed for 5 min at 37°C, to CMV (20–40 µg protein) suspended in buffer A at 37°Cina final volume of 50 µl. After 1 min, the reaction was stopped by addition of 1 ml of ice-cold buffer B. Vesicles were filtered through glass microfiber filters (Whatman 0.45 µM) and washed twice with 10 ml of ice-cold buffer B. Radioactivity on the filters was measured by using a liquid scintillation counter (Beckman LS 1801). Phosphatidylcholine at 0.2 mM final concentration alone or in combination with 20 µM phosphoinositide 3,4-bisphosphate (PI 3,4-P2) or phosphoinositide 4,5-bisphosphate (PI 4,5-P2) was sonicated in 10 mM HEPES-Tris, pH 7.4, immediately before use. CMV were incubated at 37°C for 10 min with the lipids as indicated or for 20 min with PI3K peptide. Transport assays were performed in triplicate samples.

PI3K activity assay. Assays were performed using 250 ng of CMV solubilized in 0.5% NP-40. The assay was performed as previously described in a total volume of 50 µl using 150 µM ATP, 125 mM MOPS, pH 7.0, 25 mM MgCl2, 5 mM EGTA, and 0.2 mg/ml sonicated lipids: phosphatidyl serine/PI/PI 4,5-P2 (1:1:1) in sonication buffer (25 mM MOPS, pH 7.0, and 1 mM EGTA) with 15 µCi of [{gamma}-32P]ATP per assay (42). Assays proceeded at 37°C for 20 min and were stopped with 100 µl MEOH:1 N HCl (1:1), and lipids were extracted twice with 100 µl of chloroform. The organic layer was combined, dried under nitrogen gas, and analyzed by TLC. 32P-labeled phosphoinositides were resolved in water/acetic acid/n-propanol (34:1:65) and detected by autoradiography. 32P incorporation into phosphoinositide 3,4,5-trisphosphate was quantified by liquid scintillation counting of TLC spots that were scraped and eluted in scintillation fluid.

CMV extracts and immunoprecipitation. CMV were lysed in buffer that contained 0.5% NP-40, 50 mM HEPES, pH 7.5, 50 mM sodium fluoride, 5 mM sodium orthovanadate, 0.5 mM EGTA, 10% glycerol, and protease inhibitors (leupeptin and aprotinin at 10 µg/ml, pepstatin at 5 µg/ml, and PMSF at 0.5 mM) and centrifuged at 12,000 g for 10 min. The supernatant was separated, frozen immediately in aliquots by immersion in liquid nitrogen, and stored at -70°C until use. Immune precipitates were performed using 300–400 µg of cell lysate protein from control and experimental livers in a 300-µl volume incubated with primary antibody for 2–4 h and with protein A-Sepharose beads for the last hour of incubation. After beads were washed once with lysis buffer and three times with PBS, the immune complexes were sedimented by brief centrifugation and used for PI3K assay as above.

Immunoblots. Protein (25–50 µg protein/lane) was denatured and resolved on a 10% polyacrylamide gel by using a Bio-Rad minigel apparatus. Proteins were transferred to nitrocellulose membranes and blocked for 1 h with PBS containing 7% nonfat milk, 2% BSA, and 0.1% Tween 20. Membranes were washed and probed with the primary antibodies followed by secondary antibodies conjugated to horse-radish peroxidase. Immune complexes were detected by enhanced chemiluminescence reagent according to the manufacturer's instructions and quantified by densitometry (27). Membranes were reused for blotting after neutralization with 15% H2O2 or stripping at 50°C.

Statistical analysis. Statistical analysis of data was performed by ANOVA using a Bonferroni test for multiple variants or unpaired t-test for individual variants. Results with statistical significance of P < 0.05 at 95% confidence index are indicated by asterisks in Figs. 2, 3, 4, 5, 6.



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  		Fig. 2. Effect of WM and colchicine on cAMP-mediated recruitment of bile salt excretory protein (bsep) and multidrug resistance protein (mrp2) to the canalicular membrane. The content of mrp2 (open bars) and bsep (gray bars) in canalicular membrane vesicles (CMV) was quantified by Western blotting. CMV were prepared from control or rat liver perfused with WM (A) or pretreated with colchicine (B) in addition to dBcAMP. The experimental design is described in Fig. 1. One of three representative experiments is shown. Bands visualized by Western blot were quantified by densitometry. Results were expressed as %values obtained from buffer-perfused liver (control) and are shown at bottom (n = 3). Statistical analysis of the data was performed using ANOVA and Bonferroni test of multiple variants or unpaired t-test of individual variants. *P < 0.05 at 95% confidence index. **Statistically significant differences that were calculated by t-test.

 


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  		Fig. 3. Effect of WM and colchicine on cAMP-induced phosphoinositide 3-kinase (PI3K) activity. A: total PI3K activity was measured in solubilized CMV isolated from liver after perfusion with control buffer, WM, dBcAMP, dBcAMP followed by WM, and dBcAMP after WM. B: effect of colchicine on PI3K activity in CMV from control and dBcAMP-treated rats as presented in Fig. 1C legend. PI3K activity in different preparations of CMV from control animals (n = 6) varied between 2.0 and 4.0 x l0-3 cpm/mg protein. Results are expressed as %control ± SD in 3 independent experiments performed in duplicate. Statistical analysis was performed as in Fig. 2.

 


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  		Fig. 4. Effect of dBcAMP and PI3K lipid products on ATP-dependent TC transport in CMV. A: ATP-dependent transport in CMV isolated from liver perfused with buffer (open bar), dBcAMP (gray bar), WM (closed bar), WM added following dBcAMP (hatched bar), or WM followed by cAMP (blocked bar). TC transport was also measured in the same preparations of CMV after preincubation with 20 µM phosphoinositide 3,4-bisphosphate (PI 3,4-P2)or phosphoinositide 4,5-bisphosphate (PI 4,5-P2) in 0.2 mM phosphatidylcholine-containing micelles as described in MATERIALS AND METHODS. B: ATP-dependent transport in CMV isolated from liver perfused with buffer (open bars), dBcAMP (gray bar), colchicine (closed bar), or dBcAMP after pretreatment with colchicine (hatched bar). ATP-dependent TC transport was also measured in the same preparations of CMV after preincubation with 20 µM PI 3,4-P2 in 0.2 mM phosphatidylcholine-containing micelles as above. ATP-dependent TC transport in different preparations of CMV from control animals (n = 6) varied between 22 and 27 pmol TC · mg protein-1 · min-1. Results are means ± SD; n = 3 independent experiments performed in triplicate. See legend of Fig. 2 regarding statistical analysis.

 


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  		Fig. 5. Effect of TC and cAMP on activation of PI3K isoforms. PI3K activity was measured in immunoprecipitates by using anti-phosphotyrosine (a-P-Tyr), anti-p85, or p110{alpha}, -{beta}, or -{gamma} antibodies as described in MATERIALS AND METHODS. CMV were isolated from dBcAMP- (gray bar) or TC- (white bar) perfused rat liver. Results were normalized to activity obtained in anti-phosphotyrosine immune precipitates from control CMV (0.5–1 x 10-3 cpm/mg protein) and presented as %PI3K activity in CMV obtained from control liver (means ± SD; n = 3 experiments). See legend of Fig. 2 regarding statistical analysis.

 


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  		Fig. 6. PI3K activity and ATP-dependent TC transport after addition of PI3K peptide to CMV in vitro. A: PI3K activity in CMV isolated from control (open bars), dBcAMP-treated (gray bar), and TC-treated (closed bar) rats. CMV were incubated with PBS or 20 µM PI3K peptide for 10 min before solubilization. Results are normalized to phosphatidyl inositol-3 phosphate formed in control CMV (as in legend of Fig. 3) and presented as %control values (means ± SD) from 3 independent experiments performed in duplicate samples. B: ATP-dependent TC transport in CMV. Results are presented as %control values (as in legend of Fig. 4) from 3 independent experiments performed in triplicate samples. See legend of Fig. 2 regarding statistical analysis.

 



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  		Fig. 1. Effect of wortmannin (WM) and colchicine on cAMP-induced secretion of taurocholate (TC) in rat liver perfused in situ. [3H]TC secretion was quantified in bile every 3 min and expressed as nano-moles TC per 3 min. Results are representative of 3 or more independent experiments. A: rat liver was perfused in situ with Krebs-Ringer-bicarbonate buffer containing [3H]TC (control, {square}) for 10 min before addition of dibutyryl cAMP (dBcAMP; {circ}) or WM ({blacktriangleup}). B: liver perfused with dBcAMP was treated with WM 10 ({bullet}) or 25 min ({triangleup}) after the onset of dBcAMP administration. C: 3[H]TC secretion was quantified in bile during perfusion with buffer ({square}) or dBcAMP ({circ}) or in rats pretreated with 2.5 mg/kg colchicine 2.5 h before perfusion with buffer ({triangleup}) or with dBcAMP ({blacktriangleup}).

 


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
cAMP induces rapid increase in bile acid secretion that is blocked by WM and colchicine. The effect of WM and colchicine on cAMP-mediated bile acid secretion in in situ perfused rat liver was determined. To sustain initial bile flow, the liver was perfused with Krebs-Ringer-bicarbonate buffer containing 10 µCi [3H]TC and 10 µM unlabeled TC, which resulted in a baseline TC secretion of 200–230 nmol/3 min within 10 min (Fig. 1A). Addition of dBcAMP increased TC secretion up to 10-fold (to ~2,000 nmol/3 min), which became maximal within 20–30 min of perfusion and was sustained above 1,500 nmol/3 min for the duration of the experiment. When WM was administered 10 min before treatment with dBcAMP, bile acid secretion was reduced below the baseline level. Addition of WM 10 or 25 min after perfusion with dBcAMP reduced bile acid secretion by 50–60% (Fig. 1B). These data indicate that inhibition of PI3K activity blocks cAMP-induced bile acid secretion in rat liver.

Pretreatment of rats with colchicine before liver perfusion with dBcAMP reduced TC secretion by 80–90% (Fig. 1C). Perfusion of control rat liver with dBcAMP also doubled bile flow, which was reduced by 30–40% by pretreatment with colchicine (data not shown). Thus dBcAMP-mediated bile acid secretion and, to a lesser extent, bile flow require an intact microtubular system.

Effect of WM and colchicine on cAMP-mediated recruitment of ATP-dependent transporters to the canalicular membrane. We examined the effect of WM and colchicine on dBcAMP-induced recruitment of bsep and mrp2 to the canalicular membrane (Fig. 2). CMV were prepared from dBcAMP-perfused liver with or without inhibitors under the conditions described in MATERIALS AND METHODS. As previously reported for other ABC canalicular transporters (11), dBcAMP induced a two- to threefold increase in canalicular bsep and mrp2 protein content (Fig. 2A). Perfusion of liver with WM reduced canalicular bsep by 20% and mrp2 to 50% below the level detected in control animals (Fig. 2A, bottom). However, WM, given before or after treatment with dBcAMP, had no effect on cAMP-induced accumulation of bsep or mrp2 in CMV.

Pretreatment of rats with colchicine reduced mrp2 and bsep content in CMV below baseline levels as statistically demonstrated by t-test. In contrast to the lack of effect of WM on cAMP-induced recruitment of ABC transporters to the canalicular membrane, colchicine decreased dBcAMP-induced recruitment of mrp2 but had no statistically significant effect on bsep accumulation in CMV (Fig. 2B).

Effect of dBcAMP, WM, and colchicine on PI3K activity in CMV from in situ perfused liver. To correlate ATP-dependent TC transport with PI3K activity, we quantified total PI3K activity in solubilized CMV prepared from rats treated in situ with dBcAMP with or without WM or colchicine. Perfusion of the liver with dBcAMP resulted in three- to fourfold increase in total PI3K activity in CMV (Fig. 3A), which is similar to the previously reported increase in PI3K activity in CMV in response to TC (27, 28). WM decreased baseline PI3K activity to <50% and abolished dBcAMP-mediated increase in PI3K activity. Similar inhibition of PI3K activity was observed in CMV from colchicine-treated animals (Fig. 3B).

Effect of dBcAMP on ATP-dependent TC transport in CMV isolated from liver perfused with WM or colchicine. We compared the activity of ATP-dependent TC transport in CMV isolated from control and dBcAMP-treated rats with or without WM and colchicine (Fig. 4). ATP-dependent transport of TC in CMV increased 1.5- to 1.7-fold in response to dBcAMP. WM reduced baseline TC transport in CMV by 70% and abolished dBcAMP-mediated increase in TC transport (Fig. 4A). Colchicine pretreatment also reduced ATP-dependent TC transport below baseline and blocked cAMP-mediated increase of TC transport (Fig. 4B).

Effect of PI3K lipid products on ATP-dependent TC transport in CMV from rats treated with WM or colchicine. To determine whether addition of PI3K lipid products restores TC transport in CMV isolated from rats treated with WM or colchicine, CMV from rats treated in situ were incubated with different combinations of polyphosphoinositides (Fig. 4). ATP-dependent TC transport was quantified after incubating CMV with PI 3,4-P2, a product of PI3K, or its isomer, PI 4,5-P2. Addition of PI 3,4-P2 to CMV isolated from control or dBcAMP-perfused rat increased ATP-dependent TC transport ±1.5-fold, whereas addition of PI 4,5-P2 decreased TC transport below 30%. Incubation of WM-treated CMV with PI 3,4-P2 but not with PI 4,5-P2 restored ATP-dependent TC transport (Fig. 4A). Similar results were obtained when CMV from rats treated with colchicine were incubated with PI 3,4-P2 (Fig. 4B).

dBcAMP induces phosphotyrosine-independent activation of PI3K. Activation of p85-associated PI3K has been linked to phosphotyrosine-mediated signal transduction, which is detectable in anti-phosphotyrosine immune precipitates (45, 50). In addition, activation of some receptor-associated heterotrimeric G proteins leads to activation of p110{gamma} PI3K as well as p85-associated p110{beta} subunit (29). We determined whether cAMP-mediated activation of PI3K is associated with phosphotyrosine signaling and which specific subunits of PI3K are involved. Results were compared with responses observed after TC administration. Using the same amounts of protein, we quantified PI3K activity in anti-phosphotyrosine, -P110{alpha}, -p110{beta}, and -p110{gamma} immune precipitates by using CMV from rats that had received TC or dBcAMP intravenously 45 min before preparation of CMV. Anti-phosphotyrosine immunoprecipitable PI3K activity was low and did not change following administration of dBcAMP or TC (Fig. 5). A two- to threefold increase in immunoprecipitable activity was associated with p85 and with pP110{alpha}, -{beta}, and -{gamma} subunits of PI3K in CMV from rats treated with TC. In contrast, administration of dBcAMP resulted in increased anti-p85, -p110{beta}, and -p110{gamma} but not anti-p110{alpha} immune precipitates. These results indicate that activation of PI3K in response to dBcAMP and TC involves a phosphotyrosine-independent mechanism and that different subunits of type I PI3K are involved in each process.

Effect of PI3K peptide on PI3K activity and ATP-dependent transport in CMV from rats treated with dBcAMP or TC. We used a synthetic 10-mer rhodamine-linked PI3K peptide that activates PI3K in vivo and in vitro (15, 17, 18, 28). For these experiments, rats received a single intravenous injection of PBS, 100 µmol dBcAMP, or 100 µmol TC 45 min before preparation of CMV. PI3K activity and ATP-dependent transport increased two- to fourfold (Fig. 6). These data are consistent with activities measured in CMV after perfusion of liver in situ with dBcAMP described above and with previously reported data from in situ perfusion with TC (27, 28).

Incubation of CMV with PI3K peptide for 10 min stimulated PI3K activity 2.5-fold and further increased activity in CMV from dBcAMP-treated animals fourfold. The latter effect may result from dBcAMP-induced recruitment of bsep to the canalicular membrane. PI3K peptide also enhanced ATP-dependent TC transport in control vesicles and further increased transport in CMV from dBcAMP- and TC-treated rats (Fig. 6B).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The hydrocholeretic effect of cAMP has been known for a long time and is attributed to cAMP production primarily in small bile duct cells in response to secretin and other hormones released in response to food intake (9). In addition, administration in vivo of cAMP or its soluble analog dBcAMP enhances hepatocellular bile acid secretion (2) comparable with effects observed following administration of TC (27, 33) as well as vesicular-mediated transcytosis (13). In the present study, cAMP-induced bile acid secretion was inhibited by perfusion of liver with WM before or after treatment with dBcAMP or pretreatment of animals with colchicine. These data indicate that PI3K and an intact microtubular system are required for cAMP-stimulated bile acid secretion. The effect of WM on bile acid secretion was not surprising since PI3K lipid products are required for bile acid transporter function in vitro and in vivo (27, 28). The addition of the PI3K lipid product PI 3,4-P2 but not its isomer PI 4,5-P2 to CMV isolated from rat liver perfused in situ with WM or colchicine restored ATP-dependent bile acid transport, which confirms the important role of PI3K activity in the function of bsep.

PI3K and an intact microtubular system were previously reported to participate in TC- and cAMP-induced intracellular trafficking of ABC transporters and in recruitment of PI3K regulatory subunit p85 to the canalicular membrane (11, 22, 27, 28). In the present study, perfusion of rat liver with dBcAMP resulted in accumulation of bsep and mrp2 in CMV similar to that observed after TC (11, 27, 28). However, in contrast to inhibition of TC-induced recruitment of ABC transporters, WM had no statistically significant effect on cAMP-induced recruitment of bsep or mrp2, whereas colchicine decreased cAMP-induced recruitment of mrp2 but not bsep recruitment. Addition of PI3K lipid product PI 3,4,-P2 to CMV isolated from WM and colchicine-treated rats increased ATP-dependent TC secretion above control, which confirms that bsep is functionally active in these vesicles. Furthermore, addition of PI3K peptide to CMV increased PI3K activity and ATP-dependent TC transport. These results confirm that bsep can be activated in CMV isolated from WM and colchicine-perfused liver and that PI3K is required for its function. The mechanism whereby PI3K peptide increases PI3K activity in hepatocytes is not known; however, studies performed in fibroblasts suggest that its binding to PI 4,5-P2, a substrate for PI3K, makes the substrate more available to the enzyme (15).

Previous results indicated that the effects of cAMP and TC on intracellular trafficking of canalicular ABC transporters are additive (11), which led to the proposal that the two agonists affect different trafficking routes. Results from the present studies are consistent with this hypothesis. In contrast to trafficking induced by TC, canalicular mobilization of ABC transporters in response to cAMP was independent of PI3K. Our results reveal additional differences between the trafficking routes of bsep and other canalicular ABC transporters, such as mrp2. cAMP-mediated recruitment of bsep to the canalicular membrane was unaffected by colchicine, suggesting possible independence of the microtubular system. The results do not exclude a role for microtubules, because in WIF-B9 cells nocodazole completely blocked trafficking of post-Golgi endosomes that contained green fluorescent protein (GFP)-bsep or GFP-mdr1 to the canalicular membrane (Wakabayashi Y and Arias IM, unpublished observations; and Ref. 39).

Pulse-labeling studies in intact rats (20, 22) and image-analysis studies using GFP-bsep or GFP-mdr1 in WIF-B9 cells (Wakabayashi Y and Arias IM, unpublished observations; and Ref. 39) demonstrate that ABC transporters traffic from the Golgi to the canalicular membrane and that bsep has one or more large intracellular endosomal pools from which it cycles to and from the canalicular membrane. Mrp2 also trafficks from Golgi to the canalicular membrane; however, an intracellular pool (2) for mrp2 has not been demonstrated. Thus cAMP-mediated recruitment of bsep may involve a novel unidentified pathway of trafficking in polarized cells, which differs from PI3K-dependent trafficking, which is induced by TC.

All isoforms of type I PI3K are expressed in rat liver (5, 45). Type IA PI3K is composed of a catalytic and a regulatory subunit; p110 {alpha}, -{beta}, or -{epsilon} catalytic subunits associate with regulatory subunits p85, p55, or p50. Type IA PI3K can be activated by a direct interaction of the catalytic subunits with p21ras or by specific binding of the regulatory subunits to phosphotyrosyl peptides containing a YXXM motif (3). Type IB isoform p110{gamma}, which lacks an NH2-terminal p85-binding domain, is also activated by binding to p21ras and has a putative pleckstrin homology domain that interacts with phospholipids and binds to G{beta}{gamma}. Binding of G-protein {beta} and {gamma} subunits to p110 isoforms activates PI3K (29, 41). Activation of p110{beta} but not p110{alpha} has been previously linked to G protein signaling in some cells, and synergistic activation of P110{beta}/p85 but not p110{alpha}/p85 by a combination of phosphotyrosyl peptide and G{beta}{gamma} has been reported (24, 31). Following the observation that 3'-phosphoinositides can be generated by mitogenic and nonmitogenic stimuli (3, 23, 40, 44), it has been repeatedly demonstrated that type I PI3K and its downstream target, Akt, are activated in response to many signals, including cAMP (19, 26, 34, 48, 49). Our data and previous reports demonstrate that PI3K activation in response to cAMP is phosphotyrosine independent (19, 34).

Functional differences involving PI3K isoforms occur in some cells. Activation of p110{beta} has been associated with de novo DNA synthesis, whereas p110{alpha} is implicated in cell survival (1). The role of individual isoforms in vesicular trafficking and in intracellular compartmentalization of PI3K subunits has not been described in polarized cells. In the present studies, cAMP-stimulated PI3K activity was associated with p110{beta} and -{gamma} but not p110{alpha} isoform, and these effects were not associated with tyrosine phosphorylation of intracellular proteins. These results suggest a coordinate effect of cAMP on activation of types IA and IB PI3K that is independent of tyrosine phosphorylation and, probably, p21ras.

Many effects of cAMP are PKA independent, and conflicting data have been generated based on use of PKA inhibitors (6, 19, 48). Discovery of a novel cAMP receptor, Epac, which is a cAMP-activated Rap1 guanine-nucleotide exchange factor, provides a new opportunity to elucidate cAMP-mediated cell signaling (8, 10). Many cAMP effects, which were previously attributed to PKA, may require Epac/Rap1. cAMP-mediated activation of Epac and PKA has opposite effects on Akt activation (26), which has been attributed to their different intracellular localizations (36). The role of Rap 1 and Epac in cAMP-mediated vesicular trafficking in polarized cells is the subject of our current studies.

In summary, dBcAMP and TC-induced bile secretion in rat liver involves different pathways. In contrast to effects following TC administration, cAMP-stimulated intracellular trafficking of bsep and mrp2 is PI3K independent. These observations prompt revision of current concepts regarding the role of cAMP and PI3K in intracellular trafficking, regulation of canalicular ABC transporters, and bile secretion.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This research was supported, in part, by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54785 (to I. M. Arias), DK-35652 (to I. M. Arias and L. Varticovski), and 30-DK-34928 (Digestive Disease Center), and National Cancer Institute Grant CA-53094 (to L. Varticovski).


   ACKNOWLEDGMENTS
 
We thank P. Ujhazy for assistance with Western blot analysis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. M. Arias, Dept. of Physiology, Tufts Univ. School of Medicine, 136 Harrison Ave., M&V7, Boston, MA 02111 (E-mail: iarias{at}helix.nih.gov).

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. Section 1734 solely to indicate this fact.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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