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J. Biol. Chem., Vol. 276, Issue 49, 46436-46444, December 7, 2001

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Functional Role of Protein Kinase B/Akt in Gastric Acid Secretion^*

Andrea Todisco, Nonthalee Pausawasdi, Saravanan Ramamoorthy, John Del Valle, Rebecca W. Van Dyke, and Frederick K. Askari

From the Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109

Received for publication, October 23, 2000, and in revised form, September 13, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Epidermal growth factor (EGF) stimulates gastric acid secretion and H⁺/K⁺-ATPase -subunit gene expression. Because EGF activates the serine-threonine protein kinase Akt, we explored the role of Akt in gastric acid secretion. Akt phosphorylation and activation were measured by kinase assays and by Western blots with an anti-phospho-Akt antibody, using lysates of purified (>95%) canine gastric parietal cells in primary culture. EGF induced Akt phosphorylation and activation, whereas carbachol had no effect. LY294002, an inhibitor of phosphoinositide 3-kinase, completely blocked EGF induction of Akt phosphorylation, whereas the MEK1 inhibitor PD98059 and the protein kinase C inhibitor GF109203X had no effect. We examined the role of Akt in H⁺/K⁺-ATPase gene expression by Northern blotting using a canine H⁺/K⁺-ATPase -subunit cDNA probe. The parietal cells were transduced with a multiplicity of infection of 100 of the adenoviral vector Ad.Myr-Akt, which overexpresses a constitutively active Akt gene, or with the control vector Ad.CMV--gal, which expresses -galactosidase. Ad.Myr-Akt induced H⁺/K⁺-ATPase -subunit gene expression 3-fold, whereas it failed to stimulate the gene cyclooxygenase-2, which was potently induced by carbachol in the same parietal cells. Ad.Myr-Akt induced aminopyrine uptake 4-fold, and it potentiated the stimulatory action of carbachol 3-fold. In contrast, Ad.Myr-Akt failed to induce changes in either parietal cell actin content, measured by Western blots with an anti-actin antibody or in the organization of the actin cellular cytoskeleton, visualized by fluorescein phalloidin staining and confocal microscopy. Transduction of the parietal cells with a multiplicity of infection of 100 of the adenoviral vector Ad.dom.neg.Akt, which overexpresses an inhibitor of Akt, blocked the stimulatory effect of EGF on both aminopyrine uptake and H⁺/K⁺-ATPase production, measured by Western blots with an anti-H⁺/K⁺-ATPase -subunit antibody. Thus, EGF induces a cascade of events in the parietal cells that results in the activation of Akt. The functional role of Akt appears to be stimulation of gastric acid secretion through induction of H⁺/K⁺-ATPase expression.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Epidermal growth factor (EGF)¹ is the prototypic member of a large family of peptide growth factors that are known to exert numerous biological actions in the gut (1). In the stomach, EGF affects acid secretion in a divergent fashion. Under acute conditions, EGF has a long recognized inhibitory effect on acid secretion, whereas prolonged administration of EGF increases both basal and maximal acid secretion in vivo (2-5). Similar results have been observed in vitro, using preparations of highly purified gastric parietal cells in primary culture, which have been shown to express specific receptors for EGF/transforming growth factor- (2, 4, 6, 7). In this system, prolonged exposure of the parietal cells to EGF (>16 h) has been shown to enhance the stimulatory effects of carbachol, gastrin, and histamine on gastric acid secretion (2, 4, 6). The mechanisms responsible for this phenomenon are currently only partially understood.

One hypothesis, based on in vivo observations, is that EGF stimulates the proliferation of progenitor neck cells present in the gastric glands, leading to an overall increase in the number of acid-producing parietal cells present in the gastric mucosa (5). Alternatively, recent studies performed both in isolated, cultured, parietal cells and in AGS human gastric adenocarcinoma cells have indicated that EGF is able to induce both the expression and the transcription of the H⁺/K⁺-ATPase -subunit gene (6, 8), suggesting that EGF might have a direct stimulatory effect on the expression of the gene responsible for gastric acid production.

Little is known about the intracellular signal transduction mechanisms that mediate the stimulatory effects of EGF on gastric acid production and on H⁺/K⁺-ATPase -subunit gene expression. Some studies, performed in isolated gastric parietal cells in primary culture, have suggested that the stimulatory actions of EGF on gastric acid secretion could be mediated by activation of the MAPK signal transduction pathway because they can be fully reversed either by genistein, a proteintyrosine kinase inhibitor, or by the MEK1 inhibitor, PD98059 (2, 9, 10).

However, another signal transduction pathway that has been recently shown to play an important role in the mediation of some of the physiological actions of EGF is that involving phosphoinositide 3-kinase (PI3K) and protein kinase B/Akt (11-14). Three major isoforms of Akt have been identified and described thus far (12, 14). Although Akt 1 and Akt 2 are ubiquitously expressed, Akt 3 appears to be expressed predominantly in the brain, heart, and kidney (12). Activation of Akt is known to induce cellular growth and survival and to promote the expression and the maintenance of highly differentiated cellular phenotypes (11, 12, 15, 16). In addition, Akt has also been shown to regulate the translocation of GLUT-4 to the plasma membrane (12, 16) and to contribute to the regulation of vesicular trafficking and endocytosis (12). Akt is homologous to the cAMP-dependent protein kinase and PKC families of protein kinases (12, 17). In vivo, the activity of Akt is regulated by growth factors and serum through the induction of PI3K (12, 17, 18). Phosphorylation of Akt appears to be critical for Akt activation (12, 17, 18). The major phosphorylation sites required for activation of Akt 1 have been identified as threonine 308 and serine 473, which are the targets of the phosphoinositide-dependent kinases I and II, respectively (12, 17). Similarly, Akt 2 and Akt 3 are also phosphorylated on serine and threonine residues (Thr-309 and Ser-474 in Akt 2 and Thr-305 and Ser-472 in Akt 3) by phosphoinositide-dependent kinases I and II (12, 14). Because little is known about the regulation and function of the protein kinase Akt in the stomach, we undertook these studies to examine the role of Akt in EGF-stimulated gastric acid production using highly purified gastric parietal cells in primary culture.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Adenoviral Vectors-- The replication-defective adenoviral vectors expressing wild type, HA-tagged Akt 1 (Ad.wt.Akt), dominant negative HA-tagged, Akt 1 (with serine 473 and threonine 308 mutated to alanine) (Ad.dom.neg.Akt), or HA-tagged, myristoylated, constitutively active Akt 1 (Ad.Myr-Akt), under the control of the CMV promoter, were gifts of K. Walsh (Tufts University, Boston, MA) (19). The adenoviral vector expressing the -galactosidase enzyme under the control of the CMV promoter (Ad.CMV--gal) was described previously (20).

Primary Parietal Cell Preparation and Culture-- For preparation of primary parietal cells we utilized a modification of the method of Soll (21) and others (22-26). The mucosal layer of freshly obtained canine gastric fundus was bluntly separated from the submucosa and rinsed in Hanks' balanced salt solution containing 0.1% bovine serum albumin. The cells were dispersed by sequential exposure to 0.35 mg/ml collagenase and 1 mM EDTA, and parietal cells were enriched by centrifugal elutriation using a Beckman JE-6B elutriation rotor. Our best preparations contained up to 70% parietal cells as determined by hematoxylin and eosin and periodic acid-Shiff reagent staining. The parietal cells are further purified by centrifugation through density gradients generated by 50% Percoll (Amersham Pharmacia Biotech) at 30,000 × g for 20 min. The cell fraction at  = 1.05 consists of virtually all parietal cells as determined by staining with a specific mouse monoclonal antibody against the hog H⁺/K⁺-ATPase -subunit (a gift of A. Smolka, Charleston, SC) (26). The isolated parietal cells (2 × 10⁶ cells/well) are cultured according to the methods of Ljungstrom and Chew (22) with some modifications (25, 26). Briefly, the cells are cultured in Ham's F-12/Dulbecco's modified Eagle's medium (1:1) containing 0.1 mg/ml gentamycin, 50 units/ml penicillin G, and 2% dimethyl sulfoxide (Sigma) on 6-well culture dishes (Corning Inc., Corning, NY) coated with 150 µl of H₂O-diluted (1:5) growth factor reduced Matrigel (Becton Dickinson, Bedford, MA). For our studies, the parietal cells were incubated with either EGF (Becton Dickinson) (10⁸-10⁶ M) or carbachol (Sigma) (10⁴ M), for various time periods. In some experiments PD98059 (5 × 10⁵ M) (New England Biolabs, Beverly, MA), GF109203X (3.5 × 10⁶ M) (Calbiochem, La Jolla, CA), and LY294002 (10⁵ M) (Calbiochem) were added 30 min prior to the addition of the stimulants. PD98059, GF109203X, and LY294002 were dissolved in dimethyl sulfoxide. All other test substances were dissolved in water. The parietal cells were transduced, when indicated, with a multiplicity of infection of 100 of the adenoviral vectors expressing wild type Akt, dominant negative Akt, constitutively active Akt, or -galactosidase for different time periods.

Northern Blot Analysis-- The parietal cells were lysed with TRIzol (Life Technologies, Inc.) according to the manufacturer's instructions. Northern blot hybridization assays were performed as described previously (27). Equal amounts of each RNA sample, with ethidium bromide (10 mg/ml) in a final volume of 20 µl, were electrophoresed on a 1.25% agarose gel containing formaldehyde, and the RNA was transferred from the gel to nitrocellulose filters. The ethidium-stained ribosomal RNA bands in the gel were photographed before and after transfer to ensure that equivalent amounts of RNA were loaded onto each lane and that no residual RNA was left on the gel. The canine H⁺/K⁺-ATPase -subunit and the human cyclooxygenase-2 cDNA probes were gifts of I. Song and L. Crofford, respectively (University of Michigan). The human glyceraldehyde-3-phosphate dehydrogenase cDNA probe was obtained from CLONTECH (Palo Alto, CA). The cDNAs were labeled with [³²P]dCTP by the random priming procedure, and the nitrocellulose filters were hybridized to the ³²P-labeled cDNA probes as described previously (27).

Immunoprecipitations and Kinase Assays-- Immunoprecipitations and kinase assays were performed according to techniques described previously (26, 27) with minor modifications. The parietal cells were lysed in 500 µl of lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1.5 mM MgCl₂, 1 mM Na₃VO₄, 10 mM NaF, 10 mM Na₄P₂O₇·10 H₂O, 1 mM 4-(2-aminoethyl) benzenesulfonylfluoridehydrochlorine (ICN-Biomedicals, Aurora, OH), 1 µg/ml leupeptin, and 1 µg/ml aprotinin). The lysates were transferred into microcentrifuge tubes and spun at 16,000 × g for 20 min at 4 °C. Equal amounts of protein from each treatment group (300 µg) were incubated with a monoclonal anti-Akt kinase antibody immobilized on agarose beads and recognizing all Akt isoforms (New England Biolabs). Protein concentrations were measured by the Bradford method (28). After an overnight incubation at 4 °C, the solutions were centrifuged, and the pellets were washed once with lysis buffer and twice with kinase buffer. Immunoprecipitated Akt kinase was used to phosphorylate glycogen synthase kinase 3. GSK3 phosphorylation was measured by Western blots with a specific anti-phospho-GSK3/ antibody. The reactions were carried out according to the instructions of the Akt kinase assay kit from New England Biolabs.

Western Blots-- For most blots, the parietal cells were lysed as described previously under "Materials and Methods." For the H⁺/K⁺-ATPase -subunit Western blots, the parietal cells were lysed according to a different protocol (29). Briefly, the cells were washed once in PBS and spun at 1500 RPM for 10 min at 4 °C. After removal of the supernatant, the pellets were resuspended in 500 µl of homogenization buffer (10 mM Pipes (Sigma) with Tris, pH 6.8, 0.25 M sucrose, 2 mM EDTA, 1 mM benzamidine, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 µg/ml aprotinin). The solutions were sonicated on ice (twice for 5 min each) (Braun). After sonication, the samples were homogenized in 1-ml Dounce micro-homogenizers (~15 strokes). The cell homogenates were spun at 1000 rpm for 5 min at 4 °C. The supernatants were transferred to Eppendorf tubes. Protein concentrations were measured by the Bradford method (28). 80 µg (5-10 µg for the H⁺/K⁺-ATPase -subunit Western blots) of parietal cell lysates were mixed with 5× electrophoresis buffer (for 5 ml: 2.5 ml of glycerol, 1.25 ml of 2-mercaptoethanol, 0.5 g of SDS, 1.043 ml of 1.5 M Tris, pH 6.8, and 1.25 mg of bromphenol blue), boiled for 5 min (except for the H⁺/K⁺-ATPase -subunit Western blots, in which the samples were not boiled), and loaded on 10% SDS-polyacrylamide mini-gels that were run at 200 volts for 1 h. The gels were transferred on an Immobilon-P transfer membrane (Millipore, Bedford, MA) in 25 mM Tris, 150 mM glycine, and 20% methanol. After transfer the membranes were blocked in 10 ml of TBST (20 mM Tris, 0.15 M NaCl, and 0.1% Tween) and 5% dry milk for 1 h and then incubated for 16-18 h at 4 °C in 10 ml of TBST and 5% dry milk containing either a specific anti-phospho-Akt antibody, which recognizes phosphorylated serine 473 of Akt, or an anti-phospho-AFX antibody, which recognizes phosphorylated serine 193 of the transcription factor AFX (1:1000). Control blots were performed using antibodies recognizing all isoforms of Akt kinase independent of their phosphorylation states (1:1000) (New England Biolabs). For the H⁺/K⁺-ATPase -subunit Western blots the membranes were incubated for 16-18 h at 4 °C in TBST and 5% dry milk containing a specific mouse monoclonal antibody against the hog H⁺/K⁺-ATPase -subunit (26) antibody (1:5000). In some experiments the membranes were incubated for 16-18 h at 4 °C in 10 ml of TBST and 5% dry milk, containing 20 µg of a monoclonal anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). For the GSK3 phospho-blots, the membranes were incubated for 16-18 h at 4 °C in 10 ml of TBST and 5% bovine serum albumin (Sigma) containing a monoclonal anti-phospho-GSK3/ antibody (1:1000) that recognizes phosphorylated serine 21 of GSK3 and phosphorylated serine 9 of GSK3, (New England Biolabs). At the end of the incubation periods the membranes were washed in TBST for 30 min at room temperature and then incubated for 1 h in TBST and 5% dry milk containing either protein A directly conjugated to horseradish peroxidase (Amersham Pharmacia Biotech) (1:2500) for the Akt and the AFX blots or an horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2000) for the GSK3, actin, and H⁺/K⁺-ATPase -subunit blots. The membranes were washed in TBST for 30 min at room temperature and then exposed to the Amersham ECL detection system according to the manufacturer's instructions.

Amplification and Purification of Adenoviral Vectors-- Briefly, the recombinant adenoviruses were amplified as described previously using 293 cells (20). The viruses were subsequently concentrated and purified on a cesium chloride gradient (20). The concentration of the recombinant adenoviruses was assessed on the basis of the absorbency at 260 nm and on a limiting dilution plaque assay (20).

Immunohistochemistry-- The parietal cells were transduced with the adenoviral vectors expressing HA-tagged dominant negative Akt, HA-tagged Myr-Akt, or HA-tagged wt-Akt or with the adenoviral vectors expressing the -galactosidase enzyme and cultured on slides for 16-18 h. At the end of the incubation period the cells were fixed in 4% formalin-PBS. The slides were blocked for 30 min with 20% donkey serum and incubated for 2 h with a mouse monoclonal anti-HA antibody (1:500) (Babco Berkeley Antibody Co., Richmond, CA). The cells were then rinsed with PBS, and a 1:150 dilution of a FITC-conjugated donkey anti-mouse IgG secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA) was added for 1 h. After a wash with PBS, the cells were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and visualized by confocal fluorescence microscopy. In some experiments the parietal cells were incubated with the FITC-conjugated secondary antibody without the primary antibodies. The percentage of parietal cells expressing the HA-tagged virally expressed Akt proteins was calculated by examining both the differential interference contrast and the immunofluorescence images of transduced parietal cells in three fields of each slide.

Actin Staining by Fluorescein Phalloidin in Isolated Gastric Parietal Cells-- After either stimulation with carbachol or transduction with the adenoviral vectors, the parietal cells were fixed by treatment with 4% formaldehyde in PBS for 10 min at room temperature. After a PBS wash, the cells were permeabilized by incubation in 0.2% Triton X-100 in PBS for 10 min at room temperature and blocked with three sequential 5-min incubations in PBS containing 1% bovine serum albumin and 0.2% Triton X-100. Actin was detected by incubation of the cells for 20 min in fluorescein phalloidin (Molecular Probes, Eugene, OR) in 1% bovine serum albumin with PBS at 4 °C. After three washes with PBS, the parietal cells were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and visualized by confocal fluorescence microscopy. In control experiments the cells were incubated with PBS without fluorescein phalloidin.

Detection of Adenoviral Delivered -Galactosidase-- For identification of parietal cells transduced with the adenoviral vector expressing -galactosidase, the cells were cultured on slides for 16-18 h and stained with X-gal after 24 h of infection. The cells were washed with PBS and then fixed in 0.5% glutaraldehyde at room temperature for 10 min. After two washes with 1 mM MgCl₂ in PBS, the cells were incubated overnight at 37 °C in a solution consisting of 5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, and 2 mM MgCl₂ in PBS with 0.1% X-gal. At the end of the incubation, the cells were rinsed with PBS and observed with a light transmission microscope.

Aminopyrine Uptake-- Gastric acid secretion was measured according to methods described previously (2, 6, 9). Briefly, the accumulation of the weak base [¹⁴C]aminopyrine (Amersham Pharmacia Biotech) was used as an indicator of acid production by parietal cells. Both infected and noninfected cultured parietal cells were washed once with Earle's balanced salt solution, incubated with 0.1 µCi of [¹⁴C]aminopyrine for 60 min, and then stimulated, when indicated, with carbachol (10⁴ M) for the last 30 min of aminopyrine incubation. In some experiments, the cells were cultured for 16-18 h in the presence of EGF (10⁸ M) either alone or in combination with LY294002 (10⁵ M), prior to the addition of carbachol. The parietal cells were lysed with 500 µl of 1% Triton X-100, and the radioactivity of lysate was quantified in a liquid scintillation counter as previously reported (6, 9).

Data Analysis-- The data are expressed as the means ± S.E. Statistical analysis was performed using Student's t test. p values < 0.05 were considered to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We examined the effect of EGF on Akt phosphorylation using Western blots with a specific anti-phospho-Akt antibody directed against Ser(P)-473. As shown in Fig. 1, EGF stimulated Akt phosphorylation, with a maximal effect detected after 5 min of incubation at doses ranging between 10⁸ and 10⁶ M. Akt activation returned to base line after prolonged exposure to EGF (30-60 min (Fig. 1) and 24 h (data not shown)). By contrast, the cholinergic agonist carbachol (10⁴ M) failed to have any effect (Fig. 2A). Total Akt levels were not changed by any of the treatments.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   EGF stimulates Akt phosphorylation. Phosphorylation of Akt kinase in lysates from Percoll-purified, cultured parietal cells stimulated for 5, 30, and 60 min with EGF (108-106 M) was studied by Western blots using a specific anti-phospho-Akt antibody. Total Akt kinase levels were monitored by Western blots with an antibody recognizing Akt kinase independent of its phosphorylation state. Identical results were obtained in at least three other separate experiments.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Signal transduction pathways regulating Akt phosphorylation. Phosphorylation of Akt kinase in lysates from Percoll-purified, cultured parietal cells stimulated for 5 min with either carbachol (104 M) or EGF (108 M) (A) alone or in association with LY294002 (105 M) (B), PD98059 (5 × 105 M) (C), or GF109203X (3.5 × 106 M) (C) was studied by Western blots using a specific anti-phospho-Akt antibody. Total Akt kinase levels were monitored by Western blots with an antibody recognizing Akt kinase independent of its phosphorylation state. Identical results were obtained in at least three other separate experiments.

To confirm that phosphorylation of Akt correlated with Akt activation, we examined the effect of EGF (10⁸ M) on Akt activity measured by an in vitro kinase assay using GSK3 as substrate. GSK3 phosphorylation was quantitated by Western blots with a specific anti-phospho-GSK3/ antibody. EGF exposure for 5 min induced Akt activation, but it did not alter the amount of total Akt immunoprecipitated from the cell lysates (data not shown).

Because activation of PI3K is thought to be an important step in Akt activation, we examined whether EGF induction of Akt phosphorylation, required PI3K activity. EGF-stimulated Akt phosphorylation was inhibited by 10⁵ M LY294002 (Fig. 2B), a specific and well characterized inhibitor of PI3K, indicating that in the gastric parietal cells, EGF targets Akt through PI3K-dependent signaling pathways. In contrast, both the MAPK inhibitor PD98059 (5 × 10⁵ M) and the PKC inhibitor GF109203X (3.5 × 10⁶ M) failed to have any effect on EGF-stimulated Akt phosphorylation (Fig. 2C).

To examine the role of Akt on H⁺/K⁺-ATPase gene expression and on gastric acid production, we transduced the gastric parietal cells for 18 h with a multiplicity of infection of 100 of the adenoviral vectors expressing either a hemagglutinin-tagged myristoylated, constitutively active form of Akt or a hemagglutinin-tagged dominant negative Akt gene. Control experiments were performed with the adenoviral vector Ad.CMV--gal, which expresses -galactosidase. Noninfected cells did not stain with X-gal (Fig. 3A). In contrast, as shown in Fig. 3B, histochemical staining for -galactosidase demonstrated that more than 80% of the parietal cells were transduced with the adenoviral vector expressing -galactosidase. To document expression of both dominant negative and of constitutively active Akt, parietal cells transduced with either Ad.dom.neg.Akt or Ad.Myr-Akt, were stained with an anti-hemagglutinin primary antibody and a FITC-conjugated secondary antibody. Eighteen to twenty 1-µm-thick z sections of parietal cells from three different fields of one slide of transduced cells were analyzed by confocal microscopy to examine the localization of the virally expressed kinases. As indicated by the representative images shown in Fig. 3, hemagglutinin-tagged dominant negative Akt (Fig. 3D) and hemagglutinin-tagged myristoylated Akt (Fig. 3F) were expressed in the parietal cells. Dominant negative Akt-transduced and myristoylated Akt-transduced parietal cells exhibited similar morphology. Both isoforms of virally expressed Akt appeared to be predominantly localized at the level of the plasma membrane, although cytoplasmic staining was also apparent in some preparations, particularly in the case of the dominant negative Akt protein (data not shown). Ad.CMV--gal-transduced parietal cells stained with the anti-hemagglutinin primary antibody and the FITC-conjugated secondary antibody showed no significant cell membrane staining (Fig. 3H). The nonspecific perinuclear punctate staining noted in these cells most likely represented autofluorescence of lipofuscin in lysosomes, as described previously in epithelial cells in culture (30). The percentage of parietal cells transduced with the adenoviral vectors expressing dominant negative and myristoylated Akt varied between 50 and 80%, depending on the preparation. The data represented in Fig. 3 (D and F) indicate that more than 50% of the cells were successfully transduced. Similar results were observed when the parietal cells were transduced with the adenoviral vector Ad.wt.Akt, which overexpresses a hemagglutinin-tagged wild type Akt protein (data not shown). Expression of the hemagglutinin-tagged virally expressed Akt kinases was also demonstrated by Western blot analysis of lysates from parietal cells transduced with either Ad.dom.neg.Akt or Ad.Myr-Akt using the anti-HA antibody (data not shown). Time course studies showed that expression of HA-tagged dom.neg.Akt was detectable as early as 1 h after infection (data not shown).

View larger version (66K): [in this window] [in a new window]   Fig. 3.   Transduction of cultured gastric parietal cells with adenoviral vectors. Histochemical staining for -galactosidase of nontransduced (A) and Ad.CMV--gal-transduced (B) Percoll-purified, cultured gastric parietal cells. Parietal cells transduced with either Ad.dom.neg.Akt or with Ad.Myr-Akt and stained with a mouse monoclonal primary anti-HA antibody and a donkey anti-mouse FITC-conjugated secondary antibody are shown in D and F, respectively. Control parietal cells transduced with Ad.CMV--gal and incubated with the mouse monoclonal primary anti-HA antibody and the donkey anti-mouse FITC-conjugated secondary antibody are shown in H. Images are representative 1-µm confocal fluorescence sections. Corresponding differential interference contrast images are shown in C, E, and G. The arrows indicate specific staining of the surface plasma membrane with the anti-HA antibody. Magnification, 20× in A and B and 63× in C-H. Bars, 50 µm in A and B and 10 µm in D, F, and H. Similar results were obtained in two other separate experiments.

H⁺/K⁺-ATPase mRNA was isolated from the parietal cells and quantitated by Northern blot analysis using a canine H⁺/K⁺-ATPase -subunit cDNA probe. The autoradiograms were controlled for RNA quantity by hybridization of the RNA with a glyceraldehyde-3-phosphate dehydrogenase cDNA probe. In agreement with previously published observations (6), stimulation of the parietal cells with EGF (10⁸ M) for either 7 or 16 h induced canine H⁺/K⁺-ATPase -subunit gene expression in these gastric parietal cells (data not shown). Transduction of the parietal cells with the adenoviral vector expressing constitutively active Akt for either 7 h (data not shown) or 18 h, induced H⁺/K⁺-ATPase -subunit gene expression 3-fold, whereas no effect was observed in the presence of the control vector expressing -galactosidase (Fig. 4, A and B). Similar results were observed in Western blots with a specific anti-H⁺/K⁺-ATPase -subunit antibody, suggesting that Akt increases both the H⁺/K⁺-ATPase gene and protein in the gastric parietal cells (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Akt regulates H+/K+-ATPase -subunit gene expression. Aliquots of total RNA (10 µg) extracted following infection of Percoll-purified, cultured parietal cells for 18 h with either Ad.Myr-Akt or with Ad.CMV--gal were examined by Northern blot analysis using a 32P-labeled cDNA probe for the H+/K+-ATPase -subunit gene (A). B, results obtained from densitometric analysis of H+/K+-ATPase -subunit Northern blots derived from several parietal cell preparations. The data are expressed as fold induction over control (means ± S.E.). O.D., optical density. The numbers in parentheses indicate the number of times each experiment was repeated. Aliquots of total RNA (10 µg) extracted following either infection of the cultured parietal cells for 18 h with the adenoviral vectors (C) or after stimulation for 2 h with 104 M carbachol (D) were examined by Northern blot analysis using 32P-labeled cDNA probes for either the H+/K+-ATPase -subunit gene or cyclooxygenase-2. The data were reproduced in at least one other separate experiment. The autoradiograms were controlled for RNA quantity by hybridization of the RNA with a cDNA probe encoding the ubiquitous enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

To confirm the specificity of this observation, we examined the effect of Akt on the expression of the highly inducible gene cyclooxygenase-2. Transduction of the parietal cells for 18 h with the adenoviral vector expressing constitutively active Akt failed to stimulate cyclooxygenase-2 gene expression (Fig. 4C), which, in contrast, was potently induced by stimulation of parietal cells from the same preparation with carbachol (10⁴ M) for 2 h (Fig. 4D). In addition, stimulation with EGF (10⁸ M) for 30 min, 2 h, and 24 h failed to induce cyclooxygenase-2 gene expression in the gastric parietal cells (data not shown). These results indicate that Akt exerts specific stimulatory effects on H⁺/K⁺-ATPase gene expression in the gastric parietal cells.

To examine the functional significance of Akt in gastric acid secretion, we tested the effect of Akt on [¹⁴C]aminopyrine uptake. In agreement with previously published observations (2), carbachol led to a 5-fold, statistically significant induction in [¹⁴C]aminopyrine uptake (Fig. 5). Carbachol also significantly induced [¹⁴C]aminopyrine uptake in both Ad.CMV--gal-transduced and Ad.Myr-Akt-transduced parietal cells, indicating that the adenoviral vectors did not alter the function of the parietal cells. In addition, transduction of the cells for 18 h with the adenoviral vector expressing constitutively active Akt induced basal aminopyrine uptake 4-fold and, like EGF, led to a statistically significant enhancement of the stimulatory action of carbachol (Fig. 5). Even modestly active Akt potentiated acid secretion as shown in additional experiments performed using the adenoviral vector Ad.wt.Akt. This vector overexpresses a wild type Akt protein that has been shown to exhibit a low level of kinase activity and to respond to physiological stimuli (19, 31). Transduction of the parietal cells for 18 h with Ad.wt.Akt led to a modest induction in H⁺/K⁺-ATPase gene expression (data not shown). Ad.wt.Akt also stimulated basal aminopyrine uptake by only 1.38-fold (1602 cpm ± 813.23 in the presence of Ad.CMV--gal versus 2214.5 ± 893.55 cpm in the presence of Ad.wt.Akt, means ± S.E., n = 6) compared with the effects of Ad.Myr.Akt, yet, like EGF, it enhanced the stimulatory action of carbachol on acid secretion by 30% (data not shown). Thus, activation of Akt appears to be important for stimulation of gastric acid production.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Effect of adenoviral vectors on [14C]aminopyrine uptake. Percoll-purified cultured, canine gastric parietal cells were incubated for 30 min with 104 M carbachol after infection for 18 h with either Ad.Myr-Akt or with Ad.CMV--gal. The data are expressed as cpm (means ± S.E.). the numbers in parentheses indicate the number of times each experiment was repeated. *, p < 0.05.

To confirm the involvement of Akt in the stimulatory action of EGF on gastric acid secretion, we transduced the gastric parietal cells with an adenoviral vector expressing dominant negative Akt and measured [¹⁴C]aminopyrine uptake. Transduction of the gastric parietal cells with dominant negative Akt did not affect basal [¹⁴C]aminopyrine uptake, but it blocked the stimulatory action of EGF on carbachol-induced increase in [¹⁴C]aminopyrine uptake (Fig. 6).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effect of Ad.dom.neg.Akt on EGF enhancement of gastric acid secretion. Percoll-purified, cultured, canine gastric parietal cells were infected for 18 h with either Ad.dom.neg.Akt or with Ad.CMV--gal and incubated for 30 min with 104 M carbachol, alone or after preincubation for 16 h with 108 M EGF. The data are expressed as fold induction over control (means ± S.E.). The numbers in parentheses indicate the number of times each experiment was repeated. *, p < 0.05.

Because EGF appeared to target Akt through PI3K-dependent signaling pathways (Fig. 2B), we examined the effect of LY294002 on the stimulatory action of EGF on carbachol-induced gastric acid secretion. As shown in Fig. 7, preincubation of the parietal cells with LY294002 completely reversed the stimulatory effect of EGF on [¹⁴C]aminopyrine uptake, suggesting that EGF requires the sequential activation of PI3K and Akt kinase to regulate gastric acid production.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   LY294002 inhibits EGF enhancement of gastric acid secretion. Percoll-purified, cultured, canine gastric parietal cells were incubated for 16 h with 105 M LY294002 or for 30 min with 104 M carbachol, alone or after preincubation for 16 h with 108 M EGF in the presence or absence of 105 M LY294002. The data are expressed as fold induction over control (means ± S.E.). The numbers in parentheses indicate the number of times each experiment was repeated. *, p < 0.05.

To further demonstrate that EGF stimulation of gastric acid secretion is mediated by increased production of the H⁺/K⁺-ATPase through an Akt-dependent mechanism, we performed Western blots with a specific anti-H⁺/K⁺-ATPase -subunit antibody. As shown in Fig. 8, EGF stimulated the production of the H⁺/K⁺-ATPase -subunit, whereas dominant negative Akt inhibited this effect. Thus, EGF induces Akt to regulate H⁺/K⁺-ATPase production and gastric acid secretion in the gastric parietal cells.

View larger version (6K): [in this window] [in a new window]   Fig. 8.   EGF induces the production of the H+/K+-ATPase. H+/K+-ATPase -subunit expression in lysates from Percoll-purified, cultured parietal cells transduced for 18 h with either Ad.dom.neg.Akt or with Ad.CMV--gal and treated for 16 h with 108 M EGF was studied by Western blots with an anti-H+/K+-ATPase -subunit antibody. Identical results were obtained in two other separate experiments.

Actin and the actin cytoskeleton are thought to play an important role in the regulation of gastric acid secretion (2). Furthermore, gastrin, carbachol, and histamine have been shown to induce actin gene expression (2, 23). Accordingly, we investigated whether Akt altered the amount or organization of actin in the gastric parietal cells. As indicated in the Western blot shown in Fig. 9, transduction of the parietal cells for 18 h with Ad.Myr-Akt failed to change actin protein content, although Ad.Myr-Akt was active in the same cells as the forkhead transcription factor AFX, a known substrate of Akt (12, 32) was heavily phosphorylated.

View larger version (9K): [in this window] [in a new window]   Fig. 9.   Akt does not regulate actin expression in the gastric parietal cells. Actin expression in lysates from Percoll-purified, cultured parietal cells transduced for 18 h with either Ad.Myr-Akt or with Ad.CMV--gal was studied by Western blots with an anti-actin antibody. Control blots were performed using an anti-phospho-AFX antibody. Identical results were obtained in one other separate experiment.

We also examined the role of Akt in the organization of the actin cytoskeleton. For these studies, gastric parietal cells were stained with fluorescein phalloidin. Eighteen to twenty 1-µm-thick z sections of parietal cells from three different fields of one slide of cultured cells were analyzed by confocal microscopy. As shown in the representative images depicted in Fig. 10, treatment of the cells with 10⁴ M carbachol for 2 h induced profound changes in the organization of the actin cytoskeleton, characterized by the fusion of the small intracellular canaliculi (Fig. 10A) into larger canalicular structures, as it has been demonstrated by other investigators (Fig. 10B) (33). In contrast, transduction of parietal cells with Ad.CMV--gal (Fig. 10C), Ad.Myr-Akt (Fig. 10D), or Ad.dom.neg.Akt (Fig. 10E) for 18 h failed to induce significant changes in the organization of the actin cytoskeleton. Transduction of parietal cells obtained from the same dog preparations with Ad.Myr-Akt led to a significant increase in aminopyrine uptake (data not shown), indicating that the cells were transduced with an active preparation of the adenoviral vector. The specificity of the fluorescent staining was demonstrated by incubation of the parietal cells with PBS without fluorescein phalloidin (Fig. 10F). Thus, activation of Akt alone does not lead to reorganization of the actin cytoskeleton in these isolated parietal cells.

View larger version (38K): [in this window] [in a new window]   Fig. 10.   Akt does not regulate the organization of the actin cytoskeleton in the gastric parietal cells. Images of actin staining by fluorescein phalloidin of Percoll-purified, cultured parietal cells that were left untreated (A), stimulated for 2 h with 104 M carbachol (B), or transduced for 18 h with Ad.CMV--gal (C), Ad.Myr-Akt (D), or Ad.dom.neg.Akt (E) are shown. Control parietal cells incubated with PBS without fluorescein phalloidin are shown in F. The arrows indicate the intracellular canaliculi. The images are representative 1-µm confocal fluorescence sections. Magnification, 100×. Bar, 10 µm. Identical results were obtained in two other separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Gastric acid secretion is a complex biological process that involves the coordinated interaction of hormones, growth factors and neurotransmitters (2). The importance of carbachol, gastrin, and histamine as stimulants of gastric acid secretion has been confirmed in studies with isolated gastric parietal cells (2). These agents are known to trigger a complex cascade of intracellular events that leads to parietal cell activation and gastric acid production (2, 23). Several investigations have demonstrated that the resting parietal cells are characterized by the presence of a collapsed canalicular system and of cytoplasmic tubulovesicles containing the gastric proton pump, H⁺/K⁺-ATPase. Upon stimulation with the gastric acid secretagogues for as little as 20 min, the parietal cells rapidly develop an intracellular canalicular system bulging with microvilli with concomitant fusion of the tubulovesicles with the canaliculi (2, 33). This fusion translocates the H⁺/K⁺-ATPase from the vesicular membranes to the apical plasma membrane, where it actively pumps H⁺ ions in exchange for K⁺ (2).

Of the different acid secretagogues, carbachol is the most potent inducer of gastric acid secretion in the canine parietal cells. The signal transduction pathways that mediate this action of carbachol have been the focus of numerous investigations (2, 9, 22). In particular, mobilization of intra- and extracellular calcium appears to be a crucial event for carbachol induction of acid secretion. The specific intracellular targets of these calcium-dependent signal transduction pathways have been, to date, poorly characterized.

In addition to carbachol, gastrin, and histamine, numerous other agents are known to regulate the secretory activity of the parietal cells (2). EGF, in particular, is known to interact with specific EGF receptors present on the parietal cells to modulate gastric acid production (2, 7). Several studies conducted in isolated and cultured parietal cells have indicated that EGF has a rapid inhibitory effect on gastric acid secretion that appears to be mediated by activation of PKC (3). In contrast, prolonged incubation of the cells (>16 h) with EGF leads to enhancement of the stimulatory action of carbachol, gastrin, and histamine on gastric acid production (2, 4, 5, 6, 9). Additional investigations have suggested that EGF enhancement of secretagogue-stimulated gastric acid secretion might reflect the ability of EGF to induce the expression of the H⁺/K⁺-ATPase gene (6). A study performed in the canine parietal cells has demonstrated that EGF is able to induce the transcription of the -subunit of the H⁺/K⁺-ATPase gene through a novel EGF response element located between bases 162 and 156 (5'-GACATGG-3') relative to the cap site (6). This EGF response element was found to be homologous to the 3'half-site of the c-fos serum response element.

Because Akt is known to exert numerous important physiological functions and to mediate some of the intracellular effects of EGF (13, 14), we explored the possibility that Akt could be one of the kinases that participate in the complex series of events that mediate the stimulatory action of EGF on H⁺/K⁺-ATPase gene expression and gastric acid production. In this study, we reported that EGF potently stimulates Akt activation in the gastric parietal cells. In addition, although in the parietal cells EGF induces both MAPK and PKC, we showed that EGF stimulates Akt through PKC- and MAPK-independent signaling pathways. In fact, neither the PKC inhibitor GF109203X nor the MEK1 inhibitor PD98059 had any effect on EGF stimulation of Akt activation. These observations are in agreement with other studies that have indicated that MAPK is not involved in the activation of Akt (34). Although the role of PKC in Akt activation has been poorly characterized (35), our results suggest that GF109203X-sensitive PKC isoforms do not play any role in the regulation of Akt in the parietal cells. In contrast, we confirmed the involvement of PI3K-dependent signal transduction pathways, because the PI3K inhibitor LY294002 completely blocked EGF stimulation of Akt activation.

We also reported that carbachol alone does not induce Akt in the gastric parietal cells, whereas it potently activates this kinase in the PC-12 cells (36). This observation suggests the presence of cell type-specific differences regarding the ability of muscarinic receptors to induce the Akt signal transduction pathway. Furthermore, this finding indicates that induction of Akt is a signaling event that is specifically activated by EGF but not carbachol.

To analyze the functional significance of Akt activation in the parietal cells, we took advantage of adenoviral vectors expressing the wild type, constitutively active, or dominant negative forms of Akt. Using immunocytochemistry and confocal microscopy, we confirmed that these molecules could be effectively delivered to the parietal cells. As expected, the constitutively active Akt protein, which contains a myristoylation sequence, was expressed mostly at the level of the surface membrane. Although they lacked a specific membrane localization signal, both the wild type and the dominant negative Akt proteins were also predominantly localized at the level of the plasma membrane. We hypothesize that this observation might be the result of the high intracellular level of expression of both dominant negative and wild type Akt achieved by the CMV promoter.

In this study we reported that transduction of the parietal cells with Ad.Myr-Akt led to readily detectable increases in both H⁺/K⁺-ATPase gene expression and gastric acid production. We also demonstrated that EGF increases the level of the H⁺/K⁺-ATPase protein through a signal transduction pathway that is inhibited by Ad.dom.neg.Akt. These observations together with the finding that both LY294002 and Ad.dom.neg.Akt block the stimulatory effect of EGF on gastric acid production strongly support a role for Akt in EGF regulation of gastric acid secretion.

The actin cytoskeleton is an important element in secretagogue-stimulated gastric acid secretion, because during this process the parietal cell undergoes dramatic morphological modifications (2). In addition, studies conducted in the canine parietal cells have shown that the gastric acid secretagogues are able to induce the gene encoding actin, which is required for the cellular morphological transformations involved in the acid secretory process (2, 23). Accordingly, we explored the possibility that Akt might regulate the expression and the organization of actin in the gastric parietal cells. Although we were able to reproduce the well characterized changes in the organization of the actin cytoskeleton that occur in response to stimulation of the parietal cells with gastric acid secretagogues (2, 33, 37), we did not detect any morphological differences between untreated parietal cells and cells that were transduced with Ad.Myr-Akt. Similarly, we did not observe any changes in actin expression in Ad.Myr-Akt-transduced gastric parietal cells. On the basis of these observations, we suggest that although Akt induces the H⁺/K⁺-ATPase and stimulates gastric acid production, these effects are not mediated by changes in the expression and organization of actin in the parietal cells. Interestingly, similar findings were recently reported in L6 myoblasts (38), pig aortic endothelial cells (39), and SKF5 neuroectodermal cells (40) in which actin remodeling and reorganization were not mediated by the activation of Akt.

Although Akt appears to be one of the signal transduction pathways that mediate the stimulatory action of EGF on gastric acid secretion, we noted that transduction of the parietal cells with Ad.Myr-Akt did not completely mimic the effects of EGF or carbachol alone. In fact, we observed that Ad.Myr-Akt by itself markedly increased acid secretion, as does carbachol, whereas EGF did not. Further, although Ad.Myr-Akt alone increased aminopyrine uptake, it failed to influence the organization of the actin cytoskeleton, which, in contrast, was significantly affected by treatment of the parietal cells with carbachol. Accordingly, regulation of gastric acid secretion in response to stimulation of the gastric parietal cells with growth factors and neurotransmitters appears to require the activation of a complex network of signal transduction pathways.

One possible explanation for the stimulatory effect of Ad.Myr-Akt on basal gastric acid secretion might involve differences in the intensity and duration of Akt activation by EGF and Myr-Akt. Overexpression of Akt and stimulation of H⁺/K⁺-ATPase gene expression by the virus could lead to a situation of "overproduction" of the H⁺/K⁺-ATPase with saturation of the intracellular membranes and insertion of an increased number of proton pumps to the apical plasma membrane under basal conditions. In contrast, the canine parietal cell EGF receptor might be responsible for a lower level of Akt activation that can induce the H⁺/K⁺-ATPase and enhance secretagogue-stimulated gastric acid production but is not able to affect basal gastric acid secretion to a level detectable by currently available methods. In support of this hypothesis, we observed that transduction of the parietal with the adenoviral vector Ad.wt.Akt, which overexpresses a wild type Akt protein with a low level of kinase activity (19, 31), had only a modest stimulatory effect on both H⁺/K⁺-ATPase gene expression and basal gastric acid secretion. Interestingly, in some experiments, we noted that EGF by itself could lead to modest, although not reproducible, increases in basal aminopyrine uptake (1.3-1.5-fold; data not shown), suggesting that depending on the preparation, EGF, by itself, could exhibit a small stimulatory effect on gastric acid secretion. Furthermore, in contrast to what we observed in the dog, in the rabbit EGF alone has been shown to exhibit a modest, albeit statistically significant effect on aminopyrine uptake (4), supporting the notion that EGF alone can regulate gastric acid production in the gastric parietal cells.

This study also demonstrates that carbachol-stimulated acid secretion is mediated by several interacting processes, only some of which are affected by the EGF/Akt signal transduction pathway. Our observations suggest that carbachol-induced insertion of the H⁺/K⁺-ATPase on the apical membranes of the parietal cells could be enhanced in cells that posses more H⁺/K⁺-ATPase molecules, such as those exposed to EGF or transduced with either Ad.wt.Akt or Ad.Myr-Akt. However, it is clear that carbachol stimulates actin rearrangement through an EGF/Akt-independent mechanism. Maximal stimulation of gastric acid secretion appears to require both processes.

We have previously reported that activation of the parietal cell EGF receptor induces MAPK and that incubation of the parietal cells with the MEK1 inhibitor PD98059 leads to a complete reversal of EGF enhancement of carbachol-stimulated gastric acid secretion (9). Accordingly, it appears that multiple signal transduction pathways could be involved in the stimulatory effect of EGF on gastric acid production. Although a direct link between induction of MAPK and stimulation of the H⁺/K⁺-ATPase gene has not been demonstrated, it is possible that this gene might receive input from signaling pathways involving both Akt and MAPK. It is clear that additional studies are necessary to examine in more detail the relative contribution of MAPK and Akt in the mediation of the stimulatory action of EGF on H⁺/K⁺-ATPase gene expression.

Akt is known to induce the expression of highly specialized cellular phenotypes (12, 15, 16). In particular, Akt has been shown to play an important role in the complex process of myogenesis and muscle differentiation (15). Similarly, expression of constitutively active forms of Akt can induce the differentiation of 3T3-L1 fibroblasts into adipocytes (16). Another important function of Akt is to promote cell survival (12) through its ability to phosphorylate proteins such as forkhead/winged helix transcription factors (13, 32, 41), bad (12, 34, 42), and caspase-9 (43) that appear to play an important role in the induction of apoptosis. Accordingly, the ability of Akt to induce the expression of the H⁺/K⁺-ATPase gene might also represent a more generalized effect of this kinase on parietal cell differentiation and survival. It is possible that induction of Akt might be an important signal transduction pathway activated by the parietal cell EGF receptor to promote cellular differentiation and to inhibit apoptosis.

In conclusion, we have demonstrated that in the parietal cells, EGF regulates the expression of the H⁺/K⁺-ATPase through a signal transduction pathway that involves the activation of Akt. These findings suggest a novel role for Akt in the regulation of the secretory function of the gastric parietal cells, and they shed new insight into the molecular mechanisms that regulate the complex process of gastric acid secretion.

	ACKNOWLEDGEMENTS

We thank Jung Park for assistance with parietal cells preparation, Thomas Witham, Marianne Lewis, Daniel Miller, and Jace Nielsen for technical assistance, and Chris Edwards and the University of Michigan Microscopy and Image-analysis Laboratory for assistance with confocal microscopy.

	FOOTNOTES

^* This work was supported by NIDDK, National Institutes of Health Grant RO1-DK-058312 (to A. T.), as well as funds from National Institutes of Health Grant P30DK34933 to the University of Michigan Gastrointestinal Peptide Research Center.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.

Recipient of an American Gastroenterological Association Industry Research Scholar Award, a Clinical Investigator Award from the National Institutes of Health (National Institutes of Health Grant K08DK02336), and a grant from the Charles E. Culpeper Foundation Health Program. To whom correspondence should be addressed: 6520 MSRB I, Ann Arbor, MI 48109-0682. Tel.: 734-647-2942; Fax: 734-763-2535; E-mail: atodisco@umich.edu.

Published, JBC Papers in Press, September 19, 2001, DOI 10.1074/jbc.M009645200

	ABBREVIATIONS

The abbreviations used are: EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; HA, hemagglutinin; CMV, cytomegalovirus; GSK3, glycogen synthase kinase 3; PBS, phosphate-buffered saline; Pipes, piperazine-N,N-bis[2-ethanesulfonic acid]; FITC, fluorescein isothiocyanate; X-gal, 5-bromo-4-chloro-3-indolyl -D-galactopyranoside; -gal, -galactosidase.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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