Epidermal growth factor modulates pepsinogen secretion in guinea pig gastric chief cells

GASTROENTEROLOGY 1996;111:945–958

Epidermal Growth Factor Modulates Pepsinogen Secretion in Guinea Pig Gastric Chief Cells STEFANO FIORUCCI,* LUISA LANFRANCONE,‡ LUCA SANTUCCI,* ANTONIO CALABRO,§ BARBARA ORSINI,§ BARBARA FEDERICI,* and ANTONIO MORELLI* *Dipartimento di Medicina Clinica, Patologia e Farmacologia, Clinica di Gastroenterologia ed Endoscopia Digestiva, Perugia; ‡Istituto di Medicina Interna e Scienze Oncologiche, Universita` degli Studi di Perugia, Perugia; and §Clinica di Malattie Apparato Digerente, Universita` di Firenze, Firenze, Italy

Background & Aims: Although epidermal growth factor (EGF) inhibits gastric acid secretion, the effects it exerts on gastric chief cells are unknown. The aim of this study was to investigate whether EGF modulates pepsinogen release and intracellular Ca2/ concentrations ([Ca2/]i ) and whether the effect involves mitogenactivated protein (MAP) kinase, eicosanoid generation, and nitric oxide. Methods: Chief cells were obtained by sequential digestion with collagenase and Ca2/ chelation. [Ca2/]i was measured in cells loaded with Fura2 and NO generation by the NO coproduct citrulline. Results: In situ hybridization, immunohistochemistry, and immunoblotting showed that EGF receptor and MAP kinases were constitutively expressed in chief cells. EGF caused a concentration-dependent stimulation of pepsinogen secretion and MAP kinase activity and determined a 2.5–7.0-fold increase in [Ca2/]i , inositol 1,4,5-tryphosphate, prostaglandin E2 , and leukotriene B4 . Tyrosine kinase inhibitors and cyclooxygenase and lipoxygenase inhibitors reduced pepsinogen secretion and eicosanoid generation induced by EGF. EGF increased citrulline generation and guanosine 3*,5*-cyclic monophosphate accumulation sixfold; the effect was blocked by NG monomethyl-L-arginine, which is an NO synthase inhibitor. Conclusions: EGF stimulates pepsinogen secretion by activating eicosanoid generation, tyrosine kinases, MAP kinases, Ca2/, NO, and guanosine 3*,5*-cyclic monophosphate.

E

pidermal growth factor (EGF) is a member of a large peptide family that includes transforming growth factor, amphiregulin, heparin-binding EGF–like growth factor, and betacellulin, which is characterized by similarities in structure and biological function.1 – 5 All these peptides bind to the same cell surface receptor, the EGF receptor (EGF-R), which is a transmembrane glycoprotein of 180 kilodaltons.2 – 4 Binding of EGF to its receptor induces receptor dimerization and clustering that results in the activation of the receptor’s intracellular tyrosine kinase activity. Well-characterized substrates for the receptor kinase are phospholipase C and the SH2-con/ 5e12$$0052

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taining adapter protein.6 – 10 It is believed that on EGF stimulation, both the EGF-R and SH2-containing adapter protein become tyrosine-phosphorylated and bind to each other and to the Grb2-SOS complex10 that, in turn, modulates the guanosine triphosphatase–activating protein p21ras and facilitates downstream activation of mitogen-activated protein (MAP) kinase.1 – 10 Activation of EGF-R also triggers a variety of ionic changes, including transient plasma membrane polarization and an increase in the cytoplasmatic calcium (Ca2/) concentration ([Ca2/]i ).1,5 Some of these responses have been linked to the hydrolysis of polyphosphoinositides by phospholipase C, which leads to generation of 1,2-diacylglycerol and inositol 1,4,5-trisphosphate (IP3 ) and to the release of Ca2/ from intracellular stores.9,11 – 13 Activation of EGF-R also stimulates an arachidonyl-specific cytoplasmatic phospholipase A2 (cPLA2 ) that releases arachidonic acid (AA) from the plasma membrane phospholipids and results in leukotriene (LT) and prostaglandin (PG) generation.11 – 13 EGF and the transforming growth factor regulate mucosal growth and differentiation in the gastrointestinal tract, accelerate gastric ulcer healing by stimulating cell proliferation and migration, and modulate gastric mucosal blood flow.1,5,14 – 18 Moreover, administering EGF to humans and animals exerts a potent suppressive action on acid and pepsin secretion.17,18 Although pepsin is the other harmful component of Abbreviations used in this paper: AA, arachidonic acid; BAPTA, 2bis(aminophenoxy)-ethane-N,N,N*,N*-tetraacetic acid; BW775C, 3amino-1[3-(trifluoromethyl)phenyl]-2-pyrazoline hydrochloride; [Ca2/]i , cytoplasmatic calcium concentration; cNOS, constitutive nitric oxide synthase; cPLA2 , cytoplasmatic phospholipase A2 ; ED50 , mean effective dose; EGF, epidermal growth factor; EGF-R, epidermal growth factor receptor; EGTA, ethylene glycol-bis(b-aminomethyl ether)-N,N,N*,N*-tetraacetic acid; Fura-2/AM, Fura-2-acetoxymethyl; IP3 , inositol 1,4,5-triphosphate; L-NMMA, NG-monomethyl-L-arginine; LT, leukotriene; MAP, mitogen-activated protein; MBP, myelin basic protein; SNAP, S-nitroso-n-acetylpenicllamine; TCA, trichloroacetic acid; W7, N-(6-amino-hexyl)-5-chloro-1-naphthalene sulfonamide. q 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

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gastric secretion, it is also a known ulceronic agent. Inhibition of pepsin activity plays an important role in healing peptic ulcers. The effect that EGF exerts on pepsinogen secretion has never been investigated in isolated pepsinogen-secreting chief cells.19 Agonist-induced pepsinogen release by gastric chief cells is modulated by two major biochemical cascades. PGE2 , secretin, and vasoactive intestinal peptide (VIP) activate a G protein–regulated adenylate cyclase and increase adenosine 3*,5*cyclic monophosphate,20 – 23 whereas carbamylcholine (carbachol), cholecystokinin octapeptide (CCK-8)-gastrin, and LTs activate a phospholipase C, which hydrolyzes polyphosphoinositides to yield 1,2-diacylglycerol, IP3 , and Ca2/ mobilization.22,23 Carbachol and CCK-8 also stimulate a Ca2//calmodulin-dependent constitutive nitric oxide synthase (cNOS) that results in NO generation and guanosine 3*,5*-cyclic monophosphate (cGMP) accumulation.24 – 26 Inhibition of NO generation reduces the pepsinogen secretion induced by Ca2/-mediated agonists. The present study was designed to investigate whether exposure to EGF modulates pepsinogen release and Ca2/ mobilization from isolated chief cells and whether the effect is mediated by protein tyrosine kinases.27 – 30 In addition, because the mechanism through which EGF protects the gastric mucosa is still unclear, the ability of the growth factor to regulate prostanoids’ secretion and NO generation in these cells was also investigated.

Materials and Methods Male guinea pigs (200–250 g) were obtained from Charles River (Morini, Monza, Italy). HEPES, bovine serum albumin fraction V, soybean trypsin inhibitor, collagenase (type I), carbachol, ethylene glycol-bis (b-aminomethyl ether)N,N,N*,N*-tetraacetic acid (EGTA), Fura-2-acetoxymethyl (Fura-2/AM), and 2-bis(2-aminophenoxy)-ethane-N,N,N*,N*tetraacetic acid (BAPTA) were from Sigma Chemical Co. (St Louis, MO); essential amino acid mixture, 1% essential vitamin mixture, and Dulbecco’s minimal essential medium were from GIBCO (Milan, Italy); Percoll was from Pharmacia (Milan, Italy); and secretin, CCK-8, and VIP were from Peninsula Laboratories (St. Helens, Merceyside, England). T7 and SP6 RNA polymerase and pronase were from Boehringer (Mannheim, Germany); [35S]-uridine-5*-(a-thio)-triphosphate (sp act, 1250 Ci/mmol) was from New England Nuclear (Dreieich, Germany); [3H]thymidine (sp act, 83.0 Ci/mmol) was from Amersham (Little Chalfont, Buckinghamshire, England); and plasmid pGEM1 was from Promega Biotec (Heidelberg, Germany). 3-Amino-1-[3-(trifluoromethyl)phenyl]-2-pyrazoline hydrochloride (BW775C) was from Wellcome Laboratories (Beckenham, Bromley, England); L-651,392 was from Merck Frosst Canada (Montreal, Canada); N-(6-aminohexyl)-5-chloro1-naphthalene sulfonamide (W7), NG-monomethyl-L-arginine (L-NMMA), S-nitroso-n-acetylpenicilamine (SNAP), myelin

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basic protein (MBP) peptide, genestein, staurosporin, lavendustatin, and tyrphostin 51 were from Biomol (Plymouth Meeting, PA). Human recombinant EGF from Calbiochem (Nottinghamshire, Nottingham, England), Anti–EGF-R polyclonal antibody was from Pharmingen (San Diego, CA), and anti-MAP kinase polyclonal antibody from Upstate Biotechnology Inc. (Lake Placid, NY).

Effect of EGF on [3H]thymidine Uptake on SAA Cells The biological effect of EGF was first assessed by determining the rate of [3H]thymidine incorporation on transfected NIH 3T3 fibroblasts overexpressing the EGF-R (SAA) cells.8 Cells were grown in Dulbecco’s minimal essential medium containing 10% fetal calf serum and, after serum starvation for 3 days, incubated with increasing concentration of EGF (0.06–1.4 nmol/L) in the presence of 1 mCi/well of [3H]thymidine. Twenty-four hours later, the medium was aspirated, and the cells were washed twice in ice-cold phosphatebuffered saline (PBS) at pH 7.4 and treated with 5% trichloroacetic acid (TCA) for 10 minutes at 47C. The amount of [3H]thymidine incorporated in the TCA-insoluble material was quantitated in a Beckmann beta scintillation counter.

Chief Cell Preparation Chief cells from a guinea pig stomach were prepared as previously described by Raufman et al.23 This method yields a cell population that is approximately 90% chief cells, 7% parietal cells, and 3% other cells. The purity of each chief cell preparation was verified daily using light microscopy, and the number of cells were counted. Chief cells were suspended in a standard incubation solution containing 24.5 mmol/L HEPES, 120 mmol/L NaCl, 7.2 mmol/L KCl, 1.5 mmol/L Ca2Cl, 0.8 mmol/L MgCl2 , 2.6 mmol/L KH2PO4 , 14 mmol/L glucose, 6 mmol/L Na pyruvate, 6 mmol/L glutamate, 7 mmol/L fumarate, 2 mmol/L glutamine, 0.1% (wt/vol) trypsin inhibitor, 0.1% (wt/vol) albumin, 1% (vol/vol) essential amino acid mixture, and 1% (vol/vol) essential vitamin mixture. The pH was 7.4, and all incubations were performed at 377C with 100% O2 .

In Situ Hybridization Cell suspensions (100–150 mL) containing 1.5 1 105 chief cells were centrifuged on gelatin-coated slides at 500 rpm. Cells were air-dried and fixed in 4% paraformaldehyde/ PBS for 20 minutes. After two washes in PBS, dehydration in graded ethanols, and short air-drying, slides were used for in situ hybridization. To obtain RNA probes, the 900–base paired EcoRI/HindIII fragment of the EGF-R complementary DNA clone (pHER-A64-1; ATCC 57484)14,31,32 was subcloned into the plasmid pGEM at the appropriate restriction sites. After linearization of the plasmids with either HindIII or EcoRI restriction endonuclease, T7 or SP6 RNA polymerase was used to obtain run-off transcripts of the antisense (complementary to messenger RNA) or sense (anticomplementary; negative control) strands, respectively. Transcription and labeling of

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RNA probes were performed with 60 mCi of 35S-uridine-5*(-thio)-triphosphate (1250 mCi/mmol). The specific activity routinely obtained was 1.2–1.4 1 109 cpm/mg.

Hybridization and Autoradiography Air-dried sections were treated in 0.2 N HCl, digested with 0.125 mg/mL pronase/PBS for 10 minutes at room temperature, rinsed in 0.1 mol/L glycine/PBS, fixed for 20 minutes in ice-cold 4% paraformaldehyde/PBS, and acetylated with acetic anhydride diluted at 1:400 in 0.1 mol/L trietanolamine, pH 8.0, for 10 minutes. After dehydration with graded ethanols, the sections were briefly air-dried before hybridization. Slides were incubated with 25 mL of hybridization mixture (50% formamide, 10% dextran sulfate, 10 mmol/L dithiothreitol, 0.1 mol/L Tris-HCl, pH 7.5, 0.1 mol/L sodium phosphate, 0.3 mol/L NaCl, 50 mmol/L ethylenediaminetetraacetic acid [EDTA], 11 Denhardt’s solution, 0.2 mg/mL yeast transfer RNA) containing 2 1 105 cpm of 35S-labeled RNA probe for 18 hours at 507C. Probe excess was removed by washing it in hybridization buffer for 4 hours at 527C. To decrease background, slides were digested for 30 minutes at 377C with 20 mg/mL ribonuclease A and washed for 30 minutes at 377C in TES buffer (0.1 mol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA, and 0.5 mmol/L NaCl). After a further wash in 21 standard saline citrate for 30 minutes, the slides were dehydrated in graded ethanols and air-dried. Autoradiography was performed by dipping the dehydrated slides into Ilford G5 nuclear emulsion (Ilford, Mobberley Cheshite, England). Slides were then exposed at 47C for 30 days, developed with Kodak D19 (Kodak, Milan, Italy), rinsed for 2.5 minutes in 1% acetic acid, immersed in a Kodak fixer for 3 minutes, and counterstained with Harris H&E.14,31,32

Immunoblotting of EGF-R The anti–EGF-R monoclonal antibody was absorbed on protein A Sepharose Cl-4B (Pharmacia LKB, Uppsala, Sweden) and then incubated with cell lysates for 90 minutes at 47C. Immune complexes were washed 3 times with cold NETgel buffer (50 mmol/L Tris-HCl at pH 7.5, 150 mmol/L EDTA at pH 8.0, 0.1% [vol/vol] Nonidet P-40, 0.25% gelatin, 1 mmol/L sodium orthovanadate, and proteinase inhibitors), eluted, and denatured by heating for 3 minutes at 957C in residual Laemmli buffer. Samples were then resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, according to the standard protocol.10,33 For immunoblot analysis, either specific immunoprecipitates or 20 mg total gel lysate were electrotransferred onto nitrocellulose filters (Bio-Rad Laboratories, Hercules, CA). After blocking nonspecific reactivity, filters were probed overnight at 47C with specific antibodies diluted in Tris-buffered saline-T (20 mmol/L Tris-HCl at pH 7.8, 150 mmol/L NaCl, 0.02% Tween 20) containing 5% bovine serum albumin fraction V. Filters were then washed, and immunocomplexes detected with horseradish peroxidase– conjugated species-specific secondary antiserum (Bio-Rad) followed by enhanced chemiluminescence detection reaction (ECL

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Western blotting kit; Amersham International). SAA cells were used as the control.8

Immunohistochemistry Cytospins of gastric chief cells were air-dried overnight and fixed in acetone at 47C for 5 minutes; washed in PBS; dipped in methyl alcohol with 0.3% hydrogen peroxide for 10 minutes, which blocks endogenous peroxidase activity; washed 3 times in PBS; and incubated in 0.1% albumin for 30 minutes. Smears were then incubated for 30 minutes with a mouse anti-human EGF-R polyclonal antibody at a 1:10 dilution followed by biotinylated goat anti-mouse immunoglobulin G for 30 minutes at room temperature. After another wash in PBS, smears were incubated with a peroxidase-labeled acetyl-avidin biotinylated complex for 30 minutes and color developed with 3,3*-diaminobenzidine tetrahydrochloride for 15 minutes. Smears were then counterstained with hematoxylin and mounted in Acquovitrex (Carlo Erba, Milan, Italy). Controls were obtained by omitting the primary antibody.

Effect of EGF on Pepsinogen Secretion Chief cells (300,000/mL) were suspended in the standard incubation solution for 15 minutes alone or with concentrations of EGF ranging from 10 pmol/L to 100 nmol/L. Pepsinogen released into the supernatant (250 mL) was assayed using acid-denatured hemoglobin as substrate. Each sample was both incubated and assayed in duplicate. Pepsinogen secreted during incubation is expressed as a percentage of total pepsinogen present in chief cells at the beginning of the incubation minus the pepsinogen secreted before starting incubation.23

Effect of EGF on [Ca2/]i [Ca2/]i was measured in dispersed chief cells (2 1 106/ mL) loaded with Fura-2/AM using a Hitachi 2000 (Pabisch, Milan, Italy) fluorescence spectrophotometer.24,25,34 [Ca2/]i was measured in cells preincubated alone or with 1 mmol/L BAPTA for 15 minutes or 1 mmol/L genestein for 30 minutes and then stimulated with 10 nmol/L EGF.35 Control cells were stimulated with 30 mmol/L carbachol. [Ca2/]i was calculated according to Tsien and Poenie.34 The Fura-2/AM-Ca2/ signal was calibrated at the end of each recording by adding digitonin followed by EGTA.24,25 To investigate whether EGF increased the Ca2/ influx, we used Mn2/ as a Ca2/ surrogate. Fura2–loaded cells were resuspended in a Ca2/-free buffer and stimulated with 10 nmol/L EGF. Fluorescence was excited at 360 nm, i.e., the isosbestic wavelength in which Ca2/ does not affect Fura-2 fluorescence and changes in fluorescence intensity are only caused by Mn2/ quenching. Emission was recorded at 505 nm. Maximal Mn2/ quenching was calibrated in each preparation at the end of recording with digitonin.34

Effect of EGF on IP3 Generation IP3 was measured in isolated chief cells using Amersham’s assay system, which uses [3H]-labeled D-myo-IP3 as substrate. Briefly, 1 mL of chief cell suspension (2 1 106 chief

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cells) was preincubated alone or with 10 nmol/L EGF for the indicated time; the reaction was terminated by adding 1 mL of 20% TCA. After centrifugation of the precipitate at 750g for 10 minutes, the supernatant was extracted, and the IP3 content in the aqueous phase was measured. Results were calculated from a standard curve prepared by incubating adrenal IP3-binding protein with 0.19–25 pmol/L of authentic IP3 and expressed as pmol of IP3/2 1 106 cells.24,25,35

Effect of EGF on Ca2/ Adenosine Triphosphatase Ca2/ –adenosine triphosphatase (ATPase) activity was measured on membranes obtained from chief cell sonicates after incubation with no agent, 10 nmol/L EGF, or 30 mmol/ L carbachol.25 One hundred micrograms of membrane protein was then suspended in 1 mL of buffer containing 100 mmol/ L CaCl2, 60 mmol/L K-EGTA, 100 mmol/L KCl, 5 mmol/L MgCl2, 300 mmol/L sucrose, 5 mmol/L HEPES at pH 7.4, and 2.5 mmol/L adenosine triphosphate (ATP). After incubation at 377C for 10 minutes, the reaction was stopped by adding 1 mL of 10% TCA and the precipitate removed by centrifugation at 5000g for 10 minutes. The released orthophosphate (PO430) was estimated in 1 mL of clear supernatant. The enzymatic activity was expressed as micromolar PO430 releasedrmg protein01rmin01.

MAP Kinase Identification in Unstimulated Chief Cells Isolated chief cells were pelleted and lysed in ice in 50 mmol/L Tris-HCl at pH 8.0, 150 mmol/L NaCl, 1 mmol/L EGTA at pH 8.0, 100 mmol/L NaF, 10% glycerol, 1% mmol/ L MgCl2 , 1% vol/vol Triton X-100, 1 mmol/L Na3VO4 , 1 mmol/L phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, and 5 mg/mL aprotinin. Insoluble materials were removed by centrifugation at 12,000g at 47C for 10 minutes and protein concentration determined by Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). For Western blot analysis, 50 mg of total lysates were electrophoresed on an 11% sodium dodecyl sulfate polyacrylamide gel, blotted onto nitrocellulose membrane, and incubated with anti–MAP kinase polyclonal antibody at a final dilution of 1 mg/mL.36 MAP kinase phosphorylation (and activation) by EGF was shown by a shift in the molecular mass.

MAP Kinase Activity Assay MAP kinase activity was determined using MBP peptide as substrate.37 Briefly, 10 mL of cell-free extracts (50 mg protein) obtained from cells incubated alone for 5 minutes, with increasing concentrations of EGF alone or in combination with 1 mmol/L staurosporin or 1 mmol/L genestein and tyrphostin 51 were mixed with 1.0 mg/mL MBP peptide in a solution containing 50 mmol/L Tris at pH 7.4, 2 mmol/L EGTA, 10 mmol/L MgCl2 , 70 mmol/L ATP, and 2.5 mCi [31]ATP (final volume, 25 mL) at 307C for 10 minutes. Fifteen microliters of the reaction mixture were then spotted on 1-cm2 Whatman p81 phosphocellulose paper. After washing, the amount of

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radioactivity retained on the paper was determined by liquidscintillating counting; MAP kinase activity was expressed as a percentage of control from unstimulated cells.

Effect of Cyclooxygenase and Lipoxygenase Inhibitors on EGF-Induced Pepsinogen Secretion and Eicosanoid Generation Whether or not and to what extent prostanoids affected pepsinogen release induced by EGF was established by preincubating chief cells (4 1 106/mL) for 60 minutes alone or with 0.1 mmol/L BW755C, which is a dual inhibitor of the cyclooxygenase and lipoxygenase pathways,38 or with 10 nmol/L indomethacin, which is a cyclooxygenase inhibitor, or with 1 mmol/ L L-651,392, which is a selective lipoxygenase inhibitor.38 Cells were then stimulated with increasing concentrations of EGF (1 pmol/L to 100 nmol/L) for 15 minutes, and the pepsinogen released was measured. PGE2 and LTB4 concentrations that were released into supernatants of cells incubated with 0.1 mmol/L BW775C were measured by two specific enzymelinked immunosorbent assays.38 The detection limit was 23.6 pg/mL for PGE2 and 0.3 pg/mL for LTB4 . Concentrations of PGE2 were expressed as ng/4 1 106 cells and, for LTB4 , as pg/4 1 106 cells. To further investigate whether inhibition of EGF-induced pepsinogen release by cyclooxygenase and lipoxygenase inhibitors could be reverted by bypassing the AA pathways, cells preincubated with BW775C, indomethacin, or L-651,392 alone or in combination were stimulated with 10 nmol/L EGF alone or in combination with 1 mmol/L PGE2 or 100 nmol/L LTB4 and pepsinogen released into cell supernatants was assayed.

Effect of Tyrosine Kinase Inhibitors To establish whether or not protein tyrosine kinases modulate the pepsinogen secretion induced by EGF, cells were preincubated alone or with laevendustatin, genestein, staurosporine, and tyrphostin 51 for 30 minutes and stimulated with 10 nmol/L EGF for 15 minutes; the pepsinogens LTB4 and PGE2 released into the cell supernatants were measured.27 – 30

Effect of EGF on NO Generation and cGMP Accumulation NO was measured by determining the NO coproduct citrulline.24,25 Chief cells (1.5 1 106/mL) were preincubated alone or with 1 mmol/L L-NMMA for 15 minutes and stimulated with 10 nmol/L EGF for 5 minutes, and then the pepsinogen and citrulline concentration was measured. The time course of EGFinduced citrulline generation was determined in cells preincubated alone or with 1 mmol/L L-NMMA and then stimulated with 10 nmol/L EGF for 1–15 minutes. To investigate if an NO donor could restore the ability of EGF to stimulate pepsinogen release in the presence of L-NMMA, cells were first incubated alone or with 1 mmol/L L-NMMA for 15 minutes and stimulated with 10 nmol/L EGF alone or in combination with 1 mmol/L SNAP for 5 minutes, and then the pepsinogen released was measured. cGMP concentrations were measured in chief cells (2.0 1

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106) preincubated alone or with 1 mmol/L L-NMMA and then stimulated with increasing concentration of EGF for 1 minute. The incubation was stopped by adding 1.0 mL of 10% TCA, the mixture centrifuged at 10,000g for 30 seconds, the supernatants aspirated, and the pellets mixed with 0.5 mL of iced ethanol.39 After centrifugation (12,000g per 20 minutes), cGMP was measured as previously described24,25 in 50 mL of supernatants using a specific enzyme immunoassay kit (Cayman Chemical Co., Ann Arbor, MI). The detetection limit was 0.9 pmol/mL. Values of each experimental sample are expressed as a ratio to the control value.

Statistical Analysis Data reported are the mean { SE of the number of experiments indicated. ANOVA and Student’s t test for paired data were used for statistical analyses.40

Results Effect of EGF on [3H]thymidine Uptake on SAA Cells Incubating SAA cells with EGF caused a concentration-dependent stimulation of [3H]thymidine incorporation with a mean effective dose (ED50 ) of 0.16 nmol/ L.8,41 Maximal incorporation occurred at 1 nmol/L and did not increase thereafter (Figure 1). Expression of EGF-R on Gastric Chief Cells Figure 2A shows a representative example of in situ hybridization studies showing the expression of mRNA EGF-R transcripts in gastric isolated chief cells. Western blot analysis of chief cell lysates (Figure 3) revealed a 180kilodalton protein precipitation band that comigrated with the EGF-R detected in SAA cells.8 The presence of EGFR on gastric chief cells was further confirmed by immunohistochemistry; EGF-R immunoreactivities gave a mem-

Figure 1. Concentration-response curve of [3H]thymidine incorporation induced by human recombinant EGF in proliferating SAA cells. Data are expressed as mean of three different experiments.

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brane and diffuse cytoplasmatic positivity (Figure 4A and B) and were found in 85% of chief cells. Effect of EGF on Pepsinogen Release Incubating chief cells with EGF caused a concentration- and time-dependent stimulation of pepsinogen secretion (Figure 5). Basal pepsinogen secretion of 2.3% { 0.4% increased to 9.4% { 1.1% in cells incubated with 10 nmol/L EGF. The EGF-induced pepsinogen secretion was significantly above basal values at 1 pmol/L, maximal at 10 nmol/L, and half maximal at 0.1 nmol/L. Interaction of EGF With Pepsinogen Secretion Agonists The basal pepsinogen release of 2.2% { 1.0% increased to 8.1% { 0.9% in cells exposed to 10 nmol/ L EGF for 15 minutes (P õ 0.05). Whereas the EGFstimulated secretion was almost additional to that induced by 30 nmol/L secretin or 1 mmol/L VIP that remained unchanged when EGF was combined with Ca2/-mediated agonists (Table 1). Effect of EGF on [Ca2/]i , IP3 Generation, and Ca2/-ATPase [Ca2/]i increased from 118.4 { 7.3 nmol/L to 293.4 { 13.2 nmol/L and 402.5 { 34.0 nmol/L in Fura2–loaded cells after exposure to 10 nmol/L EGF and 30 mmol/L carbachol (Figure 6A). EGF-induced Ca2/ mobilization was concentration- (data not shown) and time-dependent; [Ca2/]i peaked 60 seconds after exposure to EGF and carbachol and returned to baseline after 7 minutes of incubation (Figure 6A). Preincubating cells with 1 mmol/L BAPTA, which is an intracellular Ca2/ chelator, or 0.1 mmol/L W7, which is a calmodulin antagonist, in a Ca2/-free medium prevented pepsinogen release (Figure 6B) and Ca2/ mobilization (Figure 6C). Incubating the cells with genestein caused a slight reduction in EGF-induced Ca2/ mobilization. Incubation with 10 nmol/L EGF increased Ca2/-ATPase activity from 0.9 { 0.1 to 1.3 { 0.2 mgrmL01rmg01 PO430 (P õ 0.05; five experiments) and caused a time-dependent increase in the IP3 concentration. Maximal IP3 stimulation (7.5 { 1.2–fold more than basal) occurred after 30 seconds (Figure 7A). EGF induced a marked increase in the quenching rate of the Fura-2 signal in the presence of Mn2/, indicating a net Ca2/ influx during agonist stimulation (Figure 7B). Effect of Cyclooxygenase and Lipoxygenase Inhibitor on EGF-Induced Pepsinogen Secretion and Eicosanoid Generation Preincubating chief cells with 0.1 mmol/L BW755C caused an approximately 80% reduction in WBS-Gastro

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Figure 2. (A ) In situ hybridization of mRNA EGF-R transcripts in isolated gastric chief cells (original magnification 1001). (B ) Negative control showing negative background only (original magnification 1001).

EGF-induced pepsinogen release (Figure 8A). A similar inhibitory effect was observed in cells preincubated with indomethacin or L-651,392 alone or in combination (Figure 8B). Exposing the cells to exogenous PGE2 and LTB4 restored the ability of EGF to stimulate pepsinogen secretion in cells pretreated with BW775C (Figure 8C), indomethacin, and L-651,392 (data not shown). In supernatants of chief cells incubated alone, PGE2 and LTB4 concentrations were 25.1 { 3.0 ng/1 1 106 cells and 12.0 { 1.5 pg/1 1 106 cells. Ionomycin (100 nmol/L) provoked a threefold increase in PGE2 and a 10fold increase in LTB4 . EGF (10 nmol/L) triggered a 2– 6-fold increase (Figure 9A and B). Pretreating the cells with BW755C, indomethacin, or L-651,392 prevented the effect of EGF.

Identification and Activation of MAP Kinase Because resolution of chief cell lysates on gel electrophoresis probed with specific anti-MAP kinase antibodies yielded two major bands with molecular masses of 42 and 44 kilodaltons, at least two forms of MAP kinase must exist in isolated gastric chief cells (Figure 10). Incubating the cells with EGF caused a time- and concentration-dependent stimulation of MAP kinase activity (Figure 10 and 11A). The effect was inhibited by pretreating the cells with tyrosine kinase inhibitors (Figure 10 and 11B). Effect of Tyrosine Kinase Inhibitor on Pepsinogen Secretion As shown in Table 2, genestein, tyrphostin 51, and staurosporin inhibited pepsinogen release by 50%.41 Moreover, tyrosine kinase inhibitors hindered EGF-induced LTB4 and PGE2 releases (Figure 12). Effect of EGF on NO Generation and cGMP Accumulation

Figure 3. Western blot analysis of EGF-R in guinea pig gastric chief cells (CC) and SAA cells (SAA). The molecular mass standard is shown on the left. The blots are from a single experiment representative of five others.

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Preincubating cells with 1 mmol/L L-NMMA for 15 minutes suppressed the stimulatory effect of EGF on pepsinogen secretion (Figure 13A), indicating that EGF releases pepsinogen by activating NO pathways. The effect exerted by L-NMMA was reverted by incubating the cells with L-arginine but not by its steroisomer D-arginine, indicating a selective cNOS activation (Figure 13A). Incubating the cells with 10 nmol/L EGF caused WBS-Gastro

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a time-dependent release of citrulline (Figure 13B). Maximal citrulline generation occurred after 5 minutes of incubation, but reached approximately 80% maximum after 1 minute. The effect was prevented by incubating

the cells with 1 mmol/L L-NMMA. In cells preincubated with L-NMMA, 1 mmol/L SNAP (an NO donor) was able to stimulate pepsinogen secretion in the presence of EGF (Figure 13A). Exposure to EGF for 1 minute increased cGMP concentration dependently (Figure 13C) with an ED50 of 1 nmol/L. Again the effect was prevented by incubating the cells with 1 mmol/L L-NMMA.

Discussion The present results show that isolated guinea pig gastric chief cells express EGF-R and that binding EGF to its receptor stimulates pepsinogen secretion and eicosanoid generation by activating a number of intracellular messengers, including protein tyrosine kinase, MAP kinases, IP3 , and Ca2/. We also provided evidence that the inhibition of NO generation by L-NMMA abolishes the effect EGF exerts on pepsinogen release and that EGF activates a Ca2/ and calmodulin-dependent cNOS that leads to NO and cGMP accumulation. The presence of EGF-R in gastric isolated chief cells

Figure 4. Immunohistochemical demonstration of EGF-R on guinea pig gastric chief cells. (A ) Immunostaining of chief cells incubated with a polyclonal anti–EGF-R antibody showed both membrane and diffuse cytoplasmatic positivity. Immunoperoxidase-hematoxylin (original magnification 8001). (B ) EGF-R-positive chief cells (higher magnification 10001). (C ) Hematoxylin counterstaining (negative control; original magnification 6001).

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Figure 5. (A ) Effect of increasing concentrations of EGF on pepsinogen secretion in isolated chief cells. Cells were incubated for 15 minutes at 377C. Secretion induced by 10 mmol/L carbachol was 10.5% { 0.7%. Results are expressed as means { SE of seven experiments. (B ) Time course of pepsinogen secretion induced by 10 nmol/L EGF. Results are expressed as means { SE of six experiments.

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Table 1. Effect of EGF on Agonist-Induced Pepsinogen Secretion in Isolated Chief Cells Basal EGF (10 nmol/L) Secretin (30 nmol/L) VIP (1 mmol/L) Carbachol (30 mmol/L) Ionomycin (10 nmol/L) CCK-8 (3 nmol/L) Gastrin (1 mmol/L) EGF / secretin EGF / VIP EGF / carbachol EGF / ionomycin EGF / CCK-8 EGF / gastrin

2.2 8.1 7.6 6.6 9.5 8.5 9.2 7.6 14.2 13.2 9.7 9.1 9.2 8.0

{ { { { { { { { { { { { { {

1.0 0.9a 0.5a 0.6a 0.6a 0.7a 0.8a 1.0a 1.3b 1.1b 1.4 1.0 0.9 1.1

NOTE. Results are expressed as mean { SE of seven experiments. a P õ 0.05 vs. basal. b P õ 0.05 vs. EGF alone.

was shown by a variety of methods, including immunohistochemistry, in situ hybridization, and Western blot analysis of chief cells lysates. In situ hybridization showed that the EGF-R is constitutively expressed in gastric chief cells, which agrees with the finding of mRNA EGFR transcripts in guinea pig gastric parietal and chief cells by Beauchamp et al.15 The fact that immunohistochemistry revealed EGF-R immunoreactivities in both the membrane and cytoplasm of chief cells suggests that redistribution of EGF-R occurred during the process of cell preparation. Finally, the presence of EGF-R was further confirmed by Western blot analysis of chief cells’ lysates, which precipitated a 180-kilodalton protein band. Despite the constitutive expression of EGF-R in gastric chief cells, we failed to show its phosphorylation under conditions in which phosphorylation was readily apparent in SAA cells (data not shown).8,42 However, a similar situation has been reported in gastric parietal cells, where EGF modulates H/ secretion without inducing any detectable receptor phosphorylation.43 This finding could be explained by the low copy number of receptors expressed in these cell populations.43 Alternatively, it has been proposed that EGF-R phosphorylation is not strictly required for activating differentiated cell functions, and in line with this hypothesis, EGF has been shown to induce responses in cell lines transfected with EGF-R mutants lacking autophosphorylation sites.44,45 Incubating gastric isolated chief cells with EGF caused a concentration- and time-dependent stimulation of pepsinogen release with an ED50 of 0.1 nmol/L. Maximal EGF-induced pepsinogen secretion was 80% of that triggered by 30 mmol/L carbachol.23 Because plasma and gastric luminal EGF concentrations range from 66 to 277 nmol/mL in healthy humans, the effect this growth / 5e12$$0052

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Figure 6. (A ) Effect of 10 nmol/L EGF on [Ca2/]i in chief cells loaded with Fura-2. Results are expressed as means { SE from 65 determinations. (B ) Effect of 1 mmol/L BAPTA or 0.1 mmol/L W7 on pepsinogen secretion induced by 10 nmol/L EGF. Results are expressed as means { SE of six experiments. *P õ 0.05 vs. EGF alone; **P õ 0.05 vs. basal. (C ) Effect of 1 mmol/L BAPTA on EGF-induced Ca2/ mobilization. Results are expressed as means { SE of 46 determinations.

factor exerts on pepsinogen release by gastric isolated chief cells is likely to be physiological.5,41 The stimulatory effect exerted by EGF on pepsinogen secretion was somewhat surprising and was not predicted by results from in vivo studies. In fact, EGF administration in WBS-Gastro

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animals results in a profound inhibition of acid and gastric fluid secretion.17,18 This inhibitory effect, however, has only been partially reproduced by in vitro studies. In isolated rabbit parietal cells, inhibition of histaminestimulated H/ secretion by EGF is incomplete (40%) and requires supraphysiological concentrations: 10–40 times greater than the dissociation constant for EGF binding to isolated gastric gland and basolateral membranes.43 Moreover, in long-term cultured parietal cells, EGF potentiates, rather than inhibits, the H/ secretion induced by histamine and carbachol.43 Discrepancies between in vivo and in vitro effects have also been reported for agents that regulate pepsinogen secretion. PGE2 , like

EGF, inhibits pepsinogen release in vivo but stimulates secretion in vitro. The reason for these discrepancies are unclear. It may be speculated that EGF and PGE2 act indirectly on gastric chief cells by releasing one or more

Figure 7. (A ) Time course of IP3 generation in chief cells incubated with no agent or 10 nmol/L EGF. Results are expressed as means { SE of eight experiments. (B ) Effect of Mn2/ addition to Fura-2– loaded cells incubated without stimulation or EGF. Mn2/ was used as a substitute for Ca2/. The more pronounced the Fura-2 signal quenching, the higher is Mn2/ influx. Cells were incubated in a Ca2/free medium and then stimulated with 10 mmol/L EGF or no agent. After 2 minutes of incubation, 25 mmol/L Mn2/ was added to the cell suspension. Fluorescence intensity was normalized to 100% just before Mn2/ addition. Data are expressed as means { SE of at least 50 determinations.

Figure 8. (A ) Effect of 0.1 mmol/L BW775C on pepsinogen secretion induced by 10 nmol/L EGF. Data are expressed as means { SE of seven experiments. (B ) Effect of 10 nmol/L indomethacin and 1 mmol/L L651,392 pretreatment on EGF-induced pepsinogen release. (C ) Exposing gastric chief cells to 1 mmol/L PGE2 or 100 nmol/L LTB4 restored the ability of EGF to stimulate pepsinogen secretion in cells pretreated with BW775C. Results are expressed as means { SE of at least five experiments. (A and B ) s, Control; m, indomethacin plus EGF; l, L-651,392 plus indomethacin plus EGF; ., L-651,392 plus EGF; h, EGF alone. (C ) s, Basal; m, BW775C; ., BW775C plus EGF; l, BW775C plus EGF / LTB4 ; ●, BW775C plus EGF plus PGE2 ; h, EGF alone.

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Figure 9. Effect of BW775C, indomethacin, and L-651,392 on EGFinduced (A ) PGE2 and (B ) LTB4 generation in isolated gastric chief cells. Chief cells (4 1 106/mL) were incubated alone or with 0.1 mmol/L BW775C, 10 nmol/L indomethacin, or 1 mmol/L L-651,392 and then stimulated with 10 nmol/L EGF or 100 nmol/L ionomycin. Results are expressed as means { SE of 5–7 experiments. *P õ 0.05 vs. chief cells incubated alone. **P õ 0.05 vs. chief cells incubated with EGF alone.

inhibitory factors when treated in vivo. However, inhibition of agonist-induced pepsinogen secretion may also result from the down-regulation of secretory pathways induced by the cells’ exposure to agonists, as suggested by the finding that PGE2-induced pepsinogen secretion is abolished in chief cells preincubated with adenosine 3*,5*-cyclic monophosphate–mediated agonists.21 We have previously shown that preincubating gastric chief cells with PGE2 , VIP, or secretin reduces the pepsinogen

Figure 10. Western blot analysis of MAP kinases. (A ) Expression of MAP kinases on gastric chief and SAA cells overexpressing the human EGF-R.8 Two MAP kinases of 42 and 44 kilodaltons are shown. Molecular masses are shown on the left. (B ) Effect of EGF on MAP kinase expression on SAA cells. MAP kinase activation in the presence of EGF is shown by a complete shift in the molecular masses. (C ) Time course effect of 10 nmol/L EGF on MAP kinase expression on gastric chief cells. EGF causes an incomplete activation of MAP kinase as shown by the fact that only part of the p42 isoform migrates to p43 and only part of the p44 isoform migrates to p45. (D ) Effect of 1 mmol/ L staurosporine and 1 mmol/L genestein on MAP kinase expression induced by 10 nmol/L EGF in gastric chief cells (CC).

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Figure 11. (A ) Effect of increasing concentrations of EGF alone or in combination with 1 mmol/L staurosporine on MAP kinase activity in gastric chief cells. Results are expressed as means { SE of seven experiments; P õ 0.05. (B ) Effect of tyrosine kinase inhibitors on MAP kinase activity. Cells were preincubated alone or with 1 mmol/L staurosporine (ST), 1 mmol/L genestein (Gen), or 1 mmol/L tyrphostin 51 (Tyr) and then stimulated with no agent (basal) or 10 nmol/L EGF. Results are expressed as means { SE of six experiments; *P õ 0.05 vs. basal **P õ 0.05 vs. chief cells incubated with EGF alone.

release induced by a subsequent stimulation with these agents (heterologous desensitization) by 60%–70%.21 Therefore, interactions between stimuli at target cells may dramatically affect the pattern of secretory response. Activation of EGF-R in gastric chief cells triggers 2/ Ca mobilization from an IP3-sensitive pool.2,4,9,11–13,46–48 The following results support the hypothesis that EGFinduced pepsinogen release is a Ca2/-mediated process: (1) EGF-stimulated secretion was additive with secretion induced by secretin and VIP, but not with secretion induced by Ca2/-mediated agents; (2) BAPTA prevented pepsinogen secretion and Ca2/ mobilization; (3) W7, a calmodulin antagonist, inhibited pepsinogen release; and (4) EGF triggered a 2–2.5-fold increase of [Ca2/]i and Ca2/-dependent ATPase. Ca2/ mobilization in response WBS-Gastro

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Table 2. Effect of Tyrosine Kinase Inhibitor on EGF-Induced Pepsinogen Release Basal EGF (10 nmol/L) EGF / laevendustatin (1 mmol/L) EGF / genestein (1 mmol/L) EGF / staurosporine (1 mmol/L) EGF / tyrphostin 51 (1 mmol/L) Laevendustatin (1 mmol/L) Genestin (1 mmol/L) Staurosporine (1 mmol/L) Tyrphostin 51 (1 mmol/L)

2.2 8.1 4.5 4.4 5.2 4.6 3.3 3.2 3.5 3.4

{ { { { { { { { { {

1.0 0.9a 0.8b 1.0b 1.2b 0.7b 0.3 0.7 0.6 0.5

NOTE. Results are expressed as mean { SE of seven experiments. a P õ 0.05 vs. basal. b P õ 0.05 vs. EGF alone.

to EGF was attributable to Ca2/ release from an IP3sensitive pool and Ca2/ influx, as shown by our Mn2/ experiments. The latter mechanism was most likely attributable to an indirect effect resulting from depletion of intracellular Ca2/ pools. In fact, in other systems, depletion of intracellular Ca2/ stores releases a diffusible cell signal that activates plasma membrane Ca2/ entry pathways.35 EGF-induced Ca2/ mobilization required activation of AA metabolism. EGF has been reported to generate both

Figure 13. (A ) Effect of preincubating chief cells with 1 mmol/L LNMMA alone or with L-NMMA plus 1 mmol/L SNAP, 1 mmol/L D-arginine, or 1 mmol/L L-arginine on pepsinogen release induced by 10 nmol/L EGF. Results are expressed as means { SE of 4–6 experiments. *P ú 0.05 vs. basal. **P õ 0.05 vs. EGF alone. (B ) Time course of EGF-induced citrulline generation in chief cells preincubated alone or with 1 mmol/L L-NMMA. Results are expressed as means { SE of eight experiments. (C ) Concentration-response curve of EGFinduced accumulation of cGMP on gastric isolated chief cells. Results are expressed as means { SE of eight experiments. Basal cGMP concentration was 18 fmol/4 1 106 cells.

Figure 12. Effect of tyrosine kinase inhibitors on EGF-induced stimulation of PGE2 and LTB4 . Cells were preincubated alone or with 1 mmol/ L staurosporine (ST) or 1 mmol/L genestein (Gen) and then left unstimulated or stimulated with 10 nmol/L EGF. Results are expressed as means { SE of seven experiments. *P õ 0.05 vs. basal. **P õ 0.05 vs. cells incubated with EGF alone.

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AA and LTs by increasing cPLA2 and 5-and 12-lipooxygenase activities.48 Moreover, exposure to LT C4 mimics several effects that EGF exerts on Ca2/ channel activity and [Ca2/]i .12,13,48 We have previously reported that gastric chief cells release PGE2 and LTB4 in response to Ca2/-mediated agents and that exposure to exogenous LTs results in pepsinogen secretion and Ca2/ mobilization.24,25,38 The fact that BW775C (a dual inhibitor of cyclooxygenase and lipoxygenase pathways), indomethaWBS-Gastro

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cin (a selective cyclooxygenase inhibitor), and L-651,392 (a selective lipoxygenase inhibitor) blocked the effect EGF exerts on pepsinogen release and eicosanoid generation suggests that activation of AA metabolism is a critical step in the process of pepsinogen release induced by this growth factor. Stimulation of eicosanoid generation by EGF in isolated chief cells was Ca2/-dependent. Support for this concept comes from the observation that ionomycin increased LTB4 and PGE2 generation, and incubating the cells in a Ca2/-free medium containing EGTA or BAPTA prevented the EGF effect. Clark et al.11 have recently provided evidence that cPLA2 possesses a Ca2/-binding domain and that the enzyme is activated and translocated from cytosol to the membrane, where the substrate phospholipid is localized in a Ca2/-dependent fashion. Activation of eicosanoid generation by EGF was also prevented by tyrosine kinase inhibitors. In cells pretreated with staurosporine and genestein, EGF was unable to stimulate LTB4 and PGE2 generation, which agrees with the finding that cPLA2 activation by Ca2/dependent agonists and growth factors in CHO cells require activation of MAP kinase.49 The site of cPLA2 phosphorylation by MAP kinase is the Ser-505 amino acid. A point mutation in this amino acid results in a mutant cPLA2 , which is not a substrate for MAP kinase, with a reduced ability to generate AA.49 Several of our findings indicate that MAP kinase modulates the effect of EGF on eicosanoid generation. (1) Two isoforms of MAP kinase of 42 and 44 kilodaltons were constitutively expressed in chief cells. (2) EGF caused a concentrationdependent stimulation of MAP kinase activity. (3) Also, genestein and staurosporin inhibited both EGF-induced stimulation of MAP kinase, eicosanoid, and pepsinogen release. Exposure of gastric chief cells to EGF increased protein tyrosine kinase activity. The finding that protein kinases are involved in regulating pepsinogen secretion is chiefly based on the results of experiments with tyrosine kinase inhibitors. Lavendustatin, genestein, staurosporine, and tyrphostin 51 caused a 50% inhibition of pepsinogen release and prevented MAP kinase activation. Similar results have been reported in pancreatic acini, in which genestein and okadaic acid, which is a selective inhibitor of type 1 protein phosphatases, caused a 40% reduction in CCK-induced amylase release.27 – 30 Despite the fact that tyrosine phosphorylation induced by CCK-8 in pancreatic acini is mediated by changes in [Ca2/]i , whereas EGF directly activates tyrosine kinase pathways, the amount of enzyme secretion inhibited by genestein is similar (40%–50%) and significantly lower than that induced by BAPTA or L-NMMA.29,30 One possible interpretation of these findings would be that the tyrosine / 5e12$$0052

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kinase functions to increase the amplitude of the secretory response rather than to provide an obligate signal for this response to occur.29 NO is a putative cellular messenger synthesized from the guanidine nitrogen atom of L-arginine by the catalytic action of NO synthase.50 The primary effect of NO in target cells is to stimulate a soluble guanylate cyclase, which leads to an elevation in cGMP concentrations, that in turn regulates protein phosphorylation, ion channel conductivity, and phosphodiesterase activity.26,49,51 EGF-induced pepsinogen secretion seems to be modulated by NO-cGMP pathways because we found that EGF stimulated citrulline generation and W7, a calmodulin antagonist, and L-NMMA abolished pepsinogen release. The extent to which EGF-induced pepsinogen secretion was inhibited in the presence of L-NMMA (approximately 80%) was similar to that reported for other Ca2/-mediated agonists.24,25 We previously reported that NO synthase immunoreactivities are constitutively expressed on gastric chief cells and that NO modulates the pepsinogen release induced by carbachol, CCK-8, ionomycin, and thapsigargin.24,25 Because Ca2/ mobilization induced by ionomycin and thapsigargin is receptor-independent, these results indicate that the increase in [Ca2/]i is responsible itself for NO synthase activation, which is in agreement with the finding that fluctuations of 100 nmol/L in [Ca2/]i are required to sustain the binding of calmodulin to a cNOS in host cells.51 The mechanisms by which NO modulates11 EGFinduced pepsinogen secretion are unclear. In pancreatic acini, cGMP regulates Ca2/ influx during agonist stimulation,39 but we have been unable to show a similar pathway in isolated chief cells.25 As NO increases cyclooxygenase activity and PGE2 generation, it seems to modulate the eicosanoid production induced by Ca2/mediated agonists in gastric chief cells.52 The finding that EGF directly stimulated NO generation from gastric epithelial cells is new. In fact, although treating intact animals with EGF has been shown to protect the gastric mucosa and increase gastric mucosal blood flow and the effect is inhibited by L-NMMA treatment, the cellular source and intracellular mechanisms modulating the NO generation have not been investigated.53,54 The evidence that chief cells, under EGF stimulation, release NO, which is a powerful vasodilator and cytoprotective agent, suggests that some of the effects that EGF exerts on the gastric mucosal microcirculation are mediated by NO generated by gastric epithelial cells. In conclusion, we provided evidence that EGF stimulates pepsinogen release by activating a number of intracellular messengers. EGF-induced eicosanoid and NO generation could explain the vascular and cytoprotective effects this growth factor exerts on the gastric mucosa. WBS-Gastro

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Received September 11, 1995. Accepted May 9, 1996. Address requests for reprints to: Stefano Fiorucci, M.D., Sezione di Gastroenterologia ed Endoscopia Digestiva, Policlinico Monteluca, 06100 Perugia, Italy. Fax: (39) 75-578-3687. Supported by a grant from the Ministero dell’ Universita` e della Ricerca Scientifica e Tecnologica, Rome, Italy. The authors thank Barbara Ugolini for technical assistance, Judy Etherington for help with the manuscript, and Dr. Paolo Gresele for BW775C and L-651,392.

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