Volume 137, Issue 1, Pages 231-241.e10 (July 2009) Delineation of the Chemomechanosensory Regulation of Gastrin Secretion Using Pure Rodent G Cells  Mark Kidd, Øyvind Hauso, Ignat Drozdov, Bjorn I. Gustafsson, Irvin M. Modlin  Gastroenterology  Volume 137, Issue 1, Pages 231-241.e10 (July 2009) DOI: 10.1053/j.gastro.2009.01.005 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 G-cell isolation. FACS of F1 (Nycodenz fraction) stained with acridine orange identified 2 peaks (F2P4 and F2P5) in F2 that are distinguishable by size and density (A) despite similar acridine orange fluorescence (inset). Immunostaining identified progressive enrichment of (B) neuroendocrine cells (chromogranin A, 95%–99%) and (C) gastrin (92%–98%). Gastrin measurements identified F2 (intermediate fraction 2) had 100× gastrin levels of F1 (Nycodenz fraction, before fluorescence-activated cell sorting); F2P4 had 5× gastrin than F2P5 (D). Chromogranin A and gastrin transcripts were significantly elevated in F2, F2P4, and F2P5 (E and F), and gastrin levels were highest in F2P4 (F). Mean ± SEM (n = 6 experiments). *P < .05 vs F0 and F1, #P < .05 vs F2P5. Gastroenterology 2009 137, 231-241.e10DOI: (10.1053/j.gastro.2009.01.005) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Electron micrographs of mucosal and isolated G cells. (A) Electron microscopy demonstrating a pure (∼99%) G-cell preparation. (B) Typical ultrastructural features of an isolated G cell demonstrating punctate nucleus, several floculated secretory vesicles, and active Golgi. Immuno–electron microscopy with gold-labeled anti-gastrin antibodies confirmed G cells, identifying staining in the (C) Golgi as well as (D) secretory vesicles that access the pericellular space. N, nucleus; g, Golgi; m, mitochondrion; ps, pericellular space. Gastroenterology 2009 137, 231-241.e10DOI: (10.1053/j.gastro.2009.01.005) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Gastrin release and cAMP production in isolated G cells. (A) Forskolin-stimulated release is inhibited by 2′5′-dideoxyadenosine (2′5DDA). (B) Basal release is increased by IBMX (10 μmol/L), PMA (10 μmol/L), cholera toxin (CT; 250 μg/mL), and pertussis toxin (PT; 50 μg/mL), while H-89 (10 μmol/L), PD98059 (PD; 0.1 μmol/L), and wortmannin (WORT; 1 nmol/L) inhibit release. (C) These agents also altered intracellular cAMP levels (increased by forskolin [FORSK], IBMX, PMA, and cholera toxin and decreased by H-89). PD98059, wortmannin, and pertussis toxin were not associated with alterations in cAMP production. (D) Constituents of food including the “sweet” tasting amino acids (tyrosine), sweet and bitter tastants, and pain/heat transducers (capsaicin) stimulate gastrin secretion. (E) Bacteria also stimulate secretion through LPS. (F) All luminal activators that stimulate secretion also induce cAMP production in G cells. Mean ± SEM (n = 4 experiments). *P < .05 vs unstimulated cells, #P < .05 vs forskolin. Gastroenterology 2009 137, 231-241.e10DOI: (10.1053/j.gastro.2009.01.005) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Effect of signaling inhibitors on gastrin release. Targeting the PKA pathway with IBMX (10mM) (A) or H-89 (10mM) (D) resulted in opposing effects. IBMX (an adenosine antagonist) enhanced GRP- (0.1mM), sucralose- (0.2nM), denatonium- (50pM), and S. enteritidis LPS (0.25nM)-mediated release, while H-89 (PKA inhibitor) inhibited gastrin release. Targeting the PKC pathway with the phorbol ester, PMA < (10mM) (B), significantly stimulated gastrin release, while Wortmannin (1nM) (E) inhibited this except for GRP. Cholera toxin (250mg/ml), a stimulant of cAMP production, increased gastrin release (except for GPR and LPS) (C) while the MAPK inhibitor, PD98059 (0.1mM), inhibited release (F). Mean±SEM (n = 4 experiments). *P < .05 vs. unstimulated cells, **P < .05 vs stimulated cells. GRP, bombesin; SUCR, sucralose; DEN, denatonium; S.e. LPS, S. enteritidis LPS. Gastroenterology 2009 137, 231-241.e10DOI: (10.1053/j.gastro.2009.01.005) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Signal transduction activation in G cells. (A) GRP (0.1 μmol/L), denatonium (DEN; 50 pmol/L), sucralose (SUCR; 0.2 nmol/L), and S enteritidis LPS (S.ent; 0.25 nmol/L) all significantly stimulated ERK phosphorylation, indicating that the MAPK signaling pathway is activated in gastrin secretion. (B) AKT (PKB signaling pathway) was activated by denatonium and LPS, while NF-κB and JNK pathways were only stimulated by LPS (C and D). Mean ± SEM (n = 5 experiments). *P < .05 vs unstimulated cells. Gastroenterology 2009 137, 231-241.e10DOI: (10.1053/j.gastro.2009.01.005) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Mechanically and pH-mediated gastrin release from the antrum and isolated G cells. (A and B) Decreasing pH by 2 log (from 7.4 to 5.5) inhibited gastrin release in both the antrum and isolated G cells. Three minutes of mechanical force increased secretion at both pH levels. (C and D) Addition of a general ADORA agonist (NECA) significantly elevated gastrin release in both the antrum and isolated G cells, while the ADORA2B antagonist MRS-1754 (MRS) significantly inhibited mechanical force-mediated secretion. Mean ± SEM (n = 5 experiments). (A and B) *P < .05 shake vs control, #P < .05 pH 7.4 vs 5.5. (C and D) *P < .05 shake vs control, #P < .05 NECA/MRS vs shake, **P < .05 MRS vs NECA. Gastroenterology 2009 137, 231-241.e10DOI: (10.1053/j.gastro.2009.01.005) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 1 FACS gastric G cells. G cells comprise the majority (>65%) of antral endocrine cells. D cells, although localized close to G cells in the base of the antral gland, differ morphologically and have larger, moderately electron-dense granules but express less chromogranin A than G cells.24 A-like cells are round, have electron-dense granules of variable size, and express only chromogranin A.25 5-HT cells are extremely infrequent. These features, particularly the differences in shape and density (G cells have numerous small granules of low to moderate electron density24), indicate that the antral neuroendocrine cell populations may be separated. Preparations of rat (male, Harlan Sprague–Dawley, 120 g) G cells were obtained using modifications of previously described methodology.26,27 A total of 5 different fractions (F0–F2P5) were collected. Whole gastric mucosa was everted and ligated at the corpus/antrum border and pylorus. The everted antral “sacs” were filled with pronase E (0.7 mg/mL) and mucosal cells separated from the muscularis by alternative switching between a calcium-containing respiration medium and a chelating digestive medium (F0).26,27 Density gradient centrifugation at 1100 rpm was performed and cells collected at the second intermediate layer (density, 1.070 g/L) (F1). Collected cells were incubated with acridine orange (20 nmol/L) for 15 minutes before filtering (50 μm) and sorting. FACS analysis (high-speed FACS Aria; Becton Dickinson, San Jose, CA) was used to identify and sort cells. Excitation was at 488 nm (activation acridine orange–labeled cells), and sorting was achieved by gating on forward and side scatter and an emission of 532 ± 15 nm (F2). Two distinct acridine orange–positive fractions were identified within F2 (F2P4, F2P5) that could be separated by forward and side scatter. Cells were collected over 1 hour. Gastroenterology 2009 137, 231-241.e10DOI: (10.1053/j.gastro.2009.01.005) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 2 Real-time PCR for gastrin and contaminating cells. Identification of transcripts for parietal and chief cells in the F0/F1 preparations indicates the inadvertent inclusion of some fundic tissue during antral ligation. (A and B) Nycodenz gradient and FACS signficantly reduced levels of these contaminating cells. (C and D) Somatostatin and ghrelin transcripts were evident in F2 and F2P5 but were absent in F2P4. The contaminating cells in F2P4 were therefore predominantly pepsinogen cells. Mean ± SEM (n = 6 experiments). #P < .01 vs F0, *P < .05 vs F2P4. Gastroenterology 2009 137, 231-241.e10DOI: (10.1053/j.gastro.2009.01.005) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 3 Luminal projections and confirmation of functional taste receptors. (A) Typical G cell demonstrating villus-like structures accessing the lumen, a large number of vesicles, and the nucleus basally situated and contact with the pericellular space. Isolated G cells retain some of these features, particularly the villus-like luminal sensing structures (B). Immunostaining of whole mucosa identified coexpression of gastrin and (C) α-gustducin, (D) T1R, and (E) T2R. Isolated G cells expressed each of these proteins (inset of each panel). N, nucleus; ps, pericellular space; L, lumen. Blue, 4′,6-diamidino-2-phenylindole (nuclei); green, fluorescein isothiocyanate (gastrin), red, Cy5 (protein of interest); yellow, coexpression. Original magnification 400×. Gastroenterology 2009 137, 231-241.e10DOI: (10.1053/j.gastro.2009.01.005) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 4 Effect of signaling inhibitors on cAMP production. Targeting the PKA pathway with IBMX (10 μmol/L) or H-89 (10 μmol/L) resulted in opposing effects. IBMX (a nonspecific inhibitor of phosphodiesterases and an adenosine antagonist) enhanced GRP-mediated (0.1 μmol/L), sucralose-mediated (0.2 nmol/L), and denatonium-mediated (50 pmol/L) cAMP production, while H-89 (PKA inhibitor) inhibited cAMP production. Targeting the PKC pathway with the phorbol ester PMA (10 μmol/L) signficantly stimulated cAMP, while wortmannin (1 nmol/L) inhibited cAMP. Cholera toxin (250 μg/mL) increased cAMP production, while the MAPK inhibitor PD98059 (0.1 μmol/L) inhibited production. Mean ± SEM (n = 4 experiments). *P < .05 vs unstimulated cells, **P < .05 vs stimulated cells. GRP, bombesin; SUCR, sucralose; DEN, denatonium. Gastroenterology 2009 137, 231-241.e10DOI: (10.1053/j.gastro.2009.01.005) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 5 Effects of luminal agents on gastrin release from the antrum. Unstimulated gastrin release was 310 ± 44 pg/mg protein. GRP (1 μmol/L), denatonium (1 nmol/L), sucralose (0.1 μmol/L), and S enteritidis LPS (0.1 μmol/L) all significantly stimulated gastrin release in situ, while octreotide (1 nmol/L) inhibited secretion. Mean ± SEM (n = 4 experiments). **P < .05 vs control mucosa. GRP, bombesin; DEN, denatonium; SUCR, sucralose; S. ent, S enteritidis LPS; Octr, octreotide. Gastroenterology 2009 137, 231-241.e10DOI: (10.1053/j.gastro.2009.01.005) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 6 Effect of the somatostatin analogue octreotide on ERK signaling in G cells. Octreotide alone (30 pmol/L) significantly inhibited ERK phosphorylation and reversed GRP-mediated (0.1 μmol/L), denatonium-mediated (50 pmol/L), sucralose-mediated (0.2 nmol/L), and S enteritidis LPS-mediated (0.25 nmol/L) ERK phorphorylation. Mean ± SEM (n = 5 experiments). *P < .01 vs unstimulated cells, **P < .05 vs stimulated cells. OCTR, octreotide; GRP, bombesin; DEN, denatonium; SUCR, sucralose; S.e., S enteritidis LPS. Gastroenterology 2009 137, 231-241.e10DOI: (10.1053/j.gastro.2009.01.005) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 7 Model of G-cell regulation. Luminal agents including amino acids, sweet and bitter tastants, pH, and bacterial LPS either directly or indirectly induce ERK phosphorylation through activation of adenylate cyclase (AC) via coupling to Gαs or Gαq, which results in gastrin secretion. ACh, GRP, and β-adrenergic stimulants (G protein–coupled receptors) as well as mechanical forces (ADORA2B) and 5-HT (through 5-HT3 coupled channels) also stimulate gastrin release through this pathway. Negative regulators include somatostatin (Gαi) and histamine H3 (Gαi) receptors that directly inhibit ERK phosphorylation and may inhibit Ca2+ influx. ERK phosphorylation and 5-HT3 activation may positively affect Ca2+ influx. Other signaling pathways activated included protein kinase B (AKT) via phosphatidylinositol 3-kinase (PI3K), PKC, JNK, and NF-κB. Targeting Gαs with cholera toxin amplified gastrin release, as did inhibiting negative regulatory G protein–coupled receptors (Gαβi) with pertussis toxin. Targeting PDE with IBMX amplified gastrin release, while inhibiting PKA, ERK, or PKC pathways reduced gastrin release. AAs, amino acids; AAT, amino acid transporter; AC, adenylate cyclase; ACh, acetylcholine; βADR, β1 adrenergic ligand; GAS, gastrin; GPCR, G protein–coupled receptor; HIST, histamine; PDK, phosphoinositide-dependent kinase; SER, serotonin; SST, somatostatin. Dashed lines reflect inhibition, and solid lines reflect stimulation. *Signaling targets: PI3K, wortmannin; cAMP, 2′5′-dideoxyadenosine; pERK, PD98059. Gastroenterology 2009 137, 231-241.e10DOI: (10.1053/j.gastro.2009.01.005) Copyright © 2009 AGA Institute Terms and Conditions