Volume 115, Issue 6, Pages 1483-1493 (December 1998) Regenerating gene protein may mediate gastric mucosal proliferation induced by hypergastrinemia in rats  Hirokazu Fukui, Yoshikazu Kinoshita, Toru Maekawa, Akihiko Okada, Shinya Waki, Md Sazzad Hassan, Hiroshi Okamoto, Tsutomu Chiba  Gastroenterology  Volume 115, Issue 6, Pages 1483-1493 (December 1998) DOI: 10.1016/S0016-5085(98)70027-7 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Immunohistochemical double staining for Reg protein and HDC in normal rat fundus. (A) Reg protein immunoreactivity was visualized by fluorescein isothiocyanate, and (C) HDC immunoreactivity was visualized by tetramethylrhodamine isothiocyanate. (B) Yellow staining represents colocalization of Reg protein and HDC immunoreactivity (bar = 10 μm). Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Effects of long-term administration of lansoprazole and/or the gastrin receptor antagonist AG-041R on the number of Reg protein–positive cells. Immunohistochemistry was performed with anti-Reg antibodies on the fundic mucosae of rats treated with (A) vehicle alone, (B) lansoprazole, (C) combination of lansoprazole and AG-041R, and (D) AG-041R for 28 days (bar = 100 μm). The data are representative of 8 rats. Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Effects of long-term administration of lansoprazole and/or the gastrin receptor antagonist AG-041R on the number of Reg protein–positive cells. Immunohistochemistry was performed with anti-Reg antibodies on the fundic mucosae of rats treated with (A) vehicle alone, (B) lansoprazole, (C) combination of lansoprazole and AG-041R, and (D) AG-041R for 28 days (bar = 100 μm). The data are representative of 8 rats. Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Effects of long-term administration of lansoprazole and/or the gastrin receptor antagonist AG-041R on the number of Reg protein–positive cells. Immunohistochemistry was performed with anti-Reg antibodies on the fundic mucosae of rats treated with (A) vehicle alone, (B) lansoprazole, (C) combination of lansoprazole and AG-041R, and (D) AG-041R for 28 days (bar = 100 μm). The data are representative of 8 rats. Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Effects of long-term administration of lansoprazole and/or the gastrin receptor antagonist AG-041R on the number of Reg protein–positive cells. Immunohistochemistry was performed with anti-Reg antibodies on the fundic mucosae of rats treated with (A) vehicle alone, (B) lansoprazole, (C) combination of lansoprazole and AG-041R, and (D) AG-041R for 28 days (bar = 100 μm). The data are representative of 8 rats. Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Effects of long-term administration of lansoprazole and/or the gastrin receptor antagonist AG-041R on gene expressions of Reg, HB-EGF, HGF, and TGF-α in rat fundus. Total fundic RNA was extracted from rats treated with vehicle alone (lane 1), lansoprazole (lane 2), combination of lansoprazole and AG-041R (lane 3), or AG-041R (lane 4). (A) Equivalent loading of each sample was verified by measuring of 18 S RNA levels. Relative signal intensities of Reg, HB-EGF, HGF, and TGF-α to that of 18 S RNA were quantified with a BAS 2000 image analyzer. (B) Vertical lines represent the means ± SEM (n = 4). *Significantly greater than control (P < 0.01). §Significantly lower than control (P < 0.05). Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Effects of long-term administration of lansoprazole and/or the gastrin receptor antagonist AG-041R on gene expressions of Reg, HB-EGF, HGF, and TGF-α in rat fundus. Total fundic RNA was extracted from rats treated with vehicle alone (lane 1), lansoprazole (lane 2), combination of lansoprazole and AG-041R (lane 3), or AG-041R (lane 4). (A) Equivalent loading of each sample was verified by measuring of 18 S RNA levels. Relative signal intensities of Reg, HB-EGF, HGF, and TGF-α to that of 18 S RNA were quantified with a BAS 2000 image analyzer. (B) Vertical lines represent the means ± SEM (n = 4). *Significantly greater than control (P < 0.01). §Significantly lower than control (P < 0.05). Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Time course of the effect of short-term administration of pentagastrin on Reg mRNA expression in rat fundus. Total fundic RNA (20 μg) was extracted 0, 3, 6, 9, 12, and 24 hours after pentagastrin administration and analyzed by Northern blotting as described in Materials and Methods. (A) Equivalent loading of each sample was verified by measuring 18 S levels. Relative signal intensities of each Reg mRNA levels to that of 18 S RNA were quantified with a BAS 2000 image analyzer. (B) Vertical lines represent the means ± SEM (n = 4). *P < 0.01 vs. 0 hour. Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Time course of the effect of short-term administration of pentagastrin on Reg mRNA expression in rat fundus. Total fundic RNA (20 μg) was extracted 0, 3, 6, 9, 12, and 24 hours after pentagastrin administration and analyzed by Northern blotting as described in Materials and Methods. (A) Equivalent loading of each sample was verified by measuring 18 S levels. Relative signal intensities of each Reg mRNA levels to that of 18 S RNA were quantified with a BAS 2000 image analyzer. (B) Vertical lines represent the means ± SEM (n = 4). *P < 0.01 vs. 0 hour. Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of short-term administration of gastrin on Reg mRNA expression in the rat fundic mucosa. Photomicrographs of in situ hybridization to Reg mRNA are shown. Hybridization was performed as described in Materials and Methods. (A) Normal fundic mucosa. Hybridized signals for Reg mRNA were observed in the relatively small cells localized in the lower segment. (B) Fundic mucosa of rats 3 hours after pentagastrin administration. Both the number of Reg mRNA-positive cells and the signal intensity in each cell were markedly increased (bar = 100 μm). Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of short-term administration of gastrin on Reg mRNA expression in the rat fundic mucosa. Photomicrographs of in situ hybridization to Reg mRNA are shown. Hybridization was performed as described in Materials and Methods. (A) Normal fundic mucosa. Hybridized signals for Reg mRNA were observed in the relatively small cells localized in the lower segment. (B) Fundic mucosa of rats 3 hours after pentagastrin administration. Both the number of Reg mRNA-positive cells and the signal intensity in each cell were markedly increased (bar = 100 μm). Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Time course of the effect of rat gastrin administration (10−7 mol/L) on Reg mRNA expression in cultured ECL cells. Total RNA (10 μg) was extracted 0, 3, 6, and 12 hours after gastrin administration and analyzed by Northern blotting as described in Materials and Methods. (A) Equivalent loading of each sample was verified by measuring 18 S RNA levels. Relative signal intensities of each Reg mRNA level to that of 18 S RNA were quantified with a BAS 2000 image analyzer. (B) Vertical lines represent the means ± SEM (n = 4). *P < 0.05, **P < 0.01 vs. 0 hour. Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Time course of the effect of rat gastrin administration (10−7 mol/L) on Reg mRNA expression in cultured ECL cells. Total RNA (10 μg) was extracted 0, 3, 6, and 12 hours after gastrin administration and analyzed by Northern blotting as described in Materials and Methods. (A) Equivalent loading of each sample was verified by measuring 18 S RNA levels. Relative signal intensities of each Reg mRNA level to that of 18 S RNA were quantified with a BAS 2000 image analyzer. (B) Vertical lines represent the means ± SEM (n = 4). *P < 0.05, **P < 0.01 vs. 0 hour. Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Effects of various concentrations of recombinant Reg protein on [3H]thymidine incorporation into cultured gastric mucosal cells. Results are expressed as the mean ± SEM of 4 samples. *P < 0.05 vs. 0 nmol/L. Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Effects of various concentrations of (A) recombinant Reg protein and (B) gastrin on cultured gastric mucosal cell growth. Results are expressed as the mean ± SEM of 4 samples. *P < 0.05 vs. 0 nmol/L. Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Effects of various concentrations of (A) recombinant Reg protein and (B) gastrin on cultured gastric mucosal cell growth. Results are expressed as the mean ± SEM of 4 samples. *P < 0.05 vs. 0 nmol/L. Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 9 Time course effect of recombinant Reg protein (0.3 nmol/L) and gastrin (10−9 mol/L) on cultured gastric mucosal cell growth. Results are expressed as the mean ± SEM of 4 samples. *P < 0.01 vs. control. Gastroenterology 1998 115, 1483-1493DOI: (10.1016/S0016-5085(98)70027-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions