Volume 126, Issue 3, Pages 732-740 (March 2004) Guanylin regulates chloride secretion in the human gallbladder via the bile fluid  Hasan Kulaksiz, Thorsten Schlenker, Daniel Rost, Adolf Stiehl, Martin Volkmann, Thomas Lehnert, Yalcin Cetin, Wolfgang Stremmel  Gastroenterology  Volume 126, Issue 3, Pages 732-740 (March 2004) DOI: 10.1053/j.gastro.2003.11.053

Figure 1 RT-PCR analysis of human gallbladder (A) and Mz-Cha-1 cells (B) reveal gene expression of guanylin (lane 2), guanylate cyclase C (GC-C, lane 3), cGMP-dependent protein kinase II (cGKII, lane 4), and cystic fibrosis transmembrane conductance regulator (CFTR, lane 5). A 100-bp DNA ladder is indicated (lanes 1 and 6). (C and D) Immunoreactive 12.5 kilodalton guanylin in extracts of the rat lung (positive control; lane 1), of the human gallbladder (lane 2), and bile fluid of human origin (lane 3) detected by the region-specific guanylin antibodies K605 (C) and K42 (D). (E–G) Western blot analysis showing that GC-C (E, Ab K735), cGKII (F, Ab EG(1)-PKIIA), and CFTR (G, EG(1)-CFTR843) are present coincidently in extracts of the rat lung (positive control; lane 1) and human gallbladder (lane 2). Gastroenterology 2004 126, 732-740DOI: (10.1053/j.gastro.2003.11.053)

Figure 2 Cellular localization of guanylin in human gallbladder. (A and B) Two serial paraffin sections of the human gallbladder immunostained for guanylin by the region-specific antibodies K605 and K42 showing localization of guanylin to the same epithelial cells. (C and D) Note the intercellular differences of guanylin immunostaining intensities; beside strongly immunoreactive cells (arrow), some are totally unreactive (arrowhead) for guanylin. (Original magnification: A and B, ×120; C, ×280; D, ×520.) Gastroenterology 2004 126, 732-740DOI: (10.1053/j.gastro.2003.11.053)

Figure 3 Cellular localization of guanylin (A, Ab K42), GC-C (C, Ab K735), cGKII (E, Ab EG(1)-PKIIA), and CFTR (G, Ab EG(1)-CFTR122) in human gallbladder. Immunocytochemical stainings reveal that, in some tissues, immunoreactivity for guanylin is concentrated at the apical pole of the secretory epithelial cells. Immunoreactivity for GC-C, cGKII, and CFTR is confined to the apical membrane domain of gallbladder epithelial cells. Note that immunostaining in the gallbladder epithelial cells is completely blocked by preabsorption of the antibodies (B, guanylin; D, GC-C; F, cGKII; H, CFTR) (Original magnification: A, C, E, G, ×680; B, D, F, H, ×520.) Gastroenterology 2004 126, 732-740DOI: (10.1053/j.gastro.2003.11.053)

Figure 4 Immunocytochemical characterization of Mz-Cha-1 cells. Immunofluorescence experiments with protein-specific antisera reveal that guanylin (A), GC-C (C), cGKII (E), and CFTR (G) are highly expressed in Mz-Cha-1 cells. Note that immunostaining in Mz-Cha-1 cells is blocked by preabsorption of the antibodies (B, guanylin; D, GC-C; F, cGKII; H, CFTR). (Bar = 9 μm.) Gastroenterology 2004 126, 732-740DOI: (10.1053/j.gastro.2003.11.053)

Figure 5 Basal guanylin-, STa-, and forskolin-stimulated 125I efflux in subconfluent Mz-Cha-1 cell monolayers. (A) Control: NaCl-rich bath solution was exchanged in 3-minute intervals. (B–D) Bath solution in interval 7–9 contained either guanylin (B), STa (C), or forskolin (D). Each point represents the average of 7 studies, of which samples were measured in duplicate. Values on y-axis represent efflux rate in percentage of total 125I load (mean ± SD). The statistical analysis was evaluated by unpaired t test. The differences were considered significant at P < 0.05. Gastroenterology 2004 126, 732-740DOI: (10.1053/j.gastro.2003.11.053)

Figure 6 Second messenger measurements in Mz-Cha-1 cells. Stimulation of Mz-Cha-1 cells with guanylin or STa induces a significant increase of intracellular concentration of cGMP (A) but not of cAMP (B) as measured by enzyme immunoassay. The statistical analysis was evaluated by Student t test. The differences were considered significant at P < 0.05. Gastroenterology 2004 126, 732-740DOI: (10.1053/j.gastro.2003.11.053)