Volume 118, Issue 1, Pages 108-114 (January 2000) Differential role of cyclic GMP–dependent protein kinase II in ion transport in murine small intestine and colon  Arie B. Vaandrager, Alice G.M. Bot, Peter Ruth, Alexander Pfeifer, Franz Hofmann, Hugo R. De Jonge  Gastroenterology  Volume 118, Issue 1, Pages 108-114 (January 2000) DOI: 10.1016/S0016-5085(00)70419-7 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression of cGK II in various intestinal segments. Western blot of cGK II in isolated epithelial cells (20 μg protein) from duodenum (Du), jejunum (Je), distal ileum (DI), cecum (Ce), proximal colon (PC), and distal colon (DC) from cGK II +/+ and cGK II −/− mice. In the first lane 10 ng of histidine-tagged recombinant mouse cGK II purified from Sf9 cells was loaded as standard (St); note that standard has a slightly smaller mobility than endogenous cGK II. Apparent molecular masses (kilodaltons) are indicated on the left side. Gastroenterology 2000 118, 108-114DOI: (10.1016/S0016-5085(00)70419-7) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 In vivo ligated intestinal loop assay of net fluid transport in cGK II +/+ and −/− mice. Intestinal fluid transport in the absence (basal) or presence of STa (0.5 μmol/L) was measured in anesthetized mice as described in Materials and Methods. Data are mean ± SEM (n = 6). A plus sign denotes net fluid absorption; a minus sign denotes net fluid secretion. *P < 0.05 vs. wild type. **P < 0.05 vs. basal condition. Gastroenterology 2000 118, 108-114DOI: (10.1016/S0016-5085(00)70419-7) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of STa and cGMP on Isc across intestinal epithelium of cGK II +/+ and −/− mice. Isc was determined in stripped proximal colon and jejunum in an Ussing chamber, and 100 μmol/L 8-pCPT-cGMP was added to the serosal side (cGMP), and saturating concentrations of STa (100 nmol/L) or guanylin (1 μmol/L) to the mucosal side (STa gave a transient response in jejunum, peak value is given, and plateau phase was approximately 50%-30% of peak). Forsk denotes the maximal response recorded after addition of 10 μmol/L forskolin to the serosal side on top of either cGMP or STa. Data are mean ± SEM of 3-5 experiments. *P < 0.05 vs. wild type. Gastroenterology 2000 118, 108-114DOI: (10.1016/S0016-5085(00)70419-7) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of STa and amrinone on Isc across intestinal epithelium of cGK II +/+ and −/− mice. Isc was determined in stripped proximal colon and jejunum in an Ussing chamber. STa (100 nmol/L) was added to the mucosal side (STa gave a transient response in jejunum, peak value is given, and plateau phase was approximately 50%-30% of peak). Amrinone (200 μmol/L) was added to the serosal bath (amrin), and subsequently STa (100 nmol/L) was added to the mucosal bath (STa + amrin). Data are mean ± SEM of 3-4 experiments. *P < 0.05 vs. wild type. Gastroenterology 2000 118, 108-114DOI: (10.1016/S0016-5085(00)70419-7) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of STa and cGMP on Na+ absorption in jejunum of cGK II +/+ and −/− mice. Mucosa to serosa flux of 22Na was determined in stripped jejunum in an Ussing chamber. After a basal flux period of 20 minutes, agents were added. Following a subsequent 20-minute equilibration period, flux was determined for another 20 minutes. The effects of the agents were expressed as the difference between the 22Na flux in the last and the first (basal) period. A negative value indicates inhibition of Na+ absorption. 8-pCPT-cGMP (100 μmol/L; cGMP) and 8-Br-cAMP (1 mmol/L; cAMP) were added to the serosal side, and STa (100 nmol/L) to the mucosal side. Data are mean ± SEM of 4-8 animals. *P < 0.05 vs. wild type; **P < 0.05 vs. basal condition. Gastroenterology 2000 118, 108-114DOI: (10.1016/S0016-5085(00)70419-7) Copyright © 2000 American Gastroenterological Association Terms and Conditions