Volume 114, Issue 4, Pages 791-797 (April 1998) Guanylin stimulates regulated secretion from human neuroendocrine pancreatic cells  Mathias John, Bertram Wiedenmann, Mogens Kruhøffer, Knut Adermann, Ieva Ankorina–Stark, Eberhard Schlatter, Gudrun Ahnert– Hilger, Wolf–Georg Forssmann, Michaela Kuhn  Gastroenterology  Volume 114, Issue 4, Pages 791-797 (April 1998) DOI: 10.1016/S0016-5085(98)70593-1 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Concentration-dependent effects of guanylin and the NO donor DEANO on intracellular cGMP content of BON cells in the presence and absence of IBMX (1 mmol/L). Incubation time, 30 minutes (n = 4). Gastroenterology 1998 114, 791-797DOI: (10.1016/S0016-5085(98)70593-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Effects of ligands for different membrane-bound guanylyl cyclases (GC-A, -B, and -C) on the intracellular cGMP concentration of BON cells. Ligands for GC-A (atrial natriuretic peptide, CDD-ANP-99-126 [□]; brain natriuretic peptide, BNP [■]), for GC-B (C-type natriuretic peptide, CNP [▵]), and for GC-C (guanylin [○] and STa [●]). Basal cGMP concentration of BON cells was 0.23 ± 0.02 pmol/well (in the presence of IBMX) (n = 6–8). Gastroenterology 1998 114, 791-797DOI: (10.1016/S0016-5085(98)70593-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Effects of guanylin, STa, forskolin, IBMX, acetylcholine, and A23187 on intracellular cAMP concentration of BON cells. Basal cAMP content of BON cells was 1.49 ± 0.2 pmol/well (n = 6–8). Asterisks denote significant effects of the respective substances. P < 0.05 was considered significant. Gastroenterology 1998 114, 791-797DOI: (10.1016/S0016-5085(98)70593-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Detection of GC-C mRNA in BON and T84 cells by RT-PCR analysis. Amplified DNA was analyzed by electrophoresis on ethidium bromide–stained agarose gels (1.5%). A GC-C PCR product of the expected size of 416 base pairs was detected in both T84 and BON cells (lanes 2 and 3). The intactness of the complementary DNA was tested by amplification of β-tubulin (lanes 6 and 7). No amplification products were detected in the respective controls (lanes 4 and 5). DNA size markers were pUC18 digested with Sau3AI (lane 1) and 1-DNA digested with HindIII (lane 8). Gastroenterology 1998 114, 791-797DOI: (10.1016/S0016-5085(98)70593-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Effects of consecutive addition of guanylin, acetylcholine, and ATP on the fura-2 fluorescence ratio of BON cells (original recording). Measurements were obtained from BON cells grown on glass coverslips, passage 48, 3–5 days after seeding. Gastroenterology 1998 114, 791-797DOI: (10.1016/S0016-5085(98)70593-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of various test agents on the release of chromogranin A by BON cells. (A) Chromogranin A release on potassium depolarization and elevation of intracellular cGMP by guanylin, STa, or 8-Br-cGMP. (B) Increase of cellular cAMP levels (by forskolin or IBMX) and increase of [Ca2+]i (with Bay K8664, A23187, or acetylcholine), and subsequent chromogranin A secretion. Basal release of chromogranin A amounted to 11.8 ± 4.3 ng/mL during a 25-minute incubation period and was significantly stimulated by each of these agents. The basal release (control) or chromogranin A was set at 100%. Gastroenterology 1998 114, 791-797DOI: (10.1016/S0016-5085(98)70593-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Effects of guanylin, STa, and 8-Br-cGMP on the (A) regulated secretion and (B) uptake of GABA by BON cells. (A) BON cells released GABA in a regulated fashion when treated with either 50 mmol/L K+ or 1 μmol/L BayK 8664. Increases in cellular cGMP levels by guanylin, STa, or 8-Br-cGMP also induced a significant increase of GABA secretion. In addition, dibutyryl-cAMP induced a significant increase in GABA release from BON cells. Basal GABA release (control) under nondepolarizing conditions was set at 100%. (B) GABA uptake in untreated BON cells (control) was set at 100%. In the presence of 2 mmol/L unlabeled GABA, the uptake of labeled GABA was inhibited by about 87%, indicating that BON cells possess a specific GABA transport system. Neither 1 μmol/L guanylin and 100 μmol/L 8-Br-cGMP nor 1 μmol/L BayK 8644 inhibited GABA uptake. Gastroenterology 1998 114, 791-797DOI: (10.1016/S0016-5085(98)70593-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions