Volume 114, Issue 4, Pages 782-790 (April 1998) Stimulation of cyclic guanosine monophosphate production by natriuretic peptide in human biliary cells  Marie V. St–Pierre, Thorsten Schlenker, Jean–François J. Dufour, Douglas M. Jefferson, J.Gregory Fitz, Irwin M. Arias  Gastroenterology  Volume 114, Issue 4, Pages 782-790 (April 1998) DOI: 10.1016/S0016-5085(98)70592-X Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Time-averaged effect of bcGMP on 125I efflux from human BECs. H69 cells were loaded for 4 hours with 125I in Hank's balanced salt solution. Saline (CON) or bcGMP (1 mmol/L) was added in the presence and absence of the chloride channel inhibitor NPPB. 125I was measured in the medium and cell lysate after 5 minutes. bcGMP stimulated 125I efflux by 24% (*P < 0.05) compared with controls. This increase was abolished by NPPB. Data represent the means ± SD of three experiments in triplicate. Gastroenterology 1998 114, 782-790DOI: (10.1016/S0016-5085(98)70592-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Time-averaged effect of cyclic nucleotides on 125I efflux in human BECs. Mz-ChA-1 cells were loaded for 4 hours with 125I as described in Figure 1. Saline (CON), bcGMP (1 mmol/L), or dbcAMP (1 mmol/L) was added, and 125I was measured in the medium and cell lysate after 5 minutes. 125I efflux was stimulated by 18%–22% by all agonists (*P < 0.05) compared with controls (■) and was abolished by NPPB (▨). Data represent the means ± SD of three experiments in triplicate. Gastroenterology 1998 114, 782-790DOI: (10.1016/S0016-5085(98)70592-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Representative whole-cell recording of bcGMP-activated IK and ICl. Currents were measured at a holding potential of −40 mV and at test potentials of −80 and 0 mV. The pipette solution contained 1 mmol/L adenosine triphosphate and 1 mmol/L IBMX; 1 mmol/L bcGMP was added to the bath subsequently. IBMX alone had no effect. (B) Starting from a holding potential of −40 mV, the current-voltage relationship was assessed by stepping the potentials between −120 and +100 mV in 20-mV increments for 400 milliseconds (inset). Shown is a representative recording and the current-voltage relationship for IK. The pipette contained 500 μmol/L cGMP and 1 mmol/L IBMX. (C) Representative recording and current-voltage relationship for ICl. Methods were as described in Figure 3B. Gastroenterology 1998 114, 782-790DOI: (10.1016/S0016-5085(98)70592-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Comparison of cGMP-activated (A) ICl (at Vp, −80 mV) and (B) IK (at Vp, 0 mV) under different experimental conditions (▩) compared with initial values (■). Currents are presented as current density and were activated by inclusion of 500 μmol/L cGMP to the pipette solution (ICl, n = 4 and P < 0.05; IK, n = 4 and NS), by addition of 1 mmol/L bcGMP to the bath in the presence of a 1 mmol/L IBMX-containing pipette solution (ICl, n = 5 and NS; IK, n = 3 and P < 0.05), by inclusion of 500 μmol/L cGMP to the IBMX-containing pipette solution (ICl, n = 7 and P < 0.05; IK, n = 5 and P < 0.05), or by addition of 10 μmol/L ANP to the bath (ICl, n = 3 and P < 0.05; IK, n = 3 and NS). Gastroenterology 1998 114, 782-790DOI: (10.1016/S0016-5085(98)70592-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Comparison of cGMP-activated (A) ICl (at Vp, −80 mV) and (B) IK (at Vp, 0 mV) under different experimental conditions (▩) compared with initial values (■). Currents are presented as current density and were activated by inclusion of 500 μmol/L cGMP to the pipette solution (ICl, n = 4 and P < 0.05; IK, n = 4 and NS), by addition of 1 mmol/L bcGMP to the bath in the presence of a 1 mmol/L IBMX-containing pipette solution (ICl, n = 5 and NS; IK, n = 3 and P < 0.05), by inclusion of 500 μmol/L cGMP to the IBMX-containing pipette solution (ICl, n = 7 and P < 0.05; IK, n = 5 and P < 0.05), or by addition of 10 μmol/L ANP to the bath (ICl, n = 3 and P < 0.05; IK, n = 3 and NS). Gastroenterology 1998 114, 782-790DOI: (10.1016/S0016-5085(98)70592-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of agonists of guanylate cyclase on cGMP levels in a human intrahepatic BEC line. H69 cells were incubated for 2 hours in serum-free medium and then for 30 minutes with 0.5 mmol/L IBMX. cGMP was measured in cell lysates (■) and medium (□) after 30-minute stimulation with agonists in the presence of IBMX. Intracellular and extracellular cGMP increased in response to 1 μmol/L ANP and 1 μmol/L CNP but not in response to 1 mmol/L SNP or 250 U/mL STa. Data represent the means ± SD. Gastroenterology 1998 114, 782-790DOI: (10.1016/S0016-5085(98)70592-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of agonists of guanylate cyclase on cGMP levels in human BECs derived from a cholangiocarcinoma of the gallbladder. Mz-ChA-1 cells were stimulated with agonists as described in Figure 5. cGMP was measured in cell lysates (■) and medium (▨). Intracellular cGMP increased (10-fold) in response to 1 μmol/L ANP (*P < 0.01) but not in response to CNP and increased sixfold in response to 1 mmol/L SNP (*P < 0.01). Extracellular cGMP was increased only after stimulation with ANP (**P < 0.05). Gastroenterology 1998 114, 782-790DOI: (10.1016/S0016-5085(98)70592-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Scatchard analysis of 125I-ANP binding in human biliary cells. (A) H69 cells and (B) Mz-ChA-1 cells were incubated with 0.05 μCi 125I-ANP and increasing concentrations of unlabeled peptide (0.1–20 nmol/L) for 30 minutes at 25°C. Specific binding was calculated from the difference between total binding and nonspecific binding (measured in the presence of 1 μmol/L unlabeled ANP). Maximal binding values were 24.2 and 3.1 fmol/mg protein for H69 and Mz-ChA-1 cells, respectively. Data represent three experiments in duplicate. B/F, bound/free. Gastroenterology 1998 114, 782-790DOI: (10.1016/S0016-5085(98)70592-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Scatchard analysis of 125I-ANP binding in human biliary cells. (A) H69 cells and (B) Mz-ChA-1 cells were incubated with 0.05 μCi 125I-ANP and increasing concentrations of unlabeled peptide (0.1–20 nmol/L) for 30 minutes at 25°C. Specific binding was calculated from the difference between total binding and nonspecific binding (measured in the presence of 1 μmol/L unlabeled ANP). Maximal binding values were 24.2 and 3.1 fmol/mg protein for H69 and Mz-ChA-1 cells, respectively. Data represent three experiments in duplicate. B/F, bound/free. Gastroenterology 1998 114, 782-790DOI: (10.1016/S0016-5085(98)70592-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Expression of natriuretic peptide receptor isoforms in human biliary cell lines. PCR was performed with reverse-transcribed (+) or untranscribed (−) total RNA isolated from H69 and Mz-ChA-1 cells. (A) PCR amplification with primers specific for the GC-A receptor (lane 1, H69; lane 3, Mz-ChA-1), the clearance receptor (lane 5, H69; lane 7, Mz-ChA-1), and β-actin (lane 9, H69; lane 10, Mz-ChA-1) gave PCR products of the expected size. (B) PCR amplification of an aliquot (1/100) of PCR products obtained as described in A. Nested primers were designed to give fragments of 251 bp (lane 1, H69; lane 3, Mz-ChA-1) and 777 bp (lane 5, H69; lane 7, Mz-ChA-1) for GC-A and a fragment of 510 bp (lane 9, H69; lane 11, Mz-ChA-1) for the clearance receptor. Sequencing of the 251- and 510-bp bands confirmed the identity of GC-A and clearance receptors, respectively. Gastroenterology 1998 114, 782-790DOI: (10.1016/S0016-5085(98)70592-X) Copyright © 1998 American Gastroenterological Association Terms and Conditions