Volume 140, Issue 4, Pages 1292-1302 (April 2011) Atrial Natriuretic Factor Stimulates Efflux of cAMP in Rat Exocrine Pancreas via Multidrug Resistance–Associated Proteins  Myrian R. Rodríguez, Federico Diez, Maria S. Ventimiglia, Vanina Morales, Sabrina Copsel, Marcelo S. Vatta, Carlos A. Davio, Liliana G. Bianciotti  Gastroenterology  Volume 140, Issue 4, Pages 1292-1302 (April 2011) DOI: 10.1053/j.gastro.2010.12.053 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Extracellular cAMP accumulation after pancreatic acini exposure to secretagogues alone or combined with ANF. Isolated pancreatic acini were incubated with secretin, cholecystokinin (cck), isoproterenol, amthamine, and VIP with or without ANF. cAMP was determined as described in the Materials and Methods section. ***P < .001 vs control; †††P < .01 vs secretin; and #P < .01 vs VIP. Number of experiments, 5–7. Gastroenterology 2011 140, 1292-1302DOI: (10.1053/j.gastro.2010.12.053) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Time-course of cAMP production and extracellular accumulation in the (A and B) absence or (C and D) presence of PDE activity. Pancreatic acini were exposed to (A and C) secretin or (B and D) secretin and ANF with or without IBMX (PDE inhibitor). cAMP was measured at 0, 1, 3, 5, 10, and 30 minutes in tissues (■) and incubation media (○) as described in the Materials and Methods section. Dotted lines represent extracellular cAMP in the absence of stimuli. Number of experiments, 6–8. Gastroenterology 2011 140, 1292-1302DOI: (10.1053/j.gastro.2010.12.053) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Time-course cAMP production and extracellular accumulation in the presence of (A) isoproterenol, (B) amthamine, (C) VIP, and (D) VIP + ANF. Tissues were incubated with the secretagogues and IBMX (PDE inhibitor). cAMP was measured at 0, 1, 3, 5, 10, and 30 minutes in tissues (■) and incubation media (○) as described in the Materials and Methods section. Dotted lines represent extracellular cAMP in the absence of stimuli. Number of experiments, 6–8. Gastroenterology 2011 140, 1292-1302DOI: (10.1053/j.gastro.2010.12.053) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 (A) Intracellular and (B) extracellular cAMP in the presence of secretin (s) and ANF or ANP (4–23 amide) after (A and B) PLC or (C and D) PKC inhibition. Isolated pancreatic acini with U73122 (u) (PLC inhibitor) or GF103201X (gf) (PKC inhibitor) were incubated with secretin and ANF or ANP (4–23 amide). cAMP was measured in (A and C) tissues and (B and D) incubation media as described in the Materials and Methods section. ***P < .001 vs control; ‡‡‡P < .001; and ‡‡P < .05 vs secretin; ‡P < .05 vs secretin + ANF; †P < .05 vs 4–23 amide. Number of experiments, 6–8. Gastroenterology 2011 140, 1292-1302DOI: (10.1053/j.gastro.2010.12.053) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Role of MRPs. Effect of probenecid on (A) intracellular and (B) extracellular cAMP in the presence of secretin or VIP alone or combined with ANF. cAMP was measured in (A) acini and (B) incubation media as described in the Materials and Methods section. ***P < .001 vs control; †††P < .001 vs secretin; ‡‡‡P < .001 vs secretin and ANF; #P < .05 vs VIP; ###P < .01 vs VIP and ANF. Number of experiments, 6–8. (C) Expression of MRP4, MRP5, and MRP8 assessed by real-time polymerase chain reaction in pancreatic acini. Gastroenterology 2011 140, 1292-1302DOI: (10.1053/j.gastro.2010.12.053) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 cAMP egression in AR42J cells. (A) MRP4 protein expression. Representative Western blot of MRP4 in AR42J cells and pancreatic acini. (B) Concentration-response curve to secretin in AR42J cells. Intracellular (●) and extracellular (■) cAMP assessed in cells exposed for 15 minutes to increasing secretin concentrations. (C) Time-course cAMP production and egression without IBMX in cells pretreated (○;□) or not (●;■) with probenecid; ● and ○, intracellular cAMP; ■ and □, extracellular cAMP. (D) Representative Western blot of MRP4 in AR42J cells transfected with MRP4–siRNA or scrambled-siRNA and densitometric analysis. MRP4 knock-down was performed as detailed in the Materials and Methods section. (E) AR42J cells transfected with MRP4–siRNA or scrambled-siRNA were exposed to 100 nmol/L secretin and intracellular and extracellular cAMP (icAMP [intracellular cAMP] and ecAMP[extracelluluar cAMP]) was assessed. Results are expressed as the ratio between intracellular and extracellular cAMP. ***P < .001. Number of experiments: 3. Gastroenterology 2011 140, 1292-1302DOI: (10.1053/j.gastro.2010.12.053) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 (A) Plasma and (B) pancreatic secretion (ps) cAMP levels after secretin and ANF infusion. Anesthetized rats were infused with saline (control) (○), secretin (▾), ANF (▴), or ANF + secretin (●). cAMP was assessed as described in the Materials and Methods section. *P < .05, **P < .01, and ***P < .001, respectively, vs control; ‡P < .05 and ‡‡‡P < .001 vs secretin. Number of experiments, 5–6. Gastroenterology 2011 140, 1292-1302DOI: (10.1053/j.gastro.2010.12.053) Copyright © 2011 AGA Institute Terms and Conditions