cAMP-regulated whole cell chloride currents in pancreatic duct cells

Abstract

Using the whole cell configuration of the patch-clamp technique, we have identified an adenosine 3',5'-cyclic monophosphate (cAMP)-regulated chloride conductance in pancreatic duct cells. Basal whole cell currents in single isolated cells were very low (approximately 5 pA/pF) but could be stimulated 17-fold by elevation of intracellular cAMP. The cAMP-activated currents exhibited 1) a high chloride selectivity, 2) a near linear current-voltage relationship, 3) time and voltage independence, 4) block by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 5) an anion selectivity sequence based on permeability ratios of SCN > NO3 > Br > Cl > I > HCO3 > F > ClO4 > gluconate. Currents in single cells ran down within a few minutes; however, stable chloride currents could be recorded from duct cell clusters in which four or five cells were in electrical communication. We present evidence suggesting that these cAMP-regulated currents are carried by cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. Physiologically, these CFTR channels act in parallel with chloride-bicarbonate exchangers to facilitate bicarbonate secretion across the apical plasma membrane of the duct cell.

Molecular Cloning, Chromosomal Localization, Tissue Distribution, and Functional Expression of the Human Pancreatic Sodium Bicarbonate Cotransporter

“…The oocytes were then transferred into 1.4 ml of the following Cl Ϫ -containing solution: NaCl (100 mM), KCl (2 mM), CaCl 2 (1 mM), MgCl 2 (1 mM), NaHCO 3 (8 mM) bubbled with 1.5% CO 2 , pH 7.4. with 3.3 Ci of 36 Cl Ϫ . A 10-l aliquot was removed from the influx solution for later determination of 36 Cl Ϫ -specific activity. 36 …”

“…A 10-l aliquot was removed from the influx solution for later determination of 36 Cl Ϫ -specific activity. 36 …”

“…Functional Expression of pNBC in Xenopus Oocytes-We examined the functional properties of pNBC using measurements of pH i , 22 Na, and 36 Cl Ϫ uptake after injecting the corresponding polyadenylated cRNA into Xenopus oocytes. Polyadenylated cRNA prepared as described above was injected into oocytes and allowed to express for 72 h. Augmented 22 Na uptake was observed in oocytes injected with pNBC cRNA (Fig.…”

“…The nel on the apical membrane (5,36). Influx of H ϩ equivalents during the process of apical bicarbonate secretion requires the efflux of H ϩ or the influx of bicarbonate in the steady state.…”

The distribution and expression of CFTR restricts electrogenic anion secretion to the ileum of the brushtail possum, Trichosurus vulpecula

“…A similar model to explain HCO 3 -secretion by the pancreatic duct of non-rodent eutherian mammals has been proposed (Park et al, 2010;Steward et al, 2005). CFTR from eutherian mammals also has a relatively low P HCO3 (Gray et al, 1993;Gray et al, 1990;Linsdell et al, 1997;O'Reilly et al, 2000;Poulsen et al, 1994). However, in the pancreatic duct cells it has been suggested that HCO 3 -secretion occurs via CFTR because there is no mechanism on the basolateral membrane to accumulate Cl -, whereas there is a mechanism for HCO 3 -accumulation (Fernández-Salazar et al, 2004).…”