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CFTR functions as a bicarbonate channel in pancreatic duct cells.

Ishiguro H, Steward MC, Naruse S, Ko SB, Goto H, Case RM, Kondo T, Yamamoto A - J. Gen. Physiol. (2009)

Bottom Line: Apical HCO(3)(-) fluxes activated by cyclic AMP were independent of Cl(-) and luminal Na(+), and substantially inhibited by the CFTR blocker, CFTR(inh)-172.From the changes in pH(i), membrane potential, and buffering capacity, the flux and electrochemical gradient of HCO(3)(-) across the apical membrane were determined and used to calculate the HCO(3)(-) permeability.This suggests that CFTR functions as a HCO(3)(-) channel in pancreatic duct cells, and that it provides a significant pathway for HCO(3)(-) transport across the apical membrane.

View Article: PubMed Central - PubMed

Affiliation: Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan. ishiguro@htc.nagoya-u.ac.jp

ABSTRACT
Pancreatic duct epithelium secretes a HCO(3)(-)-rich fluid by a mechanism dependent on cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. However, the exact role of CFTR remains unclear. One possibility is that the HCO(3)(-) permeability of CFTR provides a pathway for apical HCO(3)(-) efflux during maximal secretion. We have therefore attempted to measure electrodiffusive fluxes of HCO(3)(-) induced by changes in membrane potential across the apical membrane of interlobular ducts isolated from the guinea pig pancreas. This was done by recording the changes in intracellular pH (pH(i)) that occurred in luminally perfused ducts when membrane potential was altered by manipulation of bath K(+) concentration. Apical HCO(3)(-) fluxes activated by cyclic AMP were independent of Cl(-) and luminal Na(+), and substantially inhibited by the CFTR blocker, CFTR(inh)-172. Furthermore, comparable HCO(3)(-) fluxes observed in ducts isolated from wild-type mice were absent in ducts from cystic fibrosis (Delta F) mice. To estimate the HCO(3)(-) permeability of the apical membrane under physiological conditions, guinea pig ducts were luminally perfused with a solution containing 125 mM HCO(3)(-) and 24 mM Cl(-) in the presence of 5% CO(2). From the changes in pH(i), membrane potential, and buffering capacity, the flux and electrochemical gradient of HCO(3)(-) across the apical membrane were determined and used to calculate the HCO(3)(-) permeability. Our estimate of approximately 0.1 microm sec(-1) for the apical HCO(3)(-) permeability of guinea pig duct cells under these conditions is close to the value required to account for observed rates of HCO(3)(-) secretion. This suggests that CFTR functions as a HCO(3)(-) channel in pancreatic duct cells, and that it provides a significant pathway for HCO(3)(-) transport across the apical membrane.

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HCO3− fluxes across the apical membrane of interlobular ducts isolated from the guinea pig pancreas. (A) Experimental conditions for the detection of membrane potential–evoked HCO3− fluxes across the apical membrane of microperfused interlobular ducts. It was assumed that, because of the leakiness of the ductal epithelium, changes in bath K+ concentration would bring about comparable changes in membrane potential at both apical and basolateral membranes. Concentrations of Cl− and HCO3− are indicated in mM. Basolateral HCO3− efflux was inhibited with 0.5 mM H2DIDS. HCO3− fluxes across the apical membrane were detected as changes in pHi. (B) Isolated interlobular duct from guinea pig pancreas cannulated with concentric holding and perfusion pipettes. This configuration allowed independent perfusion of the lumen and bath. Duct cells were loaded with BCECF, and a small region of the duct epithelium (indicated by the rectangle) was selected for measurement of pHi. (C and D) Membrane potential–evoked changes in pHi in ducts exposed to different bath K+ concentrations. Bath and lumen were first perfused with the standard HEPES-buffered solution, and the luminal solution was then switched to the high-HCO3− solution containing 125 mM HCO3− and 24 mM Cl−. 0.5 mM dbcAMP was present in the bath perfusate as indicated. The Na+ concentration in the bath and luminal solutions was 60 mM throughout, and [K+]B was raised or lowered (1, 5, and 70 mM) by replacement with NMDG. Each trace is representative of four experiments.
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fig1: HCO3− fluxes across the apical membrane of interlobular ducts isolated from the guinea pig pancreas. (A) Experimental conditions for the detection of membrane potential–evoked HCO3− fluxes across the apical membrane of microperfused interlobular ducts. It was assumed that, because of the leakiness of the ductal epithelium, changes in bath K+ concentration would bring about comparable changes in membrane potential at both apical and basolateral membranes. Concentrations of Cl− and HCO3− are indicated in mM. Basolateral HCO3− efflux was inhibited with 0.5 mM H2DIDS. HCO3− fluxes across the apical membrane were detected as changes in pHi. (B) Isolated interlobular duct from guinea pig pancreas cannulated with concentric holding and perfusion pipettes. This configuration allowed independent perfusion of the lumen and bath. Duct cells were loaded with BCECF, and a small region of the duct epithelium (indicated by the rectangle) was selected for measurement of pHi. (C and D) Membrane potential–evoked changes in pHi in ducts exposed to different bath K+ concentrations. Bath and lumen were first perfused with the standard HEPES-buffered solution, and the luminal solution was then switched to the high-HCO3− solution containing 125 mM HCO3− and 24 mM Cl−. 0.5 mM dbcAMP was present in the bath perfusate as indicated. The Na+ concentration in the bath and luminal solutions was 60 mM throughout, and [K+]B was raised or lowered (1, 5, and 70 mM) by replacement with NMDG. Each trace is representative of four experiments.

Mentions: The lumen of each interlobular duct segment was microperfused (Ishiguro et al., 2000). Both ends of the duct were cut open using sharpened needles, and one end was cannulated with concentric holding and perfusion pipettes (Fig. 1 B). The bath and lumen were perfused separately, and the bath was maintained at 37°C.


CFTR functions as a bicarbonate channel in pancreatic duct cells.

Ishiguro H, Steward MC, Naruse S, Ko SB, Goto H, Case RM, Kondo T, Yamamoto A - J. Gen. Physiol. (2009)

HCO3− fluxes across the apical membrane of interlobular ducts isolated from the guinea pig pancreas. (A) Experimental conditions for the detection of membrane potential–evoked HCO3− fluxes across the apical membrane of microperfused interlobular ducts. It was assumed that, because of the leakiness of the ductal epithelium, changes in bath K+ concentration would bring about comparable changes in membrane potential at both apical and basolateral membranes. Concentrations of Cl− and HCO3− are indicated in mM. Basolateral HCO3− efflux was inhibited with 0.5 mM H2DIDS. HCO3− fluxes across the apical membrane were detected as changes in pHi. (B) Isolated interlobular duct from guinea pig pancreas cannulated with concentric holding and perfusion pipettes. This configuration allowed independent perfusion of the lumen and bath. Duct cells were loaded with BCECF, and a small region of the duct epithelium (indicated by the rectangle) was selected for measurement of pHi. (C and D) Membrane potential–evoked changes in pHi in ducts exposed to different bath K+ concentrations. Bath and lumen were first perfused with the standard HEPES-buffered solution, and the luminal solution was then switched to the high-HCO3− solution containing 125 mM HCO3− and 24 mM Cl−. 0.5 mM dbcAMP was present in the bath perfusate as indicated. The Na+ concentration in the bath and luminal solutions was 60 mM throughout, and [K+]B was raised or lowered (1, 5, and 70 mM) by replacement with NMDG. Each trace is representative of four experiments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654087&req=5

fig1: HCO3− fluxes across the apical membrane of interlobular ducts isolated from the guinea pig pancreas. (A) Experimental conditions for the detection of membrane potential–evoked HCO3− fluxes across the apical membrane of microperfused interlobular ducts. It was assumed that, because of the leakiness of the ductal epithelium, changes in bath K+ concentration would bring about comparable changes in membrane potential at both apical and basolateral membranes. Concentrations of Cl− and HCO3− are indicated in mM. Basolateral HCO3− efflux was inhibited with 0.5 mM H2DIDS. HCO3− fluxes across the apical membrane were detected as changes in pHi. (B) Isolated interlobular duct from guinea pig pancreas cannulated with concentric holding and perfusion pipettes. This configuration allowed independent perfusion of the lumen and bath. Duct cells were loaded with BCECF, and a small region of the duct epithelium (indicated by the rectangle) was selected for measurement of pHi. (C and D) Membrane potential–evoked changes in pHi in ducts exposed to different bath K+ concentrations. Bath and lumen were first perfused with the standard HEPES-buffered solution, and the luminal solution was then switched to the high-HCO3− solution containing 125 mM HCO3− and 24 mM Cl−. 0.5 mM dbcAMP was present in the bath perfusate as indicated. The Na+ concentration in the bath and luminal solutions was 60 mM throughout, and [K+]B was raised or lowered (1, 5, and 70 mM) by replacement with NMDG. Each trace is representative of four experiments.
Mentions: The lumen of each interlobular duct segment was microperfused (Ishiguro et al., 2000). Both ends of the duct were cut open using sharpened needles, and one end was cannulated with concentric holding and perfusion pipettes (Fig. 1 B). The bath and lumen were perfused separately, and the bath was maintained at 37°C.

Bottom Line: Apical HCO(3)(-) fluxes activated by cyclic AMP were independent of Cl(-) and luminal Na(+), and substantially inhibited by the CFTR blocker, CFTR(inh)-172.From the changes in pH(i), membrane potential, and buffering capacity, the flux and electrochemical gradient of HCO(3)(-) across the apical membrane were determined and used to calculate the HCO(3)(-) permeability.This suggests that CFTR functions as a HCO(3)(-) channel in pancreatic duct cells, and that it provides a significant pathway for HCO(3)(-) transport across the apical membrane.

View Article: PubMed Central - PubMed

Affiliation: Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan. ishiguro@htc.nagoya-u.ac.jp

ABSTRACT
Pancreatic duct epithelium secretes a HCO(3)(-)-rich fluid by a mechanism dependent on cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. However, the exact role of CFTR remains unclear. One possibility is that the HCO(3)(-) permeability of CFTR provides a pathway for apical HCO(3)(-) efflux during maximal secretion. We have therefore attempted to measure electrodiffusive fluxes of HCO(3)(-) induced by changes in membrane potential across the apical membrane of interlobular ducts isolated from the guinea pig pancreas. This was done by recording the changes in intracellular pH (pH(i)) that occurred in luminally perfused ducts when membrane potential was altered by manipulation of bath K(+) concentration. Apical HCO(3)(-) fluxes activated by cyclic AMP were independent of Cl(-) and luminal Na(+), and substantially inhibited by the CFTR blocker, CFTR(inh)-172. Furthermore, comparable HCO(3)(-) fluxes observed in ducts isolated from wild-type mice were absent in ducts from cystic fibrosis (Delta F) mice. To estimate the HCO(3)(-) permeability of the apical membrane under physiological conditions, guinea pig ducts were luminally perfused with a solution containing 125 mM HCO(3)(-) and 24 mM Cl(-) in the presence of 5% CO(2). From the changes in pH(i), membrane potential, and buffering capacity, the flux and electrochemical gradient of HCO(3)(-) across the apical membrane were determined and used to calculate the HCO(3)(-) permeability. Our estimate of approximately 0.1 microm sec(-1) for the apical HCO(3)(-) permeability of guinea pig duct cells under these conditions is close to the value required to account for observed rates of HCO(3)(-) secretion. This suggests that CFTR functions as a HCO(3)(-) channel in pancreatic duct cells, and that it provides a significant pathway for HCO(3)(-) transport across the apical membrane.

Show MeSH
Related in: MedlinePlus