Chloride transport in microperfused interlobular ducts isolated from guinea-pig pancreas

Abstract

Isolated interlobular ducts from the guinea-pig pancreas secrete a HCO3--rich fluid in response to secretin. To determine the role of Cl- transporters in this process, intracellular Cl- concentration ([Cl-]i) was measured in ducts loaded with the Cl--sensitive fluoroprobe, 6-methoxy-N-ethylquinolinium chloride (MEQ). [Cl-]i decreased when the luminal Cl- concentration was reduced. This effect was stimulated by forskolin, was not dependent on HCO3- and was not inhibited by application of the anion channel/transporter inhibitor H2DIDS to the luminal membrane. It is therefore attributed to a cAMP-stimulated Cl- conductance, probably the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. [Cl-]i also decreased when the basolateral Cl- concentration was reduced. This effect was not stimulated by forskolin, was largely dependent on HCO3- and was inhibited by basolateral H2DIDS. It is therefore mediated mainly by Cl-/HCO3- exchange. With high Cl- and low HCO3- concentrations in the lumen, steady-state [Cl-]i was 25-35 mM in unstimulated cells. Stimulation with forskolin caused [Cl-]i to increase by approximately 4 mM due to activation of the luminal anion exchanger. With low Cl- and high HCO3- concentrations in the lumen to simulate physiological conditions, steady-state [Cl-]i was 10-15 mM in unstimulated cells. Upon stimulation with forskolin, [Cl-]i fell to approximately 7 mM due to increased Cl- efflux via the luminal conductance. We conclude that, during stimulation under physiological conditions, [Cl-]i decreases to very low levels in guinea-pig pancreatic duct cells, largely as a result of the limited capacity of the basolateral transporters for Cl- uptake. The resulting lack of competition from intracellular Cl- may therefore favour HCO3- secretion via anion conductances in the luminal membrane, possibly CFTR.

Figures

Figure 1. Calibration of MEQ fluorescence for measurement of intracellular Cl− concentrations in isolated interlobular duct segments

A, a representative experiment showing MEQ calibration performed in situ by exposing the duct to nigericin (5 μm) and tributyltin chloride (10 μm) and by varying the perfusate Cl− concentration (by substitution with gluconate). MEQ fluorescence is plotted as the relative intensity normalised to the value at the beginning of the recording. B, Stern-Volmer plot of the calibration data (means ±s.d., n = 9). F0 and F are the values of the relative intensity in the absence and presence of Cl−, respectively.

Figure 2. Changes in [Cl−]i on modifying luminal Cl− concentration

Initially the bath and lumen of the duct segments were perfused with the standard HCO3−-buffered solution containing 124 mmCl− and 25 mm HCO3−. Thereafter, the luminal perfusate was switched to a low Cl−-high HCO3− solution containing 24 mmCl− and 125 mm HCO3−, and then forskolin (1 μm) was applied to the bath. A, changes in relative intensity in a representative experiment followed by in situ calibration by exposing the duct to 0, 20 and 40 mmCl− solution in the presence of nigericin and tributyltin. B, changes in [Cl−]i which were estimated from the calibration equation. One of five experiments.

Figure 3. Effects of forskolin on Cl− flux across the luminal membrane in the presence of HCO3−/CO2

Initially the bath and lumen of the duct segments were perfused with the standard HCO3−-buffered solution. A, forskolin (1 μm) was applied to the bath and then the luminal perfusate was switched to the low Cl−-high HCO3− solution in the continued presence of forskolin. One of seven experiments. B, the luminal membrane was pretreated with H2DIDS (200 μm) and forskolin (1 μm) was applied to the bath. One of four experiments. C, the luminal perfusate was switched to the low Cl−-high HCO3− solution in the absence and presence of forskolin (1 μm). One of five experiments.

Figure 4. Effects of forskolin on Cl− flux across the luminal membrane in the absence of HCO3−/CO2

Initially the bath and lumen of the duct segments were perfused with the standard Hepes-buffered solution containing 139 mmCl−. A, the luminal perfusate was switched to the 0 Cl− Hepes-buffered solution and then forskolin (1 μm) was applied to the bath. One of five experiments. B, the luminal perfusate was switched to the 0 Cl− Hepes-buffered solution in the absence and presence of forskolin (1 μm). One of four experiments.

Figure 5. Effects of luminal inhibitors on forskolin-stimulated Cl− flux across the luminal membrane

Initially the bath and lumen of the duct segments were perfused with the standard HCO3−-buffered solution and forskolin (1 μm) was applied to the bath. The luminal perfusate was switched to the low Cl−-high HCO3− solution in the absence and presence of 100 μm glibenclamide (A), 500 μm H2DIDS (B), or 10 μm NPPB (C). Each trace is a representative of four experiments.

Figure 6. Changes in [Cl−]i on modifying basolateral Cl− concentration

A, initially the bath and lumen of the duct segments were perfused with the standard HCO3−-buffered solution. The basolateral perfusate was then switched to the low Cl−-high HCO3− solution in the absence and presence of forskolin (1 μm). One of five experiments. B, with the bath and lumen initially perfused with the standard Hepes-buffered solution, the luminal perfusate was switched to the 0 Cl− Hepes-buffered solution in the absence and presence of forskolin (1 μm). One of four experiments. C, with the bath and lumen initially perfused with the HCO3−-buffered solution, the basolateral membrane was pretreated with H2DIDS (200 μm) and then the basolateral perfusate was switched to the low Cl−-Ihigh HCO3− solution in the presence of forskolin (1 μm). One of four experiments.

Figure 7. Effects of basolateral or luminal H2DIDS on [Cl−]i

The bath and lumen of the duct segments were perfused with the standard HCO3−-buffered solution. H2DIDS (200 μm) was sequentially applied to the bath and then to the lumen (A and C) or in a reverse order (B and D) in the absence (A and B) or presence (C and D) of forskolin (1 μm). Each trace is a representative of four experiments.

Figure 8. Implications of measured [Cl−]i values for anion channel/transporter activity under different conditions

The lumen is perfused either with a high Cl− -low HCO3− solution (A and B) or with a low Cl−-high HCO3− solution (C and D) in either the absence (A and C) or presence (B and D) of a cAMP-mediated secretory stimulus.