Luminal ATP stimulates fluid and HCO3- secretion in guinea-pig pancreatic duct

Abstract

1. The location of purinoceptors in the pancreatic duct and their role in regulating ductal secretion have been investigated by applying ATP and UTP to basolateral and luminal surfaces of pancreatic ducts isolated from the guinea-pig pancreas. 2. Changes in intracellular Ca2+ concentration were measured by microfluorometry in microperfused interlobular duct segments. Fluid and HCO3- secretion were estimated by monitoring luminal pH and luminal volume in sealed duct segments microinjected with BCECF-dextran. 3. Both ATP and UTP (1 microM) caused biphasic increases in intracellular Ca2+ concentration in pancreatic duct cells when applied to either the basolateral or luminal membrane. 4. Luminal application of both ATP and UTP evoked fluid and HCO3- secretion. The maximum response to 1 microM ATP or UTP was about 75 % of that evoked by secretin. By contrast, basolateral application of ATP or UTP inhibited spontaneous secretion by 52 % and 73 %, respectively, and secretin-evoked secretion by 41 % and 38 %, respectively. 5. The data suggest that luminal nucleotides may act in an autocrine or paracrine fashion to enhance ductal secretion while basolateral nucleotides, perhaps released from nerve terminals, may have an inhibitory effect. The fact that both apical and basolateral purinoceptors elevate intracellular Ca2+, but that they have opposite effects on secretion, suggests that additional signalling pathways are involved.

Figure 1. Effects of basolateral and luminal ATP and UTP on intracellular Ca²⁺

A, interlobular duct segment isolated from guinea-pig pancreas and cannulated (from the right) for luminal microperfusion. In this image the lumen was perfused with a coloured solution which can be seen emerging from the left end of the duct where it is swept away by the flow of the bath solution from right to left. Scale bar represents 200 μm. B and C, changes in fura-2 fluorescence ratio (F₃₄₀/F₃₈₀) indicating changes in [Ca²⁺]_i in microperfused ducts stimulated with ATP and UTP. The bath and lumen were perfused separately with the standard HCO₃⁻-buffered solution. During the periods indicated, 1 μm ATP (B) and 1 μm UTP (C) were applied via the bath and/or the lumen. Each trace is representative of four similar experiments.

Figure 2. Effects of luminal ATP on luminal pH and fluid secretion

Guinea-pig pancreatic ducts filled by micropuncture with a weakly buffered HCO₃⁻-free, Cl⁻-free solution containing 20 μm BCECF-dextran were initially superfused with a Cl⁻-free Hepes-buffered bath solution. After 3 min this was switched to a Cl⁻-free, HCO₃⁻-buffered solution and, after a further 4 min, 10 nM secretin was applied via the bath. A and B, control experiments: changes in luminal pH (A) and fluid secretory rate (B) in the absence of luminal ATP (A, one of five experiments; B, means ±s.e.m. of all five experiments). C and D, effects of luminal ATP: changes in luminal pH (C) and fluid secretory rate (D) in ducts injected with 1 μm ATP (C, one of four experiments; D, means ±s.e.m. of all four experiments). E, concentration-response curve for the effects of luminal ATP on fluid secretory rate. Data are shown as means ±s.e.m. of at least four experiments.

Figure 3. Effects of luminal UTP on luminal pH and fluid secretion

Guinea-pig pancreatic ducts filled by micropuncture with a weakly buffered HCO₃⁻-free, Cl⁻-free solution containing 1 μm UTP and 20 μm BCECF-dextran were initially superfused with a Cl⁻-free Hepes-buffered bath solution. After 3 min this was switched to a Cl⁻-free, HCO₃⁻-buffered solution and, after a further 4 min, 10 nM secretin was applied via the bath. A, changes in luminal pH (one of four experiments); B, changes in fluid secretory rate (means ±s.e.m. of all four experiments).

Figure 4. Effects of basolateral ATP on luminal pH and fluid secretion

Guinea-pig pancreatic ducts filled by micropuncture with a weakly buffered HCO₃⁻-free, Cl⁻-free solution containing 20 μm BCECF-dextran were initially superfused with a Cl⁻-free, Hepes-buffered bath solution. After 3 min this was switched to a Cl⁻-free, HCO₃⁻-buffered solution and, after a further 6 min, 10 nM secretin was applied via the bath. During the periods indicated, 1 μm ATP was applied via the bath in the absence or presence of 10 nM secretin. A, changes in luminal pH; B, changes in fluid secretory rate (one of five experiments).C, concentration-dependent effects of basolateral ATP on the fluid secretory rate evoked by 10 nM secretin. Increasing concentrations of ATP (0.1 μm and 1 μm) were applied successively via the bath during stimulation with 10 nM secretin. Data are means ±s.e.m. of four experiments.

Figure 5. Effects of basolateral UTP on luminal pH and fluid secretion

Guinea-pig pancreatic ducts filled by micropuncture with a weakly buffered HCO₃⁻-free, Cl⁻-free solution containing 20 μm BCECF-dextran were initially superfused with a Cl⁻-free, Hepes-buffered bath solution. After 3 min this was switched to a Cl⁻-free, HCO₃⁻-buffered solution and, after a further 11 min, 10 nM secretin was applied via the bath. During the periods indicated, 1 μm UTP was applied via the bath in the absence or presence of 10 nM secretin. A, changes in luminal pH; B, changes in fluid secretory rate (one of four experiments).