Ethanol induces fluid hypersecretion from guinea-pig pancreatic duct cells

Abstract

Ethanol is the leading cause of pancreatitis; however, its cellular effects are poorly understood. We examined the direct effects of ethanol in the concentration range 0.1-30 mM, i.e. relevant to usual levels of drinking, on fluid secretion from guinea-pig pancreatic duct cells. Fluid secretion was continuously measured by monitoring the luminal volume of interlobular duct segments isolated from the guinea-pig pancreas. [Ca2+]i was estimated by microfluorometry in duct cells loaded with fura-2. Ethanol at 0.3-30 mM significantly augmented fluid secretion stimulated by physiological (1 pM) or pharmacological (1 nM) concentrations of secretin. It augmented dibutyryl cAMP-stimulated fluid secretion but failed to affect spontaneous or acethylcholine-stimulated secretion. Ethanol at 1 mM shifted the secretin concentration-fluid secretion response curve upwards and raised the maximal secretory response significantly by 41%. In secretin-stimulated ducts, 1 mM ethanol induced a transient increase in [Ca2+]i that was dependent on the presence of extracellular Ca2+. Ethanol failed to augment secretin-stimulated secretion from ducts pretreated with an intracellular Ca2+ buffer (BAPTA) or a protein kinase A inhibitor (H89). In conclusion, low concentrations of ethanol directly augment pancreatic ductal fluid secretion stimulated by physiological and pharmacological concentrations of secretin, and this appears to be mediated by the activation of both the intracellular cAMP pathway and Ca2+ mobilization.

Figure 1. Effects of ethanol on spontaneous fluid secretion, secretin-stimulated secretion and ACh-stimulated secretion

Effects of 1 mM of ethanol on spontaneous fluid secretion (A; n = 5), secretin (0.3 nM)-stimulated secretion (B; n = 6) and ACh (1 μM)-stimulated secretion (C; n = 4) from interlobular duct segments isolated from guinea-pig pancreas superfused with the standard HCO₃⁻ -CO₂ -buffered solution. Values are means ±s.e.m.* Significant differences (P < 0.05) from control levels before the application of ethanol (1 mM).

Figure 2. Effects of ethanol concentration on fluid secretion stimulated by high and low concentrations of secretin

Effects of ethanol concentration on fluid secretion stimulated by 1 pM (A) and 1 nM (B) secretin in the presence of HCO₃⁻-CO₂. Values are means ±s.e.m. of 4–6 experiments. * Significant differences (P < 0.05) from control (without ethanol).

Figure 3. Effects of 1 mM ethanol on the secretin concentration-fluid secretion relationship

The concentrations of secretin (10⁻¹³−10⁻¹⁰ M) were sequentially increased in the absence (A; n = 4) or presence (B; n = 5) of 1 mM ethanol. * Significant differences (P < 0.05) from control (without secretin). C, concentration-response curve of the pooled data in the absence (○) and presence (•) of 1 mM ethanol. * Significant differences (P < 0.05) from the respective controls (without ethanol). D, effects of 0.3 nM secretin (S, n = 6), 1 mM ethanol (EtOH, n = 5), 1 μM ACh (n = 10), 0.3 nM secretin + 1 mM ethanol (S + EtOH, n = 6) and 0.3 nM secretin + 1 μM ACh (S + ACh, n = 5) on fluid secretion. * Significant differences (P < 0.05) from spontaneous basal secretion in the presence of HCO₃⁻ -CO₂ (B, n = 5). Values are means + S.E.M.

Figure 4. Effects of ethanol on fluid secretion stimulated by dbcAMP, or by secretin following preincubation with H89

A, effects of 1 mM ethanol on fluid secretion stimulated by 0.5 mM dbcAMP (n = 7). B, effects of 1 mM ethanol on fluid secretion stimulated by 1 nM secretin in ducts preincubated with 30 μM H89 for 30 min just before experiments began (n = 5). Values are means ±s.e.m.* Significant differences (P < 0.05) from control levels before the application of ethanol (1 mM).

Figure 5. Effects of ethanol on intracellular Ca²⁺ concentration shown as changes in fura-2 fluorescence ratio (F₃₄₀/F₃₈₀) in guinea-pig pancreatic duct cells

A and B, effects of 1 mM ethanol and 10 μM ACh on [Ca²⁺]_i in unstimulated (A) and secretin (1 nM)-stimulated (B) ducts. C, effects of ethanol concentration (0.1–100 mM) on [Ca²⁺]_i (means ±s.e.m. of change in fluorescence ratio (ΔF₃₄₀/F₃₈₀ ratio; n = 4) in ducts stimulated with 1 nM secretin. * Significant differences (P < 0.05). D and E, effects of 100 pM secretin on [Ca²⁺]_i in the absence (D) and presence (E) of 1 mM ethanol. F, effects of 1 mM ethanol on [Ca²⁺]_i in dbcAMP (0.5 mM)-stimulated ducts. G, effects of 1 mM ethanol on [Ca²⁺]_i in secretin (1 nM)-stimulated ducts superfused with Ca²⁺-free solution. H, effects of 1 mM ethanol on [Ca²⁺]_i in secretin (1 nM)-stimulated ducts in the presence of 1 μM thapsigargin (Tg). Each trace is representative of 4 experiments.

Figure 6. Effects of ethanol on fluid secretion from ducts loaded with BAPTA

Ducts were pre-incubated with 10 μM BAPTA AM for 30 min. A, 1 μM ACh was applied to the BAPTA-loaded ducts (n = 4). B, the BAPTA-loaded ducts were first stimulated with 1 nM secretin and then 1 mM ethanol was added (n = 5). Values are means ± S.E.M.