Very high aquaporin-1 facilitated water permeability in mouse gallbladder

Abstract

Water transport across gallbladder epithelium is driven by osmotic gradients generated from active salt absorption and secretion. Aquaporin (AQP) water channels have been proposed to facilitate transepithelial water transport in gallbladder and to modulate bile composition. We found strong AQP1 immunofluorescence at the apical membrane of mouse gallbladder epithelium. Transepithelial osmotic water permeability (Pf) was measured in freshly isolated gallbladder sacs from the kinetics of luminal calcein self-quenching in response to an osmotic gradient. Pf was very high (0.12 cm/s) in gallbladders from wild-type mice, cAMP independent, and independent of osmotic gradient size and direction. Although gallbladders from AQP1 knockout mice had similar size and morphology to those from wild-type mice, their Pf was reduced by approximately 10-fold. Apical plasma membrane water permeability was greatly reduced in AQP1-deficient gallbladders, as measured by cytoplasmic calcein quenching in perfluorocarbon-filled, inverted gallbladder sacs. However, neither bile osmolality nor bile salt concentration differed in gallbladders from wild-type vs. AQP1 knockout mice. Our data indicate constitutively high water permeability in mouse gallbladder epithelium involving transcellular water transport through AQP1. The similar bile salt concentration in gallbladders from AQP1 knockout mice argues against a physiologically important role for AQP1 in mouse gallbladder.

Fig. 1.

Gallbladder morphology and aquaporin-1 (AQP1) expression. A: photographs of gallbladders from wild-type and AQP1 null mice taken in situ after 8-h fasting. B: hematoxylin and eosin-stained gallbladder. Scale bar: 50 μm. C: AQP1 immunofluorescence. Scale bar: 20 μm. D: RT-PCR of gallbladder epithelial cell cDNA from wild-type and AQP1 null mice. Representative of data from 3 sets of experiments. Control amplifications done using cDNAs from indicated tissues. Submandib, submandibular gland. E: quantitative real-time RT-PCR of gallbladder epithelial cell cDNA showing relative expression of transcripts encoding AQP1 and AQP8, normalized to β-actin transcript expression (SE, n = 4 gallbladders per genotype). *P < 0.05.

Fig. 2.

Transepithelial osmotic water permeability measured by luminal calcein self-quenching. A: principle of the measurement method, showing dilution of luminal calcein in response to osmotically driven water influx into the lumen of a calcein-filled gallbladder sac (left). Calcein dilution results in less fluorescence self-quenching and consequent increased fluorescence. Right: photograph of calcein-filled gallbladder sac. Scale bar: 1.5 mm. B: perfusion chamber for water permeability measurements. Left: schematic showing gallbladder sac immobilized by a nylon mesh. Right: photograph of perfusion chamber. C: transepithelial osmotic water permeability in gallbladder from wild-type mouse. Top: representative calcein fluorescence kinetics shown in response to indicated perfusate osmolalities. Bottom: summary of osmotic water permeability coefficients [mean ± SE, n = 5, transepithelial osmotic water permeability (P_f) at 23°C] measured as function of osmotic gradient (ΔmOsm). Differences are not significant. D: calcein fluorescence self-quenching shown as calcein fluorescence measured as a function of its concentration in physiological saline. Measurements were made by microfiberoptic fluorescence to avoid inner filter effect.

Fig. 3.

Greatly reduced water permeability in gallbladder in AQP1 knockout mice. A: calcein fluorescence for gallbladder from wild-type mouse subjected to a 100 mosmol/kg transepithelial osmotic gradient, measured before and after 10 min incubation with 10 μM forskolin (left) and summary of initial slopes following osmotic challenge (SE, n = 5) (right). Difference is not significant. B: calcein fluorescence for gallbladders from wild-type and AQP1 null mice shown for different osmotic gradients (left) and summary of deduced P_f (averaged in each gallbladder) (SE, n = 5) (right). *P < 0.001.

Fig. 4.

Apical plasma membrane water permeability in gallbladder epithelial cells. A: schematic of measurement method showing cytoplasmic calcein staining of gallbladder epithelial cells in an inverted gallbladder sac (apical surface facing outward), with the lumen filled with perfluorocarbon. Increased perfusate osmolality reduces gallbladder cell volume, resulting in a quenching of cytoplasmic calcein fluorescence. B: fluorescence micrograph showing calcein-stained cytoplasm of gallbladder epithelial cells. Scale bar: 1 mm. Inset: en-face magnified view of stained gallbladder cells. Scale bar: 10 μm. C: time course of cytoplasmic calcein fluorescence in response to changes in perfusate osmolality between 300 and 600 mosmol/kg for gallbladders (left) from 2 wild-type mice (top) and 1 AQP1 null mouse (bottom) and deduced reciprocal half-times (t_1/2⁻¹) for osmotic equilibration (right). •, Individual measurements; ○, mean and SE. *P < 0.001.

Fig. 5.

AQP1 deficiency does not alter bile osmolality or composition. Gallbladder contents were removed from mice after 8 h of fasting and were assayed for osmolality, total bile acid concentration, and GSH concentration (SE, n = 5 mice per genotype). Differences are not significant.