Volume 119, Issue 6, Pages 1672-1680 (December 2000) Perfused rat intrahepatic bile ducts secrete and absorb water, solute, and ions  Anatoly I. Masyuk, Ai–Yu Gong, Sertac Kip, Michael J. Burke, Nicholas F. LaRusso  Gastroenterology  Volume 119, Issue 6, Pages 1672-1680 (December 2000) DOI: 10.1053/gast.2000.20248 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Microperfusion of isolated IBDU. (A) Cartoon and (B) photograph of a microperfused IBDU. IBDUs isolated from rat liver were placed in a temperature-controlled chamber on the stage of an inverted fluorescence microscope. Concentric glass pipettes were used for immobilization (holding pipettes), microperfusion (perfusion and exchanger pipettes) of IBDU, and collection (collecting pipette) of perfused solutions. Gastroenterology 2000 119, 1672-1680DOI: (10.1053/gast.2000.20248) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Microperfusion of isolated IBDU. (A) Cartoon and (B) photograph of a microperfused IBDU. IBDUs isolated from rat liver were placed in a temperature-controlled chamber on the stage of an inverted fluorescence microscope. Concentric glass pipettes were used for immobilization (holding pipettes), microperfusion (perfusion and exchanger pipettes) of IBDU, and collection (collecting pipette) of perfused solutions. Gastroenterology 2000 119, 1672-1680DOI: (10.1053/gast.2000.20248) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Fluorescence measurement system to study water and ion transport in microperfused IBDUs. (A) Schematic representation of the microperfusion technique to study water and ion transport in isolated IBDU. The IBDU was perfused through its lumen at constant nanoliter per minute rates and positioned in the bath of a perfusion chamber on the stage of an inverted fluorescence microscope. The IBDU was immobilized by the holding pipette and perfused with the concentric perfusion pipette. The spot to measure relative fluorescence of the impermeable marker, fluorescein-5- (and -6-) sulfonic acid (FS) or BCECF dextran was positioned at the distal end of IBDU. See text for details. (B) Photomicrograph of a perfused IBDU with 1 mmol/L FS. Gastroenterology 2000 119, 1672-1680DOI: (10.1053/gast.2000.20248) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Fluorescence measurement system to study water and ion transport in microperfused IBDUs. (A) Schematic representation of the microperfusion technique to study water and ion transport in isolated IBDU. The IBDU was perfused through its lumen at constant nanoliter per minute rates and positioned in the bath of a perfusion chamber on the stage of an inverted fluorescence microscope. The IBDU was immobilized by the holding pipette and perfused with the concentric perfusion pipette. The spot to measure relative fluorescence of the impermeable marker, fluorescein-5- (and -6-) sulfonic acid (FS) or BCECF dextran was positioned at the distal end of IBDU. See text for details. (B) Photomicrograph of a perfused IBDU with 1 mmol/L FS. Gastroenterology 2000 119, 1672-1680DOI: (10.1053/gast.2000.20248) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Luminal FS fluorescence in microperfused IBDUs. (A) Relationship between luminal FS fluorescence and its concentration in the perfusion buffer during microperfusion of IBDU under basal conditions. IBDUs were perfused with isotonic KRB of 1–5 mmol/L FS. (B) Single tracing of luminal FS fluorescence at the distal end of microperfused IBDU in response to osmotic gradients. IBDUs were perfused with 1 mmol/L FS in isotonic KRB with a lumen perfusion rate of 40 nL/min for 25 minutes. An inward (secretory) osmotic gradient was established by changes in bath osmolality from 290 to 80 mOsm. An outward (absorptive) osmotic gradient was established by changes in bath osmolality from 290 to 560 mOsm. See text for details. Gastroenterology 2000 119, 1672-1680DOI: (10.1053/gast.2000.20248) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Initial characterization of microperfused IBDUs. (A) A significant correlation (r = 0.99, P < 0.05) between perfusion and collection rates in microperfused IBDU in the absence of osmotic gradient. (B) Bidirectional water movement across intrahepatic biliary epithelium in response to osmotic gradient. When there was no osmotic gradient (perfusate = bath buffer osmolality, bar a) there was no net fluid movement; in contrast, with an inward (secretory, bar b) or outward (absorptive, bar c) osmotic gradient across the IBDU (perfusate osmolality greater than and less than bath buffer osmolality, respectively), secretion or absorption of water occurred. Values are means ± SE of 8–9 microperfused IBDUs in each group (P < 0.05). Gastroenterology 2000 119, 1672-1680DOI: (10.1053/gast.2000.20248) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Water movement in microperfused IBDUs generated by established osmotic gradient. (A) Net water movement (Jv) in microperfused IBDUs. Jv was determined in experiments when IBDU's lumen was perfused with standard KRB (290 mOsm) and bath was perfused with hypotonic (80 mOsm, bar a) or hypertonic (560 mOsm, bar b) KRB. With an inward osmotic gradient across IBDU (perfusate osmolality greater than bath osmolality), net secretion occurs (a negative value); with an outward osmotic gradient across IBDU (perfusate osmolality less than bath osmolality), net absorption occurs (a positive value). O indicates no osmotic gradient (perfusate osmolality equal to bath osmolality). (B) Relationship of calculated Pf to lumen flow rate. IBDU were perfused with isotonic (290 mOsm) KRB with flow rates of 40 nL/min (bar a) and 80 nL/min (bar b) and bathed in hypotonic (80 mOsm) KRB. No differences in Pf were observed. (C) Relationship of calculated Pf to osmotic gradient direction. Pf was higher when transepithelial inward osmotic gradient was established (bar a) than with outward osmotic gradient (bar b; P < 0.001). Values are means ± SE of 6–8 microperfused IBDUs in each group. Gastroenterology 2000 119, 1672-1680DOI: (10.1053/gast.2000.20248) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Uptake of TCA by microperfused IBDUs. (A) IBDUs were perfused with KRB of 10–160 nmol/μL TCA. IBDUs absorbed TCA in a saturable manner. (B) Absorption of TCA was blocked by 60% (P < 0.05) if IBDUs were perfused with 0.5 mmol/L S0960, a specific inhibitor of the Na+/bile acid cotransporter ASBT. (C) TCA uptake by perfused IBDUs in the presence and absence of Na+. Removal of Na+ from perfusate and bathing buffer decreased TCA uptake by 50% (P < 0.05). Values are means ± SE of 4–7 microperfused IBDUs in each group. Gastroenterology 2000 119, 1672-1680DOI: (10.1053/gast.2000.20248) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 HCO3− transport by microperfused IBDUs. (A) Tracing for a single pH experiment in a microperfused IBDU treated with forskolin. Fluorescence intensity of BCECF dextran in the lumen of microperfused IBDU was measured continuously during the bath perfusion, either with KRB (time frame of experiments a, b, and c) or with forskolin (b). Forskolin increased fluorescence intensity at 495 nm and F495/F440 ratio. (B) Time course of the change in luminal pH (ΔpH) in IBDUs treated with forskolin under different conditions. IBDUs were perfused through the lumen with HCO3−-free buffer and bathed simultaneously in KRB (lines 1 and 2) or HCO3−/CO2-free buffer (line 3). Addition of forskolin to the bathing KRB solution for 30 minutes (b in time frame of experiment) resulted in a luminal pH increase of up to 0.68 ± 0.01 units (P < 0.05; line 2). Luminal pH did not change if forskolin was added to HCO3−/CO2-free bathing buffer (line 3). When no forskolin was added to the bathing buffer for the entire equipment, pH remained constant (line 1). Gastroenterology 2000 119, 1672-1680DOI: (10.1053/gast.2000.20248) Copyright © 2000 American Gastroenterological Association Terms and Conditions