Experiments done under Dr. Dorothee Guenzel supervision in Prof. Fromm’s lab (Dept of clinical Physiology at the Charité Berlin).
The Ussing chamber was invented by Hans Ussing in the 1950s to measure the net ion transport taking place across an epithelium clamped between two reservoirs are filled with symmetrical solutions. The equal charges in both sites of the epithelia result in an initial equilibrium at the set up potential of 0 mV. Ions that move through paracellular pathways, tight junctions or across the cell generate a voltage difference among chambers. Addition of specific drugs will promote or inhibit channel activity and modify the ionic conductances, thereby changing the voltage. The amount of current injected to clamp the voltage to 0 mV is called short circuit current (Isc) and is the measure of net ion transport across the epithelium.
Briefly, in our set up the current was applied by two silver/silver chloride (Ag/AgCh) electrodes (one for each tank,) placed in a vessel filled with KCl. Agar bridges (filled with KCl) connected the electrode vessel with the Ussing chamber and allow homogeneous current distribution. They were placed at the same distance of the epithelium in each of the two halves of the Ussing-chamber. Similarly, electrodes placed in KCl filled vessels, also connected to the Ussing chamber by KCl filled agar bridges, measured the transepithelial voltage.
This method very valuable to measure ionic permeability (conductance or resistance) and active ion transport (short circuit current, Isc).
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Mice were anesthetized and killed by inhalation of 100% CO2 . Late distal colon was dissected, cut open along the mesenteric insertion and partially stripped, where, the lamina muscularis propria was removed. The partial 'stripping' allows assessment of the epithelium contribution by removal of all subepithelial elements. The epithelium (lamina propria mucosae) was then clamped in the Ussing chamber. The epithelium was stretched over a silicon ring of 0.05 cm2, fixed with a tissue adhesive to a plastic container and then placed into the chamber. The two reservoirs were filled with 10 ml ringer solution warmed up by a closed water circuit (at 37°C) and gassed with carbogen (95% O2, 5% CO2, to maintain pH 7.4). This bubble lift facilitated the continuous chamber perfusion, and proper mix of the solution. Addition of 50 mg/l Piperacillin (Hexal AG, Holzkirchen, Germany) and 10 mg/l Zienam (MSD Sharp & Dohme GmbH Haar, Germany) effectively inhibited bacterial growth. TTX (serosal, 1 µM), indomethacin (mucosal and serosal, 10 µM) and amiloride (mucosal, 10 µM) were applied to inhibit neuronal activity, prevent prostaglandin release, and block apical Na+ channels (ENaC), respectively.
Prior to each experiment, the resistance of the bath solution between the voltage-sensing electrodes was determined and taken into account.
4.8.1.2 Measurement of Voltage, Resistance and Short Circuit Current
The transepithelial resistance was calculated following the Ohm's Law taking the transepithelial potential differences (V1 and V2) and the current injected by the pulse generator (I1-I2). Rte = (V1-V2)/(I1-I2). For the correct calculation of transepithelial resistance, the area of the tissue being studied (A=0,05 cm2) and the resistance of the empty removable disk or of the blank filter, were taken into account The short circuit current was calculated as the ratio between voltage and resistance, where the transepithelial resistance is known and the transepithhelial voltage is directly measured.
Isc = Vte/Rte.
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4.8.2 Ussing Chamber: Tracheal Epithelia
Dr. Ousingsawat carried out these experiments in the lab of Prof. Kunzelmann. Dept. of Physiology at the University of Regensburg
Tracheas were dissected, opened longitudinally on the opposite side of the cartilage free zone and were transferred immediately into an ice cold buffer solution 1 containing amiloride (10 µM). Stripped colon was put into ice-cold buffer containing amiloride (10 µM) and indomethacin (10 µM). Tissues were mounted into a micro-perfused Ussing chamber with a circular aperture of 0.785 mm2. Luminal and basolateral sides of the epithelium were perfused continuously at a rate of 5 ml/min. Bath solutions were heated to 37 °C, using a water jacket. Experiments were carried out under open circuit conditions. Data were collected continuously using PowerLab (AD-Instruments, Australia).
Values for transepithelial voltages (Vte) were referred to the serosal side of the epithelium. Transepithelial resistance (Rte) was determined by applying short (1s) current pulses (I = 0.5 µA). Rte and equivalent short circuit currents (ISC) were calculated according to Ohm’s law (Rte = Vte/I, ISC = Vte/Rte).
4.8.3 Patch Clamp of Mouse Colonic Crypt Cells
Experiments done in collaboration with Prof. Warth. Dept. of Physiology at the University of Regensburg
In this technique, the membrane potential is held constant (“clamped”) while the current flowing through the membrane is measured. Technically, a patch pipette with a tip diameter of approximately is placed in contact with the plasma membrane. When little negative pressure is applied from the interior of the pipette, a very high resistance seal (Giga-seal – resistance typically 50 GΩ) develops between the membrane patch and the glass of the pipette (“on-cell” configuration). Rupture of the membrane patch underlying the electrode surface (whole-cell configuration) allows direct communication between the interior of the electrode (filled with so called “internal solution”) and the intracellular space. Voltage-clamp of the cell, which consists in maintaining membrane voltage at a constant fixed value, is then achieved using a negative feedback loop through a high-gain differential amplifier (Hodgkin and Huxley, 1952).
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Mice were killed by cervical dislocation. A 3 cm piece of mouse distal colon was everted and rinsed with ice-cold “colon” solution. Both ends were tied to obtain a sac preparation. This sac was filled with a “Ca2+-free” solution and then incubated in
“Ca2+-free” solution for 11 min at 37° C. Isolated crypts were obtained by shaking these sacs in “colon” solution.
For patch clamp experiments, crypts were transferred into a bath chamber mounted on the stage of an inverted microscope. The crypt under study was held by a suction pipette. All experiments were performed at 37° C. Individual cells were approached on the basal cell pole (crypt base and crypt middle cells) by patch pipettes with a mean resistance of 6-10 MΩ. The patch pipettes solution and bath solution compositions are detailed in electrophysiology materials. The recordings reported here were obtained in whole-cell mode using an EPC7-like amplifier (U. Fröbe, Freiburg, Germany).
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