4.4. Ussing Chamber Experiments

Ussing chamber experiments provide insight into transepithelial transport processes with regard to flux rates and electrophysiological parameters. The experiments were conducted with duodenal and ileal tissue samples using twelve chambers for each segment. After stripping off the tunica serosa and tunica muscularis, the remaining tunica mucosa was mounted between the two halves of the incubation chambers with an exposed serosal area of 1.0 cm², dividing each chamber into a mucosal and serosal compartment. Both sides of the tissue were incubated with 10 mL of 38 °C warm buffer solution, which was continuously aerated with carbogen and maintained at pH 7.4. Both the serosal and the mucosal buffer solutions contained (mmol/L) 113.6 NaCl, 5.4 KCl, 0.2 HCl (1n), 1.2 MgCl₂·6H₂O, 21.0 NaHCO₃, 1.2 CaCl₂·2H₂O, 1.2 Na₂HPO₄·2H₂O, 1.2 mannitol and 0.01 indomethacin. The serosal buffer additionally contained (mmol/L) 10.0 glucose, 6.0 Na⁺-gluconate and 7.0 HEPES, while 6.0 NaOH (1n) and 20.0 HEPES were added to the mucosal buffer. Towards the end of the experiment, Na⁺-arsenate (5 mmol/L) or 2,4,6-triaminopyrimidine (TAP) (20 mmol/L) were added to the mucosal side of four chambers each. As a competitive inhibitor of Na⁺-dependent P_i transport, Na⁺-arsenate was used to investigate the transcellular P_i pathway [82]. TAP was used to investigate the paracellular P_i pathway as it blocks TJ proteins [83] and is supposed to inhibit paracellular P_i transport. The remaining four chambers were time controls to which no inhibitors were added.

The investigation of the transepithelial flux rates of P_i in the duodenum and ileum was based on the radioisotope tracer technique and was conducted in accordance with established protocols using ³²P und ³H(-mannitol) as radio isotopic tracers [4]. ³²P was used to trace the total transepithelial P_i transports, while (³H-) mannitol marked the paracellular P_i transport [84]. After an equilibration time of 5–10 min, 148 kBq of the respective radioisotope was added to each chamber either to the serosal or mucosal side. For a period of 60 min, samples were taken from the unlabelled side at 15 min intervals and immediately replaced by equal volumes of the respective buffer solution. Additionally, samples were taken from the radioactively labelled side at the beginning and at the end of the experiment. The sampling before and after adding of inhibitors was performed to obtain information about the respective proportion of the transcellular and paracellular P_i transport on total P_i transport. A liquid scintillation counter (Packard BioScience Company, Meriden, CT, USA) was used to determine the radioactivity of the samples. Without electrochemical gradients, unidirectional flux rates of P_i and mannitol from mucosal to serosal (J_ms) and in the opposite direction from serosal to mucosal (J_sm) were calculated from the appearance rate of the respective tracer on the unlabelled side using standard equations [85]. The net flux rates (J_net) were determined by subtracting J_sm from J_ms of paired tissues.

Using a computer-controlled voltage clamp device (Mussler Scientific Instruments, Aachen, Germany), the potential differences, tissue conductance (G_t) and short circuit currents (I_sc) were continuously monitored. The experiments were carried out under short-circuited conditions. The basal values for G_t and I_sc were determined as mean values over the time before adding the inhibitor. In order to examine the effects of Na⁺-arsenate or TAP on the transepithelial transport processes in the duodenum and ileum, changes in G_t and I_sc were observed after the addition. Tissue viability was monitored by continuously recording G_t and I_sc and by adding glucose (10 mmol/L) to the mucosal side and forskolin (0.01 mmol/L) to the serosal of all chambers at the end of the experiments. The change in I_sc in response to the addition of glucose and forskolin indicated epithelial viability and integrity.