P5640

The apical and basolateral compartments of the Ussing chambers were filled with 10 ml carbogen aerated Krebs solution containing (in mM) 117 NaCl, 4.7 KCl, 1.2 MgCl₂, 1.2 NaH₂PO₄, 20 NaHCO₃, 2.5 CaCl₂ and 11 glucose (all from Sigma-Aldrich, Taufkirchen, Germany), maintained at 37°C. For experiments with Cl^- free buffer solution, Krebs solution was replaced by a carbogen aerated buffer solution containing (in mM) 58.5 NaSO₄, 2.4 KSO₄, 1.2 MgSO₄, 1.2 NaH₂PO₄, 20 NaHCO₃, 2.5 CaSO₄, 11 glucose and 85 mannitol. For experiments with Cl^- and HCO₃^- free buffer solution, Krebs solution was replaced by an oxygen aerated buffer solution containing (in mM) 58.5 NaSO₄, 2.4 KSO₄, 1.2 MgSO₄, 1.2 NaH₂PO₄, 20 HEPES, 2.5 CaSO₄, 11 glucose and 91 mannitol. Stock solutions of TTX (Carl Roth GmbH and Co. KG, Karlsruhe, Germany), ω-conotoxin GVIA (Alomone Labs, Jerusalem, Israel) dissolved in distilled water, as well as stock solutions of Piroxicam (P-5654, Sigma-Aldrich,) dissolved in chloroform and prostaglandin E₂ (PGE₂) (P5640, Sigma-Aldrich) dissolved in DMSO, were stored at -20°C until use at indicated final concentrations in Ussing chambers. Filters used for experiments to prevent distension had a pore size of 0.45 μM and a diameter of 2.5 cm (MF-Millipore, HABP02500, Merck Millipore Ltd., Ireland, Tullagreen, Carrigtwohill, County Cork, Ireland).