Fura-2 transport in toad urinary bladder epithelium: effects of antidiuretic hormone, colchicine and osmotic gradients.

Fluorescence is transferred across the toad urinary bladder when fura-2/AM is added to the mucosal or serosal sides of the epithelium. It was now observed that: (1) Oxytocin (20 nM, serosal) increased fluorescence transfer from the mucosal to the serosal but not from the serosal to the mucosal baths. The ratio between the fluorescence intensities recorded with excitation wavelengths of 340 and 380 nm indicates that the calcium sensitive probe (free fura-2) was transferred to the serosal but not to the mucosal compartment by an oxytocin sensitive transport. (2) Preincubation with probenecid did not change fluorescence transfer in basal conditions but significantly reduced the oxytocin induced increase in free fura-2 transport. (3) Fluorescence accumulation inside the tissue was strongly reduced by oxytocin, but only when fura-2/AM was added to the mucosal side. (4) An osmotic gradient, in the presence of oxytocin, further increased the transfer of fluorescence at 380 nm but not at 340 nm. This indicated that the transfer of a calcium-insensitive fraction was being stimulated. (5) Preincubation with colchicine strongly inhibited fluorescence transfer across the tissue, at both 340 and 380 nm (the 340/380 ratio did not change). (6) Tissue accumulation was increased by colchicine. (7) Vanadate did not inhibit fura-2 transfer in the toad urinary bladder. We conclude that intracellularly-generated free fura-2 is only transported across the basolateral border, and that this transfer is stimulated by ADH. The calcium-insensitive fraction is transferred by a temperature-dependent process, sensitive to an osmotic gradient and colchicine.