Fura-2 Transport in Toad Bladder Epithelium: Effects of Antidiuretic Hormone, Colchicine and osmotic Gradients

48. PARISI, M., BORGNIA, M., DORR, R. AND ROSSI J.P.F.C.

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 1993 vol. 1151 p. 1 - 6

0005-2736

Fluorescence is transferred across the toad urinary bladder when fura-2/AM is added to the mucosal or serosal sides of theepithelium. It was now observed that: (1) Oxytocin (20 nM, serosal) increased fluorescence transfer from the mucosal to theserosal but not from the serosal to the mucosal baths. The ratio between the fluorescence intensities recorded with excitationwavelengths of 340 and 380 nm indicates that the calcium sensitive probe (free fura-2) was transferred to the serosal but not tothe mucosal compartment by an oxytocin sensitive transport. (2) Preincubation with probenecid did not change fluorescencetransfer in basal conditions but significantly reduced the oxytocin induced increase in free fura-2 transport. (3) Fluorescenceaccumulation 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 stronglyinhibited fluorescence transfer across the tissue, at both 340 and 380 nm (the 340/380 ratio did not change). (6) Tissueaccumulation was increased by colchicine. (7) Vanadate did not inhibit fura-2 transfer in the toad urinary bladder. We concludethat intracellularly-generated free fura-2 is only transported across the basolateral border, and that this transfer is stimulated byADH. The calcium-insensitive fraction is transferred by a temperature-dependent process, sensitive to an osmotic gradient andcolchicine.