Active chloride transport in rabbit thick ascending limb of Henle's loop and elasmobranch rectal gland: chloride fluxes in isolated plasma membranes

Abstract

To investigate directly whether a sodium-potassium-chloride cotransport system is operating in the mammalian thick ascending limb of Henle's loop (TALH) and in the elasmobranch rectal gland, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and from rectal glands of Squalus acanthias, and chloride uptake was measured by a rapid filtration technique. Chloride uptake into TALH vesicles in the presence of a 25 mM Na2SO4, 25 mM K2SO4 gradient reached 70% of equilibrium at 2.5 min. In the presence of both sodium and potassium, the 15 s chloride uptake was inhibited 35% by 1 mM bumetanide. When either sodium or potassium was removed from the incubation medium, chloride uptake decreased to the level observed in the presence of 1 mM bumetanide, 0.5 mM SITS had no effect on chloride uptake by the plasma membrane vesicles. This sodium and potassium dependent, bumetanide sensitive chloride uptake was also observed under tracer exchange conditions. Chloride uptake into rectal gland plasma membrane vesicles in the presence of a 50 mM Na2SO4, 50 mM K2SO4 gradient reached 80% of equilibrium at 2.5 min. 1 mM bumetanide inhibited the 15 s uptake of chloride by 34% and removal of either sodium or potassium from the incubation medium reduced chloride uptake to the level observed in the presence of bumetanide under both gradient and tracer exchange conditions. These studies provide additional support for the hypothesis that a sodium-potassium-chloride cotransport system is operating in these epithelia.