Dual effect of N-ethylmaleimide on Cl? transport across the thin ascending limb of Henle's loop

Abstract

Effects of SH reagents on Cl- transport were studied in the isolated hamster thin ascending limb of Henle's loop (TAL) perfused in vitro. Parachloromercuribenzene sulfonate (PCMBS) at 10(-4) M in the bath decreased the relative permeability for Cl-/Na+ (PCl/PNa), as determined by the transmural diffusion voltage (VT) generated under a NaCl concentration gradient, from 2.71 +/- 0.16 to 1.11 +/- 0.09 (P less than 0.001). The effect of PCMBS was prevented by the pretreatment with 10(-3) M dithiothreitol (DTT). N-Ethylmaleimide (NEM) at 10(-3) M in the bath exhibited a dual action on Cl- permeability of the TAL: It inhibited the Cl- permeability in fresh preparations, whereas it stimulated the Cl- permeability in the preparations pretreated with SH reagents including NEM, maleimide and PCMBS. The inhibitory effect was irreversible but the stimulatory effect was reversible. Both responses were prevented by DTT. Since dextran-maleimide did not show any inhibitory effect on PCl/PNa, the SH site responsible for the inhibition may be located inside of the cell. The stimulatory effect of NEM on PCl/PNa was markedly reduced when bath pH was reduced to 5.8. On the other hand, when the bathing fluid was made nominally Ca2+ free, the stimulatory effect of NEM was unaffected, although the basal level of PCl/PNa was reduced These observations suggest that the conductive Cl- pathway in the TAL is either stimulated or inhibited by modifying two distinct SH sites. The site of modulation by proton binding may exist distally to these SH sites. The regulatory mechanism involving Ca2+ may be independent of the SH regulatory sites.