The water permeability of the mammalian collecting duct is controlled by the antidiuretic hormone vasopressin (VP). When VP binds to its receptor on the basolateral membrane of the principal cells, intracellular vesicles bearing specific aquaporin-2 (AQP2) water channels are shuttled to the apical membrane where they fuse and allow water flow, a process that is mediated by cAMP and dependent on the cytoskeleton. Previous studies in the toad bladder, a functional homologue of the mammalian collecting duct, have demonstrated that the VP-induced increase in osmotic water permeabilty can be mimicked by the sulphydryl agent N-ethylmaleimide (NEM), when used at a concentration of 0.1 mM (Marples et al. 1994). Using a rat kidney tubule suspension, we have investigated the effects of NEM on AQP2 shuttling in a mammalian system.
Male Wistar rats were terminally anaesthetised with pentobarbitone sodium (4 ml kg-1 I.P.). The kidneys were perfused via the abdominal aorta with potassium gluconate solution (composition (mM): potassium gluconate 140, NaCl 10, MgCl2 1, CaCl2 2, KOH-Hepes 10, sucrose 33.5; 1 mg ml-1 collagenase, 0.3 mg ml-1 pronase), gassed with 100 % oxygen. The kidneys were then rapidly removed, and the inner medullae dissected out, minced, and suspended in the same solution. The tissue was incubated, with continuous agitation, for 60 min at 37 °C in conical flasks, top-gassed with 100 % O2. Every 15 min the suspension was triturated with a Pasteur pipette. The tubule suspension was then drawn off, washed twice, and suspended in Leibovitz culture medium supplemented with 1 % BSA. The suspension was divided into four aliquots for experiments (control and NEM treated, each ± VP), and incubated as before. The distribution of AQP2 was determined by membrane fractionation and western blotting (Marples et al. 1995). Membrane fractions enriched for plasma membrane (PM) and intracellular vesicles (ICV) were run for each experimental condition, and the ratio between them taken. The control PM : ICV ratio was normalised to 100 %, and other ratios were expressed relative to this. Results are quoted as means ± S.E.M., and Student’s unpaired t tests were used to assess significance.
Treatment with VP (1 nM) increased the PM : ICV ratio to 235 ± 42 % of control (n = 4, P < 0.03), indicating a shift of AQP2 to the plasma membrane. This shift was mimicked by NEM at a concentration of 0.1 mM (235 ± 42 %, n = 4, P < 0.03). When tubules were treated simultaneously with both VP (1 nM) and NEM (0.1 mM) the increase in the PM : ICV ratio was variable, with an average of 455 ± 168 % of control (n = 4, P = 0.1 with respect to VP alone).
These results show that NEM induces the translocation of AQP2 from intracellular vesicles to the plasma membrane at a concentration of 0.1 mM. This is in keeping with previous observations in the toad bladder, where NEM was shown to act at a site distal to the production of cAMP to cause the insertion of water channels. We plan to investigate the site of action of NEM further using this tubule preparation
- Marples, D., Bourguet, J. & Taylor, A. (1994). Exp. Physiol. 79, 775-795.
Marples, D., Knepper, M.A., Christensen, E.I. & Nielsen, S. (1995). Am. J. Physiol. 269, C655-664.