Abstract
Apical NaCl entry into the thick ascending limb (TAL) epithelium via NKCC2 is a regulated process. We studied cellular translocation of NKCC2 under short term vasopressin/PKA- and PKC-dependent stimulation in a rabbit (rb) TAL cell line. A crucial role for the small GTPase, RhoA and the actin cytoskeletal organisation is hypothesized. RbTAL cells were incubated for 1 hour with agents of the vasopressin-V2R-pathway (AVP [100nM]; cAMP agonist, 8-Br-cAMP [10nM]; cAMP antagonist, Rp-cAMP, [20nM]; PKA inhibitor, H89, [10nM], and the PKC pathway (activator PMA [100nM], and inhibitor Ro31-8220 [24nM]). Parallel incubation was done in rbTAL cells transiently transfected with myc-tagged intact (wt) or mutated (Ser188) forms of RhoA, S188E and S188A. Antibodies against various domains of NKCC2 were generated for immunohistochemistry (IHC). Translocation was studied by confocal microscopy, and Western blot (WB) of cell fractions. Apical membrane insertion of NKCC2 was 2-fold increased under both, PKA- and PKC short term stimulation as shown by IHC and WB. Transient expression of wt RhoA and S188E per se produced a roughly 2-fold increase, and S188A a marked decrease in NKCC2 insertion as shown by myc double-staining IHC. Cytosolic actin patterns were profoundly altered in S188A. PKA or PKC activation were without effect in S188A. We conclude that activation of PKA and PKC pathways both increase apical membrane insertion of NKCC2 in a RhoA-dependent manner. The effect of RhoA is mediated by the actin cytoskeleton.
Published Version
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