Short-term stimulation of the renal Na-K-Cl cotransporter (NKCC2) by vasopressin involves phosphorylation and membrane translocation of the protein.
Na-K-Cl cotransporter (NKCC2)-mediated sodium chloride reabsorption in the thick ascending limb is stimulated by the antidiuretic hormone vasopressin. We investigate the mechanisms underlying the short term activation of NKCC2 by vasopressin in vivo, finding that administration of a vasopressin analogue (deamino-Cys-d-Arg vasopressin) causes a 2-fold increase in mouse kidney NKCC2 phosphorylation, as detected with a phosphospecific antibody, R5. The subtissue localization of the activation is defined by immunofluorescence. In vasopressin-treated animals, a dramatic increase in R5 immunostaining is observed in the initial segment of the thick ascending limb located in the inner stripe of the outer medulla, the region with a higher sensitivity to vasopressin. Although a pool of NKCC2 is present in cytoplasmic vesicles, the distribution of the phosphorylated cotransporter seems to be restricted to the cell membrane compartment; morphometric analysis of electron microscope images demonstrates a 55% increase in NKCC2 molecules at the apical membrane, suggesting the administration of vasopressin induces trafficking of the cotransporter. Thus, the short term actions of vasopressin on the thick ascending limb cotransporter are mediated by both an effect on the translocation of the protein and an increase in phosphorylation of regulatory threonines in the amino terminus of NKCC2.
Giménez
Ignacio
I
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026, USA. ignacio.gimenez@yale.edu
Forbush
Biff
B
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D003894:Deamino Arginine Vasopressin / Q000494:pharmacology*; D007686:Kidney Tubules, Distal / Q000033:anatomy & histology / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D051379:Mice; D010766:Phosphorylation; D028021:Sodium-Potassium-Chloride Symporters / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1; D013912:Threonine / Q000737:chemistry
C578715:Slc12a1 protein, mouse; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D013912:Threonine; D003894:Deamino Arginine Vasopressin