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Studying Na‐K‐2Cl cotransporter 1 membrane dynamic in mouse colon in vivo
Author(s) -
Bouyer Patrice G,
Tang Xu,
Bridgmon Kenneth,
Smallwood Jesse,
Matlin Karl S,
Matthews Jeffrey B
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.856.9
Subject(s) - internalization , microbiology and biotechnology , in vivo , ex vivo , secretion , confocal microscopy , cotransporter , carbachol , ussing chamber , forskolin , protein kinase c , chemistry , biology , in vitro , biochemistry , cell , signal transduction , receptor , sodium , organic chemistry
The Na‐K‐2Cl cotransporter 1 (NKCC1) plays an important role in transepithelial chloride secretion in the colon. Previously we have shown that activation of PKCδ and PKCɛ in T84 cells causes a rapid internalization of NKCC1 thereby blunting chloride secretion. Whether similar mechanisms are at play in vivo remain unexplored. To test this hypothesis, we have generated a transgenic (Tg) mouse expressing eGFP‐NKCC1 under the control of the villin promoter to study NKCC1 membrane dynamic in vivo using confocal microscopy. In the Tg mice, eGFP‐NKCC1 is expressed at the basolateral membrane of colonic cells. Using the short‐circuit current to estimate chloride secretion in mouse colons mounted in Ussing chamber, we found that forskolin elicits a similar current in wild type (WT) and in Tg mice. Similarly results were obtained with carbachol. Next, we used confocal microscopy to image eGFP‐NKCC1 trafficking during activation of PKC in vivo . To do so a piece of colon is externalized and a small incision is performed on the opposite side of the mesenteric to preserve innervation and blood supply. The open lumen is apposed on a glass coverslip of a Petri dish filled with a physiological solution. The mouse and the Petri dish are mounted on the stage of a confocal microscope in a temperature‐controlled box. We found that adding phorbol 12‐myristate 13‐acetate (PMA) to activate PKC causes eGFP‐NKCC1 internalization in colonic cells. Similar results were observed with addition of carbachol in the Petri dish. Finally, we have tested the effect of rottlerin a PKCδ inhibitor on PMA‐induced eGFP‐NKCC1 internalization and found that rottlerin blocks internalization of NKCC1.