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Am J Physiol Cell Physiol 284: C220-C232, 2003; doi:10.1152/ajpcell.00374.2001
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Vol. 284, Issue 1, C220-C232, January 2003

Detection of ClC-3 and ClC-5 in epididymal epithelium: immunofluorescence and RT-PCR after LCM

Corinne Isnard-Bagnis1,*, Nicolas Da Silva1,*, Valérie Beaulieu1, Alan S. L. Yu3, Dennis Brown1,2, and Sylvie Breton1,2

1 Renal Unit and Program in Membrane Biology, Massachusetts General Hospital, Charlestown 02129; 2 Department of Medicine, Harvard Medical School, Boston 02215; and 3 Renal Division, Brigham and Women's Hospital, Boston, Massachusetts 02115

Epithelial cells of the epididymis and vas deferens establish an optimum luminal environment in which spermatozoa mature and are stored. This is achieved by active transepithelial transport of various ions including Cl- and H+. We investigated the localization of three closely related members of the ClC family, ClC-3, ClC-4, and ClC-5, in the epididymis and vas deferens. RT-PCR using mRNA isolated by laser capture microdissection (LCM)-detected ClC-3 and ClC-5 transcripts but did not detect any ClC-4-specific transcript. Western blot and immunofluorescence analysis demonstrated that ClC-3 and ClC-5 proteins are present in all regions of the epididymis and in the vas deferens. ClC-5 is expressed exclusively in H+-ATPase-rich cells (narrow and clear cells). Confocal microscopy showed that ClC-5 partially colocalizes with the H+-ATPase in the subapical pole of clear cells. ClC-3 is strongly expressed in the apical membrane of principal cells of the caput epididymidis and the vas deferens and is less abundant in principal cells of the body and cauda epididymidis. These findings are consistent with a potential role for ClC-3 in transepithelial chloride transport by principal cells and for ClC-5 in the acidification of H+-ATPase-containing vesicles in narrow and clear cells. ClC-5 might facilitate endosome trafficking in the epididymis, as has been proposed in the kidney.

chloride channels, vacuolar H+-ATPase, principal cells, clear cells; laser capture microdissection

* C. Isnard-Bagnis and N. Da Silva contributed equally to this work.




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