We have recently characterized the endogenous NHE3 in Caco-2 cells. This exchanger is present exclusively on the apical surface and it is maximally active after 12-14 postconfluent days (PCD). Phorbol ester (PMA, 1 uM) acutely inhibited the NHE3 activity (sodium dependent, amiloride resistant pHi recovery) by 28+5%, the effect being attenuated by PKC inhibitor H7 (65 uM). The objective of this study was to test the hypothesis that the inhibitory effect of PMA involved translocation (endocytic retrieval) of NHE3 from brush border (BB) to the subapical cytoplasmic compartment (SAC). A novel morphometric method was used for quantitative evaluation of vertical translocation of NHE3. Caco-2 cells (12-14 PCD) were exposed to PMA (1 uM) for 20 min., and NHE3 was localized by indirect immunofluorescence using our 1380 polyclonal Ab and Cy3-conjugated 2nd Ab. The BB limits were defined prior to permeabilization step by labeling with FITC-conjugated lectin (P.vulgaris PHA-E). Serial optical sections (0.3 μm) parallel to the cell surface were obtained by use of confocal fluorescent microscope, and the vertical distribution of NHE3 was analyzed using MetaMorph software. In control cells, 18.7+2.8% of the total apical NHE3 was found in the SAC. Exposure to PMA resulted in an increase of the SAC fraction to 29.2+3.5%, the effect being attenuated by H7. We conclude that: (1).PKC-mediated inhibition of endogenous NHE3 in Caco-2 cells involves translocation of the exchanger from BB to SAC, (2).Translocation seems to be only partially responsible for the PMA-induced inhibition of NHE3 activity, and (3).Since all acute regulation of NHE3 is accomplished by changes in Vmax, both endocytic retrieval and a change in the turnover number seem to be involved in the PKC-mediated inhibition of NHE3 in Caco-2 cells.
|Original language||English (US)|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology