ENDOSOMAL RECYCLING OF THE NA+ H+ EXCHANGER NHE3 ISOFORM IS REGULATEDBY THE PHOSPHATIDYLINOSITOL 3-KINASE PATHWAY/

The NHE3 isoform of the Na+/H+ exchanger localizes to both the plasmal emmal and endosomal compartments in polarized epithelial and transfect ed Chinese hamster ovary (AP-1) cells. It is unclear how the distribut ion of NHE3 between these compartments is regulated. In this study, we examined the potential involvement of phosphatidylinositol 3'-kinase (PI3-K) in regulating the activity and distribution of NHE3, as this l ipid kinase has been implicated in modulating vesicular traffic in the endosomal recycling pathway. Wortmannin and LY294002, both potent inh ibitors of PI3-K, markedly inhibited NHE3-mediated Ht extrusion across the plasma membrane in a concentration- and time-dependent manner. Th e subcellular distribution of the antiporters was monitored by transfe cting epitope-tagged NHE3 into AP-1 cells. In parallel with the inhibi tion of transport, PIS-K antagonists induced a pronounced loss of NHE3 from the cell surface and its accumulation in an intracellular compar tment, as assessed by immunofluorescence microscopy and enzyme-linked immunosorbent assays. Further analysis using cells transfected with an tiporters bearing an external epitope tag revealed that the redistribu tion reflected primarily a decrease in the rate of recycling of intrac ellular NHE3 to the cell surface. The wortmannin-induced inhibition an d redistribution of NHE3 were prevented when cells were incubated at 4 degrees C, consistent with the known temperature dependence of the en docytic process. These observations demonstrate that NHE3 activity is controlled by dynamic endocytic and recycling events that are modulate d by PI3-K.