Phenylarsine oxide inhibits phosphate uptake in human ciliary non-pigmented epithelial cells

Phenylarsine oxide (PAO), a sulfhydryl modifying reagent and a widely used inhibitor for tyrosine phosphatases and endocytosis, was tested on the leve l of phosphorylation in human nonpigmented ciliary epithelial ocular (HNPE) cells. Pretreatment with (PAO, 10 mu M) for 30 min followed by incubation with P-32(i) to stimulate endogenous phosphorylation surprisingly resulted in a total reduction in P-32(i) labeled proteins. PAO (10-50 mu M) dose-dep endently inhibited both sodium-dependent and -independent phosphate uptake in cells. p-Hydroxymercuribenzoate (pHMB, 10 mu M), another sulfhydryl modi fying reagent failed to mimic PAO effects. However, metabolic inhibitors (i odoacetamide (0.1 mM) and 2,4-dinitrophenol (DNP, 0.5 mM) also mimicked PAO effects, suggesting that the inhibition of ATP production may be responsib le for attenuation of both phosphate uptake mechanisms. However, sodium-dep endent phosphate uptake in isolated plasma membrane vesicles pretreated wit h PAO was also significantly lower than control vesicles treated with dimet hlysulfoxide (DMSO), suggesting that PAO may be directly targeting a compon ent of the sodium-dependent cotransporter. It is suggested that PAO is a no vel inhibitor of phosphate uptake in HNPE cells that acts indirectly by inh ibiting ATP production and directly by inhibiting the Na-dependent cotransp orter.