Squalamine, a novel am1nosterol antibiotic, is a spec II-U inhibitor of epithelial brush border Na /H exchanger isoform NHE3.

The observation that squalamine causes changes in endothelial cell shape suggested that transport proteins controlling cell volume or cell shape might be its potential targets To study possible effectors of squalamine, its effect on cloned mammalian Na'H exchanger isoforms. NHEl. NHE2, and NHE3, was studied in stably transfected PS12U fibroblasts. Na -'H exchange \vas measured both by spectrofluorimetnc methods using the pH sensitive dye. BCECF. and by amiloride sensitive isotopic N a uptake Squalamine (Ih pretreatment) decreased the V,njll of rabbi! NHF.3 m a concentration dependent manner ( 13%, 47%, and 57% with 1, 5, and 7 ug/ml respectively). This effect was time dependent, with a maximal effect occuring with Ih uf exposure and was lulK reversible within 3h. Squalamine had the same effect on transfected human NHE> These results were confirmed by inhibition of amiloride sensitive Na uptake into acid preloaded PS120/NHE3 cells by squalamine In addition, squalamine inhibited ileal brush border membrane vesicle (BBMV) Na'/H' exchange, again only when tissue u;is pretreated with squalamine (51% inhibition with 30 min exposure). Direct addition <>l squalamine to ileal BBMV. NHE3 transfecied, or untransfccted PS 120 fibrobiasts during measurement of exchanger activity had no effect. Squalamine had no cytotoxic effect LI-, indicated by iactate dehydrogenase release at any of the concentrations used in these experiments. In contrast to NHE3, squalamine did not affect the kinetic parameters ol human and rabbit NHEl or rabbit NHE2. These results indicate that squalamine I ) is a species-independent specific inhibitor of the brush border NHE isoform NHE3 and nut NHEl or NHE2; 2) acts in a nonloxic and fully reversible manner; 3) may influence brush border exchanger function indirectly through an mtracdlular signaling pdihwa\ or as an intrazellular modulator Supported by Magainin Pharmaceuticals.