Three molecular steps of aminoglycoside ototoxicity demonstrated in outer hair cells

Previously postulated molecular mechanisms of aminoglycoside ototoxicity were investigated in outer hair cells in vitro. Cells were isolated by microdissection from the organ of Corti of the guinea pig and maintained in Hank's balanced salt solution. (1) Approx. 100 cells (the standard number per assay) bound 432 +/- 198 pmol calcium as determined with 45Ca2+ (1.2 mM). 1 mM neomycin or gentamicin lowered this value by 28% and 45%, respectively. (2) Binding of radiolabeled gentamicin (28 fmol per 100 cells at 0.1 microM gentamicin) was reduced by 55% by 1 mM neomycin or spermine. Washing with an excess of unlabeled gentamicin displaced only 60% of the drug, the remainder being tightly bound to a less accessible compartment. Incubation at low temperature essentially abolished gentamicin uptake. (3) Phospholipids were labeled with [32P]orthophosphate which was mostly incorporated into phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate, phosphatidylinositol, and phosphatidic acid. When the lipids were chromatographed over immobilized gentamicin PIP2, in contrast to other lipids, was strongly retained. These results are compatible with the following actions of aminoglycosides in outer hair cells: (1) competition with calcium; (2) energy-dependent uptake competitive with polyamines; and (3) binding to the phospholipid PIP2.