Radioactive labeling of phospholipids and proteins by cochlear perfusion in the guinea pig and the effect of neomycin

Abstract

Phospholipids and proteins of guinea pig stria vascularis, spiral ligament and organ of Corti were radioactively labeled by perilymphatic perfusion with artificial perilymph containing [32P] orthophosphate or radioactive amino acids. Phospholipids were separated by thin-layer chromatography, proteins by disc gel electrophoresis and quantitated by liquid scintillation counting. The addition of 10-4M to 10-2M neomycin to the perfusion fluid resulted in a dose-dependent increase of tissue permeability to the radioactive precursors, and a specific decrease in the 32P-incorporation into phosphatidylinositol diphosphate in stria vascularis and organ of Corti. No effect of neomycin on protein labeling was observed using a double label approach with [3H]methionine and [35S]-methionine. In vitro, low concentrations of neomycin led to the formation of a complex and polyphosphoinositides. Much higher concentrations of the drug were needed for a comparable reaction with the acid mucopolysaccharide, chondroitin sulfate A. The implications of these findings for the mechanism of neomycin ototoxicity are discussed.