The ototoxicities of eight aminoglycoside antibiotics and fragments were measured quantitatively by cochlear perfusion in the guinea pig. Perilymphatic spaces were perfused for 1 hr with 'artificial perilymph' containing 10 mM drug, during which time continuous measurements of cochlear microphonic potentials were made. Kanamycin B and neomycin I3 caused the most rapid decline of cochlear microphonic potentials, followed by gentamicin Cla = rihostamicin > kanamycin A = G-418. Neamine and methylneobiosamine did not show significant effects. The same drugs were tested for their interaction with monomolecular films of polyphosphoinositides, and relative binding constants were determined. Neomycin B and kanamycin B had the highest affinities to the lipids. followed bv the other drugs in the order as seen for toxicity. The correlation between the in situ and in virro ac&s of the drugs was r = 0.9. These results support the hypothesis that binding to polyphosphoinositides plays an important role in the decrease of the cochlear microphonic potentials. Furthermore, the good correlation between the drug actions in the two test systems suggests that an in vitro assay may be possible for the assessment of aminoglycoside ototoxicity.
The interactions of calcium and the aminoglycosidic antibiotic, neomycin, with various lipids were investigated in monomolecular films. Lipids were spread over a subphase of 0.05 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.0, and NaCl to give an ionic strength of 0.2. Measurements of surface pressure (pi) were taken with a Wilhelmy balance. In the absence of Ca2+, 1 muM--1 mM neomycin in the subphase decreased pi (i.e. condensed films) of all acidic lipids tested. In the presence of 1 mM Ca2+, neomycin did not change pi of films of phosphatidylserine, phosphatidylinositol and phosphatidic acid while it lowered pi of cardiolipin and cerebroside sulfate films. A unique pattern of interaction was observed with polyphosphoinositide monolayers. In the absence of Ca2+, 1 muM neomycin decreased pi followed by an increase of pi at higher neomycin concentrations. Ca2+ (1 mM) condensed the film significantly more than did neomycin. However, as little as 1 muM neomycin induced expansion of the calcium/lipid film which at 1 mM neomycin reached the same pi as in the absence of Ca2+. Such expansion was observed at all pressures of the film including the collapse pressure indicating a strong 'complex' between the drug and polyphosphoinositide not antagonized by Ca2+. In the absence of possible hydrophobic interactions, both the condensation and the expansion of the film should be mediated by ionic forces. Combined in vivo and in vitro evidence is discussed to suggest the polyphosphoinositides as the physiological receptors for aminoglycosides in the mammalian cell membrane.
Neomycin and related aminoglycosidic antibiotics displace calcium from synaptosomes of guinea pig cerebral cortex and from preparations of phosphatidylinositol diphosphate. At low drug concentrations, inhibition of synaptosomal calcium binding is competitive (Ki = 3-10(-5) M), at high concentrations it is non-competitive (Ki = 4-10(-4) M). Monomolecular films of phosphatidylinositol diphosphate are contracted by low concentrations of neomycin in the subphase, and are expanded at high concentrations. This expansion perists even at the collapse pressure indicating a strong interaction between the drug and the lipid.
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