Use Of Fluorescence Probes To Study Membrane Effects Of Aminoglycoside Antibiotics.

The mechanism of membrane disturbance by aminoglycoside antibiotics was investigated in liposomes containing the fluorescent probe, 1-anilino-8-naphthalene sulfonate (ANS), an indicator of structural changes of the bilayer. Liposomes of phosphatidylcholine (PtdCho) and different anionic phospholipids (1:1 to 15:1 molar ratios) were challenged with aminoglycosides in the presence of low (1 (mu)M) and high (3 mM) concentrations of calcium. Liposomes containing phosphatidylinositol bisphosphate (PtdInsP(,2)) showed the greatest drug-induced changes in ANS fluorescence in the presence of high and low concentrations of calcium and at all PtdCho:PtdInsP(,2) molar ratios tested. Liposomes containing other anionic phospholipids (phosphatidylserine, phosphatidylinositol and phosphatidylinositol phosphate) were not reactive toward aminoglycosides in the presence of 3 mM calcium or when the ratio of PtdCho to anionic lipid was increased to 8:1. The aminoglycoside-induced changes of ANS fluorescence were not due to any changes in the emission spectrum of ANS, nor to changes in quantum yield, nor to a change in the binding affinity of ANS. It is concluded that a specific aminoglycoside-PtdInsP(,2) interaction results in phase separation of PtdCho and PtdInsP(,2) and thus increases the number of available ANS binding sites in PtdCho:PtdInsP(,2) liposomes. The experiments performed with the water-soluble fluorescent dye, carboxyfluorescein (CF), an indicator of membrane permeability, again demonstrated the uniqueness of PtdInsP(,2) among the anionic lipids tested. Both calcium and aminoglycosides induced the greatest degree of CF release from the PtdInsP(,2)-containing liposomes. Even in the presence of a high (3 mM) concentration of calcium, aminoglycosides induced further release of CF from PtdInsP(,2)-liposomes but not from the other anionic liposomes tested. Most importantly, this study demonstrated that aminoglycoside-PtdInsP(,2) interaction leads to increased permeability of the liposomes towards water-soluble marker. The relative ability of various aminoglycosides to affect the liposomal membrane monitored by ANS and CF correlated well with the intrinsic ototoxicity of the antibiotics previously established in cochlear perfusions. These results suggest that perturbation of membranes containing PtdInsP(,2) may be a key step in aminoglycoside ototoxicity and probably in nephrotoxicity as well.Ph.D.Pharmacy sciencesPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127942/2/8702675.pd