Interaction of Some Drugs, Metal Ions, and Alcohols With Rat Liver Microsomes as Studied With a Fluorescent Probe

Abstract

S U M M A R Y 1. The effects of various compounds on the fluorescence of microsome-bound 1-anilino-8-naphthalene sulphonate (ANS) have been studied.2. Cationic drugs and divalent metal cations enhance the fluorescence of microsome-bound ANS whereas anionic drugs and primary aliphatic alcohols decrease it.3. No changes were observed in the fluorescence lifetime or emission maximum of ANS and it was concluded that there were no significant changes in the quantum yield of ANS fluorescence.4. The changes in fluorescence are shown to be related to changes in ANS binding.5. The results suggest that drug-induced changes in the binding of ANS to the microsomal membrane reflect a change in the net charge on the membrane as a result of the binding of a charged drug and that changes in ANS fluorescence cannot be directly interpreted as changes in the structure of the membrane.6 . Primary aliphatic alcohols displace ANS from the microsomal membrane but cause no change in quantum yield. It is suggested that the alcohols change the net charge on the membrane either by exposure of negative charges or occlusion of positive charges.7. The changes in ANS fluorescence can be used as a measure of the interaction of various drugs with the microsomal membrane. Apparent binding constants determined by this method for various drugs fall in the concentration ranges over which the drugs are reported to induce changes in membrane function in various in vitro systems.

Application of the Fluorescent Probe 1-Anilinonaphthalene-8-Sulfonate to the Measurement of the Nonspecific Binding of Drugs to Human Liver Microsomes

“…Consistent with previous reports (Diaugustine et al, 1970;Hawkins and Freedman, 1973;Birkett, 1974), ANS fluorescence was enhanced by basic drugs that bound to HLMs, whereas acidic drugs and the neutral compound diazepam caused a decrease in ANS fluorescence. More importantly, there was a highly significant linear relationship between f u(mic) and the modulus of the fluorescence increment or decrement for the combined data set at each added drug concentration.…”

“…Thus, the binding of cations to phosphate and carboxyl groups, which decreases surface electrostatic potential, facilitates ANS binding and fluorescence (Slavik, 1982). Consistent with the known effects of membrane charge on ANS fluorescence, it has been demonstrated that several drugs that are positively charged at physiological pH enhance the fluorescence of microsome-bound ANS, whereas negatively charged drugs decrease the fluorescence of ANS in the presence of microsomes (Diaugustine et al, 1970;Hawkins and Freedman, 1973;Birkett, 1974). These data suggest that drug-induced shifts in baseline ANS fluorescence may provide a measure of the NSB of drugs to HLMs.…”

“…The occurrence of quenching was excluded for all drugs. Percentage fluorescence increment or decrement in ANS fluorescence due to added drug was calculated according to Birkett (1974) …”

Binding of fluorescent probes to microsomes with regard to the function of the mono‐oxygenase complex

“…Some reports have appeared where fluorescent probes have been used to study the reaction mechanism of microsomal oxidation of xenobiotics: 1,8-ANS has been found to decrease the binding of some substrates for microsomes [3,9] and to inhibit microsomal drug metabolism [7-91. The interpretation of these findings requires, however, a closer understanding of the action mechanism of the probes, which is not yet known in detail.…”