Binding and subcellular distribution of cyclosporine in human fibroblasts

The uptake, binding, and subcellular sites of accumulation of [3H]-cyclosporine (CS) in two human gingival fibroblast strains, GN 23 and GN 54, have been examined. GN 23 responds to CS treatment with a decrease in collagenolysis, while GN 54 does not. Binding of the drug was determined using [3H]-CS concentrations ranging from 10(-5) to 10(-8) M in the absence or presence of excess unlabeled CS (1 mM). The binding of the drug to both strains was specific and reached a plateau within 10 min, remaining at that level for up to 1 h. Scatchard analysis of the specific binding of [3H]-CS to the responsive GN 23 strain revealed two dissociation constants: KD = 5 x 10(-8) M (1.2 x 10(7) sites/cell) and KD = 1.4 x 10(-6) M (2.2 x 10(8) sites/cell). GN 54, on the other hand, had only one class of low affinity binding site (KD = 0.47 x 10(-6) M [1.2 x 10(8) sites/cell]). Unlabeled CS (0.01-1 mM) inhibited the binding of [3H]-CS in a dose-dependent manner to both strains, as did the calmodulin antagonist W-7, to a lesser extent. However, W-7 inhibited CS binding much more efficiently in GN 54 than in GN 23, suggesting that calmodulin may be the predominant CS receptor in GN 54. In both strains, 70% of the drug accumulated in the crude nuclear fraction after a 1 min incubation, with very little (< or = 4%) being membrane associated, and the remainder was in the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)