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: K_D = 5 × 10^?8 M (1.2 × 10⁷ sites/cell) and K_D = 1.4 × 10^?6 M (2.2 × 10⁸ sites/cell). GN 54, on the other hand, had only one class of low affinity binding site (K_D = 0.47 × 10^?6 M 1.2 × 10⁸ 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 (? 4%) being membrane associated, and the remainder was in the cytosol. In GN 23, CS levels in the crude nuclear fraction reached 80% by 20 min, and remained at this level for up to 1 h. In contrast, in GN 54, at incubation times of more than 1 min, the drug did not selectively accumulate in the crude nuclear fraction, but appeared to be in equilibrium between the nuclear and cytosolic fractions. These data show that the CS resistance of human gingival fibroblasts was not due to their inability to take up and bind CS. Rather, the different effects of CS on the collagenolysis of the responder and non-responder fibroblast strains may be related to the types of CS receptors they possess and differences in the cellular metabolism of CS occurring after binding, including the subcellular sites of drug accumulation. © 1993 Wiley-Liss, Inc.