High‐Affinity [3H]Choline Accumulation in Cultured Human Skin Fibroblasts

Abstract

[³H]Choline can be transported across cell membranes by high‐affinity (K_T 5 μM) and low‐affinity (K^T≫ 5 μM) systems. High‐affinity choline accumulation (HACA) has been demonstrated in synaptosomes made from cholinergic brain regions such as the hippocampus and caudate‐putamen. In cell culture. HACA has been demonstrated in glia and avian telencephalon, dissociated spinal cord, and muscle fibroblasts. We examined [³H]choline accumulation in a single normal human fibroblast line cultured from skin biopsy. [³H]Choline accumulation was temperature‐dependent and linear with incubation time up to 6 min at 0.125 μM‐choline. The apparent K^T for [³H]choline was 5 μM which is similar to that observed in avian fibroblasts. Isoosmotic replacement of Na⁺ with either Li⁺ (144 MM) or sucrose (288 mM) severely reduced [³H]choline accumulation (by 70–90%). Pre‐incubation with ouabain (100 μM), sodium orthovanadate (100 μM), or 2,4‐dinitrophenol (100 μM), or replacement of Ca²⁺ by Mg²⁺ had little or no effect on subsequent [³H]choline accumulation. [³H]Choline accumulation was inhibited by hemicholinium‐3 (HC‐3); after pre‐incubation in HC‐3 at 37°C for 10 min, the IC₅₀ (at 0.125 μM‐chohne) was 5.6 μM. The HC‐3 sensitivity, Na⁺ dependence, and low K_T suggest that human skin fibroblasts have a high‐affinity transport system for choline.