Comparison of the Uptake and Metabolism of Retinol Delivered to Primary Mouse Keratinocytes Either Free or Bound to Rat Serum Retinol-binding Protein

Serum retinol-binding protein (RBP) is believed to be responsible for the transport of retinol from its storage site in the liver to vitamin A requiring target cells such as keratinocytes. We have used primary mouse keratinocytes as a model system to compare the uptake and metabolism of [3H] retinol delivered to them either free in solution or bound to RBP. RBP was purified from rat serum, loaded with [3H]retinol, and the [3H]retinol-RBP complex purified by affinity chromatography on human transthyretin-Sepharose. Keratinocytes incubated with either free [3H]retinol or [3H]retinal-RBP complex accumulated [3H]retinol in a time and temperature dependent manner, However, cells incubated with free [3H]retinol acquired 15- to 20-fold more ligand than if the retinol was delivered via RBP. The uptake of free [3H]retinol or [3H]retinol from RBP was not inhibited by excess unlabeled free retinol. The uptake of [3H]retinol from RBP was inhibited by high concentrations of holo-RBP, with half maximal inhibition occurring at 3μM holo-RBP. However, no specific binding of 125I-labeled RBP to monolayers of keratinocytes or membranes prepared from them was found indicating the absence of a high affinity RBP receptor on keratinocytes. Surprisingly, 50% of the [3H]retinol delivered to the keratinocytes during a 30-min uptake period was released from them within 30-min irrespective of whether or not it was initially delivered to them as free [3H]retinol or bound to RBP. The remaining 50% was lost at a much slower rate, but only 20% remained 24-h after delivery. Studies on retinol metabolism demonstrated that 7%12% of the total cell-associated [3H]retinol delivered during a 90-min uptake period was esterified (mostly as retinyl palmitate) whether or not it was given free in solution or bound to RBP