327
S-10-4
Cellular
R.Blomhoff,
Uptake
H.Senoo,
of Vitamin
S.Smeland,
A
T.Bjerknes,
and K.R.NORUM
Institute P.O.Box
I.
for 1046,
Nutrition 0316 Oslo,
Research, Norway
University
of
Oslo,
INTRODUCTION
Most cells in the body are exposed continously to about 2 AM retinol bound to retinol-binding protein (RBP) [1-2]. The specific mechanism by which retinol is transferred from retinolRBP to the plasma membrane of cells is not yet fully elucidated. Retinol uptake by cells appears to involve one or both of the following processes: (a) the small amount of free retinol in equilibrium with retinol-RBP in plasma may partition into the plasma membrane; (b) retinol might enter cells as a result of receptor-mediated transmembrane transport of retinol and/or endocytosis of retinol-RBP.
II.
PARTITIONING
OF
FREE
RETINOL
INTO
THE
PLASMA
MEMBRANE
From in vitro experiments, it is known that unbound retinol will rapidly associate with cells and elicit specific biological effects when it is solubilized in a small amount of organic solvent (e.g. ethanol) and added to cells in culture. Since, in plasma, a small amount of unbound retinol is in equilibrium with retinol-RBP, this retinol might be available for cellular uptake by a nonspecific mechanism. That is, since unbound retinol is relatively hydrophobic, it could simply partition into the plasma membrane of cells without any involvement of membrane receptors. Thus, two important questions are: to what degree does retinol in plasma or interstitial fluid exist in an unbound state and could membrane partitioning of such retinol contribute significantly to cellular retinol uptake in vivo? Based on recent findings [reviewed in ref 2], it is reasonable to conclude that there may be some nonspecific transfer of retinol from retinol-RBP to cell membranes in vivo. However, other observations indicate that it is not likely to be the main mechanism by which cells obtain retinol® III.
SELECTIVE
INTERACTION
OF
RBP
WITH
SPECIFIC
CELLS
One type of evidence which supports the existence of a specific mechanism for cellular uptake of a particular ligand is data indicating that the ligand is selectively taken up in certain cells. Following this line of reasoning, our data suggest that a specific cell surface receptor for RBP exists. A selective uptake of RBP was observed in a study by Gjoen et al. in our laboratory [3]. We injected 125 I-tyramine cellobiose-labeled RBP (125I-TC-RBP) intravenously into rats and determined the plasma disappearance of radioactivity and its recovery in liver, kidneys, intestine, spleen, heart, lungs etc. at different times. The TC ligand was used since, after clearance of the TC-labeled protein by cells, labeled TC which
Symposium ED Carcinogenesis
328
reaches the lysosomes is slowly lost, compared with a rapid loss of radioactivity due to lysosomal protein degradation when the protein itself is directly labeled. Thus, this labeling technique can be used to determine the net organ uptake of RBP. Of the organs studied, liver and kidneys contained the most radioactivity: after 1 h, about 20% and 10% of the injected dose was recovered in liver and kidneys, respectively; other organs contained
S-10-4
Cellular
R.Blomhoff,
Uptake
H.Senoo,
of Vitamin
S.Smeland,
A
T.Bjerknes,
and K.R.NORUM
Institute P.O.Box
I.
for 1046,
Nutrition 0316 Oslo,
Research, Norway
University
of
Oslo,
INTRODUCTION
Most cells in the body are exposed continously to about 2 AM retinol bound to retinol-binding protein (RBP) [1-2]. The specific mechanism by which retinol is transferred from retinolRBP to the plasma membrane of cells is not yet fully elucidated. Retinol uptake by cells appears to involve one or both of the following processes: (a) the small amount of free retinol in equilibrium with retinol-RBP in plasma may partition into the plasma membrane; (b) retinol might enter cells as a result of receptor-mediated transmembrane transport of retinol and/or endocytosis of retinol-RBP.
II.
PARTITIONING
OF
FREE
RETINOL
INTO
THE
PLASMA
MEMBRANE
From in vitro experiments, it is known that unbound retinol will rapidly associate with cells and elicit specific biological effects when it is solubilized in a small amount of organic solvent (e.g. ethanol) and added to cells in culture. Since, in plasma, a small amount of unbound retinol is in equilibrium with retinol-RBP, this retinol might be available for cellular uptake by a nonspecific mechanism. That is, since unbound retinol is relatively hydrophobic, it could simply partition into the plasma membrane of cells without any involvement of membrane receptors. Thus, two important questions are: to what degree does retinol in plasma or interstitial fluid exist in an unbound state and could membrane partitioning of such retinol contribute significantly to cellular retinol uptake in vivo? Based on recent findings [reviewed in ref 2], it is reasonable to conclude that there may be some nonspecific transfer of retinol from retinol-RBP to cell membranes in vivo. However, other observations indicate that it is not likely to be the main mechanism by which cells obtain retinol® III.
SELECTIVE
INTERACTION
OF
RBP
WITH
SPECIFIC
CELLS
One type of evidence which supports the existence of a specific mechanism for cellular uptake of a particular ligand is data indicating that the ligand is selectively taken up in certain cells. Following this line of reasoning, our data suggest that a specific cell surface receptor for RBP exists. A selective uptake of RBP was observed in a study by Gjoen et al. in our laboratory [3]. We injected 125 I-tyramine cellobiose-labeled RBP (125I-TC-RBP) intravenously into rats and determined the plasma disappearance of radioactivity and its recovery in liver, kidneys, intestine, spleen, heart, lungs etc. at different times. The TC ligand was used since, after clearance of the TC-labeled protein by cells, labeled TC which
Symposium ED Carcinogenesis
328
reaches the lysosomes is slowly lost, compared with a rapid loss of radioactivity due to lysosomal protein degradation when the protein itself is directly labeled. Thus, this labeling technique can be used to determine the net organ uptake of RBP. Of the organs studied, liver and kidneys contained the most radioactivity: after 1 h, about 20% and 10% of the injected dose was recovered in liver and kidneys, respectively; other organs contained
