SEROTONIN TRANSPORT IN RECONSTITUTED ENDOTHELIAL-CELL PLASMA-MEMBRANE PROTEOLIPOSOMES - EFFECT OF HYPOXIA

To determine whether changes in the lipid dynamics of the plasma membrane bilayer are responsible for hypoxic stimulation of serotonin (5-hydroxytryptamine [5-HT]) transport in pulmonary artery endothelial cells, we solubilized and isolated phospholipid and protein fractions from plasma membrane vesicles derived from endothelial cells exposed to 20% O2 (normoxia) or 0% O2 (hypoxia) for 24 h. Four different combinations of proteoliposomes were prepared by reconstituting (1) normoxic protein and normoxic phospholipid, (2) normoxic protein and hypoxic phospholipid, (3) hypoxic protein and normoxic phospholipid, and (4) hypoxic protein and hypoxic phospholipid. Fluorescence anisotropy of diphenylhexatriene (DPH), a measure of fluidity, and 5-HT transport were evaluated in each of the four groups of reconstituted proteoliposomes. 5-HT transport by the reconstituted proteoliposomes was saturable, linear with protein (5 to 25-mu-g) and time (15 to 60 s), and optimal with a phospholipid-to-protein ratio of 3:1. There were no significant differences in intravesicular volume, phospholipid-to-protein ratio, and size distribution among the four different groups of proteoliposomes. 5-HT transport was significantly higher and fluorescence anisotropy of DPH was significantly lower in proteoliposomes made from hypoxic phospholipids irrespective of the source of protein. Hypoxia also had a direct effect on the 5-HT transporter since uptake was increased slightly in proteoliposomes from group 3. These results indicate that changes in the plasma membrane phospholipids, and to a much lesser extent changes in the 5-HT transporter, are responsible for increases in the transmembrane transport of 5-HT by hypoxic endothelial cells. Increased fluidity of the plasma membrane lipid bilayer, leading to freer movement of the 5-HT transporter and increased accessibility to substrate, is likely to be the primary mechanism responsible for increased 5-HT transport in hypoxic endothelial cells.