Transport of 5-hydroxytryptamine by endothelial cells [proceedings

Abstract

5-Hydroxytryptamine in plasma is removed from the circulation predominantly in the lungs (Gaddum et al., 1953). Although blood platelets actively transport and store 5-hydroxytryptamine, they are apparently relatively unimportant in removing the circulating compound (Axelrod & Inscoe, 1963; Thomas & Vane, 1967), and radioautographic evidence indicates that pulmonary endothelial cells are mainly responsible (Strum & Junod, 1972). In the perfused lung, 5-hydroxytryptamine is transported by a saturable high-affinity uptake system (sensitive to inhibitors such as chlorimipramine) and metabolized intracellularly by monoamine oxidase; the vasoinactive breakdown products (predominantly 5-hydroxyindolylacetic acid) are released into the circulation (Alabaster & Bahkle, 1970; Junod, 1972). Isolated and cultured aortic endothelial cells also take up 5-hydro~y['~C]tryptamine (Shepro et af., 1975), and we here present our initial findings on the characteristics of this transport process. Endothelial cells were as a rule obtained from pigs aged 1-10 days, and occasionally from adult pigs. The pattern of growth was similar in both groups, though somewhat slower in the latter. The method used for endothelial-cell culture was based on that described by de Bono (1974). Thoracic aortas were removed, cannulated and flushed with sterile growth medium (Dulbecco's modification of Eagle's medium plus 50i.u. of penicillin/ml, 50pg of streptomycin/ml and 1OOpg of kanamycin/ml) containing heparin (5i.u./ml). After clamping the intercostal branches, aortas were filled with growth medium containing 0.2 % collagenase (Aldrich Chemical Co., Gillingham, Dorset, U.K. or Sigma Chemical Co., Kingston-upon-Thames, Surrey, U.K., type II), and incubated for 15 min at 37°C. The endothelial cells loosened bythis procedure wereremoved in small sheets, by gently flushing the aorta with growth medium, then centrifuged (1OOg for 3 min), resuspended in growth medium plus20 %(v/v)foetal calf serum, and plated into 25cm2 Falcon tissue-culture flasks. Growth medium was changed every 2 or 3 days, and cells were subcultured (usually at 1 :2 split ratio) when confluent, and used after one to five subcultures. For subculturing, cells were treated with trypsin (0.10%, w/v, Difco, 1 :250) and EDTA (0.025 %, w/v) in phosphate-buffered saline for 1-2min at 37°C. Some batches of cells were stored until required for use, in growth medium plus 20% (v/v) foetal calf serum plus 10% (v/v) dimethyl sulphoxide, under liquid NZ. Viabilty of stored cells was greater than 9 %. For uptake experiments, replicate 0.2ml volumes containing 0.5 x 104-2 x 1 O4 cells in growth medium plus 20% foetal calf serum were seeded in Falcon 96-well Microtest I1 plates and used when the monolayer was confluent (approx. 2 x lo4 cells per well). Uptake experiments were performed by using 5-hydro~y[G-~H]tryptamine creatinine sulphate (1 1.2Ci/mmol), from The Radiochemical Centre, Amersham, Bucks., U.K., which was dissolved in iso-osmotic NaCl to a concentration of 2 0 p ~ , and stored in 0.5ml volumes at 4°C. It was diluted in the appropriate incubation medium before each experiment, and 0.1 ml volumes were added to Microtest wells after the original growth medium had been removed. Experiments were performed at 37C, and in some the monoamine oxidase inhibitor isoniazid (from British Drug Houses, Poole, Dorset, U.K.) was added at a concentration of 0 . 5 m ~ . At the end of the incubation, cells were rinsed 3 times with a large excess of ice-cold iso-osmotic NaCI, dissolved in 0.1 ml of formic acid (25 M), and their radioactivity was determined by liquid-scintillation counting. Cell numbers were determined by electronic counting (Coulter Counter, model B) of suspensions from replicate wells. Uptake of 5-hydro~y[~H]tryptamine was slightly increased when cells were preincubated in serum-free medium, and in the experiments reported here cells were preincubated for 30min with the media to be used for incubations.