Uptake and metabolism of adenosine by pig aortic endothelial and smooth-muscle cells in culture

Abstract

1. Adenosine, a potent vasodilator, is transported very efficiently by pig aortic endothelium in monolayer culture (approx. 50pmol/min per 10(6) cells at 2 micrometer). Uptake proceeds by diffusion at high (millimolar) substrate concentrations, and by two discrete transport processes (Km approx. 3 micrometer and 250 micrometer) at lower concentrations. Over 90% of the adenosine taken up at 10 micrometer or 100 micrometer is rapidly converted into adenine nucleotides (mainly ATP). 2. The high-affinity process is selectively inhibited by dipyridamole and by nitrobenzylthioinosine. Adenine preferentially inhibits the lower-affinity process, papapaverine inhibits both transport processes, and inosine has no significant effect. 3. Pig aortic smooth-muscle cells in culture show no high-affinity transport system for adenosine; uptake is much slower at low concentrations than that by endothelium (approx. 5pmol/min per 10(6) cells at 2 micrometer). Over 80% of the incorporated adenosine at 10 micrometer or 100 micrometer is rapidly converted into adenine nucleotides. 4. The uptake of adenosine by smooth-muscle cells is powerfully inhibited by adenine, but dipyridamole is much less potent than in endothelium. 5. We conclude that endothelial cells are mainly responsible for the removal of circulating adenosine.

Effects of neutrophil elastase and other proteases on porcine aortic endothelial prostaglandin I2 production, adenine nucleotide release, and responses to vasoactive agents.

“…Endothelial cells were isolated from porcine thoracic aorta, essentially as previously described (20) Endothelial cell columns. 0.4-0.5 ml of microcarrier beads covered with endothelial cells (1-2 X 106 cells) was packed into a l-ml plastic syringe plugged with glass wool; the syringe was closed with a rubbertipped plunger through which plastic tubing had been threaded, as previously described (7,21 The release of lactate dehydrogenase (LDH) from cells in static culture and from columns was assayed according to standard techniques (23) as follows: cells were solubilized in 0.2% Triton X-100 or column fractions were made up to 1.0 ml in 0.2% Triton X-100.…”

“…Basal release of 3H-nucleotides from unstimulated cell columns was <0.05% per min; this rose to a maximum of 0.9% per min after 100 jg/ml trypsin and 6.4% per min after 30 ,ug/ml elastase. These values were obtained by chromatography of samples using a solvent system (26) When cells were challenged a second time with ATP (100 AM) 20-40 min after the first challenge, the stimulation of PGI2 production was similar to the initial response (96.0±1.7%, n = 5). Since the proteases tested had variable effects on PGI2 production and on purine release, the effects of exposure to these proteases on subsequent PG12 responses to ATP were studied.…”

Modulation of blood flow and tissue perfusion by endothelium‐derived relaxing factor

“…Endothelial cells have the intrinsic capacity to synthesize acetylcholine and ATP (Pearson, Carleton, Hutchings & Gordon, 1978;Parnevalas, Kelly & Burnstock, 1985). They also contain heterogeneous cytoplasmic stores of agonists that stimulate EDRF synthesis, including bradykinin, vasopressin, angiotensin II, 5-HT and substance P, although it is unclear whether these are taken up or synthesized de novo (Bodin, Bailey & Burnstock, 1991;Loesch, Bodin & Burnstock, 1991;Ralevic, Lincoln & Burnstock, 1992).…”

Transport

“…Plasma adenosine levels are regulated by rapid transport into red cells and endothelial cells (Sobrevia et al 1994). Pig aortic smooth muscle cells take up adenosine by a low affinity route which is weakly inhibited by dipyridamole (Pearson et al 1978). In other species (e.g.…”