We have investigated the responsiveness of porcine endothelial cells, cultured from aorta or umbilical vein, to bradykinin and thrombin. Aortic cells studied in situ, in primary culture and in subculture responded to both agents with an increase in prostacyclin (PGI2) release; the increase was greater with bradykinin than with thrombin. Umbilical vein cells in primary culture or in subculture also responded to bradykinin and to thrombin; bradykinin was again more effective than thrombin. For both cell types there was a rapid quantitative decline in their ability to be stimulated by either agent with increasing passage number, so that by passage 8 stimulation of PGI2 release could no longer be detected from monolayers cultured under conventional conditions. The presence of a stimulatory response was still, however, easily demonstrable in subcultured cells when they were grown on microcarrier beads, packed in small columns, and perfused. Under these conditions greater than 10-fold stimulation above baseline levels was often found, while the conventional monolayer culture technique gave a smaller maximum stimulation even in primary cultures.

1. Pig aortic endothelial and smooth-muscle cells in culture rapidly catabolize exogenous ATP, ADP or AMP. 2. In both cell types catabolism is due to Mg2+-stimulated ectoenzymes. 3. Inhibition and substrate-specificity studies suggest that both cell types possess three distinct ectonucleotidases, namely nucleoside triphosphatase (EC 3.6.1.15), nucleoside diphosphatase (EC 3.6.1.6) and 5'-nucleotidase (EC 3.1.3.5), as well as nucleoside diphosphate kinase (EC 2.7.4.6). 4. These ectonucleotidase systems could be of importance in the regulation of neurotransmission, blood platelet function and vasodilation.

We previously described a model system for studying the adherence of granulocytes to cultured endothelium, and have now investigated the effects of other blood components on granulocyte adhesion in this system. Red cells enhanced adhesion, whereas blood platelets decreased adhesion, and further experiments suggested that endothelial cells secrete a proadhesive factor, particularly if incubated with plasma. We have also investigated the effects of several drugs, and attempted to localize their sites of action. Flavonoid drugs increased adhesion by an effect at the endothelial cell surface, whereas agents that increase cyclic AMP levels, including several prostaglandins, stimulated adhesion mainly by their effects on granulocytes. The effects of some agents on granulocyte adhesion to endothelium were not paralleled by their effects on adhesion to serum-coated glass. We conclude that granulocyte-endothelial interaction is a complex process, with each cell type responding to the other or to factors produced by it, and that data derived solely from studies of granulocyte adhesion to inert substrata will not always reflect granulocyte-endothelial interaction in vivo.

Using a model system with which we have previously investigated the adhesion of granulocytes to cultured endothelium, we have now shown that adherent granulocytes migrate through cultured endothelium in a manner closely resembling that found during the acute inflammatory response in vivo. The migration of granulocytes was markedly enhanced in the presence of erythrocytes, whereas blood platelets did not affect migration. Several test agents, including Paroven and some prostaglandins, had different effects upon migration and adhesion. We conclude that the adhesion of granulocytes to endothelium and their migration through it are responses that are, at least in part, separately controlled, and that migration does not depend exclusively upon extravascular stimuli.

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.

The nature, kinetics and cofactor requirements of leukocyte adhesion to cultured vascular cells have been investigated in vitro, using a model system in which leukocyte suspensions are in continuous motion over the cultured cells. Adhesion is assessed by histological examination or by using 51Cr-labelled leukocytes. Leukocytes adhere preferentially to endothelial cell monolayers rather than to smooth muscle cells, adventitial fibroblasts or serum-coated glass. Arterial and venous endothelium are equally good substrates for leukocyte adhesion, and lymphocytes and granulocytes at the same suspension density adhere to endothelial cells in similar numbers. Adherence is greatest during the first 10 min, and is inversely related to flow rate. Numbers of leukocytes adhering to the cell monolayer are proportional to the initial concentration of leukocytes in suspension, although only 2--10% of the leukocytes adhere. Leukocytes that have adhered to the cell monolayer spread over the cells and then migrate, apparently through intercellular junctions. Adhesion requires Ca2+ or Mg2+ ions, but not plasma protein cofactors. Fewer leukocytes adhere to endothelial cells grown for 48 h in rapidly stirred medium than to cells grown under conventional, static conditions.