STIMULATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL-CELL PROLIFERATION BYA(2)-ADENOSINE AND BETA(2)-ADRENOCEPTORS

1 Adenosine is known to stimulate capillary outgrowth and endothelial cell proliferation, but the underlying mechanism has not been identifi ed. In order to identify the receptor subtype involved, the effects of adenosine receptor agonists and antagonists on human umbilical vein e ndothelial cell (HUVEC) proliferation were investigated. 2 Raising int racellular adenosine levels by use of the adenosine transport inhibito r, C-nitrobenzylthioinosine (NBMPR) did not affect cell growth. This o bservation suggests that stimulation of an extracellular adenosine rec eptor generates the mitogenic signal. 3 In the presence of adenosine d eaminase (ADA), which was used to remove adenosine present in the cult ure medium, the adenosine receptor agonists N-ethylcarboxamidoadenosin e (NECA, non-selective) and CGS21680 (A(2A)-receptor-selective) stimul ated [H-3]-thymidine incorporation with a half-maximum effect at about 10 nM, while N-6-cyclopentyladenosine (CPA, A(1)-selective) was about 100 fold less potent. The adenosine receptor antagonist, xanthine ami ne congener (XAC) produced a concentration-dependent decrease in endot helial cell proliferation with a half-maximum effect at about 10 nM. H ence, stimulation of an endothelial A(2A)-adenosine receptor seems res ponsible for the mitogenic signal. 4 In the presence of ADA, isoprenal ine is also able to stimulate [H-3]-thymidine incorporation with a hal f maximal effect of about 3 nM, an effect, which is reversed by the hi ghly beta(2)-selective antagonist, ICI 118,551. In the absence of ADA, isoprenaline exerts only a minor stimulatory effect. Combination of A (2A) adenosine and beta(2)-adrenoceptor agonists did not further enhan ce [H-3]-thymidine incorporation when compared to the sole addition of each agonist. We therefore conclude that both receptors stimulate end othelial cell proliferation via a common signal transduction pathway. 5 Both receptors are coupled to stimulation of adenylyl cyclase via th e stimulatory G protein G(s). However, direct activation of downstream effecters in the cyclic AMP-signalling cascade (G(s) with cholera tox in, adenylyl cyclase with forskolin, protein kinase A with Br-8-cyclic AMP) not only failed to mimic the action of receptor-activation, but even reduced cell proliferation. 6 Similarly, pertussis toxin-treatmen t which inactivated the G(i2) protein present in HUVEC and thus inhibi ted cell proliferation per se, did not impair the ability of A(2A)-rec eptor agonists to stimulate cell proliferation. This suggests that the A(2A)-adenosine and beta(2)-adrenoceptor-mediated stimulation of endo thelial cell proliferation occurs via a mechanism that is independent of G(s) and G(i).