EFFECTS OF PURINERGIC AGENTS ON HUMAN MONONUCLEAR PHAGOCYTES ARE DIFFERENTIATION-DEPENDENT - IMPLICATIONS FOR ATHEROGENESIS

The differentiation-dependent expression of purinergic receptors for m etabolically stable analogues of adenosine and ATP was studied in huma n mononuclear phagocYtes (MNPs). Ligands of these receptors are able t o modulate cellular cholesterol metabolism. In addition, the intracell ular signal transduction pathways of the purinergic receptor system we re examined. ATPgammaS, the metabolic stable analogue of ATP, was used as a P2 ligand, and arboxyethyl)phenylethylamino-5'-N-ethylcarboxamid o adenosine (CGS 21680) and 5'-(N-ethylcarboxamido)adenosine (NECA) we re used as P1 ligands in binding studies. Binding of [S-35]ATPgammaS t o MNPs at 4-degrees-C revealed saturable low-affinity binding sites wi th a K(d) of 868 +/- 52 nmol/L and B(max) of 7.3 +/- 0.4 pmol per 10(6 ) cells in 1-day cultured human MNPs and a K(d) of 780 +/- 30 nmol/L a nd B(max) of 14.0 +/- 0.8 pmol per 10(6) cells in 7-day cultured human MNPs. The characterization of the P1 receptors on 1- and 7-day cultur ed human MNPs showed that they are expressed only on 7-day cultured hu man MNPs. The specific binding curve of the adenosine A2 receptor agon ist [H-3]CGS 21680 was biphasic, with a K(d1) of 33 +/- 15 nmol/L and a Ku of 90-10 nmol/L and with B(max1) of 0.19 +/- 0.06 pmol per 10(6) cells and B(max2) of 0.41 +/- 0.09 pmol per 10(6) cells, whereas NECA did not exhibit specific binding. The typical agonists for probing A1 receptor subtypes did not bind to 1- and 7-day cultured human MNPs, in dicating that only A2 receptors are expressed on 7-day cultured human MNPs. ATPgammaS enhanced [Ca2+]i in 1- and 7-day cultured human MNPs i n a concentration-dependent manner, whereas the P1 ligands, adenosine and CGS 21680, induced Ca2+ flux only in 7-day cultured MNPs. All thre e drugs increased intracellular cAMP levels in 7-day cultured human MN Ps at a concentration of 10(-5) mol/L, whereas no effect was observed in 1-day cultured human MNPs. The uptake of fluorescently labeled acet ylated low-density lipoprotein (LDL) in 7-day cultured human MNPs was inhibited by adenosine, CGS 21680, ATP, and ATPgammaS. No significant influence of these compounds was measured on the uptake of LDL, acetyl ated LDL, and high-density lipoprotein 3 in 1-day cultured MNPs. Our i nvestigations indicate that the expression of P2, and A2 receptors is increased during differentiation of blood monocytes to macrophages. It is shown that both purinergic ligands, ATPgammaS and CGS 21680, inhib it the uptake of acetylated LDL in a concentration-dependent manner, w hich might impair foam cell formation.