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

The differentiation-dependent expression of purinergic receptors for metabolically stable analogues of adenosine and ATP was studied in human mononuclear phagocYtes (MNPs). Ligands of these receptors are able to modulate cellular cholesterol metabolism. In addition, the intracellular signal transduction pathways of the purinergic receptor system were examined. ATPgammaS, the metabolic stable analogue of ATP, was used as a P2 ligand, and 2-p-(2-carboxyethyl)phenylethylamino-5'-N-ethylcarboxamido adenosine (CGS 21680) and 5'-(N-ethylcarboxamido)adenosine (NECA) were used as P1 ligands in binding studies. Binding of [S-35]ATPgammaS to MNPs at 4-degrees-C revealed saturable low-affinity binding sites with 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 and 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 cultured human MNPs showed that they are expressed only on 7-day cultured human MNPs. The specific binding curve of the adenosine A2 receptor agonist [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, indicating that only A2 receptors are expressed on 7-day cultured human MNPs. ATPgammaS enhanced [Ca2+]i in 1- and 7-day cultured human MNPs in a concentration-dependent manner, whereas the P1 ligands, adenosine and CGS 21680, induced Ca2+ flux only in 7-day cultured MNPs. All three drugs increased intracellular cAMP levels in 7-day cultured human MNPs at a concentration of 10(-5) mol/L, whereas no effect was observed in 1-day cultured human MNPs. The uptake of fluorescently labeled acetylated 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, acetylated LDL, and high-density lipoprotein 3 in 1-day cultured MNPs. Our investigations 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, inhibit the uptake of acetylated LDL in a concentration-dependent manner, which might impair foam cell formation.