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. ATP gamma S, 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 [35S]ATP gamma S to MNPs at 4 degrees C revealed saturable low-affinity binding sites with a Kd of 868 +/- 52 nmol/L and Bmax of 7.3 +/- 0.4 pmol per 10(6) cells in 1-day cultured human MNPs and a Kd of 780 +/- 30 nmol/L and Bmax 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 [3H]CGS 21680 was biphasic, with a Kd1 of 33 +/- 15 nmol/L and a Kd2 of 90 +/- 10 nmol/L and with Bmax1 of 0.19 +/- 0.06 pmol per 10(6) cells and Bmax2 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. ATP gamma S 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 ATP gamma S. No significant influence of these compounds was measured on the uptake of LDL, acetylated LDL, and high-density lipoprotein, in 1-day cultured MNPs. Our investigations indicate that the expression of P2y and A2 receptors is increased during differentiation of blood monocytes to macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)
Institut für Klinische Chemie und Laboratoriumsmedizin, Universität Regensburg, Germany.
G
G
Müller
C
C
Kerkhoff
J
J
Hankowitz
M
M
Pataki
E
E
Kovacs
K J
KJ
Lackner
G
G
Schmitz
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001161:Arteriosclerosis / Q000209:etiology*; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000032:analysis; D006152:Cyclic GMP / Q000032:analysis; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism; D010586:Phagocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011983:Receptors, Purinergic / Q000032:analysis* / Q000502:physiology; D015398:Signal Transduction
D008077:Lipoproteins, LDL; D011983:Receptors, Purinergic; C032629:acetyl-LDL; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D006152:Cyclic GMP; D002118:Calcium