Pharmacological and biochemical characterization of A3 adenosine receptors in Jurkat T cells.
1. The present work was devoted to the study of A3 adenosine receptors in Jurkat cells, a human leukemia line. 2. The A3 subtype was found by means of RT-PCR experiments and characterized by using the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with K(D) of 1.9+/-0.2 nM and B(max) of 1.3+/-0.1 pmol mg(-1) of protein. 3. The pharmacological profile of [3H]-MRE 3008F20 binding on Jurkat cells was established using typical adenosine ligands which displayed a rank order of potency typical of the A3 subtype. 4. Thermodynamic data indicated that [3H]-MRE 3008F20 binding to A3 subtype in Jurkat cells was entropy- and enthalpy-driven, according with that found in cells expressing the recombinant human A3 subtype. 5. In functional assays the high affinity A3 agonists Cl-IB-MECA and IB-MECA were able to inhibit cyclic AMP accumulation and stimulate Ca(2+) release from intracellular Ca(2+) pools followed by Ca(2+) influx. 6. The presence of the other adenosine subtypes was investigated in Jurkat cells. A1 receptors were characterized using [3H]-DPCPX binding with a K(D) of 0.9+/-0.1 nM and B(max) of 42+/-3 fmol mg(-1) of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a K(D) of 2.5+/-0.3 nM and a B(max) of 1.4+/-0.2 pmol mg(-1) of protein. 7. In conclusion, by means of the first antagonist radioligand [3H]-MRE 3008F20 we could demonstrate the existence of functional A3 receptors on Jurkat cells.
Gessi
S
S
Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, Italy.
Varani
K
K
Merighi
S
S
Morelli
A
A
Ferrari
D
D
Leung
E
E
Baraldi
P G
PG
Spalluto
G
G
Borea
P A
PA
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D010671:Phenylurea Compounds / Q000378:metabolism / Q000494:pharmacology; D058906:Purinergic P1 Receptor Agonists; D011743:Pyrimidines / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D043705:Receptor, Adenosine A2A; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1 / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013816:Thermodynamics; D013997:Time Factors; D014230:Triazoles / Q000378:metabolism / Q000494:pharmacology; D014316:Tritium; D014970:Xanthines / Q000378:metabolism / Q000494:pharmacology
C098657:5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine; C405605:MRE 3008-F20; D010671:Phenylurea Compounds; D058906:Purinergic P1 Receptor Agonists; D011743:Pyrimidines; D012333:RNA, Messenger; D043705:Receptor, Adenosine A2A; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1; D014230:Triazoles; D014970:Xanthines; D014316:Tritium; D006160:Guanosine Triphosphate; C051360:1,3-dipropyl-8-cyclopentylxanthine; D000242:Cyclic AMP; D002118:Calcium