Adenosine modulates various physiological effects on many cell types by activating four different subtypes of G protein coupled receptors classified as A1, A2A, A2B and A3. Recently, 5-N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)-pyrazolo-[4,3-e]1,2,4-triazolo[1,5-c]-pyrimidine, [3H]MRE3008F20, has been identified as a potent and selective radiolabelled antagonist of human recombinant A3 receptors (1). In this study we investigated the pharmacological and biochemical profile of A3 subtypes in Jurkat T cells, a human leukemia line, where the presence of A2B and A2A but not A3 receptors has been previously demonstrated. A3 receptors were first revealed by RT-PCR experiments; then saturation of [3H]MRE3008F20 binding to A3 receptors (range 0.2-2nM) showed a single high affinity binding site with Kd of 1.90.2 nM and Bmax of 1.30.1 pmol/mg of protein. Competition studies of [3H]MRE3008F20 binding (2nM) using typical adenosine ligands displayed a pharmacological profile typical of the A3 subtype. Agonist competition curves yelded both a high (30%) and a low-affinity state of A3 receptors. Cl-IB-MECA and IB-MECA were the most potent compounds with KH of 1.6, 2.7 nM and KL of 83, 150 nM, respectively. Functional studies showed that Cl-IBMECA and IB-MECA, were able to inhibit cAMP accumulations with EC50 values of 3.50.3 and 120.1 nM, respectively. The same agonists (30 M) stimulated Ca2+ release from intracellular Ca2+ pools from a basal level of 100-150 nM to a stimulated level of 250-300 nM. Finally, A3 agonists (30-40M) and antagonists (1-10M) produced a cell death effect, reducing cell number to 40% of control (p<0.05, ANOVA and Dunnett’s test) as demonstrated by different viability assays: trypan blue, JAM and MTT assays. These data are the first evidence of the presence of functional A3 adenosine receptors in Jurkat cells. 1. Varani K., Merighi S., Gessi S., Klotz K.N., Leung E., Baraldi P.G., Cacciari B., Romagnoli R., Spalluto G., and Borea P.A. (2000) Mol. Pharmacol. 57:968-975.
Pharmacological and biochemical characterization of A3 adenosine receptors in Jurkat cells, a human leukemia line.
GESSI, Stefania;VARANI, Katia;MERIGHI, Stefania;MORELLI, ANNA;FERRARI, Davide;
2001
Abstract
Adenosine modulates various physiological effects on many cell types by activating four different subtypes of G protein coupled receptors classified as A1, A2A, A2B and A3. Recently, 5-N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)-pyrazolo-[4,3-e]1,2,4-triazolo[1,5-c]-pyrimidine, [3H]MRE3008F20, has been identified as a potent and selective radiolabelled antagonist of human recombinant A3 receptors (1). In this study we investigated the pharmacological and biochemical profile of A3 subtypes in Jurkat T cells, a human leukemia line, where the presence of A2B and A2A but not A3 receptors has been previously demonstrated. A3 receptors were first revealed by RT-PCR experiments; then saturation of [3H]MRE3008F20 binding to A3 receptors (range 0.2-2nM) showed a single high affinity binding site with Kd of 1.90.2 nM and Bmax of 1.30.1 pmol/mg of protein. Competition studies of [3H]MRE3008F20 binding (2nM) using typical adenosine ligands displayed a pharmacological profile typical of the A3 subtype. Agonist competition curves yelded both a high (30%) and a low-affinity state of A3 receptors. Cl-IB-MECA and IB-MECA were the most potent compounds with KH of 1.6, 2.7 nM and KL of 83, 150 nM, respectively. Functional studies showed that Cl-IBMECA and IB-MECA, were able to inhibit cAMP accumulations with EC50 values of 3.50.3 and 120.1 nM, respectively. The same agonists (30 M) stimulated Ca2+ release from intracellular Ca2+ pools from a basal level of 100-150 nM to a stimulated level of 250-300 nM. Finally, A3 agonists (30-40M) and antagonists (1-10M) produced a cell death effect, reducing cell number to 40% of control (p<0.05, ANOVA and Dunnett’s test) as demonstrated by different viability assays: trypan blue, JAM and MTT assays. These data are the first evidence of the presence of functional A3 adenosine receptors in Jurkat cells. 1. Varani K., Merighi S., Gessi S., Klotz K.N., Leung E., Baraldi P.G., Cacciari B., Romagnoli R., Spalluto G., and Borea P.A. (2000) Mol. Pharmacol. 57:968-975.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
