Characterization of adenosine receptors in the human bladder carcinoma T24 cell line.

Abstract The molecular and pharmacological properties of adenosine receptors in the T24 human bladder epithelial carcinoma cell line were assessed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), Ca 2+ Flux, cAMP production and interleukin-8 measurements. RT-PCR experiments detected the presence of transcripts for the adenosine A 1 , A 2A and A 2B receptors but not for the adenosine A 3 subtype. Application of specific adenosine receptor ligands resulted in concentration-dependent increases in intracellular calcium ([Ca 2+ ] i ) with the following order of potency and EC 50 values: 5′- N -Ethylcarboxamidoadenosine (NECA) (1153 ± 214) > 5′-( N -Cyclopropyl)carboxamidoadenosine (CPCA) (1436 ± 186) > adenosine (4823 ± 932). This rank order of potency is typical of adenosine A 2B receptors. In addition, select adenosine receptor antagonists N -(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6 dioxo-1,3-dipropyl-1 H -purin-8-yl)phenoxy]acetamide (MRS 1706), 8-[4-[((4-Cyano[2,6-]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di( n- propyl)-xanthine (MRS 1754), 1,3-Diethyl-8-phenylxanthine (DPCPX), 1,3-Diethyl-8-phenylxanthine (DPX), Alloxazine, 8-(3-Chlorostyryl)caffeine (CSC), and Theophylline blocked the NECA-induced calcium responses. Additionally, NECA, CPCA, and adenosine stimulated cAMP formation with a rank order of potency characteristic of adenosine A 2B receptors. The select adenosine A 2A antagonist, 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine (SCH 58261) failed to antagonize the NECA response, whereas the potent and highly selective adenosine A 2B antagonists MRS 1754 and MRS 1706 inhibited NECA-stimulated cAMP production. Lastly, NECA-induced interleukin-8 secretion was inhibited by MRS 1754. Taken together, these data indicate that [Ca 2+ ] i accumulation and cAMP production as well as interleukin-8 secretion is mediated through the adenosine A 2B receptor in the T24 cell line.