Investigation of endegonous A2B adenosine receptor-evoked Ca2+ signalling in human embryonic kidney (HEK) cells using a fluorescence integrayted platre reader (FLIPR) AM Jackson1, J Stables2, SJ Hill1& SPH Alexander1, 1Institute of Cell Signalling & School of Biomedical Sciences, University of Nottingham Medical School, Nottingham, NG7 2UH & 2GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY.

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The A_2B adenosine receptor couples primarily to the stimulation of adenylate cyclase, but is also able to evoke increases in [Ca²⁺]_i (Fredholm et al., 2001). Previous investigations using HEK cells over-expressing human A_2B receptors demonstrated A_2B-mediated increases in [Ca²⁺],subi that were attributed to Ca²⁺ release resulting from phospholipase C activation (Linden et al., 1999). HEK cells express A_2B receptors endogenously (Cooper et al., 1997), so the aim of the present study was to investigate changes in [Ca²⁺]_i mediated by endogenous A_2B receptors in HEK cells.

HEK cells were grown essentially as described previously (Jackson et al., 2001). cAMP & inositol phosphates accumulations were determined using [³H]-adenine or [³H]-inositol pre-labeling, respectively, followed by column chromatography, as described previously (Jackson et al., 2001). Changes in [Ca²⁺]_i were measured using a FLIPR®, in which cells were stimulated with carbachol (CCh, 10^-8-10^-3 M) or buffer, followed after 330 seconds by NECA (5'-N-ethylcarboxamiodadenosine, 10^-9-10^-4 M), under Ca²⁺-containing or Ca²⁺-free conditions as described previously (Jackson et al., 2001). Unless otherwise stated, data represent the mean ± s.e.mean. of three experiments.

NECA evoked substantial increases in [Ca²⁺]i following pre-stimulation with CCh under both Ca²⁺-containing & Ca²⁺-free conditions. Following pre-stimulation with 1 µM CCh, pEC₅₀ values for NECA-evoked increases in [Ca²⁺]_i were 6.22 ± 0.65 & 7.04 ± 0.01, with responses to 100 µM NECA of 26 ± 3 & 27 ± 2 % of a 1 mM CCh response, under Ca2+-containing & Ca²⁺-free conditions, respectively. 100 µM NECA failed to evoke increases in inositol phosphates (IP) accumulation, or influence CCh-evoked increase in IP accumulation (116 ± 15 % of 1 mM CCh response). Pre-incubation with muscarinic receptor antagonists (1 µM) prior to the CCh pre-stimulus reduced NECA-evoked [Ca2+]_i elevations, such that peak responses to NECA (10 µM) were 29 ± 1 (control), & 3 ± 0 (4-DAMP), 12 ± 1 (pirenzepine) & 26 ± 2 % (methoctramine) of a control 1 mM CCh response.

Incubation with the ß-adrenoceptor agonist isoprenaline evoked a concentration-dependent increase in cAMP generation in HEK cells (pEC₅₀ = 7.32 ± 0.13, max. response = 24 ± 0 % of 10 µM NECA-evoked cAMP response). Isoprenaline also evoked a concentration-dependent (pEC₅₀ = 7.54 ± 0.28, maximum response = 27 ± 3 % 1 µM CCh response) increase in [Ca²⁺]_i following pre-stimulation with 1 µM CCh, but not buffer. Forskolin (1, 10 & 100 µM) also evoked significant (p<0.001) increases in [Ca²⁺]_i following pre-simulation with CCh. 100 µM, but not 1 & 10 µM, forskolin elicited a significant (p<0.05) increase in [Ca²⁺]_i following pre-stimulation with buffer alone (two-way ANOVA with Bonferroni post-hoc tests).

Taken together, these results indicate coupling of the endogenous A_2B adenosine receptor in HEK cells to increases of intracellular calcium, which appear to be mediated indirectly via elevation of cyclic AMP levels. The visualisation of the A_2B (& ß-adrenoceptor) stimulation of [Ca²⁺]_i appears to be dependent on prior stimulation of cells with carbachol, which in HEK 293 cells evokes increases in [Ca²⁺]_i via an M₃ muscarinic receptor (Jackson et al., 2001).

We thank the MRC for financial support.

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