Interaction of signalling pathways directs the functional output of many cells. This study investigated the consequences of activating adenosine and adrenergic receptors on ATP-induced Ca2+ responses in human retinal pigment epithelial (RPE) cells. Intracellular Ca2+ concentration ([Ca2+]i) of human RPE cells in primary culture was monitored using Fura-2. Cyclic adenosine monophosphate (cAMP) concentration was measured using an enzyme-linked immunosorbent assay. Both ATP and UTP (10 µm) increased [Ca2+]i in human RPE cells. Adenosine (10 nm–10 µm) had no effect on resting [Ca2+]i, but potentiated a sub-threshold response to ATP (100 nm) when ATP was added in the presence of adenosine. The potentiation occurred with other G-protein receptor agonists such as acetylcholine. Potentiation persisted in Ca-free medium, but was blocked by prior application of thapsigargin. The A1 and A2 adenosine receptor antagonists, DPCPX and MRS1706 (100 nm) respectively, inhibited potentiation in 76±7 and 23±12% of cells, respectively, but the A3 antagonist MRS1191 had no effect. Conversely, agents that activate the cAMP pathway, including isoproterenol (10 µm), forskolin (10 µm), and the protein kinase A (PKA) activator Sp-cBIMPS (1 µm), potentiated the ATP-induced response in the RPE cells. Agents that are known to inhibit the production of cAMP in other systems also caused potentiation, including clonidine (10 µm) and the Gi-activator mastoparan (10 µm). Under resting conditions, cAMP concentration in RPE cells was 7·1±0·5 pmol mg-1 protein. Isoproterenol (10 µm) and forskolin (10 µm) increased levels to 104·6±5·2 and 113·7±4·2 pmol mg-1 protein, respectively, while adenosine, clonidine, and mastoparan (all 10 µm) had no significant effect on cAMP levels. These data indicate that whilst activation of A1 and A2 adenosine receptors and a2 and ß adrenergic receptors does not influence basal Ca2+ levels, stimulation of these receptors can potentiate Ca2+ signalling by cAMP dependent and independent mechanisms in human RPE cells.
|Number of pages||1|
|Journal||Investigative Ophthalmology and Visual Science|
|Publication status||Published - 2004|