Potentiation of ATP-induced Ca2+ mobilisation in human retinal pigment epithelial cells

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 Ca (2+) responses in human retinal pigment epithelial (RPE) cells. Intracellular Ca (2+) concentration ([Ca (2+)]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 mu m) increased [Ca (2+)]i in human RPE cells. Adenosine (10 nm-10 mu m) had no effect on resting [Ca (2+)]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 A(1) and A(2) adenosine receptor antagonists, DPCPX and MRS 1706 (100 n(M)) respectively, inhibited potentiation in 76 +/- 7 and 23 +/- 12% of cells, respectively, but the A(3) antagonist MRS1191 had no effect. Conversely, agents that activate the cAMP pathway, including isoproterenol (10 mu m), forskolin (10 mu m), and the protein kinase A (PKA) activator Sp-cBIMPS (1 mu m), 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 mu m) and the Gi-activator mastoparan (10 mu m). Under resting conditions, cAMP concentration in RPE cells was 7.1 +/- 0.5 pmol mg(-1) protein. Isoproterenol (10 mu m) and forskolin (10 mu m) increased levels to 104.6 +/- 5.2 and 113.7 +/- 4.2 pmol mg(-1) protein, respectively, while adenosine, clonidine, and mastoparan (all 10 mu m) had no significant effect on cAMP levels. These data indicate that whilst activation of A(1) and A(2) adenosine receptors and alpha(2) and 0 adrenergic receptors does not influence basal Ca (2+) levels, stimulation of these receptors can potentiate Ca 2+ signalling by cAMP dependent and independent mechanisms in human RPE cells. (c) 2004 Elsevier Ltd. All rights reserved.