title: Potentiation of ATP-induced Ca2+ mobilisation in human retinal pigment epithelial cells.
abstract: 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 microM) increased [Ca2+]i in human RPE cells. Adenosine (10 nM-10 microM) 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 microM), forskolin (10 microM), and the protein kinase A (PKA) activator Sp-cBIMPS (1 microM), 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 microM) and the Gi-activator mastoparan (10 microM). Under resting conditions, cAMP concentration in RPE cells was 7.1+/-0.5 pmol mg(-1) protein. Isoproterenol (10 microM) and forskolin (10 microM) increased levels to 104.6+/-5.2 and 113.7+/-4.2 pmol mg(-1) protein, respectively, while adenosine, clonidine, and mastoparan (all 10 microM) had no significant effect on cAMP levels. These data indicate that whilst activation of A1 and A2 adenosine receptors and alpha2 and beta 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.
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK. david.collison@uea.ac.uk
authors.forename: David J
authors.initials: DJ
authors.lastname: Collison
authors.forename: Victoria E
authors.initials: VE
authors.lastname: Tovell
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Coombes
authors.forename: George
authors.initials: G
authors.lastname: Duncan
authors.forename: Julie
authors.initials: J
authors.lastname: Sanderson
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000241:Adenosine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004357:Drug Synergism; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007545:Isoproterenol / Q000494:pharmacology; D010641:Phenotype; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D058915:Purinergic P1 Receptor Antagonists; D011983:Receptors, Purinergic / Q000378:metabolism; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014665:Vasodilator Agents / Q000494:pharmacology
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D000318:Adrenergic beta-Agonists; D058915:Purinergic P1 Receptor Antagonists; D011983:Receptors, Purinergic; D018047:Receptors, Purinergic P1; D014665:Vasodilator Agents; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D000241:Adenosine; D007545:Isoproterenol; D000109:Acetylcholine; D002118:Calcium