Potentiation of ATP–induced Ca2+ mobilisation by adenosine in human retinal pigment epithelial cells

Abstract

Abstract: : Purpose: The interaction of Ca²⁺ and cAMP signalling pathways determines the functional output of many cellular systems. This study investigated the potentiating effect of adenosine on ATP–induced Ca²⁺ responses and the mechanisms involved in signalling pathway crosstalk in retinal pigment epithelium Methods:Primary human RPE cells were grown from donor RPE using explant culture techniques. The cultures were maintained at 35^oC in DMEM (pH7.4) supplemented with 10% FCS. Ca²⁺ measurements were made using ratiometric digital imaging techniques with the Ca²⁺–indicator Fura–2. Results: Both ATP and UTP (10 µM) elicited changes in [Ca²⁺]i indicating the presence of P2Y receptors coupled via Gq. Adenosine (10 nM–10 µM) had no effect on the resting level of [Ca²⁺]i, but potentiated a sub–threshold response to ATP when the two agonists were applied simultaneously. The antagonists DPCPX and MRS1706 of the adenosine A1 and A2 receptors respectively inhibited potentiation of the ATP–induced Ca²⁺ response in separate populations of the RPE cells. Potentiation was also observed by the adrenergic α₂ receptor agonist clonidine, the ß receptor agonist isoproterenol and agents that activated the adenylate cylcase pathway. These included forskolin and the protein kinase A (PKA) activator Sp–cBIMPS. The PKA antagonist H89 inhibited potentiation by adenosine, clonidine and isoproterenol. Conclusions: Surprisingly, receptors coupled both through Gs (A2–adenosine and ß–adrenergic receptors) and Gi (A1–adenosine and α₂–adenergic receptors) potentiated the ATP–induced Ca²⁺ response by PKA mediated mechanisms. This may arise due to non–conventional activation of the adenylate cyclase VII isoform, which is highly expressed in RPE.

Keywords: retinal pigment epithelium • adenosine • calcium