RPE cells express both NTPDase1 and NTPDase2: resulting ADP can increase cell calcium

Abstract

Abstract: : Purpose: ATP can stimulate P2Y receptors on the apical surface of the retinal pigment epithelium (RPE) to raise intracellular calcium and trigger fluid absorption. RPE cells release ATP and preliminary data suggests they use dephosphorylating ecto–enzymes to convert ATP into adenosine. RPE cells possess the widely distributed NTPDase1 (CD39) which converts ATP to AMP. As ADP can stimulate some P2Y receptors, we asked whether NTPDase2, which converts ATP to ADP, is present on RPE cells and whether the product ADP can alter RPE physiology. Methods: RT–PCR was performed on RNA from ARPE–19 cells with primers for NTPDases 1 and 2. Enzymes were localized to ARPE–19 cells with Western blotting and immunohistochemical techniques. Intracellular calcium was measured ratiometrically with fura–2. Purine levels in the solution bathing the apical membrane of bovine RPE eyecup were quantified by HPLC with UV detection. Results: mRNA message for both NTPDases 1 and 2 was identified with RT–PCR. NTPDase1 and NTPDase2 were localized to cells immunohistochemically, and expression was confirmed with Western blots. The NTPDase2 product ADP produced a transient rise of intracellular calcium levels followed by a sustained plateau which remained elevation throughout ADP exposure. Preliminary results suggest that 20 min after addition of ATP to the bovine eyecup, extracellular levels of ATP were low but both ADP and AMP were present in substantial quantities. Conclusions: Both NTPDase1 and NTPDase2 are present on RPE cells. NTPDase2 provides an alternative metabolic pathway for the degradation of ATP which could lead to a more sustained stimulation of P2Y receptors on RPE cells.

Keywords: adenosine • pharmacology • second messengers