Effect of Adenosine on P2X7 Receptor-mediated Ca2+ Signalling in Human Muller Cells and Retinal Neurons

Abstract

Abstract: : Purpose: The P2X7 ATP receptor is proposed to play a role in apoptosis. Functional expression of this receptor in human retinal cells in culture was investigated using the P2X7 agonist BzATP. Adenosine is a proposed neuromodulator in the retina. The effect of adenosine on BzATP-induced Ca²⁺ signalling was also investigated. Methods: Human retinas were obtained from donor eyes within 24 hours post mortem. The neural retina was dissociated using papain, plated onto laminin-coated cover slips and cultured in Ham/F12 medium supplemented with 10% FCS and 10ng/ml bFGF. Ca²⁺ measurements were made using ratiometric digital imaging techniques with the Ca²⁺-indicator Fura-2. Results: BzATP (50µ M) induced an increase in intracellular Ca²⁺ in Müller cells and in a subset of retinal neurons. This response was totally inhibited by the P2X antagonist PPADS (100µ M), while the L-type Ca²⁺ channel antagonist nifedipine (10µ M) had little effect on the BzATP-induced Ca²⁺ signal. Application of adenosine (10µ M) had no effect on resting intracellular Ca²⁺ concentration in the BzATP-sensitive cells. However, in the presence of adenosine the Ca²⁺ response to BzATP was potentiated in both Müller and neuronal cells. Moreover, it was observed that neurons responding only minimally to BzATP, gave large Ca²⁺ responses in the presence of both BzATP and adenosine. The adenosine-induced potentiation was inhibited by the A1 receptor antagonist DPCPX (1µ M), whereas the A3 receptor antagonist MRS1191 (1µ M) had no effect. Conclusions: P2X7 ATP receptors are functionally expressed in cultured human retinal Müller and neuronal cells. Adenosine potentiates the BzATP-induced Ca²⁺ signal via A1 adenosine receptors. This enhanced Ca²⁺ response may have implications for neuronal cell death, such as that occurring in glaucoma.

Keywords: retinal culture • calcium • adenosine