Identification of the A3 Adenosine Receptor in Rat Retinal Ganglion Cells

Abstract

Abstract: : Purpose: Adenosine can protect retinal ganglion cells (RGCs) from the death that accompanies NMDA and P2X₇ receptor stimulation, in addition to general ischemic challenge. While a role for the A₁ adenosine receptor is known, our recent findings suggest that the A₃ receptor can also contribute. Two variants of the A₃ receptor are known, and molecular identification is critical to maximize the neuroprotective potential. However, the receptor is present in low copy number and a previous in situ study was unable to detect any message in the retina. Here we identify A₃ adenosine receptor in rat retinal ganglion cells by combining the more sensitive quantitative–PCR (Q–PCR) technique with the specificity of laser capture microdisection (LCM). Methods: RGCs of Long–Evans rat pups were back–labeled with aminostilbamidine. Eyeballs were enucleated, embedded, frozen in OCT and sectioned to 10µm. Ganglion cells were identified by their fluorescence; 500– 1,000 RGCs from each specimen were zapped up using LCM. The RNA was isolated and linearly amplified. mRNA for A₃ adenosine receptor was analyzed by traditional RT–PCR, confirmed by DNA sequencing and compared with mRNA from whole retina using Q–PCR. Results: High purity RNA was obtained from RGCs collected using LCM. Expression of the A₃ receptor message was detected using RNA from these microdissected cells using traditional RT–PCR; the 640 bp product was 99% identical to the published sequence. The standard form of A₃ receptor message was detected, not the splice variance found in some hippocampal regions. Traditional RT–PCR also picked up A₃ receptor message in RNA from the whole retina. Q–PCR suggested copy number was 20x greater in RGCs than in the whole retina. The sequence of the Q–PCR product (125bp) was 100% identical to the published sequence. Conclusions: LCM is an efficient way to precisely select RGCs from the heterogeneous retina, while Q–PCR allows the successful detection of small amounts of mRNA from a limited number of cells. These techniques indicate the expression of the A₃ receptor is very low in the whole retina, but is concentrated in RGCs, where the standard form of the receptor is expressed. This identification may enhance the use of the A₃ receptor to protect RGCs in glaucoma.

Keywords: neuroprotection • neurotransmitters/neurotransmitter systems • adenosine