Identification of the A3 adenosine receptor in rat retinalganglion cells

[摘要] Purpose: Adenosine can protect retinal ganglion cells from the deaththat accompanies a general ischemic challenge as well as excitotoxicdeath. In other tissues, both A1 and A3 adenosine receptorsubtypes can mediate protection. While a role for the A1 adenosinereceptor in ganglion cell protection has been established, a potentialfor the A3 receptor has only recently been proposed. Although thepharmacology is promising, the molecular identity of the responsiblereceptor is unclear as previous studies were unable to detect messagefor the A3 receptor in retinal ganglion cells. We combined lasercapture microdisection (LCM) and immunopurification with traditional andreal-time PCR to unequivocally demonstrate the presence of the A3receptor message in rat retinal ganglion cells.Methods: Retinal ganglion cells of Long-Evans rat pups wereretrograde labeled with aminostilbamidine. Eyeballs were enucleated,embedded, frozen, sectioned, and fluorescent cells in the ganglion celllayer were collected with LCM. Purified ganglion cells were alsoisolated with a two-step panning procedure. cDNA for the A3receptor obtained from the microdissected ganglion cell layer,immunopurified ganglion cells, whole retina and testis was amplifiedusing RT-PCR, confirmed by DNA sequencing and compared with publishedsequences. A3 receptor message was also amplified using real-timePCR. Ca2+ levels in immunopanned ganglion cells were measuredratiometrically with fura-2.Results: RNA from immunopurified ganglion cells and from dye-loadedcells in the ganglion cell layer contained message for the A3receptor when amplified with either traditional RT-PCR or real-time PCR.The entire encoding region was sequenced and found to be 99% identicalto the published code. The sequence closely resembled the consensus formof the gene, with other sequences deviating from this default code.Molecular identification was functionally confirmed in purified ganglioncells as the A3 receptor agonist Cl-IB-MECA prevented theexcessive Ca2+ rise triggered by P2X7 agonist BzATP.Conclusions: Retinal ganglion cells express A3 adenosinereceptor mRNA. Stimulation of this receptor can reduce the Ca2+overload following excessive activation of P2X7 receptors.

[学科分类] 生物化学/生物物理