Abstract
Abstract: :
Purpose: Extracellular purines and pyrimidines act as neurotransmitters and neuromodulators by activation of P2 receptors in the mammalian nervous system. Despite the considerable evidence for signalling by ATP in other sensory systems, only few studies have been undertaken in the retina. Here we demonstrate the occurrence, cellular distribution and quantitative differences of P2Y receptors (P2Y1, P2Y2, P2Y4 and P2Y6) on mRNA and protein level in the rat retina. Methods: Expression of the four P2Y receptors was tested by RT–PCR in the adult rat retina. For detection of P2Y–mRNA in retinal ganglion cells (RGC), bipolar cells (BPC) and Müller cells (MC) single–cell RT–PCR was used. cRNA probes of P2Y1/2/4/6 receptors used in in–situ–hybridization (ISH) on retinal sections of adult rats showed the cellular distribution pattern of the complementary P2Y–mRNA. Antibodies raised against P2Y1/2/4 receptors were applied to retinal sections to detect cellular localization by immunohistochemistry. Western Blots were used to evaluate the differences of P2Y–protein levels in different developmental stage of the rat retina (P3–P44). Results: P2Y1/2/4/6–mRNA was detected in homogenates of total adult rat retina as well as in different proportions in the three retinal cell types tested: RGC (P2Y2: 100%, P2Y1: 80%, P2Y6: 50%, P2Y4: 30%), BPC (P2Y6: 80%, P2Y1: 60%, P2Y2: 40%, P2Y4: 20%), and MC (P2Y6: 100%, P2Y2: 80%, P2Y1: 60%, P2Y4: 60%). ISH revealed mRNA in the ganglion cell layer (GCL) and in the inner nuclear layer (INL) for P2Y1, P2Y4 and P2Y6, whereas labelling for the P2Y2–receptor subtype was found exclusively in the GCL. P2Y1/2/4–specific proteins were present in cells of the GCL, INL and outer plexiform layer (OPL). Additionally, for the P2Y4 receptor three broadly stained sublayers could be distinguished in the inner plexiform layer (IPL). Rat retinae of different developmental stages used in Western Blots showed that amounts of P2Y1/2/4–proteins differ between immature and mature tissue. P2Y1–protein is dominant in P3 up to P10 retinae, P2Y2 is weak in P3 and similar in all other developmental stages, and P2Y4–protein is absent in P3 retinae and is continuously upregulated from P5 to P44. Conclusions: Our data show the expression of several metabotropic purine–receptors in different retinal cell types. The different extent of P2Y–protein expression in various developmental stages of the rat retina suggest that extracellular purines may play complex roles as autocrine or paracrine mediators and/or may have neuromodulatory influences in the retina.
Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • receptors • neurotransmitters/neurotransmitter systems