Expression of Novel Secretory Phospholipases A2 in Rat Retina

Abstract

Abstract: : Purpose: Low-molecular-weight phospholipases A₂s (sPLA₂s) are a subgroup of PLA₂s, which are secreted and bind to receptors. At least ten different groups have been characterized in mammals and there is expanding evidence on the importance of sPLA₂s in neuronal signaling and survival [Kolko et al. (1996) J Biol Chem 271: 32722-32728]. Novel sPLA₂ activity has been meassured in the retina [e.g. Giusto et al., (2000) Prog Lipid Res 39: 315-391], but to date none has been cloned and characterized. Here we evaluated the existence and abundance of novel sPLA₂s in rat retina by PCR cloning and real-time PCR and explored their possible involvement in light damage. Methods: Based on a known sequence of sPLA₂ group IB, X, V, IIE, IIA, IIF, and IID in rat pancreas, rat lung, rat brain, mouse brain, rat platelets, and mouse spleen, respectively, we designed primers to identify the sPLA₂s in rat retina. RNA was isolated from adult rat retina and cDNA was produced and used for PCR cloning to identify the novel subtypes of sPLA₂s. We confirmed the products by sequencing. Primers were designed for real-time PCR based on our cloned sequence. Light damage was induced by bright fluorescent light for 5 hr followed by darkness until rats were killed after 0, 2, 4, 6, 12, and 24 hr and retinas were isolated [Gordon et al., (2002) IOVS, 43: 3511-3521]. Results: Using PCR cloning we identified sPLA2-IB, X, V, IIE, IIA, IIF, and IID in the normal rat retina. By real-time PCR we quantified four subgroups of sPLA₂s and found sPLA₂-IB to be the most abundant sPLA₂ followed by group V, IIE, and X, respectively. Finally, we showed a time-dependent induction of sPLA₂ in rats exposed to light damage. sPLA₂-IB and X were induced 4 and 12 hr after light damage by approximately 5 and 12 fold, respectively, whereas sPLA₂-V and IIE were only induced at most 3.5 fold, 4 hr after light damage. Conclusions: This study is the first to reveal the existence and abundance of specific sPLA₂s in rat retina. Furthermore, we show that the subtypes are differentially induced during light damage, suggesting different roles of the sPLA₂s in retina. These enzymes may be intercellular signaling modulators significant in retina cell function and in retinal degenerations.

Keywords: retina • gene/expression • molecular biology