Department of Experimental Zoology and Neurobiology, University of Pécs, Pécs, Hungary.
Invest Ophthalmol Vis Sci. 2012 Nov 27;53(12):7825-32. doi: 10.1167/iovs.12-10417.
The ubiquitous pituitary adenylate cyclase-activating peptide (PACAP) has a disparate array of functions in development (e.g., proliferation and apoptosis). Among three types of PACAP receptor (VPAC1, VPAC2, and PAC1), PAC1 is subject to alternative splicing that generates isoforms. Although the literature documenting the presence of PACAP receptors in the central nervous system is vast, their expression during development has not been established yet. Here, we performed quantitative analyses on the expression of PACAP receptors during the postnatal development of the rat retina.
Retinas were harvested from postnatal days 0 to 20 (P0-P20). Using a comprehensive primer system, expression changes were followed employing quantitative real-time PCR. Changes at the protein level were detected by immunoblotting using anti-VPAC1, -VPAC2, and -PAC1 receptor antibodies.
The expression of VPAC1 showed increases at P10 and P15. Peaks in VPAC2 expression were observed at P5 and P15. Using splicing variant-specific primers for PAC1 receptor, splicing regulation of Null, Hip, Hop1, and Hiphop1 variants was revealed in correlation with postnatal development. Transcript levels of the Null and Hip variants showed a decline, while Hop1 became the major PACAP receptor by P20. Hiphop1 transcript levels did not display remarkable changes except for a transient increase at P10. Immunoblotting confirmed the presence and expression level changes of the receptors.
We conclude that both VPAC1 and VPAC2 could have roles at all stages of retinal development, that PACAP acts through a specific set of PAC1 isoforms, and that Hip and Hop1 are predominantly involved in the postnatal development of rat retina.
普遍存在的垂体腺苷酸环化酶激活肽（PACAP）在发育过程中具有多种功能（例如，增殖和凋亡）。在三种类型的 PACAP 受体（VPAC1、VPAC2 和 PAC1）中，PAC1 可进行选择性剪接，从而产生同工型。尽管文献中记录了中枢神经系统中存在 PACAP 受体，但它们在发育过程中的表达尚未确定。在这里，我们对大鼠视网膜出生后发育过程中 PACAP 受体的表达进行了定量分析。
从出生后第 0 天到第 20 天（P0-P20）收获视网膜。使用全面的引物系统，通过定量实时 PCR 跟踪表达变化。通过使用抗-VPAC1、-VPAC2 和-PAC1 受体抗体的免疫印迹检测蛋白质水平的变化。
VPAC1 的表达在 P10 和 P15 时增加。VPAC2 表达的峰值出现在 P5 和 P15。使用 PAC1 受体的剪接变体特异性引物，揭示了 Null、Hip、Hop1 和 Hiphop1 变体的剪接调节与出生后发育相关。Null 和 Hip 变体的转录水平下降，而 Hop1 在 P20 时成为主要的 PACAP 受体。除了在 P10 时短暂增加外，Hiphop1 转录水平没有明显变化。免疫印迹证实了受体的存在和表达水平变化。
我们得出结论，VPAC1 和 VPAC2 都可能在视网膜发育的所有阶段发挥作用，PACAP 通过特定的 PAC1 同工型发挥作用，Hip 和 Hop1 主要参与大鼠视网膜的出生后发育。
