Borba Juliana Carrazzoni, Henze Isabela Pereira, Silveira Mariana Souza, Kubrusly Regina Célia Cussa, Gardino Patrícia Franca, de Mello Maria Christina Fialho, Hokoç Jan Nora, de Mello Fernando Garcia
Laboratórios de Neuroquímica and Neurobiologia da Retina, Instituto de Biofísica Carlos Chagas Filho-UFRJ, Centro de Ciências da Saúde-Bloco G, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, RJ 21949-900, Brazil.
Brain Res Dev Brain Res. 2005 May 12;156(2):193-201. doi: 10.1016/j.devbrainres.2005.02.016.
In the chick retina, dopaminergic cells are generated between embryonic days 3 and 7 (E3/E7). However, the expression of tyrosine hydroxylase (TH), the first enzyme in the catecholamine synthetic pathway, is only detected after E11/E12. During the interval comprising E7 to E12, signals conveyed by cAMP are important to determine the TH phenotype. The present study shows that pituitary adenylyl cyclase-activating polypeptide (PACAP), via cAMP, is a major endogenous component in defining the TH phenotype of retina dopaminergic cells during development. PACAP type 1 receptor and its mRNA were detected in retinas since E6. PACAP was also immunodetected in cells localized in the inner nuclear layer of retinas since E8. This peptide promoted greater than 10-fold increase in cAMP accumulation of retinas obtained from embryos since E8, an effect that was blocked by PACAP6-38 (PAC1 receptor antagonist). In cultured retina cells from E8 and E9, maintained for 6 days in vitro with 10 nM PACAP (for 5 days), the number of dopaminergic cells expressing tyrosine hydroxylase increased 2.4-fold. The cAMP analog, 8-Br-cAMP and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) also increased the number of tyrosine hydroxylase-positive cells by 4- to 6-fold. IBMX plus PACAP treatment resulted in 17-fold increase in the number of cells positive for tyrosine hydroxylase. Under this condition the amount of tyrosine hydroxylase expression, as detected by western blot analysis, was also increased. The protein kinase-A inhibitor, rp-cAMPS, significantly reduced the effect of PACAP. Our data show that this peptide is an important factor influencing the definition of the tyrosine hydroxylase phenotype of retina dopaminergic cells within a narrow window of development.
在鸡胚视网膜中，多巴胺能细胞在胚胎第3天至第7天（E3/E7）生成。然而，儿茶酚胺合成途径中的首个酶——酪氨酸羟化酶（TH），直到E11/E12之后才被检测到。在包含E7至E12的这段间隔期，由cAMP传递的信号对于确定TH表型很重要。本研究表明，垂体腺苷酸环化酶激活多肽（PACAP）通过cAMP，是发育过程中定义视网膜多巴胺能细胞TH表型的主要内源性成分。自E6起在视网膜中检测到PACAP 1型受体及其mRNA。自E8起在视网膜内核层定位的细胞中也检测到PACAP免疫反应信号。这种多肽使自E8起胚胎视网膜的cAMP积累增加了10倍以上，该效应被PACAP6 - 38（PAC1受体拮抗剂）阻断。在来自E8和E9的培养视网膜细胞中，用10 nM PACAP体外培养6天（前5天），表达酪氨酸羟化酶的多巴胺能细胞数量增加了2.4倍。cAMP类似物8 - Br - cAMP和3 - 异丁基 - 1 - 甲基黄嘌呤（IBMX，一种磷酸二酯酶抑制剂）也使酪氨酸羟化酶阳性细胞数量增加了4至6倍。IBMX加PACAP处理使酪氨酸羟化酶阳性细胞数量增加了17倍。在此条件下，通过蛋白质印迹分析检测到的酪氨酸羟化酶表达量也增加了。蛋白激酶A抑制剂rp - cAMPS显著降低了PACAP的作用。我们的数据表明，这种多肽是在发育的狭窄窗口期影响视网膜多巴胺能细胞酪氨酸羟化酶表型定义的重要因素。
