Lee D, Huang W, Yang Z, Copolov D L, Lim A T
Neuroendocrine Laboratory, Royal Park Hospital, Parkville, Victoria, Australia.
Endocrinology. 1992 Aug;131(2):911-8. doi: 10.1210/endo.131.2.1379168.
In the hypothalamus of the rat, the precursor of atrial natriuretic peptide (ANP) is produced and processed into its smaller congeners of 3K mol wt species, which are secreted from neurons with cell bodies in the periventricular areas and the paraventricular nuclei of the tissue. Employing long term monolayer cultures of neonatal rat hypothalamic cells, we have identified a small population of cells that stained positive for immunoreactive (ir) ANP. Seventy-two +/- 7% (mean +/- SE; n = 4 per 1000 cells) of the irANP positive cells were colocalized with the staining of neuron-specific enolase; some of the cells possessed multiple neurites and showed irANP staining in the perikarya, in the varicosities along neuronal processes, and at the terminals of long neurites. Over the range of 10(-6)-10(-4) M, forskolin, 3-isobutyl-1-methylxanthine, or 8-bromo-cAMP significantly augmented the total number of irANP-positive cells and those possessing neurites in a dose-related and time-dependent manner. At 10(-4) M, 4 days of forskolin treatment increased the number of irANP-positive neurons 4-fold (P less than 0.01) while tripling that of the cells with long neurites (P less than 0.01). Furthermore, it approximately tripled the number of cells (P less than 0.01) showing positive signals for pro-ANP mRNA, as ascertained by colorimetric in situ hybridization using a 30-basepair antisense oligonucleotide probe labeled with digoxigenin. Consistent with the above observation, forskolin, 3-isobutyl-1-methylxanthine, or 8-bromo-cAMP treatment significantly augmented the total amount of irANP present in the cultures, with an ED50 of forskolin approximating 5 x 10(-5) M. Although treatment with 10(-7) M phorbol 12-myristate 13-acetate approximately doubled the production of irANP in the cultures (P less than 0.05), phorbol 12-myristate 13-acetate had little effect on modulating the number or neurite outgrowth of irANP neurons. Thus, our present findings suggest that protein kinase-A pathways are of greater importance than protein kinase-C pathways in regulating both the functional and morphological development of ANP-producing neurons during the ontogenesis of the rat hypothalamus.
在大鼠下丘脑，心房钠尿肽（ANP）的前体被产生并加工成其3K分子量的较小同系物，这些同系物从位于组织室周区和室旁核的细胞体的神经元分泌。利用新生大鼠下丘脑细胞的长期单层培养，我们鉴定出一小群对免疫反应性（ir）ANP染色呈阳性的细胞。72±7%（平均值±标准误；每1000个细胞中n = 4）的irANP阳性细胞与神经元特异性烯醇化酶的染色共定位；一些细胞具有多个神经突，并在胞体、沿神经突的曲张部位以及长神经突的末端显示irANP染色。在10⁻⁶ - 10⁻⁴ M范围内，福斯可林、3 - 异丁基 - 1 - 甲基黄嘌呤或8 - 溴 - cAMP以剂量相关和时间依赖的方式显著增加irANP阳性细胞和具有神经突的细胞的总数。在10⁻⁴ M时，4天的福斯可林处理使irANP阳性神经元的数量增加了4倍（P < 0.01），而使具有长神经突的细胞数量增加了两倍（P < 0.01）。此外，通过使用地高辛标记的30个碱基对的反义寡核苷酸探针进行比色原位杂交确定，它使显示前ANP mRNA阳性信号的细胞数量增加了约两倍（P < 0.01）。与上述观察结果一致，福斯可林、3 - 异丁基 - 1 - 甲基黄嘌呤或8 - 溴 - cAMP处理显著增加了培养物中存在的irANP的总量，福斯可林的ED50约为5×10⁻⁵ M。虽然用10⁻⁷ M佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯处理使培养物中irANP的产生量增加了约一倍（P < 0.05），但佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯对调节irANP神经元的数量或神经突生长几乎没有影响。因此，我们目前的研究结果表明，在大鼠下丘脑的个体发生过程中，蛋白激酶 - A途径在调节产生ANP的神经元的功能和形态发育方面比蛋白激酶 - C途径更重要。
