Knauf C, Ferreira S, Hamdane M, Mailliot C, Prevot V, Beauvillain J C, Croix D
Institut National de la Santé et de la Recherche Médicale U422, Institut Fédératif de Recherches 22, Unité de Neuroendocrinologie et Physiopathologie Neuronale, 59045 Lille Cedex, France.
Endocrinology. 2001 Oct;142(10):4288-94. doi: 10.1210/endo.142.10.8443.
Recent studies from our laboratory suggested that the vascular endothelium of the median eminence was involved via nitric oxide secretion in the modulation of GnRH release during the estrous cycle. To further investigate that issue, we studied the variations of endothelial nitric oxide synthase protein and mRNA in the median eminence of female rats killed at different time points of the day and/or of the estrous cycle. Endothelial nitric oxide synthase protein levels were measured by Western blot, and endothelial nitric oxide synthase mRNA analysis was performed with semiquantitative RT-PCR (for each time point, n = 4). The results revealed that endothelial nitric oxide synthase synthesis varied markedly across the estrous cycle. Indeed, endothelial nitric oxide synthase protein (n = 20) and mRNA (n = 16) levels increase significantly on 0800 h and 1600 h proestrus compared with 1400 h diestrus II. In a second step, quantification analysis were made in median eminence obtained from ovariectomized and ovariectomized, E2 benzoate primed rat. The results show a significant increase in expression of endothelial nitric oxide synthase protein as well as endothelial nitric oxide synthase mRNA in ovx-E2 primed rat median eminence. Concurrently, the levels of the cav-1 protein, a specific endogenous inhibitor of endothelial nitric oxide synthase, were measured in median eminence during estrous cycle and in ME from ovx and ovx-E2 primed rats. A significant decrease of median eminence cav-1 was noted on 1600 h proestrus and in ovx-E2 primed rats when compared with 1400 h diestrus II and ovx, respectively. Altogether, these results strongly suggest that high NO release from median eminence observed on proestrus may be due to an increase of endothelial nitric oxide synthase expression and a decrease of the cav-1 protein levels. These findings demonstrate that E2 is able to modulate endothelial nitric oxide synthase and cav-1 expression both during the estrous cycle and in experimental conditions and consequently reinforce the idea that nitric oxide acting on GnRH release, is essentially endothelial in origin. These results may also imply that variations of endothelial nitric oxide synthase expression are essential for the pulsatile/cyclic nitric oxide median eminence release observed in a previous study.
我们实验室最近的研究表明，正中隆起的血管内皮通过一氧化氮分泌参与发情周期中促性腺激素释放激素（GnRH）释放的调节。为了进一步研究该问题，我们研究了在一天中的不同时间点和/或发情周期处死的雌性大鼠正中隆起中内皮型一氧化氮合酶蛋白和mRNA的变化。通过蛋白质免疫印迹法测量内皮型一氧化氮合酶蛋白水平，并用半定量逆转录聚合酶链反应（RT-PCR）进行内皮型一氧化氮合酶mRNA分析（每个时间点，n = 4）。结果显示，内皮型一氧化氮合酶的合成在发情周期中显著变化。实际上，与动情间期II的14:00相比，发情前期08:00和16:00时内皮型一氧化氮合酶蛋白（n = 20）和mRNA（n = 16）水平显著升高。在第二步中，对来自去卵巢以及去卵巢并用苯甲酸雌二醇预处理的大鼠的正中隆起进行定量分析。结果显示，在去卵巢并用雌二醇预处理的大鼠正中隆起中，内皮型一氧化氮合酶蛋白以及内皮型一氧化氮合酶mRNA的表达显著增加。同时，在发情周期期间以及来自去卵巢和去卵巢并用雌二醇预处理大鼠的正中隆起中测量内皮型一氧化氮合酶的特异性内源性抑制剂小窝蛋白-1（cav-1）的蛋白水平。与动情间期II的14:00以及去卵巢大鼠相比，发情前期16:00时和去卵巢并用雌二醇预处理的大鼠正中隆起中的cav-1显著降低。总之，这些结果强烈表明，发情前期在正中隆起观察到的高一氧化氮释放可能是由于内皮型一氧化氮合酶表达增加以及cav-1蛋白水平降低。这些发现表明，雌二醇在发情周期期间以及实验条件下均能够调节内皮型一氧化氮合酶和cav-1的表达，因此强化了这样一种观点，即作用于GnRH释放的一氧化氮本质上来源于内皮。这些结果还可能意味着，内皮型一氧化氮合酶表达的变化对于先前研究中观察到的正中隆起一氧化氮的脉冲式/周期性释放至关重要。
