Tables Supplemental Material.xlsx "Expression of NOS and ET-1 receptors in chronic hypoxia"

Sustained chronic hypoxia (CH)
produces morphological and functional changes in the carotid body (CB). Nitric
oxide (NO) and endothelin-1 (ET-1) plays a major role as modulators of the CB
oxygen chemosensory process. To characterize the effects of CH related to
normoxia (Nx) on gene expression, particularly on ET-1 and NO pathways, primary
cultures of rat CB cells were exposed to 7 days of CH. Total RNA was extracted,
cDNA-32P synthesized and hybridized with 1185 genes printed on a nylon
membrane Atlas cDNA Expression Array. Out of 324 differentially expressed genes,
184 genes were up-regulated, while 140 genes were down-regulated. The cluster
annotation and protein network analyses showed that both NO and ET-1 signalling
pathways were significantly enriched and key elements of each pathway were differentially
expressed. Thus, we assessed the effect of CH at the protein level of NOS
isoforms and ET-1 receptors. CH induced an increase in the expression of eNOS,
iNOS and ETB. During CH, the administration of SNAP, a NO-donor,
upregulated ETB. The treatment with Tezosentan (ET-1 receptor
blocker) during CH up-regulated all three NOS isoforms, while the NOS blocker L-NAME
induced up-regulation of iNOS and ETB and down-regulated the protein levels of ETA.
These results showed that CH for 7 days changed the cultured cells CB gene expression profile,
the NO and ET-1 signaling pathways
were highly enriched and these two signaling pathways interfered on the protein
expression of each other.

持续性慢性低氧（Sustained chronic hypoxia, CH）可诱导颈动脉体（carotid body, CB）产生形态学与功能学改变。一氧化氮（nitric oxide, NO）与内皮素-1（endothelin-1, ET-1）作为颈动脉体氧化学感受过程的核心调节因子，发挥关键调控作用。
为明确相较于常氧（normoxia, Nx）的慢性低氧对基因表达谱的影响，尤其是对ET-1与NO信号通路的调控效应，本研究将大鼠颈动脉体细胞原代培养物暴露于持续性慢性低氧环境中7天。
提取总RNA后，合成32P标记的互补脱氧核糖核酸（cDNA）探针，并与固定于尼龙膜上的Atlas cDNA表达阵列（Atlas cDNA Expression Array）中的1185个基因进行杂交。
在筛选获得的324个差异表达基因中，184个基因呈现上调表达，140个基因呈现下调表达。
聚类注释与蛋白质互作网络分析结果显示，NO与ET-1信号通路均显著富集，且两条通路中的关键元件均存在差异表达。
为此，本研究进一步在蛋白层面评估了慢性低氧对一氧化氮合酶（NOS）亚型及ET-1受体表达的影响。结果表明，慢性低氧可上调内皮型一氧化氮合酶（eNOS）、诱导型一氧化氮合酶（iNOS）及内皮素B受体（ETB）的表达水平。
在慢性低氧处理期间，给予一氧化氮供体SNAP可进一步上调ETB的表达。而在慢性低氧过程中使用特唑桑坦（Tezosentan，ET-1受体阻滞剂）进行干预，可上调全部3种NOS亚型的表达；与此同时，一氧化氮合酶抑制剂L-NAME可上调iNOS与ETB的蛋白水平，并下调内皮素A受体（ETA）的蛋白表达。
本研究结果显示，7天持续性慢性低氧可改变大鼠颈动脉体培养细胞的基因表达谱，NO与ET-1信号通路呈现高度富集，且两条信号通路可相互调控对方的蛋白表达。