Chronic hypoxia changes gene expression profile of primary rat carotid body cells: consequences on the expression of NOS isoforms and ET-1 receptors

Abstract

Sustained chronic hypoxia (CH) produces morphological and functional changes in the carotid body (CB). Nitric oxide (NO) and endothelin-1 (ET-1) play 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, and cDNA-32P was synthesized and hybridized with 1,185 genes printed on a nylon membrane Atlas cDNA Expression Array. Out of 324 differentially expressed genes, 184 genes were upregulated, while 140 genes were downregulated. The cluster annotation and protein network analyses showed that both NO and ET-1 signaling 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 nitric oxide synthase (NOS) isoforms and ET-1 receptors. CH induced an increase in the expression of endothelial NOS, inducible NOS, and ETB. During CH, the administration of SNAP, a NO donor, upregulated ETB. Treatment with Tezosentan (ET-1 receptor blocker) during CH upregulated all three NOS isoforms, while the NOS blocker L-NAME induced upregulation of iNOS and ETB and downregulated the protein levels of ETA. These results show that CH for 7 days changed the cultured cell CB gene expression profile, the NO and ET-1 signaling pathways were highly enriched, and these two signaling pathways interfered with the protein expression of each other.

Growth Factors in the Carotid Body—An Update

“…The mRNA expression of ET-1 and its receptors, ET A -R and ET B -R, into rat CB was demonstrated by both RT-PCR and qPCR [ 103 , 104 , 105 ]. Moreover, specific genes belonging to the ET-1 signaling pathway were detected into primary cultures of rat CB cells by cDNA Expression Array [ 106 ]. ELISA and enzyme immunoassay (EIA) allowed to determine ET-1 concentrations into rabbit CB lysates, as well as peptide content and release from rat CB [ 75 , 103 , 105 ].…”

“…Moreover, ET B receptor was identified in the cat CB by Western blot and into the cytoplasm of type I cells by immunohistochemistry [ 101 , 102 ]. More recently, the expression of ET A and ET B receptors into rat CB homogenates and CB cultured cells was detected by Western blot and immunoblotting analysis, respectively [ 106 , 108 ] ( Table 12 ).…”

“…Hypoxia was proved to strongly increase the expression of ET-1, which plays a pivotal role in cellular adaptation to hypoxic conditions. ET-1 signaling pathway appeared to be differentially expressed in rat CB cultured cells under chronic hypoxia [ 106 ]. Changes on ET-1 level in the CB were explored after different protocols of intermittent IH/ROX exposure in rabbits.…”

“…Following CH, ET A -R was not only overexpressed into type I cells but also detected in resident and invasive CD45 + immune cells in the tissue surrounding chemosensory cell lobules [ 67 ]. On the other hand, CH also increased ET B -R protein expression into cultured CB cells [ 106 ]. According to immunofluorescence and quantitative PCR studies, concurrent treatment with the ET A/B receptors antagonist (bosentan) blocked CH-induced macrophage invasion and the upregulation of pro-inflammatory cytokines and monocyte chemoattractant protein-1 (MCP-1).…”

G-Protein-Coupled Receptor (GPCR) Signaling in the Carotid Body: Roles in Hypoxia and Cardiovascular and Respiratory Disease

“…The main function of ET signaling appears to be in mediating CB adaptation to chronic hypoxia and CIH in adults [ 126 , 127 ] and neonates [ 128 ]. In adult rats, chronic hypoxia upregulates ET A - and ET B -receptor expression in the type I cell and ET A -selective antagonists have a strong impact on chemoafferent discharge, producing ca 11% inhibition before chronic hypoxia, increasing to ca 50% inhibition after 16 days of chronic hypoxia [ 129 , 130 ]. In chronic hypoxia, increased endothelin signaling seems to overlap with enhanced generation of nitric oxide (NO), a substance capable of both increasing and decreasing CB chemosensitivity dependent on the specific microdomain in which it is produced [ 129 , 131 ].…”