Proceedings of The Physiological Society
Kings College London (2005) J Physiol 565P, PC167
Hypoxia induces changes in gene expression pattern in carotid body cells and sensory neurons
Mosqueira, Matias ; Velasquez, Luis ; Iturriaga, Rodrigo ;
1. P Universidad Catolica de Chile, Santiago, Chile. 2. Biologia, Universidad de Santiago, Santiago, Chile.
The carotid body (CB) is composed of chemoreceptor cells that are innervated by sensory neurons. Exposure to chronic hypoxia enlarges the CB and increases its chemosensory response to acute hypoxia. Considering the time required by hypoxia to enhance CB chemoreception, changes in the pattern of gene expression in CB cells and/or sensory neurons are to be expected. Therefore, we studied the gene expression profile induced by chronic hypoxia in CB cells and sensory neurons. Cells from the CB and the petrosal-jugular-nodose (PJN) complex ganglion were obtained from 64 Sprague Dawley rats (100 g) anesthetized with sodium pentobarbitone (40 mg/kg i.p.). Each experiment was repeated 3 times. 8 CB and 8 PJN were cultured in normoxic (95% air - 5% CO2) and hypoxic conditions (95% N2 - 5% CO2) at 37°C for 1 week. The total RNA was extracted and amplified using a RiboAmp Kit (Arcturus). The amplified mRNA was used as a template to synthesized cDNA-32P, which was hybridized on macroarray membranes containing 1176 genes (Clontech), and revealed in a phosphoimager. The images obtained were analyzed with the Atlas-Image Software (Clontech). We considered a threshold ratio (RT) (hypoxia/normoxia) of 2.0 and a threshold difference (DT) of 15000 pixels of intensity to be a significant change in gene expression. The experimental protocol was approved by the Ethical Committee of the Universidad Catolica de Chile.The CB cells expressed 203 genes (17.3%) in normoxia, 252 genes (21.4%) under hypoxia, and 132 genes in both normoxic and hypoxic conditions (11.2%). In the PJN cells, 104 genes (8.8%) were expressed in normoxia, 149 genes (12.7%) in hypoxia, and 81 genes in both conditions (6.9%). After one week of hypoxia, 20 genes increased their RT or DT expression, and 11 genes decreased their expression in CB cultured cells, whereas 5 genes increased and 12 genes decreased their RT or their DT expression in the PJN cultured cells. For CB cultured cells, only 4 genes showed RT and DT above the threshold (c-jun prontooncogen, G1/S cyclin c, intrinsic factor precursor, rab GDI-β species), while 6 genes showed RT and DT below the threshold (medium chain acyl CoA dehydrogenase, cytocrome oxidase, liver arginase, puctative v-fos transformation effector protein, proteasome subunit RC7-1, gastric inhibitory polypeptide). In the PJN cultured cells, only 2 genes showed RT and DT above the threshold (G2/M cyclin specific G, serine proteinase-rPC7 precursor), while 4 genes showed RT and DT below the threshold (dopamine transporter, alcohol dehydrogenase A subunit, putative v-fos transformation effector protein). Our results suggest that CB cells and PJN neurons change their gene expression during one week of exposure to hypoxia.
Where applicable, experiments conform with Society ethical requirements