Chronic hypoxia (CH) produces changes not fully understood in morphology and function of the carotid body (CB). To characterize the effect of CH, primary 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. Out of 324 differentially expressed genes, 184 were up-regulated and 140 were down-regulated. Since data analyses showed that nitric oxide synthases (NOS) and endothelin-1 (ET-1) pathways were enriched, we studied the effect of CH at protein levels of NOS isoforms and ET-1 receptors. CH induced an increase in all NOS and in ET-1 receptor B (ETB). Combining CH and SNAP, iNOS and ETB were significantly up-regulated, whereas the ET-1 receptor A (ETA) was down-regulated, while Tezosentan up-regulated iNOS and ETA and L-NAME induced up-regulation of all NOS. These results described the changes of CH on the CB gene expression profile, affecting a possible interaction in between NOS and ET-1 receptors, as part of the adaptive CB response to CH.
Overall design: Total RNA from CB cultured primary rat cells was isolated by adding 1.0 ml of Trizol (Invitrogen) following manufacturer's instructions. The RNA with an absorbance ratio A260/A280 > 1.6 and a total RNA concentration above 0.3 microgram/ml were used and 1.0 microgram of RNA were amplified using RiboAmp (Arcturus) following the manufacturer's protocol. The amplified RNA with an absorbance relation over A260/A280 > 1.9 and concentration over 1.0 microgram/ml were used to synthesize the cDNA probes. Radiolabeled cDNA probes were synthesized from 6 μg of RNA amplified in the presence of dATP, dTTP, dGTP, [α-32P] dCTP (3000 Ci/mmol), poly T, Superscript II Reverse transcriptase (Invitrogen) and the purified (Atlas NucleoSpin Extraction Spin Columns, BD Biosciences Clontech). Atlas Rat 1.2 Array nylon membranes with 1185 genes (Atlas cDNA Expression Arrays Cat Nº 7854-1, BD Biosciences Clontech) were used according to manufacturer’s procedure. Briefly, the membrane was prehybridized in ExpressHyb hybridization buffer, containing the sheared salmon testes DNA for 30 min at 68 ºC in a rotating hybridization oven. The heat-denatured probe (95 ºC for 5 min) was hybridized overnight at 68 ºC with continuous agitation followed by three washing steps [twice at room temperature for 5 min with Sodium Chloride/ Sodium Citrate solution (1 X SSC = 150 mM NaCl/15 mM sodium citrate, pH 7.0), twice with 2 X SSC plus 1% SDS at 68 ºC for 30 min, and twice with 0.1 X SSC plus 0.5% SDS at room temperature for 30 min]. The membranes were exposed on phosphoimager up to 3 hours at room temperature. Image acquisition and quantification of the average of three membranes under Nx and three under CH were performed using the software AtlasImage V2.0 (Clontech). The normalization was performed to each experiment using a coefficient of normalization (adding the averaging of the signal of all genes to the signal values for all the genes that are above the background signal). The parameters used for the data analysis were the ratio threshold (TR) and the difference threshold (TD).
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