Figure 7
DETA/NO treatment causes down regulation of mRNAs coding for the α1 and β1 subunits of sGC. PKG inhibitors prevent this effect. (A) Gel showing the analysis of RT-PCR products for the α1- and β1-subunits of sGC and for 18S ribosomal RNA (used as internal control) by agarose gel electrophoresis stained with SYBR gold. Total RNA (200 ng) was reverse transcribed and amplified for 40 cycles. A marker (M) consisting of DNA fragments in increments of 100 bp (lower band, 100 bp) was used to estimate the size of the PCR products. (B and C) Variation in mRNA content for sGC α1 and β1 mRNAs in cells subjected to different treatments. Chromaffin cells were incubated with a vehicle (control), or 50 μmol/L DETA/NO alone or in the presence of the PKG inhibitor Rp-8-BrPET-cGMPS at the concentrations 3 μmol/L, 15 μmol/L or 30 μmol/L or 1 μmol/L KT5823, or in the presence of a PKA inhibitor (1 μmol/L H89). The protein kinase inhibitors were added 30 min before the DETA/NO. After 16 hours treatment, total RNA was isolated and used for quantitative RT-PCR using specific primers designed for mRNAs encoding bovine sGC α1 and β1 subunits. The figure shows the results of four experiments performed in triplicate given as mean ± SE. Significant differences from control are indicated by **p < 0.01; *p < 0.05 (Student's t test). ++p < 0.01;+p < 0.05 (Student's t test) indicates significant differences between cells treated with DETA/NO alone or with DETA/NO plus PKG inhibitors.