Figure 5

The half-life of sGC activity was diminished in DETA/NO treated cells. (A) Chromaffin cells were incubated for different times (0, 4, 8,16, 24,32 and 48 hours) with either 10 μmol/L cycloheximide (■), 50 μmol/L DETA/NO (●) or with both drugs (▲). Cells were then washed preincubated with 0.5 mmol/L IBMX for 30 min and stimulated with 1 μmol/L DEA/NO for 10 min and cGMP levels determined by radioimmunoassay. Data of DEA/NO-stimulated cGMP levels as a time function are represented in a linear model according to the equation logA = k.t/2.3 + logA ₀ , where A is the DEA/NO-stimulated cGMP level at each time and A₀ is the DEA/NO-stimulated cGMP level in control cells. From the slopes, the "k " and half-life (t_1/2) of sGC activity in each condition can be calculated according to the expression t_1/2 = 2.3 log2/k. (B) Immunoblotting analysis of sGC subunits (α₁ and β₁) or α-actin. Cells were pretreated with either: vehicle (control), 10 μmol/L cycloheximide (CHX), 50 μmol/L DETA/NO, or both compounds together. Proteins (30 μg) from each group were subjected to electrophoresis and transferred to PVDF membranes. The membranes were processed as described in Fig. 3. (C) Cell viability after different treatment. Cells were incubated for 48 hours with either: vehicle (control), 50 μmol/L DETA/NO, 10 μmol/L cycloheximide or 50 μmol/L DETA/NO plus10 μmol/L cycloheximide. After that cells were washed and their viability analyzed as described in Methods. The values are given as the percentage of live cells and are the mean ± SE of three experiments performed in triplicate.