Sodium Nitroprusside inhibits HEK293 Cell Growth by cGMP-Dependent and Independent Mechanisms

Abstract

The acute and chronic effects of sodium nitroprusside (SNP) are well characterized for vascular smooth muscle cells (VSMC). Stimulation of soluble guanylyl cyclase (sGC) gives a rapid elevation of intracellular cGMP levels and relaxation of VSMC. The antiproliferative effect of SNP needs days to develop. In the present study human embryonic kidney (HEK 293) cells were used to study the growth after repeated exposure to SNP. A dose-dependent antiproliferative effect was evident and after 5 days with an IC 50 value of 108 µM. Cyclic GMP was able to mimic the antiproliferative effect of SNP on HEK293 cells. When cGMP (1000 µM) was added to the cell culture medium for 5 days the cell densities were reduced with 37% below baseline and cGMP in increased from 5.3 to 195 pmol/10 7 cells. The interaction with the non-selective PDE (cyclic nucleotide phosphodiesterase) inhibitor 3-isobutyl-1-methylxanthine (IBMX) was tested after three days. IBMX alone (1000 µM) reduced cell densities with 48% and elevated cGMP in (from 5.2 to 9.3 pmol/10 7 cells). The effect of 10 µM SNP was reinforced on proliferation (from 13% to 90%) and elevation of cGMP levels (from 7.6 to 13.5 pmol/10 7 cells). A corresponding effect was observed after addition of 1000 µM cGMP and 1000 µM IBMX for 3 days. The antiproliferative effect of cGMP increased from 30% to 89% and the cGMP in increased from 240 to 480 pmol/10 7 cells. However, additional mechanisms exist for the antiproliferative effect of SNP. One of these is the intracellular oxidative effect which includes production of S-nitrosoglutathione. The fall in ratios between GSH and GSSG from 260 to 85 after 100 µM SNP exposure is compatible with such a mechanism since cGMP (1000 µM) added to the culture medium did not change the ratio. This study shows that the antiproliferative effects of SNP on HEK293 cells are mediated through cGMP-dependent and cGMP-independent mechanisms. The concentration-dependent effects develop over time. HEK293 cells had an efficient ef-

Smooth muscle: a stiff sculptor of epithelial shapes

“…Consistent with this conclusion, finite elementbased computational modelling has shown that differences in stiffness between the muscle layers and proliferating epithelium are sufficient to drive epithelial folding into the structures observed following smooth muscle differentiation [4,44,45]. This process appears to depend mostly on the static stiffness of the smooth muscle rather than its shortening because reducing gut motility with sodium nitroprusside [46] does not abrogate epithelial folding. Consistently, the computational model generates epithelial folds when the smooth muscle is treated as a stiff, static tissue.…”

The Effect of MRP5-Expression on Human Erythroleukemia (HEL) Cell Growth and cGMP Levels

“…On the other hand, a marked antiproliferative effect was obtained with an apparent IC 50 -value of 250 µM. The antiproliferative potency of cGMP was lower in HEK 293 cells since cGMP concentrations above 1000 µM were needed to reduce cell density 50% [11]. The most important observation in the present study was the reduced sensitivity to cGMP after MRP5 transfection.…”

“…Cyclic cGMP was added to the culture medium to elevate intracellular concentrations but the permeability is counteracted by the activity of MRP5 that is an effective efflux pump [8]. In agreement, low extracellular cGMP concentrations (1.0 -10 µM) did not give detectable increase of intracellular concentrations in HEK 293 cells [11] or hRBC [20]. However, these and other studies have shown that cGMP in millimolar concentrations may permeate the membrane from the ectoside.…”

“…The cGMP-analysis with Amerlex-M [ 125 I] kits was performed as recommended by the manufacturer (Amersham International plc). High extracellular cGMP concentrations were determined in a HPLC-assay with UV detection (254 nm) as previously described [11].…”

“…Cyclic AMP was not able to compete with the high affinity ATP-dependent cGMP transport [9] [10] but inhibited the low affinity transport [10]. Based on the observations in hRBC and the effects of elevating cGMP levels in HEK293 cells [11] we employed a human hemopoietic cell line; human erythroleukemia cells (HEL) [12] for the present study. Since butyrate increases cGMP efflux in some cells [13] the effect on HEL cell growth and elevation of cGMP levels was characterized.…”