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 mecha nisms. The concentration-dependent effects develop over time. HEK293 cells had an efficient ef flux system for cGMP and the use of inside-out vesicles (IOVs) showed high affinity ATP-dependent cGMP transport with a K m value of 2.3 μM. The antiproliferative effect of SNP was correlated to cGMP ex/in .