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 IC50 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 cGMPin increased from 5.3 to 195 pmol/107cells. 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 cGMPin (from 5.2 to 9.3 pmol/107cells). 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/107cells). 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 cGMPin increased from 240 to 480 pmol/107cells. 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 efflux system for cGMP and the use of inside-out vesicles (IOVs) showed high affinity ATP-dependent cGMP transport with a Km value of 2.3 μM. The antiproliferative effect of SNP was correlated to cGMPex/in.
Highlights
The primary nitric oxide receptor is soluble guanylate cyclase with stimulation of cGMP synthesis
The highest concentration was somewhat higher than the baseline value. This is compatible with the observation that the peak concentration of cGMPin occurs few minutes after sodium nitroprusside (SNP) addition to rat lung fibroblasts and with declining levels afterwards [19]
The present study showed that SNP was a potent anti-proliferative agent for HEK2933 cells
Summary
The primary nitric oxide receptor is soluble guanylate cyclase (sGC) with stimulation of cGMP synthesis. Nitric oxide donors like sodium nitroprusside (SNP) modulate various key-enzymes and S-nitrosylate intracellular proteins with direct effects on gene expression [1]. Several in vitro studies have shown that SNP and similar sGC-stimulating agents inhibit growth of VSMC in a dose dependent manner [2]-[5]. Several studies have focused on the antiproliferative effects due to stimulated cGMP synthesis and inhibition of cGMP degradation. In this study we characterized cell growth under the influence of cGMP elevating agents with relation to cGMPin, the transmembrane cGMP concentration ratio (cGMPex/in), and intracellular reduced (GSH) and oxidized glutathione (GSSG) concentrations
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