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


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.

Share and Cite:

Sager, G. , Sundkvist, E. , Jaeger, R. , Lysaa, R. and Fuskevaag, O. (2014) Sodium Nitroprusside inhibits HEK293 Cell Growth by cGMP-Dependent and Independent Mechanisms. Pharmacology & Pharmacy, 5, 262-271. doi: 10.4236/pp.2014.53033.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Thippeswamy, T., McKay, J.S., Quinn, J.P. and Morris, R. (2006) Nitric Oxide, a Biological Double-Faced Janus—Is This Good or Bad? Histology and Histopathology, 21, 445-458.
[2] Garg, U.C. and Hassid, A. (1989) Nitric Oxide-Generating Vasodilators and 8-Bromo-cyclic Guanosine Monophosphate Inhibit Mitogenesis and Proliferation of Cultured Rat Vascular Smooth Muscle Cells. Journal of Clinical Investigation, 83, 1774-1777.
[3] Kariya, K., Kawahara, Y., Araki, S., Fukuzaki, H. and Takai, Y. (1989) Antiproliferative Action of Cyclic GMP-Elevating Vasodilators in Cultured Rabbit Aortic Smooth Muscle Cells. Atherosclerosis, 80, 143-147.
[4] Pollman, M.J., Yamada, T., Horiuchi, M. and Gibbons, G.H. (1996) Vasoactive Substances Regulate Vascular Smooth Muscle Cell Apoptosis. Countervailing Influences of Nitric Oxide and Angiotensin II. Circulation Research, 79, 748-756.
[5] Guh, J.H., Hwang, T.L., Ko, F.N., Chueh, S.C., Lai, M.K. and Teng, C.M. (1998) Antiproliferative Effect in Human Prostatic Smooth Muscle Cells by Nitric Oxide Donor. Molecular Pharmacology, 53, 467-474.
[6] Wharton, J., Strange, J.W., Moller, G.M., Growcott, E.J., Ren, X., Franklyn, A.P., Phillips, S.C. and Wilkins, M.R. (2005) Antiproliferative Effects of Phosphodiesterase Type 5 Inhibition in Human Pulmonary Artery Cells. American Journal of Respiratory and Critical Care Medicine, 172, 105-113.
[7] Tantini, B., Manes, A., Fiumana, E., Pignatti, C., Guarnieri, C., Zannoli, R., Branzi, A. and Galie, N. (2005) Antiproliferative Effect of Sildenafil on Human Pulmonary Artery Smooth Muscle Cells. Basic Research in Cardiology, 100, 131-138.
[8] Pilz, R.B. and Broderick, K.E. (2005) Role of Cyclic GMP in Gene Regulation. Frontiers in Bioscience, 10, 1239-1268.
[9] Chen, Z.S., Lee, K. and Kruh, G.D. (2001) Transport of Cyclic Nucleotides and Estradiol 17-Beta-d-glucuronide by Multidrug Resistance Protein 4. Resistance to 6-Mercaptopurine and 6-Thioguanine. The Journal of Biological Chemistry, 276, 3747-33754.
[10] Jedlitschky, G., Burchell, B. and Keppler, D. (2000) The Multidrug Resistance Protein 5 Functions as an ATP-Dependent Export Pump for Cyclic Nucleotides. The Journal of Biological Chemistry, 275, 30069-30074.
[11] Wielinga, P.R., van der Heijden, I., Reid, G., Beijnen, J.H., Wijnholds, J. and Borst, P. (2003) Characterization of the MRP4-and MRP5-Mediated Transport of Cyclic Nucleotides from Intact Cells. The Journal of Biological Chemistry, 278, 17664-17671.
[12] van Aubel, R.A., Smeets, P.H., Van Den Heuvel, J.J. and Russel, F.G. (2005) Human Organic Anion Transporter MRP4 (ABCC4) Is an Efflux Pump for the Purine End Metabolite Urate with Multiple Allosteric Substrate Binding Sites. American Journal of Physiology. Renal Physiology, 288, F327-F333.
[13] Akerboom, T.P. and Sies, H. (1994) Transport of Glutathione Disulfide and Glutathione S-Conjugates in Hepatocyte Plasma Membrane Vesicles. Methods in Enzymology, 233, 416-425.
[14] New, L.S. and Chan, E.C. (2008) Evaluation of BEH C18, BEH HILIC, and HSS T3 (C18) Column Chemistries for the UPLC-MS-MS Analysis of Glutathione, Glutathione Disulfide, and Ophthalmic Acid in Mouse Liver and Human Plasma. Journal of Chromatographic Science, 46, 209-214.
[15] Stoscheck, C.M. (1990) Quantitation of Protein. Methods in Enzymology, 182, 50-68.
[16] Keppler, D., Jedlitschky, G. and Leier, I. (1998) Transport Function and Substrate Specificity of Multidrug Resistance Protein. Methods in Enzymology, 292, 607-616.
[17] Bradford, M.M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 72, 248-254.
[18] Chou, T.C. (1976) Derivation and Properties of Michaelis-Menten Type and Hill Type Equations for Reference Ligands. Journal of Theoretical Biology, 39, 253-276.
[19] Schroder, H., Leitman, D.C., Bennett, B.M., Waldman, S.A. and Murad, F. (1988) Glyceryltrinitrate-Induced Desensitization of Guanylate Cyclase in Cultured Rat Lung Fibroblasts. Journal of Pharmacology and Experimetal Therapeutics, 245, 413-418.
[20] Brune, B., von Knethen, A. and Sandau, K.B. (1998) Nitric Oxide and Its Role in Apoptosis. European Journal of Pharmacology, 351, 261-272.
[21] Sager, G. (2004) Cyclic GMP Transporters. Neurochemistry International, 45, 865-873.
[22] Radziszewski, W., Chopra, M., Zembowicz, A., Gryglewski, R., Ignarro, L.J. and Chaudhuri, G. (1995) Nitric Oxide Donors Induce Extrusion of Cyclic GMP from Isolated Human Blood Platelets by a Mechanism Which May Be Modulated by Prostaglandins. International Journal of Cardiology, 51, 211-220.
[23] Francis, S.H., Busch, J.L., Corbin, J.D. and Sibley, D. (2010) cGMP-Dependent Protein Kinases and cGMP Phosphodiesterases in Nitric Oxide and cGMP Action. Pharmacology Reviews, 62, 525-563.
[24] Flo, K., Hansen, M., Orbo, A., Kjørstad, K.E., Maltau, J.M. and Sager, G. (1995) Effect of Probenecid, Verapamil and Progesterone on the Concentration-Dependent and Temperature-Sensitive Human Erythrocyte Uptake and Export of Guanosine 3',5' Cyclic Monophosphate (cGMP). Scandinavian Journal of Clinical and Laboratory Investigation, 55, 715-721.
[25] Francis, S.H., Sekhar, K.R., Ke, H. and Corbin, J.D. (2011) Inhibition of Cyclic Nucleotide Phosphodiesterases by Methylxanthines and Related Compounds. Handbook of Experimental Pharmacology, 200, 93-133.
[26] Schultz, C., Vaskinn, S., Kildalsen, H. and Sager, G. (1998) Cyclic AMP Stimulates the Cyclic GMP Egression Pump in Human Erythrocytes: Effects of Probenecid, Verapamil, Progesterone, Theophylline, IBMX, Forskolin, and Cyclic AMP on Cyclic GMP Uptake and Association to Inside-Out Vesicles. Biochemistry, 37, 1161-1166.
[27] Sundkvist, E., Jaeger, R. and Sager, G. (2002) Pharmacological Characterization of the ATP-Dependent Low km Guanosine 3’,5’-cyclic Monophosphate (cGMP) Transporter in Human Erythrocytes. Biochemical Pharmacology, 63, 945-949.
[28] Ishida, A., Sasaguri, T., Miwa, Y., Kosaka, C., Taba, Y. and Abumiya, T. (1999) Tumor Suppressor p53 But Not cGMP Mediates NO-Induced Expression of p21(Waf1/Cip1/Sdi1) in Vascular Smooth Muscle Cells. Molecular Pharmacology, 56, 938-946.
[29] Tanner, F.C., Meier, P., Greutert, H., Champion, C., Nabel, E.G. and Luscher, T.F. (2000) Nitric Oxide Modulates Expression of Cell Cycle Regulatory Proteins: A Cytostatic Strategy for Inhibition of Human Vascular Smooth Muscle Cell Proliferation. Circulation, 101, 1982-1989.
[30] Tarr, J.M., Eggleton, P. and Winyard, P.G. (2006) Nitric Oxide and the Regulation of Apoptosis in Tumour Cells. Current Pharmaceutical Design, 12, 4445-4468.
[31] Borutaite, V. and Brown, G. (2005) What Else Has to Happen for Nitric Oxide to Induce Cell Death? Biochemical Society Transactions, 33, 1394-1396.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.