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. 1996 Jan;117(1):147–155. doi: 10.1111/j.1476-5381.1996.tb15167.x

Mechanisms of tolerance to sodium nitroprusside in rat cultured aortic smooth muscle cells.

A Papapetropoulos 1, C Y Go 1, F Murad 1, J D Catravas 1
PMCID: PMC1909386  PMID: 8825356

Abstract

1. While exposure of smooth muscle cells to sodium nitroprusside (SNP) leads to the development of tolerance to soluble guanylate cyclase (sGC) activation, the mechanisms responsible for this phenomenon in intact cells remain unclear. In the present study, possible mechanisms of tolerance were investigated in a cell culture model where sGC activity was estimated from the accumulation of cyclic GMP in response to 10 microM SNP over a 15 min period in the presence of a phosphodiesterase (PDE) inhibitor. 2. Pretreatment of rat aortic smooth muscle cells with 10-500 microM SNP led to a dose-dependent downregulation of cyclic GMP accumulation upon subsequent SNP stimulation. This effect was evident as early as 2 h following incubation with 10 microM SNP, reached a plateau at 4 h and was blocked by co-incubation with 30 microM oxyhaemoglobin. 3. Pretreatment of smooth muscle cells with the PDE inhibitor, zaprinast, resulted in downregulation of the SNP-induced cyclic GMP accumulation in a time- and concentration-dependent manner, that was first evident after 12 h. Moreover, while the zaprinast-induced downregulation of cyclic GMP accumulation was completely inhibited by the protein kinase A (PKA) inhibitor, H89, tolerance to SNP was partially reversed by H89. 4. beta 1 sGC steady state mRNA levels of S-nitroso N-acetylpenicillamine (SNAP)- or 8Br-cyclic GMP-pretreated cells were unchanged, as indicated by Northern blot analysis. However, Western blot analysis revealed that alpha 1 protein levels were decreased in zaprinast, but not in SNP, SNAP or 8Br-cyclic GMP pretreated cells. 5. While thiol depletion did not prevent the development of tolerance, pretreatment of cells with SNP in the presence of reducing agents partially or completely restored the ability of cells to respond to SNP. 6. We conclude that tolerance to SNP results from two distinct mechanisms: an early onset, NO-mediated event that is reversed by reducing agents and a more delayed, PKA-sensitive process that is mediated through increases in cyclic GMP and a decrease in sGC protein levels.

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Selected References

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  1. Axelsson K. L., Ahlner J., Ljusegren M. E. Tolerance to organic nitroesters in experimental animals. An assessment of in vitro and in vivo investigations. Acta Pharmacol Toxicol (Copenh) 1986;59 (Suppl 6):121–128. doi: 10.1111/j.1600-0773.1986.tb02557.x. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Brandwein H. J., Lewicki J. A., Murad F. Reversible inactivation of guanylate cyclase by mixed disulfide formation. J Biol Chem. 1981 Mar 25;256(6):2958–2962. [PubMed] [Google Scholar]
  4. Braughler J. M. Soluble guanylate cyclase activation by nitric oxide and its reversal. Involvement of sulfhydryl group oxidation and reduction. Biochem Pharmacol. 1983 Mar 1;32(5):811–818. doi: 10.1016/0006-2952(83)90581-6. [DOI] [PubMed] [Google Scholar]
  5. Brooker G., Terasaki W. L., Price M. G. Gammaflow: a completely automated radioimmunoassay system. Science. 1976 Oct 15;194(4262):270–276. doi: 10.1126/science.184530. [DOI] [PubMed] [Google Scholar]
  6. Buechler W. A., Nakane M., Murad F. Expression of soluble guanylate cyclase activity requires both enzyme subunits. Biochem Biophys Res Commun. 1991 Jan 15;174(1):351–357. doi: 10.1016/0006-291x(91)90527-e. [DOI] [PubMed] [Google Scholar]
  7. Chijiwa T., Mishima A., Hagiwara M., Sano M., Hayashi K., Inoue T., Naito K., Toshioka T., Hidaka H. Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem. 1990 Mar 25;265(9):5267–5272. [PubMed] [Google Scholar]
  8. Geisterfer A. A., Peach M. J., Owens G. K. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res. 1988 Apr;62(4):749–756. doi: 10.1161/01.res.62.4.749. [DOI] [PubMed] [Google Scholar]
  9. Giuili G., Scholl U., Bulle F., Guellaën G. Molecular cloning of the cDNAs coding for the two subunits of soluble guanylyl cyclase from human brain. FEBS Lett. 1992 Jun 8;304(1):83–88. doi: 10.1016/0014-5793(92)80594-7. [DOI] [PubMed] [Google Scholar]
  10. Gruetter C. A., Barry B. K., McNamara D. B., Gruetter D. Y., Kadowitz P. J., Ignarro L. Relaxation of bovine coronary artery and activation of coronary arterial guanylate cyclase by nitric oxide, nitroprusside and a carcinogenic nitrosoamine. J Cyclic Nucleotide Res. 1979;5(3):211–224. [PubMed] [Google Scholar]
  11. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  12. Harteneck C., Wedel B., Koesling D., Malkewitz J., Böhme E., Schultz G. Molecular cloning and expression of a new alpha-subunit of soluble guanylyl cyclase. Interchangeability of the alpha-subunits of the enzyme. FEBS Lett. 1991 Nov 4;292(1-2):217–222. doi: 10.1016/0014-5793(91)80871-y. [DOI] [PubMed] [Google Scholar]
  13. Henry P. J., Drummer O. H., Horowitz J. D. S-nitrosothiols as vasodilators: implications regarding tolerance to nitric oxide-containing vasodilators. Br J Pharmacol. 1989 Nov;98(3):757–766. doi: 10.1111/j.1476-5381.1989.tb14603.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Henry P. J., Horowitz J. D., Louis W. J. Nitroglycerin-induced tolerance affects multiple sites in the organic nitrate bioconversion cascade. J Pharmacol Exp Ther. 1989 Feb;248(2):762–768. [PubMed] [Google Scholar]
  15. Ignarro L. J., Kadowitz P. J. The pharmacological and physiological role of cyclic GMP in vascular smooth muscle relaxation. Annu Rev Pharmacol Toxicol. 1985;25:171–191. doi: 10.1146/annurev.pa.25.040185.001131. [DOI] [PubMed] [Google Scholar]
  16. Kamisaki Y., Waldman S. A., Murad F. The involvement of catalytic site thiol groups in the activation of soluble guanylate cyclase by sodium nitroprusside. Arch Biochem Biophys. 1986 Dec;251(2):709–714. doi: 10.1016/0003-9861(86)90380-2. [DOI] [PubMed] [Google Scholar]
  17. Katsuki S., Arnold W., Mittal C., Murad F. Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine. J Cyclic Nucleotide Res. 1977 Feb;3(1):23–35. [PubMed] [Google Scholar]
  18. Kojda G., Beck J. K., Meyer W., Noack E. Nitrovasodilator-induced relaxation and tolerance development in porcine vena cordis magna: dependence on intact endothelium. Br J Pharmacol. 1994 Jun;112(2):533–540. doi: 10.1111/j.1476-5381.1994.tb13106.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kojda G., Meyer W., Noack E. Influence of endothelium and nitrovasodilators on free thiols and disulfides in porcine coronary smooth muscle. Eur J Pharmacol. 1993 Dec 21;250(3):385–394. doi: 10.1016/0014-2999(93)90025-d. [DOI] [PubMed] [Google Scholar]
  20. Komas N., Lugnier C., Stoclet J. C. Endothelium-dependent and independent relaxation of the rat aorta by cyclic nucleotide phosphodiesterase inhibitors. Br J Pharmacol. 1991 Oct;104(2):495–503. doi: 10.1111/j.1476-5381.1991.tb12457.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kourembanas S., McQuillan L. P., Leung G. K., Faller D. V. Nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia. J Clin Invest. 1993 Jul;92(1):99–104. doi: 10.1172/JCI116604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lewicki J. A., Chang B., Murad F. Quantification of guanylate cyclase concentrations by a direct double determinant tandem immunoradiometric assay. J Biol Chem. 1983 Mar 25;258(6):3509–3515. [PubMed] [Google Scholar]
  23. Lincoln T. M., Cornwell T. L. Intracellular cyclic GMP receptor proteins. FASEB J. 1993 Feb 1;7(2):328–338. doi: 10.1096/fasebj.7.2.7680013. [DOI] [PubMed] [Google Scholar]
  24. Magrinat G., Mason S. N., Shami P. J., Weinberg J. B. Nitric oxide modulation of human leukemia cell differentiation and gene expression. Blood. 1992 Oct 15;80(8):1880–1884. [PubMed] [Google Scholar]
  25. Moncada S., Rees D. D., Schulz R., Palmer R. M. Development and mechanism of a specific supersensitivity to nitrovasodilators after inhibition of vascular nitric oxide synthesis in vivo. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2166–2170. doi: 10.1073/pnas.88.6.2166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Münzel T., Sayegh H., Freeman B. A., Tarpey M. M., Harrison D. G. Evidence for enhanced vascular superoxide anion production in nitrate tolerance. A novel mechanism underlying tolerance and cross-tolerance. J Clin Invest. 1995 Jan;95(1):187–194. doi: 10.1172/JCI117637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nakane M., Arai K., Saheki S., Kuno T., Buechler W., Murad F. Molecular cloning and expression of cDNAs coding for soluble guanylate cyclase from rat lung. J Biol Chem. 1990 Oct 5;265(28):16841–16845. [PubMed] [Google Scholar]
  28. Nakane M., Saheki S., Kuno T., Ishii K., Murad F. Molecular cloning of a cDNA coding for 70 kilodalton subunit of soluble guanylate cyclase from rat lung. Biochem Biophys Res Commun. 1988 Dec 30;157(3):1139–1147. doi: 10.1016/s0006-291x(88)80992-6. [DOI] [PubMed] [Google Scholar]
  29. Needleman P., Johnson E. M., Jr Mechanism of tolerance development to organic nitrates. J Pharmacol Exp Ther. 1973 Mar;184(3):709–715. [PubMed] [Google Scholar]
  30. Papapetropoulos A., Marczin N., Mora G., Milici A., Murad F., Catravas J. D. Regulation of vascular smooth muscle soluble guanylate cyclase activity, mRNA, and protein levels by cAMP-elevating agents. Hypertension. 1995 Oct;26(4):696–704. doi: 10.1161/01.hyp.26.4.696. [DOI] [PubMed] [Google Scholar]
  31. Papapetropoulos A., Marczin N., Snead M. D., Cheng C., Milici A., Catravas J. D. Smooth muscle cell responsiveness to nitrovasodilators in hypertensive and normotensive rats. Hypertension. 1994 Apr;23(4):476–484. doi: 10.1161/01.hyp.23.4.476. [DOI] [PubMed] [Google Scholar]
  32. Patel A., Linden J. Purification of 125I-labeled succinyl cyclic nucleotide tyrosine methyl esters by high-performance liquid chromatography. Anal Biochem. 1988 Feb 1;168(2):417–420. doi: 10.1016/0003-2697(88)90338-7. [DOI] [PubMed] [Google Scholar]
  33. Romanin C., Kukovetz W. R. Tolerance to nitroglycerin is caused by reduced guanylate cyclase activation. J Mol Cell Cardiol. 1989 Jan;21(1):41–48. doi: 10.1016/0022-2828(89)91491-0. [DOI] [PubMed] [Google Scholar]
  34. Sedlak J., Lindsay R. H. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem. 1968 Oct 24;25(1):192–205. doi: 10.1016/0003-2697(68)90092-4. [DOI] [PubMed] [Google Scholar]
  35. Shimouchi A., Janssens S. P., Bloch D. B., Zapol W. M., Bloch K. D. cAMP regulates soluble guanylate cyclase beta 1-subunit gene expression in RFL-6 rat fetal lung fibroblasts. Am J Physiol. 1993 Nov;265(5 Pt 1):L456–L461. doi: 10.1152/ajplung.1993.265.5.L456. [DOI] [PubMed] [Google Scholar]
  36. Shirasaki Y., Su C. Endothelium removal augments vasodilation by sodium nitroprusside and sodium nitrite. Eur J Pharmacol. 1985 Aug 7;114(1):93–96. doi: 10.1016/0014-2999(85)90527-8. [DOI] [PubMed] [Google Scholar]
  37. Ujiie K., Drewett J. G., Yuen P. S., Star R. A. Differential expression of mRNA for guanylyl cyclase-linked endothelium-derived relaxing factor receptor subunits in rat kidney. J Clin Invest. 1993 Feb;91(2):730–734. doi: 10.1172/JCI116255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ujiie K., Hogarth L., Danziger R., Drewett J. G., Yuen P. S., Pang I. H., Star R. A. Homologous and heterologous desensitization of a guanylyl cyclase-linked nitric oxide receptor in cultured rat medullary interstitial cells. J Pharmacol Exp Ther. 1994 Aug;270(2):761–767. [PubMed] [Google Scholar]
  39. Waldman S. A., Murad F. Cyclic GMP synthesis and function. Pharmacol Rev. 1987 Sep;39(3):163–196. [PubMed] [Google Scholar]
  40. Wu X. B., Brüne B., von Appen F., Ullrich V. Reversible activation of soluble guanylate cyclase by oxidizing agents. Arch Biochem Biophys. 1992 Apr;294(1):75–82. doi: 10.1016/0003-9861(92)90139-n. [DOI] [PubMed] [Google Scholar]
  41. Yuen P. S., Potter L. R., Garbers D. L. A new form of guanylyl cyclase is preferentially expressed in rat kidney. Biochemistry. 1990 Dec 11;29(49):10872–10878. doi: 10.1021/bi00501a002. [DOI] [PubMed] [Google Scholar]
  42. Zhang L. M., Castresana M. R., Stefansson S., Newman W. H. Tolerance to sodium nitroprusside. Studies in cultured porcine vascular smooth muscle cells. Anesthesiology. 1993 Nov;79(5):1094–1103. [PubMed] [Google Scholar]

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