Skip to main content
PMC home page
AAPS PharmSci logoLink to AAPS PharmSci
. 2002 May 7;4(2):45–55. doi: 10.1208/ps040208

cDNA Microarray analysis of vascular gene expression after nitric oxide donor infusion in rats: Implications for nitrate tolerance mechanisms

Ellen Q Wang 1,✉, Woo-In Lee 1, Daniel Brazeau 1, Ho-Leung Fung 1,✉
PMCID: PMC2751295  PMID: 12102617

Abstract

Vascular nitrate tolerance is often accompanied by changes in the activity and/or expression of a number of proteins. However, it is not known whether these changes are associated with the vasodilatory properties of nitrates, or with their tolerance mechanisms. We examined the hemodynamic effects and vascular gene expressions of 2 nitric oxide (NO) donors: nitroglycerin (NTG) and S-nitroso-N-acetylpenicillamine (SNAP). Rats received 10 μg/min NTG, SNAP, or vehicle infusion for 8 hours. Hemodynamic tolerance was monitored by the maximal mean arterial pressure (MAP) response to a 30-μg NTG or SNAP bolus challenge dose (CD) at various times during infusion. Gene expression in rat aorta after NTG or SNAP treatment was determined using cDNA microarrays, and the relative differences in expression after drug treatment were evaluated using several statistical techniques. MAP response of the NTG CD was attenuated from the first hour of NTG infusion (P<.001, analysis of variance [ANOVA]), but not after SNAP (P>.05, ANOVA) or control infusion (P> .05, ANOVA). Student t-statistics revealed that 447 rat genes in the aorta were significantly altered by NTG treatment (P <.05). An adjusted t-statistic approach using resampling techniques identified a subset of 290 genes that remained significantly different between NTG treatment vs control. In contrast, SNAP treatment resulted in the up-regulation of only 7 genes and the downregulation of 34 genes. These results indicate that continuous NTG infusion induced widespread changes in vascular gene expression, many of which are consistent with the multifactorial and complex mechanisms reported for nitrate tolerance.

KeyWords: DNA microarray, gene regulation, nitrate tolerance, nitric oxide donor, nitroglycerin

References

  • 1.Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci USA. 1987;84:9265–9269. doi: 10.1073/pnas.84.24.9265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524–526. doi: 10.1038/327524a0. [DOI] [PubMed] [Google Scholar]
  • 3.Feelisch M. The use of nitric oxide donors in pharmacological studies. Naunyn Schmiedebergs Arch Pharmacol. 1998;358:113–122. doi: 10.1007/PL00005231. [DOI] [PubMed] [Google Scholar]
  • 4.Tseng CM, Tabrizi-Fard MA, Fung HL. Differential sensitivity among nitric oxide donors toward ODQ-mediated inhibition of vascular relaxation. J Pharmacol Exp Ther. 2000;292:737–742. [PubMed] [Google Scholar]
  • 5.Fung HL, Bauer JA. Mechanisms of nitrate tolerance. Cardiovasc Drugs Ther. 1994;8:489–499. doi: 10.1007/BF00877927. [DOI] [PubMed] [Google Scholar]
  • 6.Needleman P, Johnson EM. Mechanism of tolerance development to organic nitrates. J Pharmacol Exp Ther. 1973;184:709–715. [PubMed] [Google Scholar]
  • 7.Fung HL, Piliszczuk R. Nitrosothiol and nitrate tolerance. Z Kardiol. 1986;75:25–27. [PubMed] [Google Scholar]
  • 8.Axelsson KL, Andersson RG. Tolerance towards nitroglycerin, induced in vivo, is correlated to a reduced cGMP response and an alteration in cGMP turnover. Eur J Pharmacol. 1983;88:71–79. doi: 10.1016/0014-2999(83)90393-X. [DOI] [PubMed] [Google Scholar]
  • 9.Munzel T, Sayegh H, Freeman BA, Tarpey MM, Harrison DG. Evidence for enhanced vascular superoxide anion production in nitrate tolerance: a novel mechanism underlying tolerance and crosstolerance. J Clin Invest. 1995;95:187–194. doi: 10.1172/JCI117637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Munzel T, Giaid A, Kurz S, Stewart DJ, Harrison DG. Evidence for a role of endothelin 1 and protein kinase C in nitroglycerin tolerance. Proc Natl Acad Sci USA. 1995;92:5244–5248. doi: 10.1073/pnas.92.11.5244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Munzel T, Li H, Mollnau H, et al. Effects of long-term nitroglycerin treatment on endothelial nitric oxide synthase (NOS III) gene expression, NOS III-mediated superoxide production, and vascular NO bioavailability. Circ Res. 2000;86:E7–E12. doi: 10.1161/01.res.86.1.e7. [DOI] [PubMed] [Google Scholar]
  • 12.Lander ES. Array of hope. Nat Genet. 1999;21:3–4. doi: 10.1038/4427. [DOI] [PubMed] [Google Scholar]
  • 13.Taniguchi M, Miura K, Iwao H, Yamanaka S. Quantitative assessment of DNA microarrays--comparison with Northern blot analyses. Genomics. 2001;71:34–39. doi: 10.1006/geno.2000.6427. [DOI] [PubMed] [Google Scholar]
  • 14.Gow AJ, Stamler JS. Reactions between nitric oxide and haemoglobin under physiological conditions. Nature. 1998;391:169–173. doi: 10.1038/34402. [DOI] [PubMed] [Google Scholar]
  • 15.Lipton AJ, Johnson MA, Macdonald T, Lieberman MW, Gozal D, Gaston B. S-nitrosothiols signal the ventilatory response to hypoxia. Nature. 2001;413:171–174. doi: 10.1038/35093117. [DOI] [PubMed] [Google Scholar]
  • 16.Kowaluk EA, Poliszczuk R, Fung HL. Tolerance to relaxation in rat aorta: comparison of an S-nitrosothilol with nitroglycerin. Eur J Pharmacol. 1987;144:379–383. doi: 10.1016/0014-2999(87)90392-X. [DOI] [PubMed] [Google Scholar]
  • 17.Bauer JA, Fung HL. Differential hemodynamic effects and tolerance properties of nitroglycerin and an S-nitrosothiol in experimental heart failure. J Pharmacol Exp Ther. 1991;256:249–254. [PubMed] [Google Scholar]
  • 18.Matsumoto T, Takahashi M, Nakae I, Kinoshita M. Vasorelaxing effect of S-nitrosocaptopril on dog coronary arteries: no crosstolerance with nitroglycerin. J Pharmacol Exp Ther. 1995;275:1247–1253. [PubMed] [Google Scholar]
  • 19.Schuchhardt J, Beule D, Malik A, et al. Normalization strategies for cDNA microarrays. Nucleic Acids Res. 2000;28:E47–E47. doi: 10.1093/nar/28.10.e47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Lee ML, Kuo FC, Whitmore GA, Sklar J. Importance of replication in microarray gene expression studies statistical methods and evidence from repetitive cDNA hybridizations. Proc Natl Acad Sci USA. 2000;97:9834–9839. doi: 10.1073/pnas.97.18.9834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Good P. Resampling Methods: A Practical Guide to Data Analysis. New York, NY: Springer-Verlag; 1999. [Google Scholar]
  • 22.Edgington E. Randonization Tests. New York, NY: Marcel Dekker, Inc.; 1980. [Google Scholar]
  • 23.Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39:783–791. doi: 10.2307/2408678. [DOI] [PubMed] [Google Scholar]
  • 24.Excoffier L, Smouse PE, Quattro JM. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics. 1992;131:479–491. doi: 10.1093/genetics/131.2.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Carpenter J, Bithell J. Bootstrap confidence intervals: when, which, what? A practical guide for medical statisticians. Stat Med. 2000;19:1141–1164. doi: 10.1002/(SICI)1097-0258(20000515)19:9&#x0003c;1141::AID-SIM479&#x0003e;3.0.CO;2-F. [DOI] [PubMed] [Google Scholar]
  • 26.Westfall PH, Young SS. Resampling-based Multiple Testing: Examples and Methods for p-adjustment. New York, NY: Wiley; 1993. [Google Scholar]
  • 27.Kerr MK, Churchill GA. Bootstrapping cluster analysis: assessing the reliability of conclusions from microarray experiments. Proc Natl Acad Sci USA. 2001;98:8961–8965. doi: 10.1073/pnas.161273698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA. 2001;98:5116–5121. doi: 10.1073/pnas.091062498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Herwig R, Aanstad P, Clark M, Lehrach H. Statistical evaluation of differential expression on cDNA nylon arrays with replicated experiments. Nucleic Acids Res. 2001;29:1–9. doi: 10.1093/nar/29.23.e117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Kowaluk EA, Fung HL. Spontaneous liberation of nitric oxide cannot account for in vitro vascular relaxation by S-nitrosothiols. J Pharmacol Exp Ther. 1990;255:1256–1264. [PubMed] [Google Scholar]
  • 31.Liu L, Hausladen A, Zeng M, Que L, Heitman J, Stamler JS. A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans. Nature. 2001;410:490–494. doi: 10.1038/35068596. [DOI] [PubMed] [Google Scholar]
  • 32.Yamamoto T, Bing RJ. Nitric oxide donors. Proc Soc Exp Biol Med. 2000;225:200–206. doi: 10.1046/j.1525-1373.2000.22525.x. [DOI] [PubMed] [Google Scholar]
  • 33.Miller RA, Galecki A, Shmookler-Reis RJ. Interpretation, design, and analysis of gene array expression experiments. J Gerontol A Biol Sci Med Sci. 2001;56:B52–57. doi: 10.1093/gerona/56.2.b52. [DOI] [PubMed] [Google Scholar]
  • 34.Dudoit S, Yang YH, Callow M, Speed T. Statistical methods for identifying differentially expressed genes in replicated cDNA microarry experiments. UC Berkeley, Technical report #578, 2000.
  • 35.Feelisch M. The biochemical pathways of nitric oxide formation from nitrovasodilators: appropriate choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solution. J Cardiovasc Pharmacol. 1991;17:S25–S33. doi: 10.1097/00005344-199117003-00006. [DOI] [Google Scholar]
  • 36.Bennett BM, McDonald BJ, Nigam R, Simon WC. Biotransformation of organic nitrates and vascular smooth muscle cell function. Trends Pharmacol Sci. 1994;15:245–249. doi: 10.1016/0165-6147(94)90319-0. [DOI] [PubMed] [Google Scholar]
  • 37.Xuan YT, Guo Y, Han H, Zhu Y, Bolli R. An essential role of the JAK-STAT pathway in ischemic preconditioning. Proc Natl Acad Sci USA. 2001;98:9050–9055. doi: 10.1073/pnas.161283798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Huber A, Neuhuber WL, Klugbauer N, Ruth P, Allescher HD. Cysteine-rich protein 2, a novel substrate for cGMP kinase I in enteric neurons and intestinal smooth muscle. J Biol Chem. 2000;275:5504–5511. doi: 10.1074/jbc.275.8.5504. [DOI] [PubMed] [Google Scholar]
  • 39.Wolin MS. Interactions of oxidants with vascular signaling systems. Arterioscler Thromb Vasc Biol. 2000;20:1430–1442. doi: 10.1161/01.atv.20.6.1430. [DOI] [PubMed] [Google Scholar]
  • 40.Kunsch C, Medford RM. Oxidative stress as a regulator of gene expression in the vasculature. Circ Res. 1999;85:753–766. doi: 10.1161/01.res.85.8.753. [DOI] [PubMed] [Google Scholar]
  • 41.Fox PL, Mazumder B, Ehrenwald E, Mukhopadhyay CK. Ceruloplasmin and cardiovascular disease. Free Radic Biol Med. 2000;28:1735–1744. doi: 10.1016/S0891-5849(00)00231-8. [DOI] [PubMed] [Google Scholar]
  • 42.Floris G, Medda R, Padiglia A, Musci G. The physiopathological significance of ceruloplasmin: a possible therapeutic approach. Biochem Pharmacol. 2000;60:1735–1741. doi: 10.1016/S0006-2952(00)00399-3. [DOI] [PubMed] [Google Scholar]
  • 43.Meininger CJ, Marinos RS, Hatakeyama K, et al. Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency. Biochem J. 2000;349:353–356. doi: 10.1042/0264-6021:3490353. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from AAPS PharmSci are provided here courtesy of American Association of Pharmaceutical Scientists

RESOURCES