Analysis of Vascular Hydrogen Sulfide Biosynthesis

  • Thomas J. Lechuga
  • Dong-bao ChenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2007)


With potent vasodilatory and pro-angiogenic properties, hydrogen sulfide (H2S) is now accepted as the third gasotransmitter after nitric oxide (NO) and carbon monoxide. Endogenous H2S is mainly synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). Akin to previous studies showing hormonal regulation of NO biosynthesis, we first reported that uterine and systemic artery H2S biosynthesis is regulated by exogenous estrogens in an ovariectomized sheep model of estrogen replacement therapy, specifically stimulating CBS, but not CSE, expression, in uterine (UA) and mesenteric (MA), but not carotid (CA), arteries in ovariectomized nonpregnant sheep. We have found significantly elevated H2S biosynthesis due to CBS upregulation under estrogen-dominant physiological states, the proliferative phase of menstrual cycle and pregnancy in primary human UAs. Our studies have pioneered the role of H2S biology in uterine hemodynamics regulation although there is still much that needs to be learned before a thorough elucidation of a role that H2S plays in normal physiology of uterine hemodynamics and its dysregulation under pregnancy complications can be determined. In this chapter we describe a series of methods that we have optimized for analyzing vascular H2S biosynthesis, including (1) real-time quantitative PCR (qPCR) for assessing tissue and cellular levels of CBS and CSE mRNAs, (2) immunoblotting for assessing CBS and CSE proteins, (3) semiquantitative immunofluorescence microscopy to specifically localize CBS and CSE proteins on vascular wall and to quantify their cellular expression levels, and (4) methylene blue assay for assessing H2S production in the presence of selective CBS and CSE inhibitors.

Key words

H2qPCR Immunoblotting Immunofluorescence microscopy Methylene blue assay 



The present study was supported in part by National Institutes of Health (NIH) grants RO1 HL70562, R21 HL98746, and RO3 HD84972 to D.B.C.


  1. 1.
    Wang R (2012) Physiological implications of hydrogen sulfide: a whiff exploration that blossomed. Physiol Rev 92:791–896CrossRefGoogle Scholar
  2. 2.
    Gadalla MM, Snyder SH (2010) Hydrogen sulfide as a gasotransmitter. J Neurochem 113:14–26CrossRefGoogle Scholar
  3. 3.
    Mustafa AK, Gadalla MM, Sen N, Kim S, Mu W, Gazi SK, Barrow RK, Yang G, Wang R, Snyder SH (2009) H2S signals through protein S-sulfhydration. Sci Signal 2:ra72PubMedPubMedCentralGoogle Scholar
  4. 4.
    Leffler CW, Parfenova H, Basuroy S, Jaggar JH, Umstot ES, Fedinec AL (2011) Hydrogen sulfide and cerebral microvascular tone in newborn pigs. Am J Physiol Heart Circ Physiol 300:H440–H447CrossRefGoogle Scholar
  5. 5.
    Bhatia M (2005) Hydrogen sulfide as a vasodilator. IUBMB Life 57:603–606CrossRefGoogle Scholar
  6. 6.
    Shibuya N, Mikami Y, Kimura Y, Nagahara N, Kimura H (2009) Vascular endothelium expresses 3-mercaptopyruvate sulfurtransferase and produces hydrogen sulfide. J Biochem 146:623–626CrossRefGoogle Scholar
  7. 7.
    Zhao W, Zhang J, Lu Y, Wang R (2001) The vasorelaxant effect of H(2)S as a novel endogenous gaseous K(ATP) channel opener. EMBO J 20:6008–6016CrossRefGoogle Scholar
  8. 8.
    Li Y, Zang Y, Fu S, Zhang H, Gao L, Li J (2012) H2S relaxes vas deferens smooth muscle by modulating the large conductance Ca2+ −activated K+ (BKCa) channels via a redox mechanism. J Sex Med 9:2806–2813CrossRefGoogle Scholar
  9. 9.
    Papapetropoulos A, Pyriochou A, Altaany Z, Yang G, Marazioti A, Zhou Z, Jeschke MG, Branski LK, Herndon DN, Wang R, Szabo C (2009) Hydrogen sulfide is an endogenous stimulator of angiogenesis. Proc Natl Acad Sci U S A 106:21972–21977CrossRefGoogle Scholar
  10. 10.
    Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K, Meng Q, Mustafa AK, Mu W, Zhang S, Snyder SH, Wang R (2008) H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase. Science 322:587–590CrossRefGoogle Scholar
  11. 11.
    Magness RR, Rosenfeld CR (1986) Systemic and uterine responses to alpha-adrenergic stimulation in pregnant and nonpregnant ewes. Am J Obstet Gynecol 155:897–904CrossRefGoogle Scholar
  12. 12.
    Magness RR, Rosenfeld CR (1989) Local and systemic estradiol-17 beta: effects on uterine and systemic vasodilation. Am J Phys 256:E536–E542Google Scholar
  13. 13.
    Rosenfeld CR, Chen C, Roy T, Liu X (2003) Estrogen selectively up-regulates eNOS and nNOS in reproductive arteries by transcriptional mechanisms. J Soc Gynecol Investig 10:205–215CrossRefGoogle Scholar
  14. 14.
    Naden RP, Rosenfeld CR (1985) Systemic and uterine responsiveness to angiotensin II and norepinephrine in estrogen-treated nonpregnant sheep. Am J Obstet Gynecol 153:417–425CrossRefGoogle Scholar
  15. 15.
    Killam AP, Rosenfeld CR, Battaglia FC, Makowski EL, Meschia G (1973) Effect of estrogens on the uterine blood flow of oophorectomized ewes. Am J Obstet Gynecol 115:1045–1052CrossRefGoogle Scholar
  16. 16.
    Magness RR, Phernetton TM, Gibson TC, Chen DB (2005) Uterine blood flow responses to ICI 182 780 in ovariectomized oestradiol-17beta-treated, intact follicular and pregnant sheep. J Physiol 565:71–83CrossRefGoogle Scholar
  17. 17.
    Ford SP (1982) Control of uterine and ovarian blood flow throughout the estrous cycle and pregnancy of ewes, sows and cows. J Anim Sci 55(Suppl 2):32–42PubMedGoogle Scholar
  18. 18.
    Gibson TC, Phernetton TM, Wiltbank MC, Magness RR (2004) Development and use of an ovarian synchronization model to study the effects of endogenous estrogen and nitric oxide on uterine blood flow during ovarian cycles in sheep. Biol Reprod 70:1886–1894CrossRefGoogle Scholar
  19. 19.
    Nelson SH, Steinsland OS, Suresh MS, Lee NM (1998) Pregnancy augments nitric oxide-dependent dilator response to acetylcholine in the human uterine artery. Hum Reprod 13:1361–1367CrossRefGoogle Scholar
  20. 20.
    Lang U, Baker RS, Braems G, Zygmunt M, Kunzel W, Clark KE (2003) Uterine blood flow—a determinant of fetal growth. Eur J Obstet Gynecol Reprod Biol 110(Suppl 1):S55–S61CrossRefGoogle Scholar
  21. 21.
    Lechuga TJ, Zhang H, Sheibani L, Karim M, Jia J, Magness RR, Rosenfeld CR, Chen DB (2015) Estrogen replacement therapy in ovariectomized nonpregnant ewes stimulates uterine artery hydrogen sulfide biosynthesis by selectively upregulating cystathionine beta synthase expression. Endocrinology 156:2288–2298CrossRefGoogle Scholar
  22. 22.
    O’Leary P, Boyne P, Flett P, Beilby J, James I (1991) Longitudinal assessment of changes in reproductive hormones during normal pregnancy. Clin Chem 37:667–672PubMedGoogle Scholar
  23. 23.
    Sheibani L, Lechuga TJ, Zhang H, Hameed A, Wing DA, Kumar S, Rosenfeld CR, Chen DB (2017) Augmented H2S production via cystathionine-beta-synthase upregulationplays a role in pregnancy-associated uterine vasodilation. Biol Reprod 96:664–672.CrossRefGoogle Scholar
  24. 24.
    Chen DB, Westfall SD, Fong HW, Roberson MS, Davis JS (1998) Prostaglandin F2alpha stimulates the Raf/MEK1/mitogen-activated protein kinase signaling cascade in bovine luteal cells. Endocrinology 139:3876–3885CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Obstetrics and Gynecology & PathologyUniversity of California IrvineIrvineUSA

Personalised recommendations