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Analysis of Vascular Hydrogen Sulfide Biosynthesis

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Vascular Effects of Hydrogen Sulfide

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2007))

Abstract

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.

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Acknowledgments

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.

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Authors and Affiliations

  1. Department of Obstetrics and Gynecology & Pathology, University of California Irvine, Irvine, CA, USA

    Thomas J. Lechuga & Dong-bao Chen

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  1. Thomas J. Lechuga
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  2. Dong-bao Chen
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Corresponding author

Correspondence to Dong-bao Chen .

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Editors and Affiliations

  1. Department of Pathophysiology, Medical University, Lublin, Poland

    Jerzy Bełtowski

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Lechuga, T.J., Chen, Db. (2019). Analysis of Vascular Hydrogen Sulfide Biosynthesis. In: Bełtowski, J. (eds) Vascular Effects of Hydrogen Sulfide. Methods in Molecular Biology, vol 2007. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9528-8_3

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  • DOI: https://doi.org/10.1007/978-1-4939-9528-8_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9527-1

  • Online ISBN: 978-1-4939-9528-8

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