UC Irvine

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.