The Role of Regulatory Proteins and S-nitrosylation of Endothelial Nitric Oxide Synthase in the Human Clitoris: Implications for Female Sexual Function

Abstract

Introduction During female sexual arousal, clitoral blood flow is controlled by endothelial nitric oxide synthase (eNOS) and its product, nitric oxide (NO). The mechanisms regulating eNOS activity and NO bioavailability in the clitoris are largely unknown. Aim To identify proteins involved in regulation of eNOS activity within the clitoris and to evaluate the effects of S-nitrosoglutathione reductase (GSNO-R) and eNOS nitrosylation/denitrosylation on clitoral blood flow. Methods Immunohistochemistry for eNOS, caveolin-1 (Cav1), heat shock protein-90 (Hsp90), phosphodiesterase type 5 (PDE5), GSNO-R, and soluble guanylate cyclase (sGC) was performed on human and murine clitoral tissue. Western blot analysis was performed for eNOS, phosphorylated eNOS (phospho-eNOS, Ser1177), Cav1, Hsp90, sGC, PDE5, phosphoinositide 3-kinase (PI3K), Akt (protein kinase B), and GSNO-R on protein from human clitoral tissue. A biotin switch assay was used to analyze the S-nitrosylation of eNOS, nNOS, and GSNO-R. Clitoral blood flow was measured in wild-type and GSNO-R-/- mice at baseline and during cavernous nerve electrical stimulation (CNES). Main Outcome Measures Localization of eNOS regulatory proteins and clitoral blood flow. Results eNOS and GSNO-R co-localized to the vascular endothelium and sinusoids of human clitoral tissue. Immunohistochemistry also localized Cav1 and Hsp90 to the endothelium and PDE5 and sGC to the trabecular smooth muscle. Expression of S-nitrosylated (SNO)-eNOS and SNO-GSNO-R was detected by biotin switch assays. Wild-type control mice exhibited increased clitoral blood flow with CNES whereas GSNO-R-/- animals failed to show an increase in blood flow. Conclusions Several key eNOS regulatory proteins are present in the clitoral tissue in a cellular specific pattern. S-nitrosylation of eNOS may also represent a key regulatory mechanism governing eNOS activation/deactivation since mice deficient in GSNO-R failed to increase clitoral blood flow. Additional studies are necessary to define the role of S-nitrosylation in the genital vascular response and its subsequent impact on female sexual function.

Protective effects of irbesartan and benazepril against vaginal vascular remodeling and fibrosis in female spontaneously hypertensive rats

“…3 Although the mechanism of FSD is still poorly understood, vaginal vascular function is known to play a significant role in female sexual function, and increased blood flow to the vagina, clitoris, and labia is responsible for the vasocongestion, engorgement, and lubrication involved in the sexual arousal response. 6 Endothelial dysfunction in the female lower genital organs 7 and fibrosis might thus be the important mechanisms of FSD in women with hypertension.…”

Transnitrosylation: A Factor in Nitric Oxide–Mediated Penile Erection

“…First, we did not directly measure eNOS S -nitrosylation. However, eNOS S -nitrosylation has been measured directly in human clitoral tissue [37], suggesting that eNOS may be nitrosylated similarly in penile tissue. Second, we measured total NO production, produced by all 3 NOS isoforms.…”

NADPH diaphorase detects S-nitrosylated proteins in aldehyde-treated biological tissues

“…This is most likely to be due to the SNO-mediated nitrosylation of NBT, which is then able to directly accept electrons from α-or β-NADPH. As such, this new view of NADPH diaphorase as defining the presence of SNOs and nitrosylated proteins including NOS itself [62][63][64][65] can be seen as a strong redefinition of the current concept that NADPH diaphorase defines the tissue distribution of NOS. In particular, we suggest that NADPH diaphorase is an invaluable tool for the visualization of S-nitrosylated proteins and preformed pools of SNOs in biological tissues.…”