The role of regulatory proteins and S-nitrosylation of endothelial nitric oxide synthase in the human clitoris: implications for female sexual function.
BACKGROUND
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.


OBJECTIVE
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).


METHODS
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.
Department of Urology, University of Virginia, Charlottesville, VA, USA.
Janine L
JL
Oliver
Parviz K
PK
Kavoussi
Ryan P
RP
Smith
Robin I
RI
Woodson
Sean T
ST
Corbett
Raymond A
RA
Costabile
Lisa A
LA
Palmer
Jeffrey J
JJ
Lysiak
D000445:Aldehyde Oxidoreductases / Q000502:physiology; D000818:Animals; D051242:Caveolin 1 / Q000378:metabolism; D002987:Clitoris / Q000098:blood supply / Q000201:enzymology*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000378:metabolism; D004727:Endothelium / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D005260:Female; D006162:Guanylate Cyclase / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008810:Mice, Inbred C57BL; D009130:Muscle, Smooth / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D000071756:Soluble Guanylyl Cyclase
D051242:Caveolin 1; D018841:HSP90 Heat-Shock Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent; D019869:Phosphatidylinositol 3-Kinases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase
Clitoral Blood Flow; Clitoris; Female Sexual Function; Nitric Oxide; Nitrosylation; eNOS