Nitric oxide (NO) signaling through S-nitrosylation has emerged as an important mechanism for post-translational regulation of proteins. Reversible protein S-nitrosylation represents soluble guanylyl cyclase/cyclic guanosine monophosphate (cGMP)-independent NO signaling. It is accomplished by a covalent addition of an NO group to a reactive free thiol of proteins to form S-nitrosothiols (SNOs). Once formed, nitrosothiols can be transferred to free thiols on cysteine residues of proteins via transnitrosation reactions [1]. Transnitrosation allows transfers of NO signaling to a site distant from the NO source. The most abundant endogenous SNO and the major stable intracellular NO reservoir and NO transporter is S-nitrosoglutathione (GSNO), generated by S-nitrosylation of the key antioxidant glutathione (GSH). S-nitrosylation/denitrosylation depends on activities of nitric oxide synthases (NOSs) and denitrosylases. S-nitrosoglutathione reductase (GSNOR) is the enzyme that selectively metabolizes GSNO, and thereby depletes the levels of S-nitrosylated proteins, which are in equilibrium with GSNO [2]. The role of S-nitrosylation in mediating penile erection is, however, not well understood. The findings reported in this issue of the International Journal of Impotence Research in the article entitled “S-Nitrosylation of Endothelial Nitric Oxide Synthase Impacts Erectile Function” [3] offer additional support for S-nitrosylation operating in the penis possibly as a regulator of erectile function.

The authors performed experiments combining molecular and physiologic approaches to evaluate the role of S-nitrosylation in erectile function, suggesting it may serve as an alternative pathway leading to detumescence. Co-localization of eNOS and GSNOR in the mouse and human cavernosal tissue, presented in this study, confirms previous findings in the rodent [4, 5] and elevates these findings to human level. To assess the role of global S-nitrosylation, and specifically eNOS S-nitrosylation, in erectile function, the authors used the GSNOR−/− mouse, and intracavernosal injection of a cell permeable glutathione analog glutathione ethyl ester, respectively. Global S-nitrosylation in GSNOR−/− mice resulted in the inability of these mice to maintain elevated intracavernosal pressure (ICP), while decreased eNOS S-nitrosylation resulted in improved tumescence, suggesting that this post-translational protein modification limited normal erectile function. These findings importantly add to previous reports of the presence of S-nitrosylated eNOS in the penis and the role of S-transnitrosylation in the homeostatic eNOS function of the penis [4, 5].

S-nitrosylation of proteins is involved in the regulation of a number of physiologic and pathologic processes. More than 3000 proteins are S-nitrosylated, including NOSs themselves. In resting endothelial cells, eNOS targeting to the plasma membrane results in its constitutive S-nitrosylation and tonic inhibition. Translocation of eNOS from the membrane to the cytosol upon stimulation results in rapid and transient eNOS denitrosylation concomitant with the increase in eNOS phosphorylation at Ser-1177 and enzyme activation [6]. The source of NO for nitrosylation is eNOS itself localized in the membranes, implying that this modification may be one of the final steps in returning the enzyme to its basal state after its activation and translocation.

In this study, the authors found a marked increase in eNOS S-nitrosylation in the penis of healthy WT mice during prolonged penile erection elicited by continuous electrical stimulation of the cavernous nerve. The authors speculate that the increase in eNOS S-nitrosylation (inactivation) observed with tumescence may begin a cycle leading to detumescence. However, it is questionable whether this increased eNOS S-nitrosylation during erection contributes to detumescence, or whether continuous electrical stimulation of the cavernous nerve exhausts NO molecular signaling resulting in return of eNOS function to baseline and increased eNOS nitrosylation. Thus, eNOS S-nitrosylation may represent a consequence, rather than the cause, of decreased eNOS function and detumescence under these experimental parameters. The authors missed the opportunity to use well controlled short-term electrical stimulation of the cavernous nerve to stimulate penile erection, which would mimic more closely physiologic erectile response, in order to answer this important question. Reportedly, peak ICP recordings is most consistently obtained within 30- to 60-s intervals after starting electrical stimulation [7]. Indeed, a recent study demonstrated that global S-nitrosylation results in the failure of eNOS activation by phosphorylation in the penis in response to shear stress generated by electrical stimulation of the cavernous nerve, contrasting with findings in the current study [4]. Also, while S-nitrosylated eNOS has been reported to be nonfunctional, it remains unknown whether S-nitrosylated eNOS is truly inhibited in the penis during erection.

Systemic blood pressure drives penile erection, and ICP measurements depend on systemic blood pressure. The authors compared erectile function of WT, GSNOR−/−, and eNOS−/− mice, but they did not measure their blood pressure. These mouse groups have different cardiovascular functions, which may affect their ICP measurements. The lack of blood pressure measurement in this study, both at baseline and during erectile function evaluation, is a significant limitation of the study. Indeed, comparative evaluation of erectile function in GSNOR−/− and eNOS−/− mice in this study demonstrated decreased erectile function in both mouse groups. These data contrast with previous findings, which showed unchanged and increased erectile responses, respectively, in these mice [4, 7, 8]. It is also questionable whether eNOS−/− mice are a good choice for comparison with GSNOR−/− mice, as in the former erectile function is influenced by downregulated PDE5, a cGMP degrading enzyme, while in the latter erectile function is influenced by global protein S-nitrosylation. These two different signaling pathways potentially represent distinct experimental paradigms.

In summary, this study offers additional support for eNOS S-nitrosylation/denitrosylation molecular signaling in the penis and its potential role in erectile function. However, it is uncertain whether the authors established the operation of eNOS S-nitrosylation as an alternative pathway, in addition to PDE5, in governing detumescence. Caution must be exercised in interpreting these data and drawing a conclusion. It is hoped that future study by the investigator group will provide further mechanistic insight into the role of eNOS S-nitrosylation in erectile function.