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Effects of Hydrogen Sulfide on Erectile Function and Its Possible Mechanism(s) of Action
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REVIEWS Effects of Hydrogen Sulfide on Erectile Function and Its Possible Mechanism(s) of Action jsm_2279
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Roeswita Leono Liaw, BSc, Balasubramanian Srilatha, MD, PhD, and P. Ganesan Adaikan, PhD, DSc Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Hospital, National University of Singapore, Singapore DOI: 10.1111/j.1743-6109.2011.02279.x
ABSTRACT
Introduction. The current pharmacotherapy for erectile dysfunction (ED) relies significantly on the use of phosphodiesterase type 5 (PDE5) inhibitors, but quite a proportion of ED patients are resistant to this therapy, necessitating a search for an alternative treatment. We reviewed available published data to analyze current evidence of hydrogen sulfide (H2S) as a novel pharmacotherapeutic agent with supportive role in sexual function. Aim. To discuss the role of H2S in erectile function, its possible mechanism of action, and how this knowledge may be exploited for therapeutic use. Methods. Pubmed and Medline search was conducted to identify original articles and reviews. Main Outcome Measures. Data from peer-reviewed publications. Results. Animal studies using different species, including in vitro study done in humans, show evidence of H2S’s pro-erectile effects. The mechanism behind is still unclear, but evidence in literature points out the involvement of K+ATP channel, modulation of protein with anti-erectile effects, as well as involvement of the nitrergic pathway through a complex cross-talk. A new drug called H2S-donating sildenafil (ACS6), which incorporated an H2Sdonating moiety in sildenafil, has been developed. While more studies are still needed, this heralded a new pharmacotherapeutical approach, which is multipronged in nature. Conclusions. Given the mounting evidence of H2S’s role in erectile function and how it appears to achieve its pro-erectile effects through different mechanisms, H2S represents a potentially important treatment alternative or adjunct to PDE5 inhibitors. Liaw RL, Srilatha B, and Adaikan PG. Effects of hydrogen sulfide on erectile function and its possible mechanism(s) of action. J Sex Med 2011;8:1853–1864. Key Words. Hydrogen Sulfide; Nitric Oxide; Gasotransmitter; Corpus Cavernosum; Erectile Function
Introduction
I
n light of recent discoveries, the scientific community’s perspective of hydrogen sulfide (H2S) has undergone a paradigm shift. This toxic pollutant with rotten egg smell is now considered an important biological mediator. H2S is currently placed as a member of the “gasotransmitter” family together with other members, viz., carbon monoxide (CO) and nitric oxide (NO), the term conceived to distinguish their action from the classical signaling molecules [1]. There is a growing number of evidence that H2S can exert a multitude © 2011 International Society for Sexual Medicine
of biological effects, e.g., in inflammation, antinociception, myocardial ischemia–reperfusion, cardiovascular pathology, shock/sepsis, pulmonary hypertension, and diabetes. H2S is a weak acid with a pKa of 6.76 at 37°C, dissociating in aqueous solution as follows: H2S ↔ HS- + H+ ↔ S2- + 2H+. At physiological pH of 7.4, about 18.5% of the total sulfide has been shown to exist as H2S and 81.5% as HS- [2]; however, it is unknown as to which of the H2S moiety (H2S, HS-, or S2-) is the “active” component responsible for the net biological effects observed in different systems [3]. H2S is endogenously J Sex Med 2011;8:1853–1864
1854 produced from L-cysteine by the activity of two enzymes, cystathionine b-synthase (CBS)— existing predominantly in the brain and central nervous system—and cystathionine g-lyase (CSE)— expressed mainly in the liver, and vascular and nonvascular smooth muscles [4]. Systemically, H2S can be oxidized to form sulfate or sulfite in mitochondria; it can be scavenged by methemoglobin or glutathione, and it can also be methylated [5]. A lot of studies have employed the use of CSE inhibitors, viz., DL-propargylglycine (PAG) and b-cyano-Lalanine (BCA), to delineate H2S effects. They are the only pharmacological agents known today to inhibit H2S production by CSE; however, it is important to note that they are of modest potency and selectivity, and have limited membrane permeability [6]. Therefore, the results from such studies have been less useful in investigating the physiological role of H2S compared with NO synthase (NOS) inhibitors such as L-NG-monomethyl arginine in delineating the physiology of the NO pathway. Erectile Physiology
Erectile physiology is an intricate interplay of vascular, neurologic, and endocrine factors. Disturbances to any of these system can give rise to erectile dysfunction (ED), making it a multifactorial disorder that is rather difficult to treat. As it is known thus far, relaxation of the corpus cavernosum (CC) smooth muscle brings about penile erection through an increase in arterial flow and restriction of venous outflow; detumescence is associated with the a-adrenoceptor activity, whereas tumescence is attributed to both cholinergic and nitrergic involvement, with the nitrergic (NO/cyclic guanosine monophosphate [cGMP]) pathway being the primary mediator [7]. As an important prerequisite for erectile process, NO is endogenously produced from L-arginine by the NOS isoforms: endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and inducible nitric oxide synthase (iNOS) [8]. The released NO activates soluble guanylyl cyclase to result in an increased conversion of the guanosine triphosphate to second messenger cGMP (which, through its interaction with cGMP-dependent protein kinases, cyclic nucleotide-gated ion channels, or cyclic nucleotide phosphodiesterases, governs many aspects of cellular function in the body). In the cavernosum, cGMP stimulates the protein kinase G, which in turn initiates phosphorylation of membrane-bound proteins at K+ channels. This will lead to K+ ion outflow into the extracellular J Sex Med 2011;8:1853–1864
Liaw et al. space resulting in hyperpolarization, closure of L-type Ca2+ channels, and decrease in intracellular Ca2+ ion concentration. Together with these changes, there is decreased activation of myosin light chain (MLC) kinase, decreased phosphorylation of MLC chains, and, subsequently, reduced actin– myosin interaction to result in cavernosal relaxation and physiological erection [9] (Figure 1). Pro-Erectile Effects of H2S
Considering the substantial endogenous production of H2S by mammalian tissues and the growing evidence that H2S can act as a regulatory mediator similar to NO, it can be expected that H2S may exert important biophysiological effects in erectile function. In fact, onion—with its complex sulfur compound biochemistry—was considered to be a popular, natural remedy for impotence [12]. In a pilot study by Srilatha et al., it was demonstrated that administration of sodium hydrosulfide hydrate (NaHS.xH2O, a stable H2S donor) increased penile length, perfusion, and intracavernosal pressure (ICP) in vivo in a nonhuman primate model [13]; this serves as the first direct evidence for the pro-erectile effect of H2S in CC. Such facilitatory effects on erectile function have also been observed in other animal models. In particular, administration of PAG decreased ICP in rats [13]. Organ bath study showed that NaHS can dose-dependently relax pre-contracted rabbit and human CC [14,15]. As expected, L-cysteine (H2S precursor and CBS/ CSE substrate) has similar effects to NaHS in increasing ICP, and this effect is blocked by PAG. Adenylyl cyclase (AC) inhibitor MDL 12,330A was shown to block the H2S-induced relaxation of pre-contracted rabbit cavernosum; however, this inhibition was incomplete [14], suggesting that relaxation mediated by H2S is only partially dependent on cAMP pathway, highlighting the presence of other mechanisms contributing to its relaxant effect, possibly via K+ATP channel. Inhibition of H2S production with PAG or aminooxyacetic acid (a CBS inhibitor) is also able to significantly increase electrical field stimulationinduced contraction (associated with detumescence) at different frequencies in both rabbit and human CC [14,15]. Therefore, endogenous H2S’s role is possibly twofold: (i) relaxation of CC smooth muscle; and (ii) inhibition of basal tone in penis. This finding is significant considering that both impaired relaxation and increased contractility can contribute to ED. However, it is still
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Figure 1 (A and B) Relaxation of penile smooth muscle via cyclic guanosine monophosphate (cGMP) pathway [9–11]. NO = nitric oxide; PKA = protein kinase A; PKG = protein kinase G; PLB = phospholipase B; GC = guanylyl cyclase; PLC = phospholipase C; DAG = diacyl glycerol; NE = norepinephrine; ET-1 = endothelin-1; MLC = myosin light chain; IP3 = inositol triphosphate.
B
unclear at this point as to the actual site of action of H2S, i.e., whether it is molecular, cellular, or neurovascular in nature. A lot of the evidence for H2S’s physiological role was based on the observation that it is endogenously produced in tissues that are pertinent to its proposed role as vasorelaxant or gasomodulator. This means that rigorous assessment of methodologies used to measure very small concentrations of this labile gas accurately is important in order to avoid potential artifacts. Unfortunately, unlike NO that can be measured using its stable oxidation products (NO2- and NO3-) [16], H2S has no known stable or specific end product from its biosynthesis (Figure 2). Interestingly, a majority of the studies—using different analytical techniques—reported plasma H2S in the range of 25–80 mM in rat and humans, with few exceptions [3], indicating thereby that the results are likely to be credible. Similarly, H2S production has also been detected in different tissues such as penis, liver, aorta, and ileum in vitro [13,20]. The assay involved supplying the tissue with L-cysteine and pyridoxal5′-phosphate (substrate and cofactor, respectively, for CBS/CSE), and detecting the H2S synthesized by the tissue [21]. Srilatha et al. acknowledged that this assay has a limitation in that it only proves the presence/activity of the enzyme but does not reflect the actual in vivo H2S production, because the
amount of substrate and cofactors available in the tissue is unknown [14]. Even so, the assay has generally succeeded in demonstrating that penile erectile tissue expresses CBS and CSE enzymes. d’Emmanuele di Villa Bianca et al. showed that in the human CC, these enzymes were localized mostly in the smooth muscle (vascular and trabecular) [15]. With an organ bath study, they further demonstrated that the H2S’s relaxant effect seen in different animal models also applies to human CC, and this relaxant effect is indeed endothelium independent (noninvolvement of endothelium was verified through a test of the tissue’s responsiveness to acetylcholine). This finding has a significant implication considering that endothelial dysfunction is a major contributing factor to vascular pathology of the penis leading to ED [22]. This suggests that the novel H2S pathway, by virtue of its lack of dependence on endothelial integrity (which may be compromised in patients with ED), for its production, may aid cavernosal relaxation and complement NO signaling (in particular, with respect to the NO contribution by eNOS) in erectile physiology. H2S as K+ATP Channel Opener
H2S was shown to cause vasoconstriction at lower concentrations but vasodilatation at higher conJ Sex Med 2011;8:1853–1864
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L-Homocysteine + L-Cysteine L-Homocysteine + L-Serine
L-Cysteine + H2S L-Cystathionine + H2O
CBS
Aminoacetic Acid(AOAA) − CBS Inhibitor
H2S + Pyruvate + NH3
CSE
DL-Propargylglycine (PAG) − Irreversible Inhibitor B-Cyano-L-Alanine (BCA) − Reversible Inhibitor
CSE
L-Cysteine + NH3 + α-Ketobutyrate L-Cysteine + H2O L-Cysteine
3-Mercaptopyruvate
Pyruvate
CDO
AAT
L-Cysteine Sulfinate
AAT
CSD
MPST Hypotaurine
Sulfinyl Pyruvate
H2S Taurine
Decomposition Sulfite (SO32-)
SO CBS:Cystathionine β Synthase CSE:Cystathionine γ Lyase AAT:Aspartate Aminotransferase CDO:Cysteine Deoxygenase CSD:Cysteine Sulfinate Decarboxylase MPST:3-Mercaptopyruvate Sulfurtransferase SO:Sulfite Oxidase
Pyruvate
Sulfite (SO42-)
Figure 2 Hydrogen sulfide—biosynthesis and metabolism. SO32- and SO42- cannot be used to measure hydrogen sulfide production as they can also be formed from direct oxidation of L-cysteine with cysteine deoxygenase. Compiled and adapted from references [5,6,17–19].
centration [23]. This vasorelaxant effect seems to involve K+ channel conductance, particularly K+ATP channel but not other K+ channel (e.g., KCa or KV) [24]. Exogenously applied, as well as endogenous, H2S was shown to increase K+ATP channel currents, causing hyperpolarization (leading to closure of voltage-dependent Ca2+ channel and decreasing intracellular Ca2+, thereby causing vasodilation [25]), and dramatically improves the open probability (OP) of K+ATP channel from 0.53 to 2.67 in rat vascular smooth muscle cells [26]. Although the majority of the studies reporting the involvement of K+ATP channel in mediating H2S action was done in vascular tissue, they are still relevant because (i) K+ATP channels play a functional role in penile resistance arteries [27]; and (ii) K+ATP channels are physiologically important in modulating corporal smooth muscle tone (K+ATP channel in human CC has been characterized [28], please see Figure 3) and could serve as targets for neurotransmitters [29]. In fact, glibenclamide (a K+ATP channel blocker), which has been reported to inhibit NaHS-induced vasorelaxation, was also shown to inhibit NaHS-induced relaxation in preJ Sex Med 2011;8:1853–1864
contracted human CC. The activity of K+ATP channel in CC smooth muscle is quite low in the absence of endogenous neurotransmitter-induced relaxation at physiological membrane potential (40–50 mV) with OP of approximately <0.01, but this OP can be increased by as much as 40¥ by pinacidil, a K+ATP channel opener [30]. Despite their apparent quiescence during flaccidity, physiologically relevant stimulus can increase the channel’s activity. The ability of H2S to open K+ATP channel (as demonstrated in human CC through the use of glibenclamide) and potentially increase its activity from its low basal activity makes H2S an attractive molecular target for the treatment of ED. In fact, prostaglandin E1 (which has been known to elevate cAMP through protein kinase A [PKA] and K+ATP channel [27]) is one of the effective treatments for ED [9]. However, no information is currently available in the literature regarding the exact mechanism with which H2S opens the K+ATP channel, i.e., whether it involves direct interaction with K+ATP channel proteins or other possible mediators. It is pertinent here to note that H2S-induced vasorelaxation is only
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Figure 3 (A and B) How K+ATP channels modulate corporal smooth muscle tone [29].
partially blocked by glibenclamide (at 10 mM—the appropriate concentration for K+ATP specificity [24]), implying that other K+ATP-independent mechanism(s) are probably involved. In the aortic tissue, it was reported that H2S’s relaxant activity can also be mediated via Cl-/HCO3- channels and metabolic inhibition [31], but whether this applies to CC smooth muscle is still unclear at this stage. H2S as Potential Modulator of RhoA/Rho-Kinase Contractile Mechanism
With respect to H2S’s effect in reducing contractility, there is evidence that it may interfere with RhoA/Rho-kinase signaling because it more readily relaxed the cavernosum, which was precontracted with agents that relied strongly on this contractile mechanism, e.g., endothelin-1 and U46619 (stable thromboxane A2 analogue), than with phenylephrine [15]. While Rho-kinase phosphorylates the regulatory subunit of MLC phosphatase (myosin phosphatase target subunit 1
[MYPT1]) [32], thereby enhancing contractile response at constant intracellular Ca2+ concentration (a process also known as Ca2+ sensitization), inhibition of this pathway by Y27632, a known Rho-kinase inhibitor, can essentially reverse the contraction—giving rise to relaxation [33]. Further studies are required to understand the underlying mechanism of H2S’s interference in this pathway; however, this knowledge is intriguing for several reasons. First, RhoA/Rho-kinase signaling has been associated with ED related to aging and diabetes [34,35]. Second, there has been evidence that NO produced by eNOS facilitates the inhibition of the RhoA/Rho-kinase pathway in mice [36]. Considering that H2S and NO are two closely related gasotransmitters, it is possible that H2S’s interference with this pathway is mediated through a complex cross-talk with NO. H2S as an Inhibitor of Superoxide Formation
One of the known etiologies of ED is increased superoxide (O2-) formation in the cavernosum J Sex Med 2011;8:1853–1864
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Factors associated with ED e.g. TxAz, hypoxia, cytokines, angiotensin II
NADPH oxidase
PKA/cAK
Hydrogen sulfide (H2S)
KATP channel (one of PKA substrates)
Maxi Kchannel Nitric oxide (NO)
Superoxide anion (O2-)
Oxygen (O2)
Hyperpolarization Reactive nitrogen species (e.g.ONOO)
PDE5 expression
“Quenching” of NO
cGMP
[intracellular Ca2+]
Corpus cavernosum relaxation
NO drive
Erection
Figure 4 How hydrogen sulfide may inhibit superoxide formation and how this is pro-erectile in effect. (+) denotes stimulation, whereas (-) denotes inhibition. Dotted lines represent possible hypothetical pathways that have not been proven. ED = erectile dysfunction; TxA = thromboxane A2; NADPH = nicotinamide adenine dinucleotide phosphate; cAMP = cyclic adenosine monophosphate; PKA = protein kinase A; cAK = cyclic AMP-dependent protein kinase; PDE5 = phosphodiesterase type 5; cGMP = cyclic guanosine monophosphate.
[37]. Superoxide anions can react with NO to form reactive oxygen species, e.g., peroxynitrite (ONOO), which can not only cause alteration in vascular tone and tissue injury, but can also reduce the bioavailability of NO, therefore interfering with the “NO drive” and the erectile capacity [38]. A major intravascular source of superoxide in CC smooth muscle is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which reduces oxygen to superoxide. The resultant superoxide (produced by NADPH oxidase) also upregulates PDE5 expression, leading to detumescence (Figure 4). Indeed, many factors associated with ED increased NADPH oxidase expression, e.g., cytokines, thromboxane A2, angiotensin II, and hypoxia [39,40]. There is now evidence that H2S can inhibit the expression and activity of NADPH oxidase in pulmonary arterial endothelial cells [41] and human vascular smooth muscle cells J Sex Med 2011;8:1853–1864
(hVSMCs) [42]. In the rabbit CC, H2S, and ACS6 (sildenafil with H2S-donating moiety) were shown to be potent inhibitors of tumor necrosis factor (TNF)a-induced superoxide formation and upregulation of p47phox (a subunit of the NADPH oxidase complex) [43]. Therefore, it appears that by blocking the upregulation of NADPH oxidase’s subunit in the presence of stimulus such as TNFa, H2S may block the formation of superoxide to facilitate an erection. The concentration at which H2S exerts this inhibitory effect appears to be much lower than what causes relaxation, suggesting that H2S’s potential lies not only in its acute effect on erection but also in its longer-term effect in controlling the expression of proteins that are usually upregulated in ED (e.g., NADPH oxidase and PDE5). This is especially beneficial because identifying the therapeutic window for H2S is going to be a challenge given that its vasorelaxant
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Hydrogen Sulfide in Erectile Function effect at higher dose may also affect blood pressure. Although Shukla et al. showed that ACS6 was equipotent with sildenafil alone in relaxing pre-contracted rabbit CC [43], it was not shown how much H2S was released per 1 M of ACS6. The effective concentration of H2S released is of importance because while H2S can cause relaxation, it can also bring about contraction at lower dose, and therefore, the result from this study does not necessarily contradict earlier findings on H2S’s relaxant effects. An important point to note is that H2S only affects the superoxide level in cells that have been induced by TNFa, suggesting thereby its pathophysiological role in ED. The mechanism underlying H2S’s inhibitory effect on p47phox and superoxide is still unclear, but it seems to involve AC/PKA activation [43]. Whether this inhibition acts at the transcriptional or translational level is still unknown, but PKA has been known to phosphorylate other mediators/protein kinases that can influence transcription and translation [44]. H2S has also been shown to increase cAMP level, at least in hVSMCs [42], and some primary neuronal cultures. For a summary of the effects of H2S and its mechanism of action, please refer to Table 1. Possible Interaction Between H2S and Testosterone
Bucci et al. reported that testosterone increased the production of H2S from L-cysteine in rat aortic tissue and that this effect is blocked by PAG and BCA [45]. In the same study, PAG and BCA were also found to inhibit testosterone-induced relaxation. Interestingly, the testosterone-induced relaxation was also blocked by glibenclamide (a striking coincidence because H2S’s vasorelaxant effect is also inhibited by glibenclamide). It therefore appears that testosterone-induced relaxation in vascular tissue involved the synthesis of H2S, which then activated K+ATP channels. While this direct relationship has not been demonstrated in CC, Yildiz et al. showed that testosterone-induced relaxation in human CC involved the opening of K+ATP channels [46]. It is not far-fetched to suggest that the H2S pathway may also be implicated in testosterone’s action in CC. If testosterone can modulate H2S production, the decline in testosterone level with aging may also affect H2S biosynthesis. Given the mounting evidence of H2S’s pro-erectile effects and the fact that aging has been closely tied to ED, further implication for the H2S pathway in the pathophysiology of
ED appears apparent. Therefore, studies on H2S’s functional/molecular characteristics and mode of action should take into account the possible changes that come with aging in the body’s internal milieu. Possible Interaction Between H2S and NO— The Cross-Talk
There is evidence of a “cross-talk” between NO and H2S, but while the idea is intriguing, considering the importance of NO pathway in erectile physiology, the exact nature of the interaction has been proven to be difficult to characterize accurately. Recent studies show that H2S can inhibit NO production in vascular tissues by (i) decreasing eNOS expression at transcriptional level; (ii) blocking eNOS activity [23] in a process that seems to involve K+ATP channel opening [47] and/or interference with tetrahydrobiopterin (the cofactor of NOS) binding [48]; and (iii) modulating NOS substrate availability, e.g., reducing L-arginine uptake by downregulating its transporter [47]. Whether H2S can affect the NO pathway downstream, e.g., by altering K+Ca or cGMP’s sensitivity to NO, has not been explored so far. However, NO also modulates endogenous production of H2S [20]. As such, NO has been shown to increase CSE activity [20], possibly by acting directly on CSE because it contains 12 cysteines that are potential substrates for nitrosylation or possibly by modulating CSE substrates’ availability, considering that NO has been shown to stimulate cysteine (one of CSE substrates) uptake [49]. Furthermore, NO can also upregulate CSE expression at transcriptional level [24]. However, in terms of relaxant effect at the level of smooth muscle cells, H2S was reported to have either additive/synergistic [50] or antagonistic [51] effect with NO. Most of the studies that attempted to investigate the cross-talk between H2S and NO are done in the vascular system; there is currently very limited information of this cross-talk on smooth muscle cells and absolutely no information available on the penile tissue, in particular. Hence, it would be premature at this junction to be specific about the cross-talk between H2S and NO, and its role in erectile physiology—because the effects seen in other systems may be organ-specific. Conclusion
Since H2S was first discovered to be synthesized in human tissues a decade ago, it has generated J Sex Med 2011;8:1853–1864
J Sex Med 2011;8:1853–1864 [31] 2008
1. Vasorelaxant effect of H2S seems to be dependent on the contracting agent used but independent of prostaglandin/NO synthesis. 2. H2S’s vasorelaxant effect seems to be more potent at low O2. → Hypothesis: this was because of H2S competing with O2 at the level of cytochrome c. 3. Hypothesis: H2S stimulates cell to switch to anaerobic glycolysis, leading to a drop in ATP and pH, and promoting an increase in K+ATP currents, which subsequently brings about relaxation. 1. H2S’s vasorelaxant effect is more pronounced in aortic rings pre-contracted with epinephrine than with K-Krebs solution. 2. In absence of H2S’s concentration–response curve shifted left. 3. H2S-induced relaxation was blocked by Cl-/HCO3channel inhibitor, but not by atropine, lidocaine, or indomethacin. 4. ATP level dropped in aortic rings after administration of H2S, HCN, and 2,4-DNP at equivalent dose.
• Organ bath study on epinephrine-pre-contracted aortic rings • ATP measurement
Model: thoracic aorta of male Wistrar rats Modulator target: Cl-/HCO3- exchanger Secondary messenger: not studied
[26] 2005
1. Exogenous H2S increased KATP channel currents. This is inhibited by glibenclamide. 2. PAG and BCA inhibited whole-cell KATP channel currents. 3. H2S-induced KATP channel currents were unaffected by 8-Br-cGMP.
• Whole-cell patch clamp technique
Model: Single VSMC from mesenteric arteries of male Sprague Dawley rats Modulator target: KATP channels Secondary messenger: cGMP not implicated
1. Exogenous H2S increased whole-cell K+ATP channel currents and hyperpolarized membrane potential of rat VSMC. 2. H2S increased single-channel activity of K+ATP channel by increasing single K+ATP channel open probability. 3. Direct effects of H2S on K+ATP channel probably are not mediated by cGMP pathway.
1. HCC can produce H2S. This was inhibited by PAG and AOAA CBS and CSE’s mRNA, and protein was detected in HCC. 2. NaHS and L-cysteine relaxed HCC in an endothelium-independent manner. NaHS relaxed to a greater degree; HCC pre-contracted with U46619 and h-ET1 than with PE. 3. PAG and AOAA increased HCC’s contractile response to electrical stimulation.
• Zinc Acetate/NNDPD method to quantify H2S production in HCC • Real-time PCR and IHC for CBS and CSE expression/ localization • Organ bath study on pre-contracted HCC
Model: human corpora cavernosa (HCC) Modulator target: KATP channels, RhoA/ Rho-kinase pathway Secondary messenger: not studied
[24] 2001
[15] 2009
1. H2S’s hypotensive effect likely to be mediated through the relaxation of resistance blood vessels through KATP channel opening. 2. H2S’s mechanism of action involves KATP but not KCa and Kv channel. 3. Effect of KATP channel openers was reduced in presence of high ATP concentration, highlighting the importance of the effect of cells’ ATP metabolism on H2S action as KATP channel opener.
1. H2S-induced ↓ in b.p. in rats was mimicked by pinacidil and reduced by glibenclamide pretreatment. 2. H2S-induced vasorelaxation not affected by iberiotoxin or 4-aminopyridine, but inhibited by glibenclamide. 3. H2S and pinacidil increased KATP channel currents and caused cell membrane hyperpolarization. The increase in H2S-induced KATP channel currents was reduced by 3 mM ATP.
• Patch clamp technique to record both K+ATP channel currents • Organ bath study on PE-pre-contracted aortic rings
Model: rings of thoracic aorta from male Sprague Dawley rats Modulator target: KATP channels Secondary messenger: not studied
[23] 2007
Reference/year
1. HCC can synthesize H2S from L-cysteine through the action of CBS and CSE, and this is sufficient to cause relaxation. 2. At vascular level, CSE is the predominant enzyme involved in H2S generation. 3. H2S may interfere with RhoA/RhoA-kinase contractile mechanism. 4. L-cysteine/H2S pathway is most likely involved in maintenance of basal tone in penis.
1. Relaxant effect of H2S involves both K ATP channel-dependent and independent mechanism in rat aorta
1. NaHS caused vasoconstriction at low concentrations and relaxation at high concentrations. Glibenclamide pretreatment enhanced contractile activity of NaHS but inhibited relaxant activity of NaHS in rat aorta.
+
• Organ bath study on aortic rings pre-contracted with phenylephrine (PE)
Remarks
Model: aorta of Wistrar rats Modulator target: K+ATP channels Secondary messenger: not studied
Study finding
Method
Summary table of H2S effects and its possible mechanism(s) of action
Model/system
Table 1
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[41] 2008
1. NaHS inhibited TNFa-induced superoxide formation and gp91phox in PAEC. → NaHS can block upregulation/activation of NADPH oxidase by blocking the expression of its subunit 2. This effect is mediated by adenylyl. cyclase–cAMP–PKA pathways. However NaHS is a relatively weak stimulator of cAMP formation → direct activation of PKA by NaHS cannot be ruled out. 3. H2S release from ACS6 is important for biological action of ACS6.
1. ACS6 inhibited PDE5 activity. 2. ACS6 and NaHS inhibited TNFa-induced superoxide formation. Combination of NaHS and Sildenafil at suboptimal concentration inhibited superoxide formation, similar ACS6 effect. 3. Inhibitory effects of NaHS on superoxide generation was blocked by PKA inhibitor but not by PKG inhibitor. 4. Inhibitory effects of ACS6 was blocked by both PKA and PKG inhibitors. 5. ACS6 and NaHS increased cAMP release in PAECs. 6. NaHS and ACS6 blocked TNFa-induced gp91phox expression; this effect was reversed by PKA inhibitor. PKG inhibitor only reversed the inhibitory effect of ACS6. 7. ACS6 that has been depleted off its H2S had no effect on superoxide formation.
NaHS = sodium hydrosulfide; b.p. = blood pressure; NNDPD = N-dimethyl-p-phenylenediamine sulphate; PCR = polymerase chain reaction; IHC = immunohistochemistry; CBS = cystathionine b-synthase; CSE = cystathionine g-lyase; PAG = propargylglycine; AOAA = aminooxyacetic acid; VSMC = vascular smooth muscle cell; cGMP = cyclic guanosine monophosphate; BCA = b-cyano-L-alanine; ICP = intracavernosal pressure; HCN = hydrogen cyanide; DNP = dinitrophenol; CC = corpus cavernosum; cAMP = cyclic adenosine monophosphate; ODQ = 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; NADPH = nicotinamide adenine dinucleotide phosphate; PDE5 = phosphodiesterase type 5; PKA = protein kinase A; BSO = buthionine sulphoximine.
• Superoxide formation measurement • Measurement of PDE5 activity using two-step isotopic procedure • Western blot measurement of cAMP concentration • Note: synthesis of ACS6 is detailed in this article
Model: pulmonary arterial endothelial cells (PAECs) obtained from white landrace male pigs Modulator target: NADPH oxidase; PDE5 Secondary messenger: cAMP/PKA
[43] 2009
1. NaHS, sildenafil citrate, and ACS6 relaxed PE-pre-contracted rabbit CC and inhibited superoxide formation induced by TNFa, U46619, and 8-IPF2a. 2. ACS6 inhibited p47phox and PDE5 expression after incubation with TNFa. 3. Inhibitory action of ACS6 on TNFa-induced superoxide formation and p47phox expression were blocked by PKA and PKG inhibitors, but the inhibitory action of sildenafil was only blocked with PKG but not PKA inhibitors. 4. BSO decreased cavernosal cGMP and cAMP. ACS6 and sildenafil increased this BSO-induced decrease in cGMP and cAMP.
1. H2S-donating moiety does not diminish the capacity of sildenafil to relax CC smooth muscle. 2. ACS6, sildenafil, and NaHS are all potent inhibitors of superoxide formation. 3. ACS6 has dual effect superior to sildenafil/NaHS alone. 4. The H2S-donating moiety of ACS6 exerts inhibited superoxide formation and p47phox expression through activation of PKA.
• Organ bath study • Superoxide release measurement Western blot measurement of concentration of cGMP and cAMP
Model: male New Zealand white rabbits; male Wistrar rats (for in vivo studies) Modulator target: NADPH oxidase; PDE5 Secondary messenger: PKA of cAMP messenger system
[14] 2007
• Rabbit CC expresses functional H2S-synthesizing enzyme. • H2S-mediated relaxation is partially dependent on cAMP system. • H2S is probably involved in maintenance of basal tone.
• L-cysteine was converted to H2S by rabbit CC homogenates. NaHS relaxed rabbit CC. • MDL 12,300A (adenylate cyclase inhibitor), but not ODQ (soluble guanylyl cyclase inhibitor), inhibited NaHS-induced relaxation. • AOAA, BCA, and PAG had no effect on nitrergic relaxation but accentuated contraction slightly.
• Organ bath study
[13] 2006
This is the first literature evidence detailing pro-erectile effects of H2S • Exogenous H2S contributes to increase in ICP and blood flow in primates. • Endogenous H2S also seems to play a role in erectile pathway in rats.
• Measurement of ICP • Intracavernosal injection of NaHS increased penile response, penile blood flow, perfusion and penile length in primates in vivo. penile length in vivo • Prior treatment with PAG decreased the amplification of ICP pressure induced by nerve stimulation in rats in vivo.
Model: rabbit CC Modulator target: cAMP pathway Secondary messenger: cAMP (partial involvement)
Model: male nonhuman primates; male Sprague Dawley rats Modulator target: not studied Secondary messenger: not studied
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1862 substantial interest. While H2S has been implicated in a multitude of physiological processes in different systems, it is only recently that it has emerged as a potential endogenous gasomodulator in erectile physiology, being involved in enhancing corporal smooth muscle relaxation and also in suppressing its contractility (coincidentally, it was also found to exert a facilitatory role in female sexual function [52]). H2S’s pro-erectile effect seems to extend beyond its acute relaxant activity in the penis: at the cellular level, H2S also seems to be involved in modulating the level of proteins or factors with anti-erectile effects that are pathophysiological in nature. Further studies are needed to elucidate H2S’s exact mechanism of action, and given its multiple roles in erectile physiology, it will not be surprising if H2S uses more than one pathway in achieving its effects. Evidence in the literature so far points to the involvement of ATP-sensitive potassium channel (KATP) channels, Cl-/HCO3- exchangers, and AC/cAMP/PKA pathway in the mechanism of action of H2S. As to whether they all form components of the same pathway or not also requires further investigation. Evidence of cross-talk between H2S and NO points mainly to two issues: H2S’s ability to inhibit NO production and NO’s ability to upregulate H2S synthesis. It is possible that H2S and NO serve to regulate each other’s production; if this negative feedback system were true, any accumulation of NO is going to enhance H2S’s release, which would then suppress further production of NO or vice versa, ultimately achieving a dynamic “equilibrium” and in essence answering the question of why a biological system produces two agents with almost identical functions. From the perspective of a sex organ like the penis, this kind of negative feedback system—if it exists—is of particular interest because it may be involved in the maintenance of basal contraction and the non-erect state for normal erectile physiology. In vivo studies may throw some light into this complex cross-talk. Looking at the plethora of evidence available in the literature, it is not too presumptuous to suggest that H2S or its analogs may have a potential to be used as a pharmacological agent for the therapeutic management of ED, perhaps as an alternative or an adjunct to existing pharmacotherapy. H2S’s effects, in general, are better characterized in vascular tissues as they are used in the majority of the studies done so far to investigate its mechanism of action. While the result from these studies may give important clues as to what is likely happening in the CC, they need to be interpreted with caution because J Sex Med 2011;8:1853–1864
Liaw et al. there could be some tissue-specific differences in H2S’s mechanism of action.
Acknowledgments
We wish to thank the National Medical Research Council, Singapore for the award of the NMRC project grant R-174-000-104-213 for work related to this review. Corresponding Author: P. Ganesan Adaikan, PhD, DSc, Department of Obstetrics & Gynaecology, Yong Loo Lin School of Medicine, National University Hospital, National University of Singapore, NUHS Tower Block Level 12, 1E Kent Ridge Road, Singapore 119228. Tel: 065-67759240; Fax: 065-67794753; E-mail: [email protected] Conflict of Interest: None.
Statement of Authorship
Category 1 (a) Conception and Design P. Ganesan Adaikan; Balasubramanian Srilatha (b) Acquisition of Data P. Ganesan Adaikan; Balasubramanian Srilatha (c) Analysis and Interpretation of Data NA
Category 2 (a) Drafting the Article Roeswita Leono Liaw (b) Revising It for Intellectual Content Balasubramanian Srilatha; P. Ganesan Adaikan
Category 3 (a) Final Approval of the Completed Article P. Ganesan Adaikan
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1863 28 Insuk SO, Chae MR, Choi JW, Yang DK, Sim JH, Lee SW. Molecular basis and characteristics of KATP channel in human corporal smooth muscle cells. Int J Impot Res 2003;15:258– 66. 29 Christ GJ. K channels as molecular targets for the treatment of erectile dysfunction. J Androl 2002;23:S10–9. 30 Lee SW, Wang HZ, Christ GJ. Characterization of ATPsensitive potassium channels in human corporal smooth muscle cells. Int J Impot Res 1999;11:179–88. 31 Kiss L, Deitch EA, Szabó C. Hydrogen sulphide decreases adenosine triphosphate levels in aortic rings and leads to vasorelaxation via metabolic inhibition. Life Sci 2008;83:589– 94. 32 Gratzke C, Angulo J, Chitaley K, Dai YT, Kim NN, Paick JS, Simonsen U, Uckert S, Wespes E, Andersson KE, Lue TF, Stief CG. Anatomy, physiology, and pathophysiology of erectile dysfunction. J Sex Med 2010;7:445–75. 33 Chitaley K, Wingard CJ, Clinton Webb R, Branam H, Stopper VS, Lewis RW, Mills TM. Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway. Nat Med 2001;7:119–22. 34 Jin L, Liu T, Lagoda GA, Champion HC, Bivalacqua TJ, Burnett AL. Elevated RhoA/Rho-kinase activity in the aged rat penis: Mechanism for age-associated erectile dysfunction. FASEB J 2006;20:536–8. 35 Bivalacqua TJ, Champion HC, Usta MF, Cellek S, Chitaley K, Webb RC, Lewis RL, Mills TM, Hellstrom WJ, Kadowitz PJ. RhoA/Rho-kinase suppresses endothelial nitric oxide synthase in the penis: A mechanism for diabetes-associated erectile dysfunction. Proc Natl Acad Sci U S A 2004;101:9121–6. 36 Priviero FB, Jin LM, Ying Z, Teixeira CE, Webb RC. Up-regulation of the RhoA/Rho-kinase signaling pathway in corpus cavernosum from eNOS, but not nNOS, null mice. J Pharmacol Exp Ther 2010;333:184–92, 629. 37 Jeremy JY, Jones RA, Koupparis AJ, Hotston M, Angelini GD, Persad R, Shukla N. Re: Oxidative stress in arteriogenic erectile dysfunction: Prophylactic role of antioxidants. J Urol 2006;175:1175–6. 38 Jones RWA, Rees RW, Minhas S, Ralph D, Persad RA, Jeremy JY. Oxygen free radicals and the penis. Expert Opin Pharmacother 2002;3:889–97. 39 Muzaffar S, Shukla N, Jeremy JY. Nicotinamide adenine dinucleotide phosphate oxidase: A promiscuous therapeutic target for cardiovascular drugs? Trends Cardiovasc Med 2005;15:278–82. 40 Hotston MR, Jeremy JY, Bloor J, Koupparis A, Persad R, Shukla N. Sildenafil inhibits the up-regulation of phosphodiesterase type 5 elicited with nicotine and tumour necrosis factor-alpha in cavernosal vascular smooth muscle cells: Mediation by superoxide. BJU Int 2007;99:612–8. 41 Muzaffar S, Jeremy JY, Sparatore A, Del Soldato P, Angelini GD, Shukla N. H2S-donating sildenafil (ACS6) inhibits superoxide formation and gp91phox expression in arterial endothelial cells: Role of protein kinases A and G. Br J Pharmacol 2008;155:984–94. 42 Muzaffar S, Shukla N, Bond M, Newby AC, Angelini GD, Sparatore A, Del Soldato P, Jeremy JY. Exogenous hydrogen sulphide inhibits superoxide formation, NOX-1 expression and Rac1 activity in human vascular smooth muscle cells. J Vasc Res 2008;45:521–8. 43 Shukla N, Rossoni G, Hotston M, Sparatore A, Del Soldato P, Tazzari V, Persad R, Angelini GD, Jeremy JY. Effect of hydrogen sulphide-donating sildenafil (ACS6) on erectile function and oxidative stress in rabbit isolated corpus cavernosum and in hypertensive rats. BJU Int 2009;103:1522–9. 44 Cho-Chung YS, Nesterova M, Becker KG, Srivastava R, Park YG, Lee YN, Cho YS, Kim MK, Neary C, Cheadle C. Dissecting the circuitry of protein kinase A and cAMP signaling in
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1864 cancer genesis: Antisense, microarray, gene overexpression, and transcription factor decoy. Ann N Y Acad Sci 2002;968:22–36. 45 Bucci M, Mirone V, Di Lorenzo A, Vellecco V, Roviezzo F, Brancaleone V, Ciro I, Cirino G. Hydrogen sulphide is involved in testosterone vascular effect. Eur Urol 2009;56:378–83; Srilatha B, Adaikan PG. Re: Bucci M, Mirone V, Di Lorenzo A et al. Hydrogen sulphide is involved in testosterone vascular effect. Eur Urol 2010;57:e4. 46 Yildiz O, Seyrek M, Irkilata HC, Yildirim I, Tahmaz L, Dayanc M. Testosterone might cause relaxation of human corpus cavernosum by potassium channel opening action. Urology 2009;74:229–32. 47 Geng B, Cui Y, Zhao J, Yu F, Zhu Y, Xu G, Zhang Z, Tang C, Du J. Hydrogen sulfide downregulates the aortic L-arginine/ nitric oxide pathway in rats. Am J Physiol Regul Integr Comp Physiol 2007;293:R1608–18.
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REVIEWS Effects of Hydrogen Sulfide on Erectile Function and Its Possible Mechanism(s) of Action jsm_2279
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Roeswita Leono Liaw, BSc, Balasubramanian Srilatha, MD, PhD, and P. Ganesan Adaikan, PhD, DSc Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Hospital, National University of Singapore, Singapore DOI: 10.1111/j.1743-6109.2011.02279.x
ABSTRACT
Introduction. The current pharmacotherapy for erectile dysfunction (ED) relies significantly on the use of phosphodiesterase type 5 (PDE5) inhibitors, but quite a proportion of ED patients are resistant to this therapy, necessitating a search for an alternative treatment. We reviewed available published data to analyze current evidence of hydrogen sulfide (H2S) as a novel pharmacotherapeutic agent with supportive role in sexual function. Aim. To discuss the role of H2S in erectile function, its possible mechanism of action, and how this knowledge may be exploited for therapeutic use. Methods. Pubmed and Medline search was conducted to identify original articles and reviews. Main Outcome Measures. Data from peer-reviewed publications. Results. Animal studies using different species, including in vitro study done in humans, show evidence of H2S’s pro-erectile effects. The mechanism behind is still unclear, but evidence in literature points out the involvement of K+ATP channel, modulation of protein with anti-erectile effects, as well as involvement of the nitrergic pathway through a complex cross-talk. A new drug called H2S-donating sildenafil (ACS6), which incorporated an H2Sdonating moiety in sildenafil, has been developed. While more studies are still needed, this heralded a new pharmacotherapeutical approach, which is multipronged in nature. Conclusions. Given the mounting evidence of H2S’s role in erectile function and how it appears to achieve its pro-erectile effects through different mechanisms, H2S represents a potentially important treatment alternative or adjunct to PDE5 inhibitors. Liaw RL, Srilatha B, and Adaikan PG. Effects of hydrogen sulfide on erectile function and its possible mechanism(s) of action. J Sex Med 2011;8:1853–1864. Key Words. Hydrogen Sulfide; Nitric Oxide; Gasotransmitter; Corpus Cavernosum; Erectile Function
Introduction
I
n light of recent discoveries, the scientific community’s perspective of hydrogen sulfide (H2S) has undergone a paradigm shift. This toxic pollutant with rotten egg smell is now considered an important biological mediator. H2S is currently placed as a member of the “gasotransmitter” family together with other members, viz., carbon monoxide (CO) and nitric oxide (NO), the term conceived to distinguish their action from the classical signaling molecules [1]. There is a growing number of evidence that H2S can exert a multitude © 2011 International Society for Sexual Medicine
of biological effects, e.g., in inflammation, antinociception, myocardial ischemia–reperfusion, cardiovascular pathology, shock/sepsis, pulmonary hypertension, and diabetes. H2S is a weak acid with a pKa of 6.76 at 37°C, dissociating in aqueous solution as follows: H2S ↔ HS- + H+ ↔ S2- + 2H+. At physiological pH of 7.4, about 18.5% of the total sulfide has been shown to exist as H2S and 81.5% as HS- [2]; however, it is unknown as to which of the H2S moiety (H2S, HS-, or S2-) is the “active” component responsible for the net biological effects observed in different systems [3]. H2S is endogenously J Sex Med 2011;8:1853–1864
1854 produced from L-cysteine by the activity of two enzymes, cystathionine b-synthase (CBS)— existing predominantly in the brain and central nervous system—and cystathionine g-lyase (CSE)— expressed mainly in the liver, and vascular and nonvascular smooth muscles [4]. Systemically, H2S can be oxidized to form sulfate or sulfite in mitochondria; it can be scavenged by methemoglobin or glutathione, and it can also be methylated [5]. A lot of studies have employed the use of CSE inhibitors, viz., DL-propargylglycine (PAG) and b-cyano-Lalanine (BCA), to delineate H2S effects. They are the only pharmacological agents known today to inhibit H2S production by CSE; however, it is important to note that they are of modest potency and selectivity, and have limited membrane permeability [6]. Therefore, the results from such studies have been less useful in investigating the physiological role of H2S compared with NO synthase (NOS) inhibitors such as L-NG-monomethyl arginine in delineating the physiology of the NO pathway. Erectile Physiology
Erectile physiology is an intricate interplay of vascular, neurologic, and endocrine factors. Disturbances to any of these system can give rise to erectile dysfunction (ED), making it a multifactorial disorder that is rather difficult to treat. As it is known thus far, relaxation of the corpus cavernosum (CC) smooth muscle brings about penile erection through an increase in arterial flow and restriction of venous outflow; detumescence is associated with the a-adrenoceptor activity, whereas tumescence is attributed to both cholinergic and nitrergic involvement, with the nitrergic (NO/cyclic guanosine monophosphate [cGMP]) pathway being the primary mediator [7]. As an important prerequisite for erectile process, NO is endogenously produced from L-arginine by the NOS isoforms: endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and inducible nitric oxide synthase (iNOS) [8]. The released NO activates soluble guanylyl cyclase to result in an increased conversion of the guanosine triphosphate to second messenger cGMP (which, through its interaction with cGMP-dependent protein kinases, cyclic nucleotide-gated ion channels, or cyclic nucleotide phosphodiesterases, governs many aspects of cellular function in the body). In the cavernosum, cGMP stimulates the protein kinase G, which in turn initiates phosphorylation of membrane-bound proteins at K+ channels. This will lead to K+ ion outflow into the extracellular J Sex Med 2011;8:1853–1864
Liaw et al. space resulting in hyperpolarization, closure of L-type Ca2+ channels, and decrease in intracellular Ca2+ ion concentration. Together with these changes, there is decreased activation of myosin light chain (MLC) kinase, decreased phosphorylation of MLC chains, and, subsequently, reduced actin– myosin interaction to result in cavernosal relaxation and physiological erection [9] (Figure 1). Pro-Erectile Effects of H2S
Considering the substantial endogenous production of H2S by mammalian tissues and the growing evidence that H2S can act as a regulatory mediator similar to NO, it can be expected that H2S may exert important biophysiological effects in erectile function. In fact, onion—with its complex sulfur compound biochemistry—was considered to be a popular, natural remedy for impotence [12]. In a pilot study by Srilatha et al., it was demonstrated that administration of sodium hydrosulfide hydrate (NaHS.xH2O, a stable H2S donor) increased penile length, perfusion, and intracavernosal pressure (ICP) in vivo in a nonhuman primate model [13]; this serves as the first direct evidence for the pro-erectile effect of H2S in CC. Such facilitatory effects on erectile function have also been observed in other animal models. In particular, administration of PAG decreased ICP in rats [13]. Organ bath study showed that NaHS can dose-dependently relax pre-contracted rabbit and human CC [14,15]. As expected, L-cysteine (H2S precursor and CBS/ CSE substrate) has similar effects to NaHS in increasing ICP, and this effect is blocked by PAG. Adenylyl cyclase (AC) inhibitor MDL 12,330A was shown to block the H2S-induced relaxation of pre-contracted rabbit cavernosum; however, this inhibition was incomplete [14], suggesting that relaxation mediated by H2S is only partially dependent on cAMP pathway, highlighting the presence of other mechanisms contributing to its relaxant effect, possibly via K+ATP channel. Inhibition of H2S production with PAG or aminooxyacetic acid (a CBS inhibitor) is also able to significantly increase electrical field stimulationinduced contraction (associated with detumescence) at different frequencies in both rabbit and human CC [14,15]. Therefore, endogenous H2S’s role is possibly twofold: (i) relaxation of CC smooth muscle; and (ii) inhibition of basal tone in penis. This finding is significant considering that both impaired relaxation and increased contractility can contribute to ED. However, it is still
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Figure 1 (A and B) Relaxation of penile smooth muscle via cyclic guanosine monophosphate (cGMP) pathway [9–11]. NO = nitric oxide; PKA = protein kinase A; PKG = protein kinase G; PLB = phospholipase B; GC = guanylyl cyclase; PLC = phospholipase C; DAG = diacyl glycerol; NE = norepinephrine; ET-1 = endothelin-1; MLC = myosin light chain; IP3 = inositol triphosphate.
B
unclear at this point as to the actual site of action of H2S, i.e., whether it is molecular, cellular, or neurovascular in nature. A lot of the evidence for H2S’s physiological role was based on the observation that it is endogenously produced in tissues that are pertinent to its proposed role as vasorelaxant or gasomodulator. This means that rigorous assessment of methodologies used to measure very small concentrations of this labile gas accurately is important in order to avoid potential artifacts. Unfortunately, unlike NO that can be measured using its stable oxidation products (NO2- and NO3-) [16], H2S has no known stable or specific end product from its biosynthesis (Figure 2). Interestingly, a majority of the studies—using different analytical techniques—reported plasma H2S in the range of 25–80 mM in rat and humans, with few exceptions [3], indicating thereby that the results are likely to be credible. Similarly, H2S production has also been detected in different tissues such as penis, liver, aorta, and ileum in vitro [13,20]. The assay involved supplying the tissue with L-cysteine and pyridoxal5′-phosphate (substrate and cofactor, respectively, for CBS/CSE), and detecting the H2S synthesized by the tissue [21]. Srilatha et al. acknowledged that this assay has a limitation in that it only proves the presence/activity of the enzyme but does not reflect the actual in vivo H2S production, because the
amount of substrate and cofactors available in the tissue is unknown [14]. Even so, the assay has generally succeeded in demonstrating that penile erectile tissue expresses CBS and CSE enzymes. d’Emmanuele di Villa Bianca et al. showed that in the human CC, these enzymes were localized mostly in the smooth muscle (vascular and trabecular) [15]. With an organ bath study, they further demonstrated that the H2S’s relaxant effect seen in different animal models also applies to human CC, and this relaxant effect is indeed endothelium independent (noninvolvement of endothelium was verified through a test of the tissue’s responsiveness to acetylcholine). This finding has a significant implication considering that endothelial dysfunction is a major contributing factor to vascular pathology of the penis leading to ED [22]. This suggests that the novel H2S pathway, by virtue of its lack of dependence on endothelial integrity (which may be compromised in patients with ED), for its production, may aid cavernosal relaxation and complement NO signaling (in particular, with respect to the NO contribution by eNOS) in erectile physiology. H2S as K+ATP Channel Opener
H2S was shown to cause vasoconstriction at lower concentrations but vasodilatation at higher conJ Sex Med 2011;8:1853–1864
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L-Homocysteine + L-Cysteine L-Homocysteine + L-Serine
L-Cysteine + H2S L-Cystathionine + H2O
CBS
Aminoacetic Acid(AOAA) − CBS Inhibitor
H2S + Pyruvate + NH3
CSE
DL-Propargylglycine (PAG) − Irreversible Inhibitor B-Cyano-L-Alanine (BCA) − Reversible Inhibitor
CSE
L-Cysteine + NH3 + α-Ketobutyrate L-Cysteine + H2O L-Cysteine
3-Mercaptopyruvate
Pyruvate
CDO
AAT
L-Cysteine Sulfinate
AAT
CSD
MPST Hypotaurine
Sulfinyl Pyruvate
H2S Taurine
Decomposition Sulfite (SO32-)
SO CBS:Cystathionine β Synthase CSE:Cystathionine γ Lyase AAT:Aspartate Aminotransferase CDO:Cysteine Deoxygenase CSD:Cysteine Sulfinate Decarboxylase MPST:3-Mercaptopyruvate Sulfurtransferase SO:Sulfite Oxidase
Pyruvate
Sulfite (SO42-)
Figure 2 Hydrogen sulfide—biosynthesis and metabolism. SO32- and SO42- cannot be used to measure hydrogen sulfide production as they can also be formed from direct oxidation of L-cysteine with cysteine deoxygenase. Compiled and adapted from references [5,6,17–19].
centration [23]. This vasorelaxant effect seems to involve K+ channel conductance, particularly K+ATP channel but not other K+ channel (e.g., KCa or KV) [24]. Exogenously applied, as well as endogenous, H2S was shown to increase K+ATP channel currents, causing hyperpolarization (leading to closure of voltage-dependent Ca2+ channel and decreasing intracellular Ca2+, thereby causing vasodilation [25]), and dramatically improves the open probability (OP) of K+ATP channel from 0.53 to 2.67 in rat vascular smooth muscle cells [26]. Although the majority of the studies reporting the involvement of K+ATP channel in mediating H2S action was done in vascular tissue, they are still relevant because (i) K+ATP channels play a functional role in penile resistance arteries [27]; and (ii) K+ATP channels are physiologically important in modulating corporal smooth muscle tone (K+ATP channel in human CC has been characterized [28], please see Figure 3) and could serve as targets for neurotransmitters [29]. In fact, glibenclamide (a K+ATP channel blocker), which has been reported to inhibit NaHS-induced vasorelaxation, was also shown to inhibit NaHS-induced relaxation in preJ Sex Med 2011;8:1853–1864
contracted human CC. The activity of K+ATP channel in CC smooth muscle is quite low in the absence of endogenous neurotransmitter-induced relaxation at physiological membrane potential (40–50 mV) with OP of approximately <0.01, but this OP can be increased by as much as 40¥ by pinacidil, a K+ATP channel opener [30]. Despite their apparent quiescence during flaccidity, physiologically relevant stimulus can increase the channel’s activity. The ability of H2S to open K+ATP channel (as demonstrated in human CC through the use of glibenclamide) and potentially increase its activity from its low basal activity makes H2S an attractive molecular target for the treatment of ED. In fact, prostaglandin E1 (which has been known to elevate cAMP through protein kinase A [PKA] and K+ATP channel [27]) is one of the effective treatments for ED [9]. However, no information is currently available in the literature regarding the exact mechanism with which H2S opens the K+ATP channel, i.e., whether it involves direct interaction with K+ATP channel proteins or other possible mediators. It is pertinent here to note that H2S-induced vasorelaxation is only
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Figure 3 (A and B) How K+ATP channels modulate corporal smooth muscle tone [29].
partially blocked by glibenclamide (at 10 mM—the appropriate concentration for K+ATP specificity [24]), implying that other K+ATP-independent mechanism(s) are probably involved. In the aortic tissue, it was reported that H2S’s relaxant activity can also be mediated via Cl-/HCO3- channels and metabolic inhibition [31], but whether this applies to CC smooth muscle is still unclear at this stage. H2S as Potential Modulator of RhoA/Rho-Kinase Contractile Mechanism
With respect to H2S’s effect in reducing contractility, there is evidence that it may interfere with RhoA/Rho-kinase signaling because it more readily relaxed the cavernosum, which was precontracted with agents that relied strongly on this contractile mechanism, e.g., endothelin-1 and U46619 (stable thromboxane A2 analogue), than with phenylephrine [15]. While Rho-kinase phosphorylates the regulatory subunit of MLC phosphatase (myosin phosphatase target subunit 1
[MYPT1]) [32], thereby enhancing contractile response at constant intracellular Ca2+ concentration (a process also known as Ca2+ sensitization), inhibition of this pathway by Y27632, a known Rho-kinase inhibitor, can essentially reverse the contraction—giving rise to relaxation [33]. Further studies are required to understand the underlying mechanism of H2S’s interference in this pathway; however, this knowledge is intriguing for several reasons. First, RhoA/Rho-kinase signaling has been associated with ED related to aging and diabetes [34,35]. Second, there has been evidence that NO produced by eNOS facilitates the inhibition of the RhoA/Rho-kinase pathway in mice [36]. Considering that H2S and NO are two closely related gasotransmitters, it is possible that H2S’s interference with this pathway is mediated through a complex cross-talk with NO. H2S as an Inhibitor of Superoxide Formation
One of the known etiologies of ED is increased superoxide (O2-) formation in the cavernosum J Sex Med 2011;8:1853–1864
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Factors associated with ED e.g. TxAz, hypoxia, cytokines, angiotensin II
NADPH oxidase
PKA/cAK
Hydrogen sulfide (H2S)
KATP channel (one of PKA substrates)
Maxi Kchannel Nitric oxide (NO)
Superoxide anion (O2-)
Oxygen (O2)
Hyperpolarization Reactive nitrogen species (e.g.ONOO)
PDE5 expression
“Quenching” of NO
cGMP
[intracellular Ca2+]
Corpus cavernosum relaxation
NO drive
Erection
Figure 4 How hydrogen sulfide may inhibit superoxide formation and how this is pro-erectile in effect. (+) denotes stimulation, whereas (-) denotes inhibition. Dotted lines represent possible hypothetical pathways that have not been proven. ED = erectile dysfunction; TxA = thromboxane A2; NADPH = nicotinamide adenine dinucleotide phosphate; cAMP = cyclic adenosine monophosphate; PKA = protein kinase A; cAK = cyclic AMP-dependent protein kinase; PDE5 = phosphodiesterase type 5; cGMP = cyclic guanosine monophosphate.
[37]. Superoxide anions can react with NO to form reactive oxygen species, e.g., peroxynitrite (ONOO), which can not only cause alteration in vascular tone and tissue injury, but can also reduce the bioavailability of NO, therefore interfering with the “NO drive” and the erectile capacity [38]. A major intravascular source of superoxide in CC smooth muscle is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which reduces oxygen to superoxide. The resultant superoxide (produced by NADPH oxidase) also upregulates PDE5 expression, leading to detumescence (Figure 4). Indeed, many factors associated with ED increased NADPH oxidase expression, e.g., cytokines, thromboxane A2, angiotensin II, and hypoxia [39,40]. There is now evidence that H2S can inhibit the expression and activity of NADPH oxidase in pulmonary arterial endothelial cells [41] and human vascular smooth muscle cells J Sex Med 2011;8:1853–1864
(hVSMCs) [42]. In the rabbit CC, H2S, and ACS6 (sildenafil with H2S-donating moiety) were shown to be potent inhibitors of tumor necrosis factor (TNF)a-induced superoxide formation and upregulation of p47phox (a subunit of the NADPH oxidase complex) [43]. Therefore, it appears that by blocking the upregulation of NADPH oxidase’s subunit in the presence of stimulus such as TNFa, H2S may block the formation of superoxide to facilitate an erection. The concentration at which H2S exerts this inhibitory effect appears to be much lower than what causes relaxation, suggesting that H2S’s potential lies not only in its acute effect on erection but also in its longer-term effect in controlling the expression of proteins that are usually upregulated in ED (e.g., NADPH oxidase and PDE5). This is especially beneficial because identifying the therapeutic window for H2S is going to be a challenge given that its vasorelaxant
1859
Hydrogen Sulfide in Erectile Function effect at higher dose may also affect blood pressure. Although Shukla et al. showed that ACS6 was equipotent with sildenafil alone in relaxing pre-contracted rabbit CC [43], it was not shown how much H2S was released per 1 M of ACS6. The effective concentration of H2S released is of importance because while H2S can cause relaxation, it can also bring about contraction at lower dose, and therefore, the result from this study does not necessarily contradict earlier findings on H2S’s relaxant effects. An important point to note is that H2S only affects the superoxide level in cells that have been induced by TNFa, suggesting thereby its pathophysiological role in ED. The mechanism underlying H2S’s inhibitory effect on p47phox and superoxide is still unclear, but it seems to involve AC/PKA activation [43]. Whether this inhibition acts at the transcriptional or translational level is still unknown, but PKA has been known to phosphorylate other mediators/protein kinases that can influence transcription and translation [44]. H2S has also been shown to increase cAMP level, at least in hVSMCs [42], and some primary neuronal cultures. For a summary of the effects of H2S and its mechanism of action, please refer to Table 1. Possible Interaction Between H2S and Testosterone
Bucci et al. reported that testosterone increased the production of H2S from L-cysteine in rat aortic tissue and that this effect is blocked by PAG and BCA [45]. In the same study, PAG and BCA were also found to inhibit testosterone-induced relaxation. Interestingly, the testosterone-induced relaxation was also blocked by glibenclamide (a striking coincidence because H2S’s vasorelaxant effect is also inhibited by glibenclamide). It therefore appears that testosterone-induced relaxation in vascular tissue involved the synthesis of H2S, which then activated K+ATP channels. While this direct relationship has not been demonstrated in CC, Yildiz et al. showed that testosterone-induced relaxation in human CC involved the opening of K+ATP channels [46]. It is not far-fetched to suggest that the H2S pathway may also be implicated in testosterone’s action in CC. If testosterone can modulate H2S production, the decline in testosterone level with aging may also affect H2S biosynthesis. Given the mounting evidence of H2S’s pro-erectile effects and the fact that aging has been closely tied to ED, further implication for the H2S pathway in the pathophysiology of
ED appears apparent. Therefore, studies on H2S’s functional/molecular characteristics and mode of action should take into account the possible changes that come with aging in the body’s internal milieu. Possible Interaction Between H2S and NO— The Cross-Talk
There is evidence of a “cross-talk” between NO and H2S, but while the idea is intriguing, considering the importance of NO pathway in erectile physiology, the exact nature of the interaction has been proven to be difficult to characterize accurately. Recent studies show that H2S can inhibit NO production in vascular tissues by (i) decreasing eNOS expression at transcriptional level; (ii) blocking eNOS activity [23] in a process that seems to involve K+ATP channel opening [47] and/or interference with tetrahydrobiopterin (the cofactor of NOS) binding [48]; and (iii) modulating NOS substrate availability, e.g., reducing L-arginine uptake by downregulating its transporter [47]. Whether H2S can affect the NO pathway downstream, e.g., by altering K+Ca or cGMP’s sensitivity to NO, has not been explored so far. However, NO also modulates endogenous production of H2S [20]. As such, NO has been shown to increase CSE activity [20], possibly by acting directly on CSE because it contains 12 cysteines that are potential substrates for nitrosylation or possibly by modulating CSE substrates’ availability, considering that NO has been shown to stimulate cysteine (one of CSE substrates) uptake [49]. Furthermore, NO can also upregulate CSE expression at transcriptional level [24]. However, in terms of relaxant effect at the level of smooth muscle cells, H2S was reported to have either additive/synergistic [50] or antagonistic [51] effect with NO. Most of the studies that attempted to investigate the cross-talk between H2S and NO are done in the vascular system; there is currently very limited information of this cross-talk on smooth muscle cells and absolutely no information available on the penile tissue, in particular. Hence, it would be premature at this junction to be specific about the cross-talk between H2S and NO, and its role in erectile physiology—because the effects seen in other systems may be organ-specific. Conclusion
Since H2S was first discovered to be synthesized in human tissues a decade ago, it has generated J Sex Med 2011;8:1853–1864
J Sex Med 2011;8:1853–1864 [31] 2008
1. Vasorelaxant effect of H2S seems to be dependent on the contracting agent used but independent of prostaglandin/NO synthesis. 2. H2S’s vasorelaxant effect seems to be more potent at low O2. → Hypothesis: this was because of H2S competing with O2 at the level of cytochrome c. 3. Hypothesis: H2S stimulates cell to switch to anaerobic glycolysis, leading to a drop in ATP and pH, and promoting an increase in K+ATP currents, which subsequently brings about relaxation. 1. H2S’s vasorelaxant effect is more pronounced in aortic rings pre-contracted with epinephrine than with K-Krebs solution. 2. In absence of H2S’s concentration–response curve shifted left. 3. H2S-induced relaxation was blocked by Cl-/HCO3channel inhibitor, but not by atropine, lidocaine, or indomethacin. 4. ATP level dropped in aortic rings after administration of H2S, HCN, and 2,4-DNP at equivalent dose.
• Organ bath study on epinephrine-pre-contracted aortic rings • ATP measurement
Model: thoracic aorta of male Wistrar rats Modulator target: Cl-/HCO3- exchanger Secondary messenger: not studied
[26] 2005
1. Exogenous H2S increased KATP channel currents. This is inhibited by glibenclamide. 2. PAG and BCA inhibited whole-cell KATP channel currents. 3. H2S-induced KATP channel currents were unaffected by 8-Br-cGMP.
• Whole-cell patch clamp technique
Model: Single VSMC from mesenteric arteries of male Sprague Dawley rats Modulator target: KATP channels Secondary messenger: cGMP not implicated
1. Exogenous H2S increased whole-cell K+ATP channel currents and hyperpolarized membrane potential of rat VSMC. 2. H2S increased single-channel activity of K+ATP channel by increasing single K+ATP channel open probability. 3. Direct effects of H2S on K+ATP channel probably are not mediated by cGMP pathway.
1. HCC can produce H2S. This was inhibited by PAG and AOAA CBS and CSE’s mRNA, and protein was detected in HCC. 2. NaHS and L-cysteine relaxed HCC in an endothelium-independent manner. NaHS relaxed to a greater degree; HCC pre-contracted with U46619 and h-ET1 than with PE. 3. PAG and AOAA increased HCC’s contractile response to electrical stimulation.
• Zinc Acetate/NNDPD method to quantify H2S production in HCC • Real-time PCR and IHC for CBS and CSE expression/ localization • Organ bath study on pre-contracted HCC
Model: human corpora cavernosa (HCC) Modulator target: KATP channels, RhoA/ Rho-kinase pathway Secondary messenger: not studied
[24] 2001
[15] 2009
1. H2S’s hypotensive effect likely to be mediated through the relaxation of resistance blood vessels through KATP channel opening. 2. H2S’s mechanism of action involves KATP but not KCa and Kv channel. 3. Effect of KATP channel openers was reduced in presence of high ATP concentration, highlighting the importance of the effect of cells’ ATP metabolism on H2S action as KATP channel opener.
1. H2S-induced ↓ in b.p. in rats was mimicked by pinacidil and reduced by glibenclamide pretreatment. 2. H2S-induced vasorelaxation not affected by iberiotoxin or 4-aminopyridine, but inhibited by glibenclamide. 3. H2S and pinacidil increased KATP channel currents and caused cell membrane hyperpolarization. The increase in H2S-induced KATP channel currents was reduced by 3 mM ATP.
• Patch clamp technique to record both K+ATP channel currents • Organ bath study on PE-pre-contracted aortic rings
Model: rings of thoracic aorta from male Sprague Dawley rats Modulator target: KATP channels Secondary messenger: not studied
[23] 2007
Reference/year
1. HCC can synthesize H2S from L-cysteine through the action of CBS and CSE, and this is sufficient to cause relaxation. 2. At vascular level, CSE is the predominant enzyme involved in H2S generation. 3. H2S may interfere with RhoA/RhoA-kinase contractile mechanism. 4. L-cysteine/H2S pathway is most likely involved in maintenance of basal tone in penis.
1. Relaxant effect of H2S involves both K ATP channel-dependent and independent mechanism in rat aorta
1. NaHS caused vasoconstriction at low concentrations and relaxation at high concentrations. Glibenclamide pretreatment enhanced contractile activity of NaHS but inhibited relaxant activity of NaHS in rat aorta.
+
• Organ bath study on aortic rings pre-contracted with phenylephrine (PE)
Remarks
Model: aorta of Wistrar rats Modulator target: K+ATP channels Secondary messenger: not studied
Study finding
Method
Summary table of H2S effects and its possible mechanism(s) of action
Model/system
Table 1
1860 Liaw et al.
[41] 2008
1. NaHS inhibited TNFa-induced superoxide formation and gp91phox in PAEC. → NaHS can block upregulation/activation of NADPH oxidase by blocking the expression of its subunit 2. This effect is mediated by adenylyl. cyclase–cAMP–PKA pathways. However NaHS is a relatively weak stimulator of cAMP formation → direct activation of PKA by NaHS cannot be ruled out. 3. H2S release from ACS6 is important for biological action of ACS6.
1. ACS6 inhibited PDE5 activity. 2. ACS6 and NaHS inhibited TNFa-induced superoxide formation. Combination of NaHS and Sildenafil at suboptimal concentration inhibited superoxide formation, similar ACS6 effect. 3. Inhibitory effects of NaHS on superoxide generation was blocked by PKA inhibitor but not by PKG inhibitor. 4. Inhibitory effects of ACS6 was blocked by both PKA and PKG inhibitors. 5. ACS6 and NaHS increased cAMP release in PAECs. 6. NaHS and ACS6 blocked TNFa-induced gp91phox expression; this effect was reversed by PKA inhibitor. PKG inhibitor only reversed the inhibitory effect of ACS6. 7. ACS6 that has been depleted off its H2S had no effect on superoxide formation.
NaHS = sodium hydrosulfide; b.p. = blood pressure; NNDPD = N-dimethyl-p-phenylenediamine sulphate; PCR = polymerase chain reaction; IHC = immunohistochemistry; CBS = cystathionine b-synthase; CSE = cystathionine g-lyase; PAG = propargylglycine; AOAA = aminooxyacetic acid; VSMC = vascular smooth muscle cell; cGMP = cyclic guanosine monophosphate; BCA = b-cyano-L-alanine; ICP = intracavernosal pressure; HCN = hydrogen cyanide; DNP = dinitrophenol; CC = corpus cavernosum; cAMP = cyclic adenosine monophosphate; ODQ = 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; NADPH = nicotinamide adenine dinucleotide phosphate; PDE5 = phosphodiesterase type 5; PKA = protein kinase A; BSO = buthionine sulphoximine.
• Superoxide formation measurement • Measurement of PDE5 activity using two-step isotopic procedure • Western blot measurement of cAMP concentration • Note: synthesis of ACS6 is detailed in this article
Model: pulmonary arterial endothelial cells (PAECs) obtained from white landrace male pigs Modulator target: NADPH oxidase; PDE5 Secondary messenger: cAMP/PKA
[43] 2009
1. NaHS, sildenafil citrate, and ACS6 relaxed PE-pre-contracted rabbit CC and inhibited superoxide formation induced by TNFa, U46619, and 8-IPF2a. 2. ACS6 inhibited p47phox and PDE5 expression after incubation with TNFa. 3. Inhibitory action of ACS6 on TNFa-induced superoxide formation and p47phox expression were blocked by PKA and PKG inhibitors, but the inhibitory action of sildenafil was only blocked with PKG but not PKA inhibitors. 4. BSO decreased cavernosal cGMP and cAMP. ACS6 and sildenafil increased this BSO-induced decrease in cGMP and cAMP.
1. H2S-donating moiety does not diminish the capacity of sildenafil to relax CC smooth muscle. 2. ACS6, sildenafil, and NaHS are all potent inhibitors of superoxide formation. 3. ACS6 has dual effect superior to sildenafil/NaHS alone. 4. The H2S-donating moiety of ACS6 exerts inhibited superoxide formation and p47phox expression through activation of PKA.
• Organ bath study • Superoxide release measurement Western blot measurement of concentration of cGMP and cAMP
Model: male New Zealand white rabbits; male Wistrar rats (for in vivo studies) Modulator target: NADPH oxidase; PDE5 Secondary messenger: PKA of cAMP messenger system
[14] 2007
• Rabbit CC expresses functional H2S-synthesizing enzyme. • H2S-mediated relaxation is partially dependent on cAMP system. • H2S is probably involved in maintenance of basal tone.
• L-cysteine was converted to H2S by rabbit CC homogenates. NaHS relaxed rabbit CC. • MDL 12,300A (adenylate cyclase inhibitor), but not ODQ (soluble guanylyl cyclase inhibitor), inhibited NaHS-induced relaxation. • AOAA, BCA, and PAG had no effect on nitrergic relaxation but accentuated contraction slightly.
• Organ bath study
[13] 2006
This is the first literature evidence detailing pro-erectile effects of H2S • Exogenous H2S contributes to increase in ICP and blood flow in primates. • Endogenous H2S also seems to play a role in erectile pathway in rats.
• Measurement of ICP • Intracavernosal injection of NaHS increased penile response, penile blood flow, perfusion and penile length in primates in vivo. penile length in vivo • Prior treatment with PAG decreased the amplification of ICP pressure induced by nerve stimulation in rats in vivo.
Model: rabbit CC Modulator target: cAMP pathway Secondary messenger: cAMP (partial involvement)
Model: male nonhuman primates; male Sprague Dawley rats Modulator target: not studied Secondary messenger: not studied
Hydrogen Sulfide in Erectile Function 1861
J Sex Med 2011;8:1853–1864
1862 substantial interest. While H2S has been implicated in a multitude of physiological processes in different systems, it is only recently that it has emerged as a potential endogenous gasomodulator in erectile physiology, being involved in enhancing corporal smooth muscle relaxation and also in suppressing its contractility (coincidentally, it was also found to exert a facilitatory role in female sexual function [52]). H2S’s pro-erectile effect seems to extend beyond its acute relaxant activity in the penis: at the cellular level, H2S also seems to be involved in modulating the level of proteins or factors with anti-erectile effects that are pathophysiological in nature. Further studies are needed to elucidate H2S’s exact mechanism of action, and given its multiple roles in erectile physiology, it will not be surprising if H2S uses more than one pathway in achieving its effects. Evidence in the literature so far points to the involvement of ATP-sensitive potassium channel (KATP) channels, Cl-/HCO3- exchangers, and AC/cAMP/PKA pathway in the mechanism of action of H2S. As to whether they all form components of the same pathway or not also requires further investigation. Evidence of cross-talk between H2S and NO points mainly to two issues: H2S’s ability to inhibit NO production and NO’s ability to upregulate H2S synthesis. It is possible that H2S and NO serve to regulate each other’s production; if this negative feedback system were true, any accumulation of NO is going to enhance H2S’s release, which would then suppress further production of NO or vice versa, ultimately achieving a dynamic “equilibrium” and in essence answering the question of why a biological system produces two agents with almost identical functions. From the perspective of a sex organ like the penis, this kind of negative feedback system—if it exists—is of particular interest because it may be involved in the maintenance of basal contraction and the non-erect state for normal erectile physiology. In vivo studies may throw some light into this complex cross-talk. Looking at the plethora of evidence available in the literature, it is not too presumptuous to suggest that H2S or its analogs may have a potential to be used as a pharmacological agent for the therapeutic management of ED, perhaps as an alternative or an adjunct to existing pharmacotherapy. H2S’s effects, in general, are better characterized in vascular tissues as they are used in the majority of the studies done so far to investigate its mechanism of action. While the result from these studies may give important clues as to what is likely happening in the CC, they need to be interpreted with caution because J Sex Med 2011;8:1853–1864
Liaw et al. there could be some tissue-specific differences in H2S’s mechanism of action.
Acknowledgments
We wish to thank the National Medical Research Council, Singapore for the award of the NMRC project grant R-174-000-104-213 for work related to this review. Corresponding Author: P. Ganesan Adaikan, PhD, DSc, Department of Obstetrics & Gynaecology, Yong Loo Lin School of Medicine, National University Hospital, National University of Singapore, NUHS Tower Block Level 12, 1E Kent Ridge Road, Singapore 119228. Tel: 065-67759240; Fax: 065-67794753; E-mail: [email protected] Conflict of Interest: None.
Statement of Authorship
Category 1 (a) Conception and Design P. Ganesan Adaikan; Balasubramanian Srilatha (b) Acquisition of Data P. Ganesan Adaikan; Balasubramanian Srilatha (c) Analysis and Interpretation of Data NA
Category 2 (a) Drafting the Article Roeswita Leono Liaw (b) Revising It for Intellectual Content Balasubramanian Srilatha; P. Ganesan Adaikan
Category 3 (a) Final Approval of the Completed Article P. Ganesan Adaikan
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