Endogenous hydrogen sulfide insufficiency as a predictor of sexual dysfunction in aging rats

Abstract

Objective: Our earlier studies showed that endogenous hydrogen sulfide (H₂S) pathway contributed significantly to erectile function. In this study, we tested the hypothesis that age-dependent changes in the bioavailability of H₂S increased the risk of erectile dysfunction (ED). Methods: Young, adult (3-month) and older (18-month) male Sprague-Dawley rats (n = 6−8/group) were treated daily with sodium hydrosulfide hydrate (NaHS), DL-propargylglycine, sildenafil or l-NAME for 10 weeks. Subsequent to cavernous nerve electrical stimulation, intracavernosal pressure (ICP) responses were determined, and the samples were collected and processed for hormonal (plasma) and gaseous parameters (plasma and erectile corpus cavernosum [CC]) using standard assay protocols. Results: Aging significantly reduced the ICP response (35.9 ± 2.0 mmHg vs. 45.2 ± 1.9 mmHg in young controls), which was countered by NaHS (53.5 ± 6.0) or sildenafil (52.8 ± 9.8) treatment. In these rats, marked increments to testosterone (T) or estradiol resulted from NaHS supplementation. Similar to age-dependent decline in NO, the plasma and CC level of H₂S was significantly lower in senescent rats when compared with young animals (p < 0.05). Conclusion: Our results confirm that ED with aging may be linked to a derangement in the H₂S pathway accompanied by low T levels. It is likely that a pharmacologic intervention delivering H₂S will provide additional benefits to sexual function from an improved T milieu.

Keywords::

• Aging
• erectile dysfunction
• gasotransmitter
• hydrogen sulfide
• testosterone

Introduction

Hydrogen sulfide (H₂S), a member in the family of gasotransmitters besides nitric oxide (NO) and carbon monoxide, is endogenously produced from l-cysteine [1]. The reaction is catalyzed by cystathionine-β synthase (CBS) or cystathionine-γ lyase (CSE), using pyridoxal-5′ phosphate as cofactor [2]. Indeed, CBS is the primary H₂S-forming enzyme in the central nervous system (CNS), while in smooth muscle and vascular tissues, varying quantities of CSE and CBS are found [3]. Another enzyme, 3-mercaptopyruvate sulfurtransferase also contributes to H₂S biosynthesis from the precursor cysteine in the presence of α-ketoglutarate, particularly in vascular and nervous tissues [4]. As an important signaling molecule, H₂S has been known to play a significant role in the physiological responses of the CNS, vascular or nonvascular and visceral systems [5]. Essentially, H₂S is eliminated from the body through the kidneys as conjugated or free sulfate − catabolism being facilitated by thiol S-methyltransferase to methanethiol and dimethylsulfide [3]. Other enzymatic and nonenzymatic conversions include oxidation to thiosulfate by superoxide dismutase and mitochondrial respiratory electron transport [2], respectively.

The erectile process in the highly vascularized penile corpus cavernosum (CC) is mediated through a complex interplay of the autonomic nervous system and several well-regulated pathways of neurotransmitters and modulators [6]. It is also known that erectile dysfunction (ED), as a peripheral organic disorder, can serve as a predictor for cardiovascular diseases [7]. Together with the identified role of H₂S in the erectile response (vasodilatory and relaxant effects in the CC mediated through NO–cyclic 3′,5′-guanosine monophosphate (cGMP), cyclic adenosine monophosphate and K⁺ adenosine triphosphate pathways) [8–10], it is presently implicated as a modulator of the normal sexual function in either gender [11].

Aging in man is associated with testosterone (T) decline and an absolute and/or relative imbalance with estradiol (E₂); this can contribute to subjective and objective impairments in sexual functioning [12,13]. The present understanding is that in addition to its known cross-talk with the NO–cGMP pathway, T may also influence H₂S production by modulating the level of S-adenosyl-l-methionine, a CBS activator in the brain [14]. T has also been implicated in vasorelaxation of the rat aortic strips, which was mediated through its ability to increase H₂S release from the known precursor, l-cysteine [15]. It appears, therefore, that if T modulated the endogenous H₂S biophysiology, an age-dependent decline in T levels may impair the H₂S pathway and effects. Given the mounting evidence for H₂S pro-erectile mechanism of action and the fact that aging has been closely tied to ED, it follows that H₂S derangement is likely to play a role in the loss of erectile capacity in the aging male. Therefore, in this study using appropriate animal models, we tested the hypothesis that age-dependent changes in the bioavailability of H₂S increased the risk of erectile impairment in the elderly.

Methods

The study design used the following treatment groups given sodium hydrosulfide hydrate (NaHS.xH₂O-stable H₂S donor), dl-propargylglycine (PAG, inhibitor of CSE), sildenafil citrate (classical PDE-5 inhibitor) and l-n (G)-nitroarginine methyl ester (l-NAME, nonspecific NOS inhibitor) to evaluate the in vivo effect of H₂S and NO modulation with aging. Essentially, the intracavernous pressure (ICP) response (an objective index of penile erection) was correlated to H₂S and NO levels in plasma and CC tissue and contrasted with the plasma hormone levels in the younger (adult) and aged male rats.

Animal treatment

Young, adult- (3-month) and older- (18-month) male Sprague-Dawley (SD) rats (n = 6–8/group) were treated daily with NaHS (freshly prepared; Sigma, St. Louis, MO, USA; 0.8 mg/kg, i.p), or PAG (Sigma; 50 mg/kg, i.p), or sildenafil citrate (Pfizer, Singapore; 0.7 mg/kg, oral) or l-NAME (Sigma; 30 mg/kg, oral) [16] for 10 weeks. Vehicle-treated age-matched SD rats served as controls. All the experimental protocols were executed in accordance with the international guidelines for animal research under due approval from Institutional Animal Care and Use Committee (IACUC), National University of Singapore.

ICP evaluation

At the end of 10-weeks’ treatment, the animals were anesthetized with pentobarbitone sodium (Sanofi, Hannover, Germany, 45 mg/kg i.p with 10 mg/kg i.v supplement when necessary). Left external jugular vein and right carotid artery were cannulated with PE-10 and -50 tubing, respectively, for saline infusion and mean arterial pressure (MAP) monitoring. Tracheotomy was done to aid ventilation. Through a lower median incision, lateral prostate was exposed and the cavernous nerve was carefully isolated and suspended through platinum wire electrode for electrical stimulation. A 27-G needle filled with 250 i.u./mL of heparinized saline and connected to PE-10 tubing was inserted into the penile crus to capture the changes in the ICP during nerve stimulation. Optimal increase from baseline response was initially obtained at the stimulation criteria of 2 V and 20 Hz for 30 s, the ICP and MAP changes were measured and recorded with the respective pressure transducers (Ugo Basile, Comerio VA, Italy) and MacLab analogue-digital converter (AD instruments, Sydney, Australia) [8]. The changes in erectile response were expressed as ICP normalized to MAP (in view of the possible drug effects on blood pressure), which is calculated ICP/MAP ratio − a more objective parameter of the erectile capacity compared with ICP given alone.

Blood biochemical parameters

Plasma samples were collected from the carotid artery at the end of pressure recordings, centrifuged at 3000 rpm at 4°C for 10 min and stored at −70°C until assays for T, E₂, H₂S and NO levels were done.

T and E₂ assays

T and E₂ levels were measured using automated solid-phase competitive chemiluminescent immunoassay (Immulite 1000 analyzer; Siemens Medical Solution Diagnostics, Los Angeles, CA, USA). All analyses were performed using an aliquot of 100-μL plasma sample. The analytical protocol of these methods was within the specifications of the analyzers in duplicate, and the treatment group’s samples were run concurrently [13].

Assay of plasma H₂S concentration

Thawed plasma was centrifuged at 3000 rpm for 5 min at 4°C. Aliquot of 100 μL of the supernatant was added to a tube containing 0.25 mL of 1% zinc acetate and 0.4 mL of distilled water. This reaction mixture was incubated for 10 min at room temperature with 133 μL of 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride (NNDPD) in 7.2 M HCl and 133 μL of 30 mM FeCl₃ in 1.2 M HCl. The reaction was stopped and the samples were deproteinated by an addition of 0.25 mL of 10% TCA. The samples were then centrifuged at 14,000 rpm for 10 min at 4°C. The absorbance of the supernatant was read spectrophotometrically at 670 nm. The plasma H₂S concentration was calculated from a standard curve of NaHS (3.125–250 μM) and expressed in μM [17].

Assay of cavernous tissue H₂S biosynthesis

Tissue level of H₂S was measured in the harvested CC samples using a modified protocol [18]. After thawing, 0.1 g of cavernous tissue was homogenized in 2 mL of 100 mM ice-cold potassium phosphate buffer (pH 7.4). Centrifugation of the homogenate was done at 14,000 rpm for 30 min at 4°C and the supernatant was assayed. A reaction mixture with total volume of 500 μL − containing 430 μL of tissue homogenate, 20 μL of 10 mM l-cysteine, 20 μL of 2 mM pyridoxal-5-phosphate and 30 μL of saline − was set up in a tightly sealed eppendorf tube. Subsequently, 133 μL of NNDPD in 7.2 M HCl and 133 μL FeCl₃ in 1.2 M HCl were added. The mixture was transferred to a water bath at 37°C to initiate the reaction, and after 30 min, 250 μL of 1% zinc acetate was added to trap the evolved H₂S. Concurrent addition of 250 μL of 10% TCA deproteinated the sample and the reaction was terminated. Absorbance of the final reaction mixture was measured spectrophotometrically at 670 nm using a 96-well microplate reader (Tecan Systems, San Jose, CA, USA). The tissue H₂S biosynthesis was calculated from a standard curve of NaHS (3.125–250 μM) and expressed in μmol/g/hr, and the protein concentration was measured using the Nanodrop.

Assay of plasma and cavernous tissue NO concentration

The plasma and CC tissue levels of NO were measured as described by Tracey et al. [19]. After thawing, the plasma was centrifuged at 14,000 rpm for 30 min and the supernatant was diluted five times with phosphate buffer (pH 7.4). About 0.1 g of CC tissue was homogenized in ice-cold phosphate buffer, centrifuged at 14,000 rpm for 30 min and the supernatant was prepared for the assay protocol as follows. Aliquot of 80 μL of plasma/tissue homogenate was incubated (for 30 min at 37°C, protected from light) together with 20 μL of master mix containing: nitrate reductase (2U/mL) and cofactors β-nicotinamide adenine dinucleotide phosphate (NADPH) (2 mM) and flavin adenine dinucleotide (FAD) (0.1 mM). Then, 200 μL of Griess reagent containing 1:1 (v/v) solution of 0.2% w/v N-1 napthylethylenediamine and 2% w/v sulfanilamide in 5% v/v phosphoric acid was added to the samples and incubated for 15 min at 37°C. The nitrite formed a chromophoric diazo compound from reaction with the Greiss reagents, which was then read at 540 nm using the spectrophotometer (Tecan Systems) as described earlier. Using the standard curve of NaNO₂ (0–87.5 μM), the nitrite concentration in plasma and cavernosal tissue was extrapolated and expressed as μM (plasma) and μmol/g (tissue), respectively.

Statistical analysis

The significant differences between the sets of data from treated and control groups were determined by two-way analysis of variance and t-test for multiple comparisons using SPSS software (SPSS Inc., Chicago, IL). Results were expressed as mean ± standard error of mean from six to eight animals, and a p value of less than 0.05 was considered to be statistically significant.

Results

During in vivo experimentation, an overall increase (including baseline pressure) in ICP response of 54.1 ± 2.4 mmHg was obtained in the younger control rats at the standard stimulation criteria used in the study. Aging per se significantly reduced the net ICP (pressure recorded at nerve stimulation − baseline ICP prior to stimulation), which was 35.9 ± 2.0 mmHg in the older rats (18 month) as opposed to 45.1 ± 1.9 mmHg in the young controls (3 month). This age-dependent reduction in cavernosal perfusion pressure was not evident in groups treated with NaHS (53.5 ± 6.0 mmHg) or sildenafil (52.8 ± 9.8 mmHg), as further confirmed by significant increments in the calculated pressure (ICP/MAP) ratio. Noteworthy changes in the arterial pressure included a marked increase in the l-NAME-treated group: 138.3 ± 9.6 mmHg (younger rats) and 155.8 ± 9.2 mmHg (older rats) and a similar trend (increased MAP) in the PAG-administered rats. Well conceivably, PAG or l-NAME pre-treatment attenuated the ICP response to significantly lower values, 34.8 ± 2.2 mmHg and 28.3 ± 3.2 mmHg, respectively, with concomitant decreases in the calculated ICP/MAP ratio (Figure 1).

Figure 1.  ICP/MAP ratio in the young and aged male rat groups (n = 6–8) treated with sodium hydrosulfide hydrate (NaHS), dl-propargylglycine (PAG), sildenafil or l-NAME compared with the calculated ratio in untreated control animals. p < 0.05.

Aging was associated with a significant decline in plasma total T level in the rat model studied. In the young and older rats, weighing 328.6 ± 5.4 g and 919.4 ± 43.7 g, respectively, significant increments to total T and E₂ followed daily NaHS administration. For instance, the measured T and E₂ levels increased by 28.8% and 43.2%, respectively, in the NaHS-treated young adults. Similarly, the increases for the older rats were 57.9% (T) and 2.5-folds for E₂, at the end of 10-weeks’ NaHS treatment (Table I). No significant changes were seen in the tested hormonal parameters with PAG or l-NAME administration.

Table I.  Summary of results from experimental parameters in the young (3-month) and aged (18-month) male rat groups (n = 6–8).

Experimental parameters	Young rats (3 mo)	Aged rats (18 mo)	Comments
Baseline ICP (mm Hg)
Prior to cavernous nerver stimulation	9 ± 0.3	7.4 ± 0.6
With cavernous nerve stimulation	54.3 ± 1.7	43.3 ± 3.1*	p < 0.05 (decrease)
ICP with cavernous nerve stimulation
Control	45.2 ± 1.9	35.9 ± 2.0*	p < 0.05 (decrease)
After treatment with NaHS	53.5 ± 6.0*	49.4 ± 3.6*	p < 0.05 (increase)
After treatment with Sildenafil	52.8 ± 9.8*	52.9 ± 2.1*	p < 0.05 (increase)
After treatment with PAG	34.8 ± 2.2*	32.6 ± 2.2*	p < 0.05 (decrease)
After treatment with l-NAME	28.3 ± 3.2*	30.1 ± 0.8*	p < 0.05 (decrease)
MAP (mm Hg)
Control	115.2 ± 6.7	12.8 ± 9.0	–
After treatment with NaHS	95.8 ± 6.9*	111.5 ± 15.5	p < 0.05 (decrease)
After treatment with Sildenafil	119.9 ± 4.4	113.6 ± 16.0	–
After treatment with PAG	115.5 ± 5.9	127.1 ± 5.0	–
After treatment with l-NAME	138.3 ± 9.6*	155.8 ± 9.2*	p < 0.05 (increase)
Plasma total T (ng/ml)
Control	2.36 ± 0.6	1.14 ± 0.2	–
After treatment with NaHS	3.0 ± 0.2*	1.8 ± 0.5*	p < 0.05 (increase)
After treatment with Sildenafil	2.4 ± 0.4	1.1 ± 0.6	–
After treatment with PAG	2.2 ± 0.1	0.9 ± 0.1	–
After treatment with l-NAME	2.1 ± 0.2	0.9 ± 0.3	–
Plasma E2 (pg/ml)
Control	30.2 ± 3.4	24.3 ± 3.8	–
After treatment with NaHS	53.2 ± 3.7*	61.0 ± 0.1*	p < 0.05 (increase)
After treatment with Sildenafil	28.2 ± 1.4	23.0 ± 2.6	–
After treatment with PAG	34.6 ± 2.1	24.7 ± 2.2	–
After treatment with l-NAME	32.7 ± 3.5	22.5 ± 2.1	–
Plasma H2S (µM)
Control	24.7 ± 1.1	15.4 ± 1.0*	p < 0.05 (decrease)
After treatment with NaHS	31.8 ± 2.1*	22.2 ± 1.7*	p < 0.05 (increase)
After treatment with Sildenafil	21.2 ± 0.7	19.7 ± 2.1	–
After treatment with PAG	20.4 ± 0.3	14.3 ± 1.6	–
After treatment with l-NAME	22.6 ± 0.6	15.3 ± 1.2	–
Plasma NO (µM)
Control	25.0 ± 2.8	17.0 ± 2.7*	p < 0.05 (decrease)
After treatment with NaHS	27.8 ± 3.2	18.4 ± 0.9	–
After treatment with Sildenafil	29.4 ± 3.4	20.4 ± 0.9	–
After treatment with PAG	23.6 ± 1.7	17.9 ± 1.1	–
After treatment with l-NAME	19.8 ± 2.1*	14.4 ± 0.8	p < 0.05 (decrease)
CC tissue H2S (µM/g)
Control	4.3 ± 0.0	2.7 ± 0.4*	p < 0.05 (decrease)
After treatment with NaHS	4.9 ± 0.5	3.1 ± 0.7	–
After treatment with Sildenafil	2.8 ± 0.3	2.4 ± 0.3	–
After treatment with PAG	1.4 ± 0.1*	2.5 ± 0.8	p < 0.05 (decrease)
After treatment with l-NAME	2.7 ± 0.2	2.1 ± 0.2	–
CC tissue NO (µM/g)
Control	8.1 ± 3.3	3.7 ± 0.4*	p < 0.05 (decrease)
After treatment with NaHS	5.9 ± 0.7	3.5 ± 0.6	–
After treatment with Sildenafil	8.7 ± 1.7	3.4 ± 0.1	–
After treatment with PAG	6.6 ± 0.7	3.5 ± 0.8	–
After treatment with l-NAME	3.7 ± 1.9	2.6 ± 0.4	–

*p < 0.05.

In the presence of l-cysteine (10 mM) and pyridoxal-5-phosphate (2 mM), detectable amount of H₂S was biosynthesized in all the tested CC samples. Together with the spectrophotometrically derived data for the plasma H₂S/NO and tissue NO (Griess reaction) in the respective samples, a statistical comparison was made to evaluate the possible changes in the older rats. Interestingly, both NO and H₂S showed a well-defined decline in older rats compared with young animals. As such, the measured values for plasma and tissue NO were 68% and 45.7% of the levels in young rats; they seem to coincide with the significant decrease (76.23% and 62.8%) in plasma and tissue levels of H₂S in this study, p < 0.05 (Table I).

Discussion

The results from this study indicate that endogenous H₂S may also function as a signaling marker of erectile impairment with aging. The quantified plasma level of H₂S in our aged rats (15.4 μM) compared with reported values of 28.9 ± 1.4 μM/38.2 ± 2.07 μM in the younger SD and Wistar rat models, respectively [20], shows a significant downward trend in H₂S biosynthesis with advancing age. Furthermore, this finding correlates well with the more-than-a-third reduction in CC tissue levels of H₂S in our older rats (2.7 μmol/g protein vs. 4.3 μmol/g in younger control). Interestingly, the estimated plasma levels of NO in the young and aged rats, while somewhat conforming to the concentration in human plasma (25–31 μM) [21], also echoes the declining trend in NO release in the cavernosum seen together with aging and ED changes [22]. Taken together, this study gives an important insight into the gasotransmitter − H₂S and NO deficiencies behind the etiopathogenesis of age-related ED − albeit in the absence of the established nature of their cross-regulation/mutual contribution at this stage. Also, it is logical to postulate that endogenous H₂S deficiency may be a factor, marker or even predictor of age-related ED, independent of NO decline.

Thus far, the ICP response in rats has been considered to provide the quantitative index of the central and peripheral integrative mechanisms of penile erection and also to categorize the effects of any investigated novel pathway or mechanism for extrapolating to human response [23]. The decrease in this neuronal NO-mediated response with age in this study (seen as significant ICP reduction compared with young controls), together with low circulating and tissue levels of H₂S, characterizes the objective change that can be correlated with the importance of H₂S in maintaining male sexual health over the life span. Of noteworthy mention is the improved ICP response with NaHS pretreatment, which accounts for the likely direct and facilitatory effect (of H₂S) on the neuronal NO levels; in this context, H₂S may function as a co-modulator of NO synthesis and/or release. An indirect confirmation is the negative influence of PAG toward the cavernous pressure rise, coupled with the lack of possibility of any independent effect on the NO pathway [5]. Classically, cGMP-targeted PDE5-Is prevented the hydrolysis of cGMP while allowing its accumulation and the resultant NO–cGMP-mediated relaxation [24]. Indeed, the nitrergic pressure response in the sildenafil group was statistically significant; recent findings also account for an advantageous pharmacologic combination of sildenafil with H₂S [25]. However, the observed blockade by the NOS inhibitor− l-NAME, which hindered the guanylate cyclase activation and cGMP production − is expected to be incomplete since l-NAME is unlikely to have any effect on the H₂S-mediated relaxant response [3,5]. As such, correlations between the amplitude of ICP response to nerve electrical stimulation and the estimated MAP have been demonstrated in a number of animal models previously [23]. Apparently, a decrease in systemic pressure resulted in the reduced perfusion pressure of the cavernosal sinusoids [26]. However, in this study, the ICP changes to NaHS treatment were unrelated to systemic pressure fluctuations, which did not reach a statistical significance in the drug treatment group(s). Therefore, the obtained data not only support the previous identity of the pro-erectile nature of H₂S-mediated effects [8,10] but also stress the need for measures to tackle the age-dependent changes in this newly emerging pathway, in order to conserve the sexual quality of life.

As an important endocrine change associated with aging and ED [27], the kinetics of circulating T levels was also evaluated in the study groups, and interestingly, our results indicate a significantly higher T (and E₂) levels in the aged rats exposed to daily NaHS. It is pertinent here to mention that measured total T is not absolutely reflective of its functional level and where possible, estimating the concentrations of bioavailable or free T would be useful for better clinical translation. As for E₂ measurement, the technical constraints were minimized by keeping the sampling time constant for all experimental groups and by using an in-house standardized protocol with sensitivity to detect low E₂ levels in the male test samples. Our postulated mechanisms, therefore, for the observed elevation in plasma total T and E₂ in the NaHS-treated rat groups are as follows. (1) H₂S may promote the synthesis of GnRH (LHRH) at the hypothalamus by a central effect, which can in turn increase signals for LH release in the pituitary; an earlier study points to the role of H₂S in activating the hypothalamo–pituitary–adrenal axis [28]. (2) Peripherally, H₂S may facilitate the testicular biosynthesis of T; a corollary here is the accentuated H₂S release in T-incubated vascular tissues [15]. (3) The E₂ rise may stem from an increased aromatization of T or due to a hitherto unknown paracrine effect of H₂S [29,30]. Available literature indicates that the testicular and adrenal production of T declines with aging. Therefore, its interdependence to endogenous H₂S pathway, if any, is worthy of exploration. Furthermore, the concurrent increase in T following NaHS administration would be expected to improve the compromised sexual functioning in the aging male through a positive modulation of both the central and peripheral components. Although ED can simply co-exist with T decline, the altered balance of E₂ with T, seen as lower T/E₂ ratio in the older rats both at baseline and after treatment, conforms to its negative impact on sexual function [13]. Notwithstanding the limitations in extrapolating results from the animal study to human male, it is still possible to derive some insights into the complexity of gasotransmitter(s) − and hormone(s) − mediated sexual biology for future research.

Conclusions

The findings indicate that T may be involved in the possible crosstalk between H₂S and NO in the eventual mediation of the nitrergic neurotransmission. Treatment-induced improvement in ICP, as the objective criterion of erectile function, also shows that H₂S exhibited a similar facilitatory role as NO with its insufficiency in aging, a likely predictor for ED in the elderly. Further investigations of the relevant interaction of T with H₂S in the cavernosum or other bodily systems will establish the basis for the endocrine cross-talk at the cellular and molecular levels. Presently, our findings throw some light on the age-related ED in terms of the potential mechanism(s) as well as approaches to prevent or treat the disorder in the elderly. In the event that ED in the aging male is linked to a derangement in the endogenous H₂S pathway or bioavailability, a pharmacologic intervention delivering H₂S will be expected to provide significant improvement of sexual function, supplemented by a positive T milieu.

Acknowledgments

Authors wish to acknowledge the award of the research grant R-174-000-104-213 by the National Medical Research Council (NMRC), Ministry of Health, Singapore for the conduct of this work. The technical help provided by Ms. Jameelah SM and Ms. Liaw RL is also gratefully acknowledged.

Declaration of Interest: The authors report no conflicts of interest.