What’s known on the subject? and What does the study add?

One of the most frequent diabetic complications is urinary bladder dysfunction, which is associated with increased bladder capacity and impaired smooth muscle contractions. It has been shown that smooth muscle tissues isolated from diabetic rats have altered contractile responses. However, the mechanisms responsible for altered smooth muscle contractility remain poorly understood. Increased production of reactive oxygen species (ROS) plays a role in bladder disorders, and H₂S has a cytoprotective effect that might have a scavenging effect on ROS. It is important to determine the effect of H₂S on impaired detrusor contractility caused by ROS in order to develop new treatment principles.

Introduction

Urinary bladder dysfunction, which is one of the most common diabetic complications, is associated with bladder overactivity, increased bladder capacity, and impaired bladder smooth muscle contraction. The prevalence of dysfunction is between 43% and 87%. It is not life threatening but considerably affects life quality (1). Evaluation of bladder smooth muscle contractility is important for understanding the mechanisms underlying diabetic dysfunction. Alterations in contractile responses have been reported in smooth muscle tissues isolated from diabetic models. Increased carbachol-induced contraction was observed in bladder smooth muscle in streptozotocin-induced diabetic rats (2). Pretreatment of bladders under high glucose (HG) conditions enhanced carbachol-induced contraction in control animals (3). Nobe et al. (4) showed that glucose-dependent enhancement of contraction in the diabetic bladder is involved in the activation of the Rho kinase and calcium-independent PKC pathways. The increased vascular smooth muscle contraction, which was enhanced under HG conditions, was also reported in a type II diabetic mouse model (5,6). However, the mechanisms responsible for altered smooth muscle contractility remain poorly understood.

The involvement of hydrogen sulfide (H₂S) in pathological disorders such as diabetes mellitus has been suggested although its physiological role is still not known. Increased formation of H₂S and expression of endogenous H₂S-synthesizing enzymes, cystathionine g-lyase (CSE) and cystathionine b-synthase (CBS), have been demonstrated in liver and pancreas of streptozotocin (STZ)-induced diabetic rats (7). Inhibition of CSE, a synthase of endogenous H₂S, promotes endothelial cell dysfunction induced by hyperglycemia (8) and reduces H₂S levels in streptozotocin-induced diabetic rats (9). In diabetic mice, treatment with H₂S can restore nitric oxide efficacy and decrease oxidative stress in the mouse aorta (10). However, possible accompanying changes in the functional effects of H₂S have not been enlightened.

The cytoprotective effect of H₂S may also be attributed to its scavenging effect on reactive oxygen species (ROS). ROS are produced in increased concentrations in pathological conditions such as cardiac ischemia, reperfusion or sepsis and cause tissue damage. ROS can alter smooth and striated muscle contraction by affecting many intracellular pathways associated with excitation-contraction coupling. It has been shown that carbachol and potassium-induced contractions reduced in the presence of hydrogen peroxide in rat urinary bladder detrusor muscle (11). In our previous study, H₂S reduced carbachol-induced contraction in the permeabilized guinea pig tenia cecum and that intracellular hydrogen peroxide formation and calcium storage mitochondria are responsible for this response (12). It is also known that superoxide anions reduce the release of calcium by preventing the opening of the calcium channels of the sarcoplastic reticulum in the myocardium (13).

The aim of this study was to investigate the role of H₂S and ROS scavengers in alterations of carbachol-induced detrusor smooth muscle contraction under HG conditions.

Materials and Methods

The study was approved by Hacettepe University Animal Ethics Committee (no: 2023/06-06, date: 23.08.2023). Male New Zealand albino rabbits (4-6 months old) were used in this study.

Tissue Preparation

Rabbits were euthanized with high-dose anesthesia (Ketamine/Xylazine, 50/5 mg/kg, i.p.) and the urinary bladders were isolated. Bladder strips were isolated and mounted in 5 mL3 organ baths containing Krebs’ Henseleit solution under a resting tension of 800 mg. Tissues were equilibrated for 1 h and washed with Krebs’ Henseleit solution every 15 min before each experimental procedure. Isometric changes in tension were recorded using an isometric force transducer (MP 150-Transducer Data Acquisition System; BIOPAC Systems).

Experimental Protocol

Sodium hydrogen sulfide (NaHS) is used as an H₂S donor, and its aqueous solution is introduced directly into the organ bath by an automated pipette. NaHS dissociates to Na⁺ and HS^- in aqueous solution and then HS^- associates with H⁺ to form H₂S (Hosoki et al., 1997).

At the beginning of each experiment, KCl (80 mM)-induced contractions were elicited in bladder strips. After a 30-min washout period, cumulative (10 nM-30 µM) carbachol-induced contraction responses were obtained in bladder strips in the control group and under HG conditions. The HG condition means 30 min incubation of bladder strips in Krebs’ Henseleit solution with, 4.7; MgSO₄, 1.2; CaCl₂, 2.5; KH₂PO₄, 1.2; NaHCO₃, 25.0; glucose, 11.6 and this was gassed with 95% O₂-5% CO₂ at 37°C and pH 7.4. The Krebs’ Henseleit solution with HG contains 44 mM glucose. The cumulative carbachol-induced contraction responses were elicited in the presence of ROS scavengers catalase (1000 U/mL), superoxide dismutase (SOD; 150 U/mL), H₂S donor sodium hydrosulfide (NaHS, 300 µM), H₂S-synthesizing enzyme inhibitors propargylglycine (PAG; 300 mM) and aminooxyacetic acid (AOAA; 1 mM) in control and under HG condition.

Drugs and Solutions

Drugs used were carbamylcholine chloride (carbachol), catalase, SOD, NaHS, PAG and AOAA from Sigma (St. Louis, Missouri). All drugs and solutions were prepared by using distilled water.

Statistical Analysis

Contractions were expressed as a percentage of KCl (80 mM)-induced contraction. Data are represented as mean ± standard error of the mean. Statistical analysis was done by ANOVA/Newman-Keuls and Student’s t-test by using GraphPad Prism9 software. P<0.05 was accepted as statistically significant.

Results

The Effect of High Glucose in Bladder Cumulative Carbachol Contraction

Cumulative contractile responses were elicited with carbachol (10 nM-30 µM) in control group. Bladder strips were incubated with HG (Krebs’ Henseleit solution with 44 mM glucose). The contraction responses were significantly increased under HG conditions compared with the control group (Figure 1).

The Effects of ROS Scavengers Catalase and SOD on Cumulative Carbachol Contraction

Increased carbachol contractile responses under HG were significantly reduced in the presence of hydrogen peroxide (H₂O₂) scavenger catalase (1000 U/mL) and superoxide (O₂^-) scavenger SOD (150 U/mL) (Figure 2). There was no difference in the control group between the absence and presence of catalase or SOD (Table 1).

The Effects of H₂S Donor NaHS on Cumulative Carbachol Contraction

Cumulative carbachol (10 nM-30 µM) contractile responses were obtained in the presence of H₂S donor NaHS (300 mM) in control and under HG conditions. Increased carbachol contractile responses under HG were significantly reduced in the presence of NaHS. Contractile responses were also significantly decreased in the presence of NaHS in control group (Figure 3).

Effects of Combination of H₂S Donor NaHS and ROS Scavenger Catalase or SOD on Cumulative Carbachol Contraction

To investigate the interaction between H₂S and ROS, bladder strips were incubated with NaHS and catalase or NaHS and SOD together. There is no further inbibition in cumulative carbachol (10 nM-30 µM) contractile responses incubated with the combination of NaHS and catalase or NaHS and SOD compared to incubation with NaHS alone in control group. In contrast, further inhibition was observed in carbachol contraction responses under HG conditions when bladder strips were incubated with the combination of NaHS and catalase or NaHS and SOD compared with incubation with NaHS alone (Table 1).

Effects of H₂S-synthesizing enzyme inhibitors PAG and AOAA on cumulative carbachol contraction

Increased carbachol contractile responses under HG conditions did not change in the presence of CSE enzyme inhibitor PAG (300 mM) and CBS enzyme inhibitor AOAA (1 mM) (Figure 4).

Discussion

Many neurological, cardiovascular, urological, gastrointestinal and biochemical complications develop in diabetic patients due to increased glucose. Bladder dysfunction is one of the most common diabetic complications associated with bladder overactivity, increased bladder capacity, and impaired bladder smooth muscle contractile function. Investigating the mechanism of impaired bladder smooth muscle contractility is important for understanding the underlying mechanisms of diabetes complications. Moreover, bladder dysfunction problems, especially those more common in women, lead to social problems as well. Our aim in this study was to elucidate the effect of ROS scavengers and H₂S on impaired contractile functions under HG conditions.

Changes in contractile responses in bladder smooth muscle experimentally induced or incubated with HG have been reported (2-4). In our study; cumulative carbachol contractile responses were significantly increased in the HG group compared with the control group. An increase in carbachol-induced contractile responses was demonstrated in bladder strips isolated from streptozotocin-induced diabetic rats and in tissues pretreated with HG (2,3). Many mechanisms are believed to be responsible for the impaired contractile response due to high glucose. The consequence of hyperglycemic stimulation is the increase of ROS, thus initiating oxidative stress, which causes bladder smooth muscle damage, resulting in impaired bladder function (14-16). Growing evidence has shown that high-glucose-related oxidative stress has an essential role in the remodeling of smooth muscle function that eventually results in the decompensation of the detrusor muscle (17-19). The complications of diabetes are thought to be the result of oxidative stress associated with HG in several tissues (20) including the detrusor smooth muscle (21). It has been reported that repeated stimulation of rabbit bladder strips leads to increased lipid peroxidation and impaired smooth muscle contractility in the ischemic and hypoxic media as well as in the normal physiological media (22).

Samples from rats with STZ-induced type 1 diabetes showed that genes involved in the production or enhancement of ROS and oxidative pathways are upregulated in the bladder of these rats, whereas antioxidative enzymes are downregulated (17-20). Xue et al. (23), 2021 showed that the viability of bladder smooth muscle cells significantly decreased and apoptotic cells increased after HG treatment, at the same time, the SOD level decreased and MDA increased. SOD is an important antioxidant enzyme, and its level decreases, suggesting a decline in antioxidant capacity. MDA is a lipid oxidative damage marker, and its increased level indicates a higher level of oxidative stress (23). In this study, we investigated the effects of ROS scavengers, O^2- radical scavenger SOD and H₂O₂ scavenger catalase, on increased carbachol contractile responses under high glucose. In the present study, we observed that contractile response under HG conditions was decreased in the presence of  catalase and SOD. There was no difference in the control group in the presence of catalase and SOD. Consistent with previous studies, our results indicate that HG causes impaired contractile responses in the detrusor smooth muscle through oxidative stress.

Exogenous H₂S significantly prevented cell death, decreased the generation of apoptotic markers, and suppressed mitochondrial ROS production in rat aortic endothelial cells under HG conditions (24). NaHStreatment can distinctly reduce HG-induced cytotoxicity, apoptosis, oxidative stress, and inflammation in HUVECs (25). In diabetic mice, treatment with H₂S can restore nitric oxide efficacy and decrease oxidative stress in the mouse aorta (10). H₂S may act as a cytoprotective hormone in mouse islets and in MIN6 cells exposed to high glucose, fatty acids, or a mixture of cytotoxic cytokines (26,27). The effects of H₂S on increased carbachol contractile responses under HG conditions were also investigated. According to our results; increased carbachol contractile responses were significantly reduced under HG conditions in the presence of NaHS. Contractile responses were also significantly decreased in the control group in the presence of NaHS. Inhibition was seen in 14% (the control group) and 24% (HG group) ratios. In parallel with previous studies, our results suggest that H₂S reduces the oxidative stress caused by HG and, as a result, improves the impaired contractile responses. Moreover; when the combined effects of H₂S and ROS scavengers were examined under HG conditions, after incubation of NaHS and catalase or NaHS and SOD together, a further reduction 32% and 31% in carbachol contractile responses were obtained, respectively. According to studies examining the possible interaction of H₂S and ROS, Muzaffar et al. (28) showed that H₂S suppressed O^2- production in pulmonary artery endothelial cells. In another study, NaHS infusion decreased O^2- production in hypertensive rats (29).

H₂S-synthesizing enzyme inhibitors PAG and AOAA were examined to support the regulating effect of H₂S on deteriorated contractile responses in bladder smooth muscle under HG conditions. Increased carbachol contractile responses under HG were not change in the presence of CSE enzyme inhibitor PAG and CBS enzyme inhibitor AOAA.

Study Limitations

The fact that our study is an animal study is an important limitation. It is difficult to mimic hyperglycemia in animal tissues. Studies on the effects of H₂S and ROS on human should be conducted to strengthen these findings.

Conclusion

The study identified alterations in contractile responses in bladder smooth muscle under HG conditions. Cumulative carbachol-induced contractile responses were significantly increased in HG-incubated bladder detrusor muscle. These increased contractile responses decreased in the presence of catalase, SOD, and NaHS. Therefore, we can suggest that agonist-induced contractile functions in diabetes are related to H₂S and ROS such as H₂O₂ and O₂^-. In conclusion, these results may become a valuable source for assessing the probable benefits of H₂S donors and ROS scavengers in treating diabetic complications such as urinary bladder dysfunction.

Ethics

Ethics Committee Approval: The study was approved by Hacettepe University Animal Ethics Committee (no: 2023/06-06, date: 23.08.2023).

Informed Consent: Male New Zealand albino rabbits (4-6 months old) were used in this study.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Concept: M.D., N.T.D.K., Design: M.D., N.T.D.K., Data Collection or Processing: M.D., Analysis or Interpretation: M.D., N.T.D.K., Literature Search: M.D., Writing: M.D., N.T.D.K.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declare that they have no relevant financial.