Dimethyl Sulfoxide Enhances Acetylcholine-Induced Contractions in Rat Urinary Bladder Smooth Muscle by Inhibiting Acetylcholinesterase Activities

Abstract

Dimethyl sulfoxide (DMSO) has been used not only as an experimental solvent, but also as a therapeutic agent for interstitial cystitis. The therapeutic effects of DMSO on interstitial cystitis are presumed to involve anti-inflammatory and analgesic effects. However, the effects of DMSO on urinary bladder smooth muscle (UBSM) have not been fully investigated. Thus, in this study, we investigated the effects of DMSO on rat UBSM contractions, and these effects were compared with those of acetone, which has a structure in which the sulfur of DMSO is replaced with carbon. DMSO (0.5–5%) enhanced acetylcholine (ACh)-induced contractions, whereas acetone (3 and 5%) suppressed them. Additionally, DMSO (5%) suppressed carbachol-induced contractions. DMSO/acetone (0.5–5%) inhibited 80 mM KCl-induced contractions in a concentration-dependent manner; however, the inhibitory effects of DMSO were weaker than those of acetone. The enhancing/suppressing effects of DMSO and acetone were almost completely abolished by wash out. DMSO and acetone (0.5–5%) inhibited recombinant human acetylcholinesterase (rhAChE) activity in a concentration-dependent manner. At 0.5 and 1%, the inhibitory effects of DMSO on rhAChE activity were more potent than those of acetone. These findings suggest that DMSO can enhance ACh-induced UBSM contractions and promote urinary bladder motility by inhibiting acetylcholinesterase (AChE), although DMSO also inhibits Ca²⁺ influx-mediated UBSM contractions. In addition, the sulfur atom in DMSO might play an important role in its enhancing effect on ACh-induced contractions by inhibiting AChE, as acetone did not enhance these contractions.

INTRODUCTION

Dimethyl sulfoxide (DMSO) is a commonly used solvent for dissolving many compounds. DMSO is also approved as a therapeutic drug for interstitial cystitis; an intravesical instillation containing 50% DMSO was approved in the United States in 1978 and in Japan in 2021.¹⁾ Although the mechanism of the therapeutic effects of DMSO on interstitial cystitis has not been fully elucidated, the therapeutic efficacy is believed to be due to its anti-inflammatory effects, such as suppression of inflammatory cytokine production at inflammatory sites, and its analgesic effects by suppressing nerve activity.¹⁾ In addition, DMSO has been reported to elicit various pharmacological effects, such as inhibition of platelet aggregation, cell differentiation effects, vasodilation, and smooth muscle (SM) relaxation.²⁾ Therefore, DMSO is assumed to have pharmacological effects on the contractile function of urinary bladder (UB) SMs (UBSMs). However, the pharmacological effects of DMSO on UBSM contractile function have not been fully investigated.^3–5) Here, we found that DMSO enhances acetylcholine (ACh)-induced rat UBSM contractions and inhibits recombinant human acetylcholinesterase (rhAChE) activities. Furthermore, the sulfur in DMSO was suggested to play an important role in its augmentation of ACh-induced contractions by inhibiting acetylcholinesterase (AChE) because acetone, in which the sulfur in DMSO is replaced with carbon (Fig. 1), did not enhance these contractions.

Fig. 1. Structures of Dimethyl Sulfoxide (DMSO) and Acetone

MATERIALS AND METHODS

Animals

Male Wistar rats (age, 8–10 weeks; weight, 170–270 g; Japan SLC, Hamamatsu, Japan) were housed under controlled conditions (21–22 °C, 50 ± 5% relative air humidity) and a fixed 12/12 h light/dark cycle (08:00–20:00), with food and water available ad libitum. This study was approved by the Toho University Animal Care and User Committee (Approval Nos. 18-54-294, 19-55-294, and 22-53-444) and was conducted in accordance with the guidelines of the Laboratory Animal Center of the Faculty of Pharmaceutical Sciences, Toho University.

Assessment of Effects of DMSO and Acetone on ACh- and Carbachol (CCh)-Induced UBSM Contractions

The effects of DMSO and acetone on UBSM contractions were assessed as previously described.⁶⁾ Briefly, isolated rat UBSM strips were equilibrated under a 0.5 g resting tone for 20 min in a 20 mL organ bath containing Locke–Ringer solution of the following composition (mM): NaCl, 154; KCl, 5.6; CaCl₂, 2.2; MgCl₂, 2.1; NaHCO₃, 5.9; and glucose, 2.8. The solution was equilibrated with 95% O₂ and 5% CO₂ at 32 ± 1 °C. The UBSM preparation was contracted using 10⁻⁴ M ACh or CCh at least three times at ≥5 min intervals (preliminary procedures). After a 20 min equilibration period, ACh or CCh was cumulatively added to the bath medium, and the contractile response was recorded twice at 30 min intervals. Following this procedure, ACh or CCh was cumulatively added to the bath medium after 30 min of pre-incubation with DMSO or acetone (0.5–5%). After drug wash out (w/o) and a 30 min equilibration period, ACh or CCh was cumulatively added to the bath medium again. All contractile experiments were performed in the presence of indomethacin (3 × 10⁻⁶ M).

Assessment of Effects of DMSO and Acetone on 80 mM KCl-Induced UBSM Contractions

After the preliminary procedures, an inhibitor cocktail (atropine (10⁻⁶ M), phentolamine (10⁻⁶ M), and propranolol (10⁻⁷ M)) was added to the bath medium. After a 30 min equilibration period, to produce sustained contractions, the strip was contracted with 80 mM KCl solution (containing inhibitor cocktail) of the following composition (mM): NaCl, 79.6; KCl, 80; CaCl₂, 2.2; MgCl₂, 2.1; NaHCO₃, 5.9; and glucose, 2.8. When the contractile response reached a steady state, DMSO or acetone (0.5–5%) was cumulatively added to the bath medium. At the end of the experiment, the UBSM preparations were treated with verapamil (10⁻⁵ M).

When measuring the pre-treatment effects of DMSO and acetone, the strip was contracted with 80 mM KCl solution in the presence of an inhibitor cocktail and in the absence or presence of 5% DMSO or acetone to obtain maximum contraction.

Assessment of the Effects of DMSO and Acetone on rhAChE Activity

rhAChE (≥1000 U/mg protein; C1682) was purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.) and was dissolved in 0.1 M phosphate buffer (pH 7.4) to obtain a 0.1 mg/mL stock solution, which was cryopreserved. The thawed rhAChE stock solution was diluted 200-fold with modified Hanks’ balanced salt solution of the following composition (mM): NaCl, 136.9; KCl, 5.37; CaCl₂, 1.26; MgCl₂, 0.81; Na₂HPO₄, 0.77; KH₂PO₄, 0.44; NaHCO₃, 4.17; and glucose, 5.55.

The effects of DMSO and acetone on rhAChE activity were assessed as previously described.⁷⁾ Briefly, rhAChE (50 µL, 0.025 µg/well) and 5 mM 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB, 140 µL) were added to each well of a 96-well plate and incubated for 10 min at 30 °C. Subsequently, 10–100% DMSO or acetone (10 µL) was added to the corresponding wells and incubated for 30 min at 30 °C.

After incubation, 20 mM acetylthiocholine (ATCh, 10 µL) was added to each well and the absorbance at 415 nm was measured every 1 min for 10 min using an Infinite^® F200 PRO plate reader (Tecan, Männedorf, Switzerland). The absorbance measurements were performed in duplicate.

Drugs

The following drugs were used in this study: DMSO and acetone (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); ACh chloride (Daiichi Sankyo Co., Ltd., Tokyo, Japan); CCh chloride, atropine sulfate, indomethacin, propranolol hydrochloride, and (±)-verapamil (Sigma-Aldrich Co.); phentolamine mesylate (Novartis Pharma K.K., Tokyo, Japan); and ATCh iodide and DTNB (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). All other chemicals were of reagent grade and commercially available.

ATCh and DTNB were dissolved in 0.1 M phosphate buffer (pH 7.4). Indomethacin was dissolved in ethanol to obtain a 10⁻² M solution. All other drugs were prepared as aqueous stock solutions and were diluted with distilled water.

Data Analysis and Statistics

Concentration-response curves for ACh- and CCh-induced contractions were plotted using GraphPad Prism™ (GraphPad Software Inc., San Diego, CA, U.S.A.), as previously described.⁶⁾ AChE inhibition (%) was calculated using GraphPad Prism™, as previously described.⁷⁾ All values are presented as the mean ± standard error of the mean (S.E.M.) for different numbers (n) of experiments. Statistical analyses were performed using GraphPad Prism™ with post-hoc Tukey’s tests and Šidák’s tests after one-way and two-way ANOVA. Statistical significance was set at p < 0.05.

RESULTS

Effects of DMSO and Acetone on ACh- and CCh-Induced UBSM Contractions

DMSO (0.5–3%, Figs. 2Aa–c, Supplementary Table 1) enhanced ACh-induced contractions in a concentration-dependent manner. Five percent DMSO (Fig. 2Ad, Supplementary Fig. 1, Supplementary Table 1) also enhanced ACh-induced contractions, but its effect was similar to that of 3% DMSO. As shown in Fig. 2B and Supplementary Table 2, 5% DMSO significantly suppressed CCh-induced contractions. Acetone (0.5 and 1%, Figs. 2Ca, b, Supplementary Table 3) hardly affected ACh-induced contractions, whereas 3 and 5% acetone (Figs. 2Cc, d, Supplementary Table 3) significantly suppressed ACh-induced contractions in a concentration-dependent manner. Both the enhancing and suppressing effects of DMSO and acetone on ACh- and CCh-induced contractions were almost completely abolished after w/o.

Fig. 2. Effects of DMSO, Acetone, and Their Wash Out (w/o) on the Acetylcholine (ACh)- and Carbachol (CCh)-Induced Contractions in Rat Urinary Bladder Smooth Muscle

A: Effects of DMSO (0.5%, Aa; 1%, Ab; 3%, Ac; 5%, Ad) and its w/o on the concentration–response curves (CRCs) for ACh-induced contractions. B: Effects of DMSO (5%) and its w/o on the CRCs for CCh-induced contractions. C: Effects of acetone (0.5%, Ca; 1%, Cb; 3%, Cc; 5%, Cd) and its w/o on the CRCs for ACh-induced contractions. Data are presented as the mean ± S.E.M. (n = 4). * p < 0.05, ** p < 0.01: control vs. DMSO or acetone; ^# p < 0.05, ^## p < 0.01: DMSO vs. after w/o; ^$ p < 0.05, ^$$ p < 0.01: control vs. after w/o (Tukey's test followed by two-way ANOVA). DMSO, dimethyl sulfoxide.

Effects of DMSO and Acetone on 80 mM KCl-Induced UBSM Contractions

As shown in Figs. 3A and C, post-treatment with 0.5–5% DMSO or acetone inhibited 80 mM KCl-induced contractions in a concentration-dependent manner; however, the inhibitory effect of acetone was stronger than that of DMSO. As shown in Figs. 3B and D, pretreatment with 5% DMSO or acetone significantly inhibited 80 mM KCl-induced contractions, and both inhibitory effects were almost completely abolished after w/o.

Fig. 3. Effects of DMSO, Acetone, and Their Wash Out (w/o) on 80 mM-KCl-Induced Contractions in Rat Urinary Bladder Smooth Muscle

A, C: Effects of post-treatment of DMSO (A) and acetone (C) (0.5–5%) on the 80 mM-KCl-induced sustained contractions. B, D: Effects of pre-treatment of 5% DMSO (B) and acetone (D) and their w/o on the 80 mM-KCl-induced maximum contractions. Data are presented as the mean ± S.E.M. (n = 4). ** p < 0.01: control vs. DMSO or acetone; ^## p < 0.01: after w/o vs. DMSO or acetone (Tukey’s test followed by one-way ANOVA). Ver, verapamil (10⁻⁵ M); DMSO, dimethyl sulfoxide; ACh, acetylcholine.

Effects of DMSO and Acetone on rhAChE Activity

Both DMSO (Fig. 4A) and acetone (Fig. 4B) at 0.5–5% inhibited rhAChE activity in a concentration-dependent manner. At 0.5 and 1%, the inhibitory effects of DMSO on rhAChE activity were significantly more potent than those of acetone. At ≥2%, DMSO and acetone were deemed to inhibit rhAChE activity to the same extent although the inhibitory effects of acetone were slightly stronger than those of DMSO.

Fig. 4. Inhibitory Effects of DMSO (A) and Acetone (B) (0.5–5%) on Recombinant Human Acetylcholinesterase (rhAChE) Activity

Data are presented as the mean ± S.E.M. (n = 8, DMSO; n = 4, acetone). * p < 0.05, ** p < 0.01 vs. acetone (Šidák's test followed by two-way ANOVA). DMSO, dimethyl sulfoxide.

DISCUSSION

DMSO inhibited rhAChE activity and enhanced ACh-induced UBSM contractions. Although DMSO also suppressed Ca²⁺ influx-mediated UBSM contractions, the enhancing effect of 0.5–5% DMSO on ACh-induced contractions by AChE inhibition was considered to be superior to its suppressive effect. These findings suggest that DMSO (0.5–5%) promotes UB motility. In contrast, acetone suppressed ACh-induced UBSM contractions, although it inhibited rhAChE activity. Since acetone suppressed Ca²⁺ influx-mediated UBSM contractions more potently than DMSO, the suppressive effect of acetone was thought to be superior to its potential enhancing effect on ACh-induced contractions by AChE inhibition. Thus, the sulfur in DMSO might play an important role in enhancing ACh-induced contractions.

DMSO (0.5–5%) enhanced ACh-induced UBSM contractions, suggesting that intravesical DMSO instillation promotes UB motility. In fact, frequent urination is reported as a side effect of intravesical DMSO instillation.¹⁾ Smith et al. observed that the contractile function of porcine UB changed when 50% DMSO was added only on the epithelial side of the UB to reproduce the clinical effect of intravesical DMSO instillation in an in vitro system.⁵⁾ Epithelial-side DMSO treatment potentiated ACh- and ATP-mediated contractions induced by electrical field stimulation (EFS),⁵⁾ suggesting that the DMSO concentration used in this study (0.5–5%) is close to the clinical concentration that can reach the UBSM upon intravesical instillation. In addition, the following results suggest that the potentiating effect of DMSO on ACh-induced UBSM contractions is attributed to its AChE-inhibitory effect: 1) DMSO (0.5–5%) inhibited rhAChE; 2) DMSO was reported to inhibit AChE from rat brain, Electrophorus electricus, and human erythrocytes^8–10); and 3) DMSO did not enhance the UBSM contractions induced by CCh, which is not degraded by AChE.

In contrast, DMSO suppressed CCh- and 80 mM KCl-induced UBSM contractions, suggesting that DMSO suppresses Ca²⁺ influx-mediated UBSM contractions. In fact, ≥30% DMSO was reported to potently inhibit rat UBSM contractions induced by EFS, ACh, or 120 mM KCl.³⁾ In addition, 10% DMSO was reported to completely inhibit rabbit UBSM contractions induced by 60 mM KCl or CCh without affecting intracellular Ca²⁺ concentrations, and these effects could involve suppression of phosphorylation.⁴⁾ Therefore, the inhibitory effects of DMSO on UBSM contractions are possibly due to inhibitory effects on kinases such as myosin light chain kinase, and not on extracellular Ca²⁺ influx. However, even the AChE inhibitory effect of 5% DMSO was believed to be stronger than this suppressive effect; as a result, DMSO enhanced ACh-induced UBSM contractions.

In addition, the enhancing/suppressing effects of DMSO were abolished by w/o, suggesting that these effects temporarily occur upon intravesical DMSO instillation. The inhibitory effects of 25 and 10% DMSO on UBSM contractions were also reported to disappear after w/o.³⁾

To determine the structural cause of the AChE inhibitory effects of DMSO, we compared its inhibitory effects with those of acetone, which has a structure in which the sulfur in DMSO is replaced with carbon. The inhibition of AChE was stronger with DMSO than with acetone in the low concentration ranges (0.5 and 1%), suggesting that the sulfur in DMSO plays an important role in AChE inhibitory effects at these concentrations. DMSO is a polar solvent that undergoes nucleophilic substitution reactions and is thought to react with nucleophilic amino acids present in the active sites of enzymes.⁸⁾ The dipole moment of DMSO is larger than that of acetone, and the bond between the sulfur and oxygen in DMSO is not a double bond but a covalent single bond between the positively charged sulfur and negatively charged oxygen.¹¹⁾ Therefore, the AChE inhibitory effect of DMSO at low concentrations may be caused by the binding of the positively charged sulfur to the nucleophilic serine residue in the active site of AChE.¹²⁾ However, DMSO and acetone similarly inhibited rhAChE activity at high concentrations (≥2%). Acetone has an unshared electron pair on its oxygen and is known to act as a nucleophile.⁸⁾ Furthermore, the oxygen in DMSO is negatively charged. Therefore, the AChE inhibitory effects of DMSO and acetone at high concentrations may be caused by the action of their oxygen on the electrophilic site present in the active site of AChE.⁸⁾ Wiesner et al. predicted that the structure of rat AChE should be very similar to that of human AChE and should have identical active-site properties because various primary and tertiary alignments showed that AChEs are very evolutionarily conserved enzymes.¹³⁾

In contrast, the inhibitory effects of DMSO on 80 mM KCl-induced contractions were weaker than those of acetone. This may be due to the activating action of DMSO on tyrosine kinases.¹⁴⁾ Since the tyrosine kinase and myosin light chain kinase pathways influence each other,¹⁵⁾ tyrosine kinase activation by DMSO might attenuate the inhibitory effects of myosin light chain phosphorylation, which could be involved in the anticontractile effects of DMSO and acetone.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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