• HIGHLIGHTS
- Antispasmodic action of eugenol (EUG) on detrusor smooth muscle.
- By employing in silico and in vitro techniques, EUG exhibits binding to M3 muscarinic acetylcholine receptor and voltage-operated calcium channels, highlighting its potential to regulate these targets.
- EUG exhibits greater pharmacological potency in blocking the phasic component of K+-induced contraction (electromechanical coupling).
INTRODUCTION
Overactive bladder (OAB) is a clinical condition that arises from hyperactivity of the detrusor muscle. It is characterized by lower urinary tract dysfunction that manifests as urinary urgency. This symptom is generally accompanied by increased urinary frequency and nocturia, occurring without metabolic, infectious, or local changes [1]. OAB is highly prevalent in both European countries and Brazil, and it profoundly impacts quality of life; the frequent need to urinate interrupts daily activities and sleep, reducing productivity [2, 3]. Currently, the first-line medications for treating OAB are antimuscarinics and antispasmodics. These drugs inhibit the M3 muscarinic acetylcholine receptor (M3R) and L-type calcium channels, although their use is limited due to undesirable side effects [4].
Among the molecules with potential modulating effects on bladder contractility, eugenol (EUG) is a noteworthy natural compound. It is extracted from the stems, leaves, and flowers of various plant species, including Syzygium aromaticum L. (Myrtaceae), Ocimum gratissimum L. (Lamiaceae), and Myristica fragrans Houtt. (Myristicaceae) [4, 5]. EUG has been used since ancient times as a mild anesthetic [6] and for the relief of headaches and toothaches [7]. Additionally, it possesses antioxidant [8], antimicrobial [9], antifungal [10], antispasmodic, and relaxing effects on the smooth muscles of the trachea, ileum, and blood vessels. These effects are exerted through its action on voltage-operated calcium channels (VOCCs) and receptor-operated calcium channels (ROCCs) [11–15]. Given these properties, EUG likely also exerts an antispasmodic effect on the bladder. However, the detrusor muscle has unique characteristics and distinct signaling pathways, and no evidence has yet confirmed the antispasmodic action of EUG on this muscle. While in vitro studies are essential, they have limited clinical applicability. The absence of in vivo research and specific clinical trials represents a considerable gap in the evidence validating the use of EUG for the treatment of OAB. The present investigation was designed to elucidate the effects of EUG on isolated rat bladder using experimental and in silico approaches.
MATERIALS AND METHODS
Drugs and Solutions
EUG was prepared by diluting the compound in Krebs-Henseleit (KH) solution containing Tween 80 (≤0.2%). All drugs and salts used were of analytical grade. EUG (4-allyl-2-methoxyphenol), carbamylcholine chloride (carbachol; CCh), ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), and Tween 80 were sourced from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). BaCl2, NaCl, KCl, MgSO4, KH2PO4, NaHCO3, CaCl2, and glucose were procured from Neon Comercial (São Paulo, Brazil).
Experimental Animals
Eight male Wistar rats, 3 months old with an average mass of 299.2±1.096 g, were used in the study. These animals were purchased from the UniChristus Bioterium and housed in the Bioterium of the State University of Ceará (UECE). They were kept under conditions of 60% humidity and a controlled temperature range of 22°C to 25°C, with a 12-hour light/dark cycle, and had free access to water and food. All experiments were conducted in compliance with the Brazilian Guideline for the Care and Use of Animals in Teaching or Scientific Research Activities, as stipulated by the National Council for the Control of Animal Experimentation (CONCEA), Normative Resolution No. 55/2022, and in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.
Measurement of Detrusor Smooth Muscle Reactivity
After the animals were euthanized, their urinary bladders were removed. Longitudinal strips of bladder tissue measuring 2 mm by 6 mm were vertically mounted in organ baths with a 5-mL capacity. These baths contained KH solution, which was bubbled with a gas mixture of 95% O2 and 5% CO2 at 37°C and a pH of 7.4. Changes in tension were recorded using isometric force transducers (model F03; Grass Instruments, Quincy, MA, USA) connected to a Grass chart recorder (model 5D). In all experiments, after a 1-hour equilibration period, tissue viability was confirmed by inducing 2 contractions with the addition of 60mM K+ to the bath. Subsequently, the bladder strips were maintained at 1 gF for 60 minutes to allow the preparations to adapt to the new conditions and establish a basal resting tone before the introduction of any substances. All experimental series were conducted in parallel, comparing the effects of EUG and a vehicle control.
Inhibitory Effect of EUG on Contraction Induced by 60mM K+ (Electromechanical Coupling) and by 1μM CCh (Pharmacomechanical Coupling) in Detrusor Smooth Muscle
To evaluate the effect of EUG on contractions induced by 60mM K+, increasing concentrations of EUG (from 0.01 to 3mM) were administered in a noncumulative manner. Initially, 2 contractions were elicited using 60mM K+ without EUG, and the arithmetic mean of these contractions was established as the 100% reference value. Subsequently, the tissue preparations were preincubated with a specific concentration of EUG for 5 minutes. Following this period, the KH solution containing EUG was replaced with a modified KH solution that included both 60mM K+ and the same concentration of EUG used during preincubation. Once the contraction force reached equilibrium, the preparation was rinsed with KH solution to re-establish its resting tone. After a 5-minute interval, the preparation was exposed to the next concentration of EUG for another 5 minutes, followed by the induction of a new contraction. The contractile response to EUG was quantified as a percentage of the response to 60mM K+ in the absence of EUG.
To evaluate the impact of EUG on contractions induced by pharmacomechanical coupling, the same concentrations of EUG were used. As with the contractions induced by 60mM K+, the arithmetic mean of 2 contractions elicited by CCh without EUG was set as 100%. Following 5 minutes of incubation with a specified concentration of EUG, 1μM CCh was introduced. The resulting contractile response was then quantified as a percentage of the response observed in the absence of EUG.
For the control preparations (external controls), contractions were induced using either 60mM isotonic K+ or 1μM CCh with Tween 80, in the absence of EUG—that is, with only vehicle present.
Effect of EUG on Contractions Induced by BaCl2
Intracellular calcium stores were depleted through contractions induced by CCh (10μM) in a calcium-free KH solution with 2mM EGTA. This procedure was repeated until CCh no longer elicited contractions. Subsequently, the preparation was maintained in a calcium-free KH solution containing 80mM K+ to maintain isotonic conditions. Then, EUG was added at concentrations of 0.3, 1.0, and 3.0mM. For control preparations, only the Tween 80 solution was added at concentrations equivalent to those used for the EUG solutions. After 5 minutes of incubation with a specific concentration of EUG, increasing and cumulative concentrations of BaCl2 (from 0.3 to 30mM) were added.
Molecular Structure Analysis and Molecular Docking Studies
L-type voltage-dependent calcium channel (α1s subunit)
Initially, we obtained the amino acid sequence of the L-type voltage-dependent calcium channel α1s (ID: rno:682930) from Rattus norvegicus in FASTA format from the Kyoto Encyclopedia of Genes and Genomes (https://www.genome.jp/kegg/). The native Ca2+ channel sequence of Oryctolagus cuniculus (NCBI Reference Sequence: NP_001095190.1) was used as the bait sequence. We then predicted a model of the Ca2+ channel (α1s) from R. norvegicus using the Swiss-Model server (https://swissmodel.expasy.org/) based on the coordinates from PDB ID: 5GJV, a Ca2+ channel from O. cuniculus, following a structural modeling approach. The predicted model underwent structural optimization using WinCoot software, available at https://bernhardcl.github.io/coot/wincoot-download.html, and was subsequently energy-minimized using the Yet Another Scientific Artificial Reality Application program, available at https://www.yasara.org/minimizationserver.htm. The optimized and minimized model was then validated through root mean square deviation calculations using the PyMOL Molecular Graphics System (ver. 1.7.4; Schrodinger LLC, New York, NY, USA). The physicochemical properties were validated using the MolProbity server (http://molprobity.biochem.duke.edu/), while overall structural quality assessment was conducted using the QMEAN (https://swissmodel.expasy.org/qmean/) and VoroMQA (https://bioinformatics.lt/wtsam/voromqa ) servers.
M3 muscarinic acetylcholine receptor
The structure of M3R (PDB ID: 4FFW) was downloaded in PDB format from the RCSB Protein Data Bank database ( https://www.rcsb.org/).
EUG
The structure of the EUG ligand (Compound CID: 3314) was obtained in PDB format from PubChem, an open chemistry database managed by the National Institutes of Health ( https://pubchem.ncbi.nlm.nih.gov/).
Molecular docking studies
To obtain more reliable theoretical results, we utilized AutoDock Vina v. 1.2.0 [16]. Initially, we conducted simulations of site-directed redocking, allowing all torsional bonds of the ligands to rotate freely while maintaining the receptors in a rigid state. Subsequently, we performed additional simulations using the site-directed flexible redocking method, which permitted free rotation of both the ligands’ torsional bonds and the amino acids within the receptors. The validation process was applied to the following models: (I) for the L-type voltage-dependent calcium channel α1s, we used the crystal structure (PDB ID: 6JP5) co-crystallized with the ligand nifedipine; and (II) for M3R, we used the crystal structure (PDB ID: 4U14) co-crystallized with the ligand tiotropium.
After AutoDock Vina was validated, simulations employing the site-directed flexible docking method were performed. The structures of the K+ channel (α1s subunit) and M3R, both from R. norvegicus, served as receptors with EUG as the ligand. For these simulations, all polar hydrogen atoms were added to the receptor structures and then parameterized with Gasteiger charges. The EUG ligand was prepared by adding Kollman charges. All simulations were performed under the following conditions: the number of conformations was set to 50, the exhaustiveness to 33, and the seed to 2009. For the K+ channel (α1s subunit, predicted model) and EUG system, the box dimensions were uniformly set at XYZ=30 Å, with center coordinates of X=−10.839, Y=7.562, and Z=−4.064. In contrast, for the M3R and EUG system, the box dimensions were X=20 Å, Y=20 Å, and Z=30 Å (PDB ID: 4U14), with center coordinates of X=7.67, Y=27.962, and Z=341.413.
Statistical Analysis
Data are presented as the mean±standard error of the mean. Findings that presented a 5% or less probability of the null hypothesis (P≤0.05) were deemed statistically significant. For the comparison of points and curves, 1-way and 2-way analysis of variance (ANOVA) were utilized, followed by the Holm-Sidak test. Comparisons between 2 groups were made using the Student t-test. The concentration of the substance required to inhibit 50% of the contraction (IC50) was determined through semilogarithmic interpolation. Graphing and statistical analyses were performed using SigmaPlot 11.0 (Systat Software Inc., San Jose, CA, USA).
Methodological Limitations
Limitations of this study include the exclusive use of male Wistar rats, which may not adequately represent the effects of EUG on both sexes or across different mammalian species. The experiments were conducted in vitro using isolated bladder strips, which might not accurately replicate the complex physiological environment found in vivo. Furthermore, the concentrations of Tween 80 used as a vehicle could have influenced the results, despite the inclusion of controls. While molecular docking simulations provide insight into potential interactions with M3R, they are merely in silico models and do not necessarily replicate receptor behavior in living organisms. Consequently, these aspects of the study should be interpreted with caution, as they may not fully translate to effects observed in a clinical setting.
RESULTS
In response to both 60mM K+ (electromechanical excitation-contraction coupling) and 1μM CCh (pharmacomechanical excitation-contraction coupling), the rat detrusor muscle exhibited biphasic contractions. These were characterized by an initial rapid phase followed by a sustained steady-state phase, hereafter referred to as the phasic and tonic components, respectively. EUG inhibited the contractions evoked by 60mM K+ and by CCh, affecting both the phasic and tonic components of contraction (Figs. 1 and 2).
Inhibitory Effect of EUG on Electromechanical Coupling
EUG exhibited a concentration-dependent inhibitory effect on contractions induced by 60mM K+ (Fig. 1). This effect reached statistical significance at concentrations of 0.3mM and higher for both components, relative to control contractions (vehicle only; P<0.001, as determined by 1-way ANOVA followed by the Holm-Sidak test). At a concentration of 3mM, EUG demonstrated maximal pharmacological efficacy, completely blocking the contraction. It also displayed significantly higher pharmacodynamic potency in blocking the phasic component (IC50= 0.80±0.07mM) compared to the tonic component (IC50= 1.05±0.06mM), with a P-value of 0.018 (Student t-test).
Inhibitory Effect of EUG on Pharmacomechanical Coupling
Like the inhibition of contraction induced by electromechanical coupling, EUG exhibited a concentration-dependent inhibitory effect that displayed significance starting at 1mM (P< 0.001, as determined by 1-way ANOVA followed by the Holm-Sidak test) for both components analyzed. EUG completely blocked contraction at a concentration of 3mM. The effect of EUG in inhibiting contractions evoked by CCh (1μM) was comparable between phasic and tonic components, with IC50 values of 1.29±0.12mM and 1.18±0.17mM, respectively (P= 0.608, Student t-test).
Inhibitory Effect of EUG on BaCl2-Induced Contractions
In Fig. 3A–C, it shows that EUG at 1.0 and 3.0mM significantly inhibited the contraction induced by the addition of Ba2+ in a concentration-dependent manner (P=0.014 and P<0.001, respectively, as determined by 2-way ANOVA followed by the Holm-Sidak test). Complete inhibition was observed with 3mM EUG (Fig. 3D and E).
Molecular Docking Studies
A total of 20 simulations were conducted for both the M3R-EUG and CCsα1S-EUG systems. The molecular docking experiments for M3R-EUG and CCsα1S-EUG yielded the best performance with ΔG values of −6.5 kcal/mol (Fig. 4) and −5.4 kcal/mol (Fig. 5), respectively. Notably, ΔG values represent the combined result of van der Waals dispersion forces and electrostatic interaction energy, which together indicate the approximate binding energy between the ligand and the receptor/channel.
DISCUSSION
In the present study, we demonstrated for the first time that EUG exerts an antispasmodic effect on detrusor smooth muscle—known for its distinctive properties—in rats. Based on our in vitro findings, EUG inhibits both the phasic and tonic components of detrusor contraction, with a notably higher pharmacodynamic potency affecting the phasic component. Additionally, the in silico data suggest that EUG interacts with the M3R and VOCCs, releasing Gibbs energy at levels comparable for both structures. The magnitude of this energy release suggests that EUG has preferential binding sites on these macromolecules.
The antispasmodic activity of EUG on the bladder, as documented here, is not entirely unexpected, given its known capacity to inhibit contractions in the ileum, basilar artery, and tracheal smooth muscle [8, 9, 17]. However, certain aspects of this inhibitory activity could not have been predicted based on previous findings in smooth muscle and thus required experimental investigation. This necessity arises from the distinct characteristics of bladder muscle contraction. The bladder muscle resembles the myocardium in its relatively high dependence on intracellular calcium release [10]. While the bladder lacks a sarcoplasmic reticulum with a histological structure akin to that of striated and myocardial muscles, it possesses organelles that serve a similar function to those found in the myocardium [11]. Prior evidence also supports the physiological importance of the intracellular calcium reservoir in the bladder [12].
Smooth muscle contraction requires the influx of Ca2+ and can be initiated solely by membrane depolarization, known as electromechanical excitation-contraction coupling (e.g., KCl-induced contraction), or it can also involve the interaction of an agonist with a membrane receptor, termed pharmacomechanical coupling (e.g., acetylcholine-induced contraction) [13]. This study demonstrates that EUG inhibits rat detrusor muscle contraction induced by KCl, implicating its action in electromechanical coupling. This inhibition suggests that EUG targets VOCCs, key components of electromechanical coupling. The hypothesis that EUG affects VOCCs is further supported by the observation that EUG also blocks Ba2+-induced contractions in a Ca2+-free medium, with similar pharmacodynamic potency. This is notable because Ba2+ enters cells almost exclusively through VOCCs [14].
Detrusor contractions initiated by the cholinergic neurotransmitter acetylcholine, through mechanisms beginning with the stimulation of muscarinic receptors and ROCCs, were also inhibited by EUG. Among the receptors in the urinary bladder, the M2 and M3 types are key, with the former predominating in number and the latter being responsible for the direct contractile effects of acetylcholine on the detrusor muscle [15, 18]. Consequently, we hypothesized that EUG-induced inhibition of CCh-induced contraction may involve interference with M3R activation.
Electrophysiological studies have indicated that EUG can interfere with the functioning of ion channels, including by inhibiting VOCC activation in vascular smooth muscle [19]. Muscle contraction results from a multi-step cascade, and the blocking action of EUG may occur downstream of both M3R and VOCC. Consequently, using in silico resources, we also investigated whether EUG interference with these macromolecules was likely, and the findings supported this possibility.
Regarding VOCCs, the in silico data indicate that EUG interacts with the α1S subunit. This interaction involves key amino acids and includes 2 stacking interactions with the aromatic rings of residues Leu621 and Thr581 (Fig. 4). These interactions occur in the fourth segment of CCsα1S (ΔG=−5.54 kcal/mol), a site essential for voltage-dependent modulation, as observed in co-crystal structures with nifedipine (PDB ID: 6JP5), diltiazem (PDB ID: 6JPB), and verapamil (PDB ID: 6JPA). The interaction impedes calcium influx, decreasing detrusor smooth muscle contraction [20].
In silico molecular analysis of M3R indicates that EUG interacts with the receptor with a relatively large ΔG (−6.5 kcal/mol), underscoring the importance of this interaction. The central energy of EUG’s aromatic ring facilitates 3 stacking interactions with the aromatic rings of the residues Trp503, Tyr148, and Tyr506 in the M3 receptor (Fig. 5). Additional interactions, including van der Waals forces, hydrogen bonds, carbon bonds, alkyl interactions, and pi-alkyl interactions, are also observed. These interactions between EUG and the residues Trp503, Tyr148, and Tyr506 could result in antagonism, potentially disrupting the natural binding of acetylcholine to M3R. This is analogous to the binding observed for the selective antagonist tiotropium in its co-crystal with this receptor (PDB ID: 4U14). This interaction blocks the activation of M3R by acetylcholine, thus inhibiting the pharmacomechanical cholinergic contraction of the detrusor muscle [21].
While these data suggest that the activities of EUG on VOCCs and M3 receptors likely vary, their combined effects may synergistically promote the blockade of detrusor contraction. However, intracellular effects remain a hypothetical consideration. This is because EUG has been shown to directly release Ca2+ from the sarcoplasmic reticulum in skinned fibers of skeletal muscle. This hypothesis was not explored in the present study; thus, additional aspects of EUG’s mechanism of action may remain to be clarified.
Several studies have shown that EUG can interfere with the functioning of ion channels, including VOCC and cholinergic transmission. Our experimental results from both analyzed pathways—electromechanical and pharmacomechanical—support these findings observed in other smooth muscles. Leal-Cardoso et al. [17] demonstrated that EUG relaxed the isolated ileum and inhibited the contractions induced by both electromechanical and pharmacomechanical mechanisms. Damiani and colleagues indicated that EUG acts as a blocker of VOCCs and ROCCs, inhibiting contractions in the thoracic aorta [22]. Furthermore, 100μM EUG was shown to reduce tracheal smooth muscle contraction [23] and to relax arteries in a concentration-dependent manner, both in the presence and absence of endothelium, leading to vasorelaxation and a reduction in systemic blood pressure [19, 24] in electrophysiological experiments using the patch-clamp technique. These findings suggest that EUG exerts a myogenic antispasmodic effect; it induces direct relaxation in the smooth muscle cells of the rat cerebral artery by inhibiting VOCCs [19].
Several low-molecular-weight terpenes and terpenoids can interfere with the functioning of ion channels [16, 25, 26], including VOCCs. For instance, 1,8-cineole has been shown to block VOCCs in tracheal smooth muscle [27]. This lends further credibility to the hypothesis that EUG acts on VOCCs.
Until now, we have analyzed this process as if it were phaseless. However, detrusor contraction in response to nervous control during urination occurs in 2 distinct phases [28]. The first, known as the phasic component, is primarily characterized by the release of calcium stores from the sarcoplasmic reticulum and is typically represented as a contraction peak. The second, termed the tonic component, involves the influx of calcium through the plasma membrane via VOCCs and ROCCs, which open in response to neurotransmitter stimulation. This phase relies on an extracellular source of calcium to maintain the sustained effect of contraction [29]. We observed that EUG exhibited greater pharmacological potency in the phasic component of electromechanical coupling (IC50=0.80±0.07mM) compared to the phasic component of pharmacomechanical coupling (1.29±0.12mM). However, we noted no significant difference in the IC50 values for the tonic components of contraction induced by K+ 60mM and by CCh 1μM. The phasic component occurs through the release of calcium stores from the sarcoplasmic reticulum. In electromechanical coupling, this release is triggered by Ca2+, thus constituting calcium-induced calcium release (CICR). Notably, in the urinary bladder, the CICR mechanism is not exclusive to electromechanical coupling but is also a feature of pharmacomechanical coupling [30].
In most mammalian species, including humans, bladder emptying via urination involves the cholinergic activation of muscarinic receptors in the smooth muscle of the urinary bladder [31]. Inhibiting the initial large phasic contraction of the detrusor muscle can prevent the involuntary onset of urination [10]. Thus, the capacity of EUG to inhibit this contraction suggests its promise as a therapeutic agent for OAB.
In conclusion, this study demonstrates that EUG exerts an antispasmodic effect on the smooth muscle of the urinary bladder, likely via a mechanism involving VOCC and/or M3R blockade. This effect can achieve maximal efficacy depending on the concentration, and along with EUG’s antioxidant and anti-inflammatory properties, it suggests that EUG has therapeutic potential for the treatment of OAB. However, this study had limitations. It exclusively utilized rat detrusor muscle, which may not fully replicate the effects of EUG in humans. Additionally, the experiments were conducted in vitro and did not account for the complexity of in vivo conditions, where multiple factors influence bladder function. Although in silico analysis indicated potential interactions of EUG with M3R and VOCCs, these findings require validation through in vivo studies and clinical trials to establish their relevance in therapeutic settings.
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