2024 Volume 47 Issue 12 Pages 2011-2020
Sudachitin, a polymethoxyflavone found in sudachi peel, has been reported to improve hyperlipidemia in humans, and is thus attracting research attention. However, its effect on cardiac function remains unclear. We investigated the mechanisms underlying the chronotropic and inotropic effects of sudachitin on rat atria. Sudachitin (0.3–30 µM) produced concentration-dependent positive chronotropic and inotropic effects. Other polymethoxyflavones, including demethoxysudachitin (0.3–30 µM) and nobiletin (0.3–30 µM), also produced positive chronotropic and inotropic effects; however, the maximum efficacy of all polymethoxyflavones, including sudachitin, was lower than that of isoproterenol. Propranolol (0.1 µM) did not affect the positive chronotropic and inotropic effects of sudachitin. The concentration–response curves for the chronotropic and inotropic effects of dibutyryl-cAMP (1–100 µM) were shifted to the left upon pretreatment with sudachitin (3, 10 µM). Phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine 1 µM or milrinone 10 µM) alone, sudachitin alone (10, 30 µM), and a combination of phosphodiesterase inhibitors and sudachitin exhibited positive chronotropic and inotropic effects, whereas the lack of any interaction between each phosphodiesterase inhibitor and sudachitin indicated an additive effect of the two substances. These results suggest that sudachitin-induced positive chronotropic and inotropic effects similar to those of other polymethoxyflavones, but its maximum efficacy was lower than that of isoproterenol. Both demethoxysudachitin and nobiletin exhibited similar positive chronotropic and inotropic effects, indicating that these effects are not specific to sudachitin, but are common to polymethoxyflavones. The mechanism of action of sudachitin was associated with the enhancement of cAMP-dependent pathways, without the involvement of β-adrenoceptors.
Citrus peels contain various polymethoxyflavones (PMFs),1) which have been demonstrated to possess a range of pharmacological functions, including anti-dementia,2) anti-inflammatory,3) anticancer,4) hepatoprotective,5) and lipolysis-promoting effects.6) As a result, research on citrus fruits with high PMF contents for the development of health food products is gaining more attention.
Sudachi (Citrus sudachi Hort. ex Shirai), a traditional Japanese fruit, is popular in Tokushima Prefecture, Japan. Its peel contains PMFs, the most abundant of which is sudachitin (Fig. 1), followed by demethoxysudachitin7) (Fig. 1). Both PMFs are unique to Citrus sudachi, as they are not present in other citrus fruits. Sudachi peel extract powder is a food ingredient with a broad safety profile, as demonstrated in toxicity studies.8)
In a clinical study, sudachitin-containing sudachi peel extract powder was shown to improve visceral fat content in humans at risk of developing diabetes; thus, the preventive effect of sudachitin on lifestyle-related diseases has been attracting attention.9) In experiments using rat aorta preparations, we recently reported that sudachitin induces endothelium-independent relaxation and that the mechanism of vasorelaxation induced by sudachitin involves the enhancement of cAMP and the guanosine 3′,5′-cyclic monophosphate (cGMP) signaling pathway10); however, the effects of sudachitin on cardiac function, including chronotropic and inotropic effects, have not been clarified.
Therefore, the purpose of this study was to evaluate the chronotropic and inotropic effects of sudachitin on isolated rat atrial preparations and investigate the underlying mechanism of action of sudachitin.
Sudachitin (purity 98.8%) and demethoxysudachitin (purity 99.6%) were provided by Ikeda Yakusou Co., Ltd. (Tokushima, Japan). Isoproterenol hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Nobiletin (purity >98.0%), 3-isobutyl-1-methylxanthine (IBMX), and milrinone were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Propranolol hydrochloride was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Dibutyryl-cAMP sodium salt (bucladesine sodium salt) was purchased from Cayman Chemical Co. (Ann Arbor, MI, U.S.A.). All other chemicals used were of the highest commercially available grade.
Sudachitin, demethoxysudachitin, nobiletin, IBMX, and milrinone were dissolved in 100% dimethyl sulfoxide (DMSO) and added to the organ bath at concentrations not exceeding 0.3%. All other chemical stocks were prepared as aqueous solutions and were diluted with distilled water.
AnimalsMale Wistar rats (8–10 weeks old; weighing 170–324 g; total number, 36; Japan SLC, Hamamatsu, Japan) were housed under controlled conditions (21–23 °C and a relative humidity of 50 ± 15%) with a fixed 12/12 h light/dark cycle (8:00–20:00) and food and water available ad libitum. This study was approved by the Animal Care and Use Committee of Mukogawa Women’s University (Approval Nos. P-17-2021-02, P-17-2022-02, and P17-2023-02), and conducted in accordance with the guidelines of the Laboratory Animal Center of the School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University.
Preparation of Rat Atrial TissuesAll the rats were anesthetized with 5% isoflurane and euthanized by exsanguination from the carotid artery. Subsequently, hearts were excised and placed in a well-oxygenated (95% O2, 5% CO2) modified Krebs–Henseleit solution (KHS) (118.4 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 24.9 mM NaHCO3, and 11.1 mM glucose; pH 7.4). The right and left atria were carefully cleaned to remove attached fat and connective tissue.
Recording of Heart Rate and Contractile Force ChangesThe right atrial and left atrial preparations were mounted using stainless hooks under the optimal in a 20 mL organ bath containing well-oxygenated KHS at 36 °C. Spontaneous beating rate of the right atrium was measured using a cardiotachometer (AT-601G; Nihon Kohden, Tokyo, Japan). The left atria were stimulated using a pair of platinum plate electrodes (field stimulation) with rectangular current pulses (1 Hz, 5 ms, approximately 1.2× threshold voltage) generated by an electronic stimulator (SEN-2201, Nihon Kohden). The developed tension was measured isometrically using a force-displacement transducer (TB-612T, Nihon Kohden). All data were recorded using a mini polygraph (RM-6100, Nihon Kohden) and LabScribe® software via LAB-TRAX-4® (iWorx Systems, Dover, NH, U.S.A.).
The preparations were incubated for more than 60 min to equilibrate and were then stimulated with isoproterenol (1 µM) KHS to ensure the functional integrity of the preparations. Subsequently, the preparations were washed by replacing the isoproterenol solution with KHS for complete recovery. After confirming a stable heart rate and contractile force in the atria, the effects on chronotropism and inotropism were assessed using the following methods.
Evaluation of Chronotropic and Inotropic Effects of PMFIn the experiments shown in Figs. 2 and 3, after confirmation of a steady-state heart rate (HR) and contractile force (CF), sudachitin (0.3–30 µM), demethoxysudachitin (0.3–30 µM), or nobiletin (0.3–30 µM) was added cumulatively to the bath medium. The maximum efficacy (Emax) of each compound was determined.
Representative examples of the chronotropic effects of SDC (A) and isoproterenol (ISO; B) on the right atria. Representative examples of the inotropic effects of SDC (D) and ISO (E) on the left atria. Concentration–response curves for the chronotropic and inotropic effects of SDC (C, F). The symbols and vertical bars represent the mean ± standard error of the mean (S.E.M.) of four preparations for each group.
The symbols and vertical bars represent the mean ± S.E.M. of four to six preparations for each group.
As shown in Fig. 4, isoproterenol (0.1–1000 nM) or sudachitin (0.3–30 µM) was added cumulatively as a control, and changes in the HR and CF were observed. The atria were incubated with propranolol (0.1 µM; which concentration is about 30 times higher than propranolol’s pA2 value 8.33)11) for 20 min and then isoproterenol (0.1–1000 nM) or sudachitin (0.3–30 µM) was added cumulatively and changes in HR and CF were observed. These results were compared with the concentration–response curve for the control. As shown in Fig. 5, dibutyryl-cAMP (1–100 µM) was added cumulatively as a control, and changes in the HR and CF were observed. The atria were incubated with sudachitin (3 µM for HR or 10 µM for CF); these concentrations had positive chronotropic and inotropic effects of approximately 10% (Figs. 2C, F) for 15 min, and then dibutyryl-cAMP (1–100 µM) was added cumulatively, and changes in HR and CF were observed. Furthermore, in the experiments shown in Figs. 6–8, DMSO (0.01% or 0.03%) was added to the bath medium for 5 min, followed by sudachitin (10 µM for HR or 30 µM for CF), and changes in HR and CF were observed. After washout with KHS to ensure steady-state HR and CF, phosphodiesterase (PDE) inhibitors (1 µM IBMX or 10 µM milrinone, which had positive chronotropic and inotropic effects of approximately 20% in our preliminary study) were added, followed by sudachitin (10 µM for HR or 30 µM for CF), and changes in HR and CF were observed.
Concentration–response curves for the chronotropic (A, B) and inotropic (C, D) effects of ISO (A, C) and SDC (B, D), with or without 0.1 µM PRO. The symbols and vertical bars represent the mean ± S.E.M. of four preparations for each group. * p < 0.05 vs. control (Welch’s t-test).
Concentration–response curves for the chronotropic (A) and inotropic (B) effects of Db-cAMP, with or without 3 or 10 µM SDC. The symbols and vertical bars represent the mean ± S.E.M. of five to six preparations for each group. * p < 0.05 vs. control (Welch’s t-test).
Representative examples of the chronotropic effects of SDC and IBMX (A). Chronotropic effects of dimethyl sulfoxide (DMSO; 0.01%, Control) alone, IBMX (1 µM) alone, SDC (10 µM) alone, and the combination of IBMX (1 µM) and SDC (10 µM, B). Results of a two-way repeated measures ANOVA with IBMX and SDC as factors (C). NS: not significant. The bars and symbols represent the mean ± S.E.M. of four preparations.
Representative examples of inotropic effects of SDC and IBMX (A). Inotropic effects of DMSO (0.03%, Control) alone, IBMX (1 µM) alone, SDC (30 µM) alone, and the combination of IBMX (1 µM) and SDC (30 µM, B). Results of a two-way repeated measures ANOVA with IBMX and SDC as factors (C). NS: not significant. The bars and symbols represent the mean ± S.E.M. of four preparations.
Chronotropic effects of DMSO (0.01%, Control) alone, Mil (10 µM) alone, SDC (10 µM) alone, and a combination of Mil (10 µM) and SDC (10 µM) on the right atria (A). Results of a two-way repeated measures ANOVA of the chronotropic effects with Mil and SDC as factors (B). Inotropic effects of DMSO (0.03%, Control) alone, Mil (10 µM) alone, SDC (30 µM) alone, and a combination of Mil (10 µM) and SDC (30 µM) on the left atria (C). Results of a two-way repeated measures ANOVA of the chronotropic effects with Mil and SDC as factors (D). NS: not significant. The bars and symbols represent the mean ± S.E.M. of four preparations.
The percentage changes in HR (%ΔHR) and CF (%ΔCF) were defined relative to the maximum increase in HR and contractility obtained using 1 µM isoproterenol, minus the baseline levels before isoproterenol addition. The data were plotted as a function of the drug concentration and fitted to the following equation:
 |
where E is the % response at a given concentration, Emax is the maximum response, C is the drug concentration, nH is the Hill coefficient (slope function), and EC50 is the effective drug concentration that caused a 50% response. Logistic curve fitting was performed using ImageJ software (version 1.53; NIH, Bethesda, MD, U.S.A.; https://imagej.nih.gov/ij/). The EC50 values of the compounds were determined for each preparation.
Statistical AnalysisAll values in the text and illustrations are expressed as the mean ± standard error of the mean (S.E.M.) of the data obtained from different numbers (n) of preparations. However, the S.E.M. is included only when it exceeds the dimension of the symbol used. Statcel-the Useful Addin Forms on Excel-4th ed. (OMS Publications, Tokyo, Japan), and Microsoft® Excel® (Microsoft 365 version) were used for statistical analysis. EC50 values were log-transformed prior to statistical analysis. Welch’s t-test or post hoc Bonferroni test after ANOVA was used to evaluate the statistical significance between two groups, and p-values less than 0.05 were considered significant. To detect additive or synergistic effects between sudachitin and PDE inhibitors, a two-way repeated measures ANOVA with sudachitin and PDE inhibitors as two factors was performed, and p < 0.05 was considered to indicate significant interaction effects.
Sudachitin (0.3–30 µM) induced a positive chronotropic effect on spontaneous beating right atrial preparations in a concentration-dependent manner (Figs. 2A, C). The maximum efficacy of sudachitin was 40.1 ± 3.6% (n = 4, Fig. 2C), which was lower than the maximum efficacy (100%) elicited by 1 µM isoproterenol, a β-adrenoceptor (β-AR) full agonist. isoproterenol (0.1–1000 nM) also induced a concentration-dependent positive chronotropic effect (Fig. 2B). Both sudachitin (0.3–30 µM) and isoproterenol (0.1–1000 nM) induced positive inotropic effects on electrically stimulated left atrial preparations in a concentration-dependent manner (Figs. 2D–F), with the maximum efficacy of sudachitin being 17.7 ± 5.3% (n = 4, Fig. 2F), which was lower than the maximum efficacy elicited by isoproterenol. The positive chronotropic and inotropic effects of isoproterenol were observed quickly after its addition (Figs. 2B, E), whereas the effects of sudachitin developed more slowly (Figs. 2A, D).
Chronotropic and Inotropic Effects of Demethoxysudachitin and NobiletinWe examined whether PMF also exhibits chronotropic and inotropic effects using demethoxysudachitin and nobiletin. The results revealed that both demethoxysudachitin (0.3–30 µM) and nobiletin (0.3–30 µM) induced a positive chronotropic effect in a concentration-dependent manner (Figs. 3A, B). The maximum efficacy of demethoxysudachitin and nobiletin were 30.3 ± 4.6% (n = 5) and 32.4 ± 3.8% (n = 5), respectively, which were lower than the maximum efficacy elicited by isoproterenol. Additionally, both demethoxysudachitin (0.3–30 µM) and nobiletin (0.3–30 µM) caused a concentration-dependent positive inotropic effect on left atrial preparations (Figs. 3C, D), with the maximum efficacy of demethoxysudachitin and nobiletin being 11.3 ± 0.6% (n = 4) and 39.6 ± 12.7% (n = 6), respectively.
Influence of Propranolol on the Positive Chronotropic and Inotropic Effects of Isoproterenol and SudachitinTo clarify the potential involvement of β-AR in the positive chronotropic and inotropic effects of sudachitin, we evaluated the effects of propranolol, a β-AR antagonist. The concentration–response curves for the positive chronotropic effects of isoproterenol, which showed an EC50 value of 1.31 ± 0.24 nM (n = 4) were shifted approximately 100-fold to the right by pretreatment with 0.1 µM propranolol (Fig. 4A). Similarly, the concentration–response curves for the positive inotropic effects of isoproterenol, which showed an EC50 value of 6.07 ± 0.93 nM (n = 4) were shifted approximately 30-fold to the right by pretreatment with 0.1 µM propranolol (Fig. 4C). In contrast, the concentration–response curves for the chronotropic and inotropic effects of sudachitin were not affected by 0.1 µM propranolol (Figs. 4B, D).
Influence of Sudachitin on the Positive Chronotropic and Inotropic Effects of Dibutyryl-cAMPWe investigated the effects of dibutyryl-cAMP, a cell-permeable cAMP analog, to examine the involvement of the cAMP-dependent pathway in the sudachitin-induced chronotropic and inotropic effects. Dibutyryl-cAMP (1–100 µM) caused concentration-dependent positive chronotropic and inotropic effects (Fig. 5). The maximum efficacies for the chronotropic and inotropic effects of dibutyryl-cAMP were 36.9 ± 4.0% (n = 6) and 154.2 ± 12.7% (n = 5), respectively. The concentration–response curves for the chronotropic effect of dibutyryl-cAMP were significantly shifted by approximately 10-fold to the upper left after pretreatment with 3 µM sudachitin (Fig. 5A). Similarly, the concentration–response curves for the inotropic effect of dibutyryl-cAMP were significantly shifted by approximately 3-fold to the left by pretreatment with 10 µM sudachitin (Fig. 5B).
Chronotropic and Inotropic Effects of Sudachitin in Combination with a Phosphodiesterase InhibitorTo examine the involvement of cAMP-dependent pathways in the positive chronotropic and inotropic effects of sudachitin, we investigated the combined effects of IBMX (1 µM), a non-selective PDE inhibitor. IBMX alone or the combination of sudachitin (10 or 30 µM) with IBMX (1 µM) produced positive chronotropic (Figs. 6A, B), and inotropic effects (Figs. 7A, B). A two-way ANOVA was performed with sudachitin and IBMX as factors, and no significant interaction was observed for HR (Fig. 6C, p = 0.74, n = 4) or CF (Fig. 7C, p = 0.98, n = 4).
We also investigated the combined effects of milrinone, a PDE3 selective inhibitor. Compared with DMSO (0.01 or 0.03%, control) alone, sudachitin (10 or 30 µM) alone, milrinone (10 µM) alone, and the combination of sudachitin (10 or 30 µM) with milrinone (10 µM) produced chronotropic and inotropic effects (Figs. 8A, B). Two-way ANOVA was performed with sudachitin and milrinone as factors, and no significant interaction was observed for HR (Fig. 8B, p = 0.17, n = 4) or CF (Fig. 8D, p = 0.64, n = 4).
In the present study, using isolated rat right and left atrial muscle preparations, sudachitin exhibited positive chronotropic and inotropic effects that were smaller than those of isoproterenol (Fig. 2). One possible mechanism is voltage-dependent Ca2+ channel (VDCC) antagonism by sudachitin. In our previous study, we reported that sudachitin suppresses high-K+-induced depolarizing contractions, which depend on VDCC activity in rat thoracic aortas, at concentrations of 30–100 µM.10) The increase in sinus rate and myocardial contractility depends on the increase in intracellular Ca2+ concentrations associated with the activation of VDCCs.12) Therefore, if inhibition of vascular VDCC by sudachitin acts similarly on the heart, it may prevent excessive positive chronotropism and inotropism, as with the β-AR full agonist isoproterenol. Further studies are required to confirm the inhibitory effects of sudachitin on VDCC in the myocardium and to elucidate the underlying mechanism.
The positive chronotropic and inotropic effects of isoproterenol were both rapid (Figs. 2B, E), whereas the effects of sudachitin were slower than those of isoproterenol (Figs. 2A, D). Therefore, we speculated that the site of action of sudachitin was not on the surface of the plasma membrane but on an intracellular site.
There was no selectivity for the positive chronotropic or inotropic effects of sudachitin, and both effects were observed in the micromolar concentration range (Fig. 2). This suggests that sudachitin may not act specifically on ion channels expressed in the sinoatrial node, which controls cardiac rhythm, or on contractile proteins in the atrial muscle, but rather on common targets that influence both cardiac rhythm and contraction. To clarify this, further investigations are needed to examine the mechanism of action of sudachitin, including studies of sinoatrial node action potential waveforms and intracellular Ca2+ dynamics.
Positive Chronotropic and Inotropic Effects of Demethoxysudachitin and NobiletinIn our previous study, demethoxysudachitin was found to exhibit vasorelaxant effects similar to those of sudachitin, with an EC50 value of 28.1 µM, which is approximately twice that of sudachitin (EC50: 15.0 µM), indicating that demethoxysudachitin is less potent than sudachitin.10) In this study, demethoxysudachitin also showed positive chronotropic and inotropic effects (Figs. 3A, C), but its maximum efficacy (%⊿HR: 30.3, %⊿CF: 11.3) was less than that of sudachitin (%⊿HR: 40.1, %⊿CF: 17.7). This potency relationship is consistent with the vasodilatory effects.10)
Nobiletin, the most abundant PMF in Citrus depressa Hayata, has been reported to exhibit various pharmacological effects, including neuroprotection, cardiovascular protection, antimetabolic-disorder, anticancer, anti-inflammation, and antioxidation effects.13) Furthermore, recent studies have suggested that nobiletin, such as sudachitin, regulates circadian clock genes and may have anti-aging effects. Therefore, it has attracted attention as a functional ingredient in health foods.14,15)
In the present study, both demethoxysudachitin and nobiletin induced positive chronotropic and inotropic effects (Fig. 3), suggesting that these effects may not be unique to sudachitin, but could be a common characteristic of PMFs, including demethoxysudachitin and nobiletin.
Although our study focused on PMF and its effects on cardiac function, it is noteworthy that positive chronotropic and inotropic effects have also been reported for other compounds, such as flavanone-derived naringin16) and polyphenol-derived resveratrol,17) both of which are gaining global attention as health food components. This indicates that PMF is not the only health food constituent that has direct cardiac effects.
Although the relaxant effect of nobiletin has been reported in isolated rat aorta preparations,18) its chronotropic and inotropic effects have not yet been reported. Similarly, the chronotropic and inotropic effects of sudachitin and demethoxysudachitin have not been previously reported, making these findings novel. In the future, we plan to investigate other PMFs, such as tangeretin19) and sinensetin,20) to determine whether these effects are common to all PMFs.
β-AR Involvement in the Positive Chronotropic and Inotropic Effects of SudachitinThe concentration–response curves for the positive chronotropic and inotropic effects of isoproterenol were shifted 30- to 100-fold to the right by pretreatment with 0.1 µM propranolol, a β-AR antagonist, and the maximum response was approximately 100% (Figs. 4A, C). Thus, 0.1 µM propranolol is an appropriate concentration for effectively antagonizing β-AR in this experimental system. In addition, antagonism by propranolol was confirmed to be competitive.
We examined the positive chronotropic and inotropic effects of sudachitin in the presence of 0.1 µM propranolol, which effectively antagonizes β-AR binding. Both concentration–response curves for sudachitin were unaffected by propranolol treatment (Figs. 4B, D). These results suggest that β-ARs are not involved in the positive chronotropic and inotropic effects of sudachitin.
Influence of Sudachitin on the Positive Chronotropic and Inotropic Effects of Dibutyryl-cAMPTo examine the involvement of cAMP-dependent signaling pathways in the positive chronotropic and inotropic effects of sudachitin, we used dibutyryl-cAMP, a membrane-permeable cAMP analog21) after pretreatment with propranolol.
Under the conditions of this study, treatment with propranolol blocked β-AR-mediated stimulation, and dibutyryl-cAMP increased intramyocardial cAMP levels to produce positive chronotropic and inotropic effects (Fig. 5). Intracellular cAMP is degraded to 5′-AMP by PDE activation.22) If sudachitin inhibited PDE, the concentration–response curves for dibutyryl-cAMP-induced positive chronotropism and inotropism would be expected to shift to lower concentrations when sudachitin is used in combination.
In this experiment, the concentration–response curves for the positive chronotropic and inotropic effects of dibutyryl-cAMP significantly shifted to the left after pretreatment with sudachitin (Fig. 5). These results suggest that sudachitin may enhance cAMP-dependent signaling pathways, involving PDEs downstream of β-AR.
Combined Effects of Sudachitin and PDE Inhibitors on Chronotropism and InotropismPDEs are ubiquitously expressed enzymes in vivo, and are classified into 11 subtypes (PDE1–11). They modulate intracellular cAMP and cGMP levels.23) PDEs can be further categorized into three groups based on their substrate specificity: cAMP-specific (PDE4, 7, 8), cGMP-specific (PDE5, 6, 9), and cAMP-specific (PDE1, 2, 3, 10, 11) PDEs.
The expression of these PDEs varies across different tissues, allowing them to influence a wide range of biological processes, including ion channel regulation, the production of inflammatory mediators, memory, differentiation, apoptosis, and lipogenesis, as well as roles in cardiac chronotropism and inotropism.23)
PMFs possess numerous pharmacological actions, including the inhibition of PDEs.24) Specifically, sudachitin has been shown to inhibit PDE1, 3A, 4, 5A, 8A, and 10A2.25–27) Among the PDE subtypes that are expressed in the myocardium, PDE3 and PDE4 are predominant in mediating basal cardiac pacemaker and excitation-contractile coupling functions, and their activation leads to the degradation of cAMP.28,29) Therefore, if sudachitin inhibits PDE3 and PDE4, it would result in an increased amount of cAMP in cardiomyocytes. In this study, we investigated whether sudachitin inhibits PDE by examining the combined, potentially additive or synergistic, effects of sudachitin with IBMX, a broad PDE inhibitor with IC50 values of 6.7 and 26.3 µM for PDE3 and PDE4, respectively,30) and milrinone, a selective PDE3 inhibitor with an IC50 value of 2.0 µM for PDE3,31) on chronotropism and inotropism. Milrinone is a highly selective inhibitor of PDE3 and is used clinically for the treatment of acute heart failure32) and in basic physiology and pharmacology research.33)
The results indicated no significant interaction between sudachitin and IBMX, with either positive or negative inotropic effects, confirming an additive effect between the two compounds (Figs. 6, 7). Similarly, no significant interactions were observed between sudachitin and milrinone, further confirming their additive effect (Fig. 8). These findings suggested that PDE inhibition is one of the mechanisms of action of sudachitin. This hypothesis is supported by the results of previous studies showing that 10 and 30 µM of sudachitin, as used in the present study, inhibit PDE3 activity in cell-based assays by 68 and 91%, respectively.27) Moreover, 30 µM sudachitin increases cAMP levels and inhibits protein kinase A in adipocytes,34) which further confirms the involvement of sudachitin in PDE inhibition. Additionally, our results support the discussion presented in the section “Influence of Sudachitin on the Positive Chronotropic and Inotropic Effects of Dibutyryl-cAMP” which proposed that sudachitin may enhance cAMP-dependent signaling pathways, involving PDEs downstream of β-AR.
The tension measurement method has limitations with regard to assessing the effects of sudachitin on cAMP and protein kinase A (PKA) activity. To clarify the precise mechanism of action of sudachitin in the heart, further studies are required to investigate the relationship between cAMP levels and PKA activity in myocardial cells using molecular biology approaches.
Cardiovascular Targets of Sudachitin and Benefits to HumansWe consider the cardiocirculatory targets of sudachitin to be primarily vascular smooth muscle and, secondarily, the heart (Fig. 9). We previously reported that sudachitin exhibits endothelium-independent vasorelaxation, with the enhancement of cAMP- and cGMP-dependent signaling pathways in smooth muscle playing a key role in this mechanism.10) The fact that the relaxing effect of sudachitin is endothelium-independent suggests that sudachitin does not act directly on endothelial cells and is highly selective of vascular smooth muscle cells. In our previous study, the EC50 value of the relaxant effect of sudachitin on phenylephrine-induced contraction in the rat aorta without endothelium was 15.0 µM, and the maximum relaxation (Emax) value was approximately 100%, indicating complete relaxation. In contrast, the maximum efficacies of the positive chronotropic and inotropic effects of sudachitin were 40.1 ± 3.6 and 17.7 ± 5.3%, respectively (Figs. 2C, F, 9), which are clearly lower than the maximum efficacy of isoproterenol (100%). Applying a concentration of 10 µM, which is close to the EC50 value for vasorelaxation mentioned above, to myocardial effects, sudachitin had a positive chronotropic effect of 23.5 ± 1.6% and a positive inotropic effect of 7.3 ± 3.4% (Figs. 2C, F, 9). Thus, at concentrations sufficient to induce vasodilation, the stimulatory effects of sudachitin on the heart are relatively mild. This pharmacological profile differs from that of β-AR full agonists, such as isoproterenol.
Sudachitin activated both cAMP- and cGMP-dependent pathways in vascular smooth muscle, leading to vasorelaxation, with an Emax of approximately 100%; however, no effects were observed in the endothelium. In contrast, sudachitin activated the AMP-dependent pathway in the myocardium, resulting in a mild positive chronotropic effect, with an Emax of approximately 40%, and a weaker positive inotropic effect, with an Emax of less than 20%. AC: adenylate cyclase; GC: guanylate cyclase; AA: arachidonic acid; COX: cyclooxygenase; PGI2: Prostaglandin I2; L-Arg: L-Arginine; L-Cit: L-Citrulline; NO: nitric oxide; NOS: nitric oxide synthetase.
This mild cardiostimulating effect of sudachitin may contribute to increased cardiac output through a moderate increase in HR, enhanced blood flow, and improved peripheral circulation. Consequently, the intake of Citrus sudachi may help prevent lifestyle-related diseases by promoting overall cardiovascular health.
We thank Ikeda Yakusou Co., Ltd. for providing us with sudachitin and demethoxysudachitin samples.
The authors declare no conflict of interest.
