Animal care and experiments were conducted in accordance with the guidelines issued by the ethics committee of Pusan National University (Busan, South Korea; approval no. PNU-2015-1036) and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23; 1996 revision).

BALB/c mice (age, 3–7 days weight, 1.9–2.2 g; Samtako Bio Korea Inc., Osan-si, Korea) were anesthetized with 0.1% ether and euthanized by cervical dislocation. They were maintained under controlled conditions (temperature, 21±3°C; humidity 50±6%; 12 h light/dark cycles) and were allowed free access to food and water. Small intestines were excised from 1 cm below the pyloric ring to the cecum, and were opened along the mesenteric border. Luminal contents were removed using Krebs Ringer bicarbonate solution, tissues were pinned to the bases of Sylgard dishes, and mucosae were removed by sharp dissection. Small tissue strips of intestinal muscle, which consist of circular and longitudinal muscles, were equilibrated for 30 min in Ca²⁺-free Hank's solution containing 5.36 mM KCl, 125 mM NaCl, 0.34 mM NaOH, 0.44 mM Na₂HCO₃, 10 mM glucose, 2.9 mM sucrose, and 11 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); pH 7.4. Cells were then dispersed in an enzyme solution containing collagenase (1.3 mg/ml; Worthington Biochemical Corporation, Lakewood, NJ, USA), bovine serum albumin (2 mg/ml; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), trypsin inhibitor (2 mg/ml; Sigma-Aldrich; Merck Millipore) and ATP (0.27 mg/ml), and were plated onto sterile glass coverslips coated with murine collagen (2.5 mg/ml; BD Biosciences, Franklin Lakes, NJ, USA) in 35 mm culture dishes. Subsequently, the cells were cultured at 37°C in an atmosphere containing 95% oxygen/5% carbon dioxide in smooth muscle growth medium (Clonetics™; Lonza, Basel, Switzerland) supplemented with 2% antibiotics/antimycotics (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and murine stem cell factor (5 ng/ml; Sigma-Aldrich; Merck Millipore). All experiments on ICC clusters were performed following 12 h of culture. ICCs were identified immunologically by incubating with a phycoerythrin-conjugated rat anti-mouse c-Kit monoclonal antibody (cat. no. 12-1172; eBioscience, Inc., San Diego, CA, USA), at a dilution of 1:50 for 20 min at 37°C. Since the ICC morphology differed from the other cell types in culture, identification was possible under a phase contrast microscope following incubation with the anti-c-Kit antibody.

Physiological salt solution [5 mM KCl, 135 mM NaCl, 2 mM CaCl₂, 10 mM glucose, 1.2 mM MgCl₂, and 10 mM HEPES (adjusted to pH 7.4 with NaOH)] was used to bathe cultured ICC clusters (Na⁺-Tyrode). The pipette solution used to examine pacemaker activity consisted of the following reagents: 140 mM KCl, 5 mM MgCl₂, 2.7 mM dipotassium ATP, 0.1 mM sodium guanosine-5′-triphosphate, 2.5 mM creatine phosphate disodium, 5 mM HEPES and 0.1 mM ethylene glycol tetra-acetic acid (adjusted to pH 7.2 with KOH). Patch clamp techniques were conducted in whole-cell configuration to record potentials (i.e., current clamp mode) from cultured ICCs using Axopatch I-D and Axopatch 200B amplifiers (Axon Instruments, Inc., Foster, CA, USA). Command pulses were applied using an IBM-compatible personal computer (Compaq Computer Corporation, Houston, TX, USA) and pClamp software (versions 6.1 and 10.0; Axon Instruments, Inc.). Data were filtered at 5 kHz and displayed on an oscilloscope, a computer monitor, and/or a pen recorder (Gould 2200; Gould Instruments, Inc., Valley View, OH, USA). Results were analyzed using pClamp and Origin software (version 6.0; MicroCal, Northampton, MA, USA). All experiments were performed at 30–33°C.

ICCs were preincubated with 100 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich; Merck Millipore) for 30 min at 37°C to inhibit cGMP degradation, and were then incubated with berberine (50 μM; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) for 10 min. Following homogenization in a buffer containing 4 mM ethylenediaminetetraacetic acid to prevent degradation of enzymatic cGMP, homogenates were heated for 5 min in a boiling water bath to coagulate proteins, and were then centrifuged at 3,950 × g for 5 min at 4°C. The super-natants subsequently obtained were transferred to fresh tubes and were stored at 4°C. Samples were assayed for cGMP using cGMP enzyme-linked immunosorbent assay kits (Enzo Life Sciences, Inc., Farmingdale, NY, USA). These assays were conducted according to the manufacturer's protocol.

Opioid receptor antagonists were purchased from Tocris Bioscience (Minneapolis, MN, USA); all other drugs were obtained from Sigma-Aldrich (Merck Millipore). Stock solutions were prepared and stored in accordance with the manufacturers' protocols. Chemicals were dissolved in Na⁺-Tyrode solution to obtain their final concentrations immediately prior to use. Berberine was dissolved in methanol to produce a 50 mmol/l stock solution, which was subsequently added to the bathing solution at a final concentration of 50 μM on the day of the experiment for 5 min. The final concentration of methanol in the bathing solution was <0.1% and preliminary experiments confirmed that this concentration of methanol did not affect results. In addition, glibenclamide (Sigma-Aldrich; Merck Millipore) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck Millipore) to produce a 10 mmol/l stock solution, which was added to the bathing solution at a final concentration of 10 μM on the day of the experiment for 5 min. Next, nor-Binaltorphimine dihydrochloride (Tocris Bioscience) was dissolved in distilled water to produce a 25 mmol/l stock solution, which was added to the bathing solution at a final concentration of 100 nM on the day of the experiment. ICI 174,864 (Tocris Bioscience) and CTOP (Tocris Bioscience) was directly added to the bathing solution at a concentration of 20 μM and 10 μM on the day of the experiment. Both SQ-22536 (Sigma-Aldrich; Merck Millipore) and ODQ (Sigma-Aldrich; Merck Millipore) were dissolved in DMSO to produce a 100 mmol/l stock solution, which was added to the bathing solution at a final concentration of 100 μM on the day of the experiment for 5 min. Also, KT-5720 (Sigma-Aldrich; Merck Millipore) and KT-5823 Sigma-Aldrich; Merck Millipore) were dissolved in DMSO to produce 1 mmol/l stock solutions, which were added to the bath solution at a final concentration of 1 μM on the day of the experiment for 5 min.

The data are presented as the mean ± standard error of the mean. Student's t-test and one-way analysis of variance followed by Bonferroni's post-hoc test were used to determine significance. P<0.05 was considered to indicate a statistically significant difference. For statistical analyses, Prism version 5.0 (GraphPad, Software Inc., La Jolla, CA, USA) and Origin version 8.0 (OriginLab Corporation, Northampton, MA, USA) were used. The n values reported in the text refer to the number of cells used in patch clamp experiments. Experiments were repeated 6–8 times.

The ICCs generated PPs under whole cell patch current clamp mode (I=0) (Fig. 1) at a mean frequency of 24.1±2.1 cycles/min and a mean amplitude of 23.8±1.5 mV. Treatment with berberine (10–50 μM) inhibited PPs and decreased their amplitudes in a concentration-dependent manner (Fig. 1A–C). In the presence of berberine, mean PP frequencies were 23.1±3.2 cycles/min at 10 μM, 22.2±3.1 cycles/min at 30 μM, and 2.2±1.3 cycles/min at 50 μM (Fig. 1D, n=18), whereas mean amplitudes were 23.5±1.4 mV at 10 μM, 12.3±1.5 mV at 30 μM, and 1.8±0.9 mV at 50 μM (Fig. 1E, n=18). These results suggest that berberine may dose-dependently inhibit the PPs of ICCs.

In our previous study, pinacidil (an ATP-sensitive K⁺ channel opener) decreased the frequency and amplitude of PPs, and these pinacidil-induced effects were reversed by treatment with glibenclamide (an ATP-sensitive K⁺ channel blocker) (14). In the present study, berberine-induced ICC PP inhibition was suppressed by glibenclamide (Fig. 2A), and following glibenclamide pretreatment, berberine exerted no effects on PPs (Fig. 2B). The effects of berberine and glibenclamide on PPs are presented in Fig. 2C and D. These results suggest that berberine may inhibit PPs via ATP-sensitive K⁺ channels in ICCs.

To investigate the association between berberine and its receptors in cultured ICCs, the opioid receptors were investigated, since they are known to be involved in the regulation of GI motility (15–17). There are three major classes of opioid receptor (mu, delta and kappa) in the GI tract (15–17). To identify the opioid receptor subtypes associated with the effects of berberine, ICCs were pretreated with opioid receptor antagonists, followed by treatment with berberine. Nor-binaltorphimine dihydrochloride (a kappa opioid receptor antagonist), ICI 174,864 (a delta opioid receptor antagonist) and CTOP (a mu opioid receptor antagonist) were used to pretreat the cells for 5 min, followed by berberine treatment (50 μM) (Fig. 3). Treatment with these opioid receptor antagonists alone had no effect on PPs; however, pretreatment with ICI 174,864 or CTOP suppressed berberine-induced PP inhibition (Fig. 3B and C). In the presence of ICI 174,864 or CTOP, the mean amplitudes of berberine-induced PPs were 14.6±1.1 and 24.1±1.2 mV, respectively (n=6; Fig. 3E). Conversely, pretreatment with nor-binaltorphimine dihydrochloride did not suppress berberine-induced PP inhibition (Fig. 3A). In the presence of nor-binaltorphimine dihydrochloride, the mean frequency and amplitude of berberine-induced PPs were 3.1±1.4 cycles/min and 2.4±0.5 mV, respectively (n=6; Fig. 3D and E). These results suggest that berberine may affect ICCs via mu and delta opioid receptors.

To determine whether berberine-induced PP inhibition is mediated by a cyclic nucleotide-dependent pathway, an adenylate cyclase inhibitor (SQ-22536) and guanylate cyclase inhibitor (1H-[1,2,4] oxadi-azolo [4,3-a] quinoxalin-1-one; ODQ) were used to treat the ICC clusters. Preincubation with SQ-22536 (100 μM) alone for 5 min had no effect on PPs, and in the presence of SQ-22536, berberine (50 μM) still inhibited PPs (Fig. 4A). However, ODQ (100 μM) suppressed berberine-induced PP inhibition (Fig. 4B). In the presence of SQ-22536, the mean frequency and amplitude of berberine-induced PPs were 1.8±0.9 cycles/min and 1.6±0.9 mV, respectively (n=6; Fig. 4C and D); the ODQ corresponding values were 23.7±1.0 cycles/min and 23.8±0.8 mV, respectively (n=6; Fig. 4C and D). In addition, the effects of a protein kinase A inhibitor (KT-5720) and a PKG inhibitor (KT-5823) were detected. Preincubation of ICCs with KT-5720 or KT-5823 alone had no effect on PPs. Furthermore, in the presence of KT-5720 (1 μM), berberine (50 μM) inhibited PPs (Fig. 5A); however, preincubation with KT-5823 (1 μM) suppressed berberine-induced PP inhibition (Fig. 5B). In addition, intracellular cGMP contents were measured under basal and berberine-stimulated conditions, and berberine was revealed to stimulate cGMP production [Fig. 6; control (12.1±0.6 pmol/mg protein) vs. berberine (14.3±0.9 pmol/mg protein)]. These results suggest that cGMP and PKG may have roles in berberine-induced PP inhibition.