Mechanism of resveratrol-induced relaxation in the human gallbladder

All procedures were performed in accordance with relevant laws and institutional guidelines. In addition, the Institutional Review Board / Ethics Committee of Tzu Chi Hospital approved the protocol for this work (approval number: IRB097-55). The human specimens of gallbladder were acquired from patients undergoing cholecystectomy for gallstone (excluding acute cholecystitis) or hepatoma, and informed consent was obtained from every patient. Human gallbladder specimens were acquired from 39 patients (The ratio of male to female was 22:17; the age range was from 28 to 80 years; the median age was 58 years; the mean age was 56.8 ± 1.9 years). A standard incubation solution was made up of the following composition (in mM): 118 NaCl, 25 NaHCO₃, 4.7 KCl, 14 glucose, 1.2 NaH₂PO₄, 1.8 CaCl₂, pH 7.4. Immediately after surgical removal of the gallbladder, an area of 3 × 5 cm was excised from the middle portion of each gallbladder corpus. The human specimens were placed in oxygenated standard incubation solution gassed with 95% O₂ + 5% CO₂ for transportation to the laboratory, where the relaxation experiment was at once initiated. The period of transportation was less than 30 min. The human gallbladder strips were used to study the effects of resveratrol on gallbladder. The muscle strips, which were 3 mm wide and 10 mm long, were trimmed and hanged in 7 ml organ baths containing a standard incubation solution, incubated at 37 °C, and continuously gassed with 95% O₂ + 5% CO₂. Subsequently, the muscle strips were joined to isometric force transducers (FT.03; Grass Technologies, West Warwick, RI, USA), which were linked to amplifiers and a computer recording system (BIOPAC Systems, CA, USA). The basal tension of the muscle strips was set at 1.0 g. After a 30 min equilibration period, carbachol (1 μM) was added into the organ bath, the muscle strip contractions were measured, and the carbachol was washed out. For measurements of relaxation in carbachol-precontracted strips, resveratrol was added to muscle strips 15 min after the addition of carbachol. Resveratrol was added in a noncumulative fashion, i.e., with single dose administration (1 μM, 10 μM, 30 μM, 100 μM, or 1 mM, n is at least ≥4), and the isolated gallbladder muscle strip relaxations were measured. After the final step of the experiments, papaverine (100 μM) was added, and the muscle strip relaxations were measured. The difference between papaverine-induced relaxation and carbachol-induced contraction served as a reference (100%) for the relaxation response to resveratrol.

In order to determine whether resveratrol triggers neurally-mediated human gallbladder relaxation, 1 μM TTX (a neuronal Na⁺ channel blocker) and 1 μM CTX (a neuronal Ca²⁺ channel blocker) were added 15 min before the addition of 100 μM resveratrol to test the effects of resveratrol on neurally-mediated human gallbladder relaxation.

In order to investigate the mechanism of resveratrol-induced human gallbladder relaxation, 1 μM KT 5720 (a cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) inhibitor), 1 μM KT 5823 (a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor), and 100 μM L-NNA (a competitive nitric oxide synthase (NOS) inhibitor) were added 30 min before the addition of 100 μM resveratrol to test the involvement of cAMP, cGMP, and NO on resveratrol-induced human gallbladder relaxation [12, 13].

In order to investigate whether potassium channels are involved in resveratrol-induced human gallbladder relaxation, 1 mM TEA (a non-selective potassium channel blocker), 200 nM iberiotoxin (an inhibitor of large conductance calcium-activated potassium (BKCa) channels), 100 nM charybdotoxin, 200 nM apamine, and 10 μM glibenclamide (an adenosine triphosphate (ATP)-sensitive potassium channel blocker) were added 30 min before the addition of 100 μM resveratrol to test the effects of potassium channels on resveratrol-induced human gallbladder relaxation [14‐17].

In muscle strips isolated from human gallbladder specimens, 1 μM carbachol induced a marked and long-duration muscle contraction and 1 mM resveratrol induced an obvious muscle relaxation of the carbachol precontracted gallbladder strips (Fig. 1). The resveratrol-induced relaxation in human gallbladder was dose-dependent (Fig. 2). Resveratrol caused detectable relaxation of human gallbladder muscle strips at 10 μM and maximal relaxation at 1 mM. The maximal relaxation caused by 1 mM resveratrol was 90.8 ± 5.1% (n = 6) of the relaxation caused by 100 μM papaverine.

As shown in Fig. 3, neither 1 mM TTX nor 1 mM CTX had inhibitory effects on the relaxation in human gallbladder induced by resveratrol (p > 0.05, n = 6 and 3, respectively). These results indicate that resveratrol-induced human gallbladder relaxation does not involve the activation of the enteric nervous system.

As shown in Fig. 4, 1 μM KT 5720 (n = 6) had no inhibitory effects on the relaxation in human gallbladder induced by 100 μM of resveratrol (p > 0.05). However, both 1 μM KT 5823 and 100 μM L-NNA had significant inhibitory effects on resveratrol-induced human gallbladder relaxation (p < 0.05, n = 5 and 6 respectively). These results indicate that the relaxation in human gallbladder induced by resveratrol is associated with cGMP and NO production.

As shown in Fig. 5, 1 mM TEA (n = 5) and 200 nM apamine (n = 4) did not inhibit the relaxation in human gallbladder induced by 10 μM of resveratrol (p > 0.05, n = 12 in the resveratrol group). However, 100 nM charybdotoxin (n = 5) had a trend to inhibit relaxation in human gallbladder induced by 10 μM of resveratrol (p = 0.093). Furthermore, 10 μM of glibenclamide (n = 6) and 200 nM iberiotoxin (n = 6) induced a significant inhibitory effect on resveratrol-induced human gallbladder relaxation (p < 0.05). These results indicate that the relaxant effects in human gallbladder induced by resveratrol are related to ATP-sensitive potassium channels and BKCa channels.