Elsevier

Fitoterapia

Volume 139, November 2019, 104367
Fitoterapia

trans-δ-Viniferin inhibits Ca2+-activated Cl channels and improves diarrhea symptoms

https://doi.org/10.1016/j.fitote.2019.104367Get rights and content

Abstract

Ca2+-activated Cl channels (CaCCs) wildly exist in many tissues which play an important role in ion transport and excitation conduction, especially fluid secretion and smooth muscle contraction in epithelial tissues. TMEM16A as a classic CaCC expresses in the intestine, and has become a potential target of intestinal physiological and pathological researches and therapeutic drug screening. In this study, we identified trans-δ-viniferin (TVN), a resveratrol dimmer, could inhibit TMEM16A activity in TMEM16A expressed FRT cells with IC50 of 19.7 μM, it also prevented Ca2+-activated Cl current in HT-29 cells with IC50 of 4.65 μM and in colonic mucosa. In the mechanism studies, TVN showed no significant inhibition on CFTR and basal Na+/K+-ATPase in both intestinal epithelial cells and colonic tissues, except for inhibition of calcium concentration and Ca2+-activated K+ channel to some degree. In anti-diarrheal studies, TVN could effectively prevent diarrhea caused by rotavirus infection and reduce the pellet number in IBS-D mice. These physiological effects are at least partially attributed to the inhibitory effect of TVN on CaCC-mediated intestinal fluid secretion and the reduction of smooth muscle contraction force by inhibiting TMEM16A. Collectively, the present study identified a new pharmacological target of TVN which provided the theoretical basis for the application of TVN in the treatment of rotavirus-infected diarrhea and IBS-D.

Introduction

Ca2+-activated Cl channels (CaCCs) are widely distributed in various tissues of species ranging from invertebrates to mammals, and play diverse roles in physiological functions. Cl secretion in secretory epithelia such as airway, salivary gland and intestines is one of known functions of CaCCs [1,2]. In addition, CaCCs are also found in smooth muscles, olfactory sensory neurons, cardiac muscles, and various excitable tissues, and involved in the regulation of smooth muscle contraction, neuronal and cardiac excitability, sensory transduction, and nociception [[3], [4], [5], [6], [7]].

The molecular identity of CaCCs is subject to various speculations until TMEM16A was identified by three independent groups with different strategies [[8], [9], [10]]. TMEM16A has typical electrophysiological and pharmacological characteristics resemble to those of native CaCCs such as calcium sensitivity, outward rectification, anion selectivity, and drug sensitivity [[10], [11], [12]]. TMEM16A has been demonstrated to constitute the native CaCCs in multiple epithelial cells [13,14], smooth muscle cells [15,16], sensory neurons [17], and interstitial cells of Cajal (ICC) [18,19].

In secretory epithelia, TMEM16A as an alternative pathway of CFTR Cl secretion becomes the primary target for the study of Cl secretion in several tissues. TMEM16A has been detected in both rodent and human airway epithelium, and its mediated Cl secretion plays a crucial role in the maintenance of periciliary liquid layer and the protection against airway microbial infection. Studies have shown that TMEM16A expression in airway epithelial cells was substantially up-regulated by cytokines IL-4 and IL-13 stimulation [14,20]. Small interfering RNA treatment could down-regulate TMEM16A expression which inhibited Ca2+-activated Cl secretion in epithelial cells processed by IL-4 [8]. Therefore, TMEM16A may play critical roles in bronchial epithelial cells under pathological conditions. TMEM16A is also considered responsible for fluid secretion in the intestinal epithelium for the reasons that TMEM16A is expressed in intestinal epithelial cells, and Ca2+-activated Cl secretion was significantly suppressed in the colon of mice with intestinal epithelial TMEM16A gene knockout [21]. In contrast, TMEM16A inhibitors minimally inhibited Ca2+-activated Cl currents in colon cancer epithelial cells [21,22]. On the other hand, TMEM16A was detected highly expressed in ICC cells distributed in the gastrointestinal muscle layers [23]. As gastrointestinal pacemaker cells, the major physiological functions of the ICC are regulation of the slow wave with contractions [24], neuromodulation and mechanotransduction [25,26]. The slow wave of intestinal smooth muscle disappeared in TMEM16A knockout mice, and the rhythmic contraction of smooth muscle was abnormal [19]. Pharmacological inhibition of TMEM16A function has been shown effective in the inhibition of intestinal motility, it also increased intestinal fluid absorption, and improved the symptoms of diarrhea and cramps [27,28], therefore, TMEM16A inhibitors may become potential agents for the treatment of diarrheas induced by bacteria and viruses and functional bowel disease such as irritable bowel syndrome.

Resveratrol and its polymers as a kind of natural polyphenols have been proved in-vivo and in-vitro experiments to obtain a variety of physiological activities beneficial to body health. Among these activities, some are related to the regulation of ion channel activities. Resveratrol promoted gallbladder relaxation by activating ATP-sensitive potassium channels and BKCa channels [29]. Resveratrol exerted antinociception by regulating calcium channels and calcium/caffeine-sensitive pools [30]. Epsilon-viniferin modulated transepithelial ion transport and permeability in the intestinal mucosa, and decreased mucosal sensitivity to short-chain fatty acids [31]. CaCCs as an important class of chloride channels participated in the regulation and maintenance of various physiological functions, therefore, we speculated that the pharmacological activity of resveratrol and its polymers might be related to the regulation of CaCC. In fact, two resveratrol oligomers were identified to have inhibitory activity on intestinal epithelial CFTR and CaCCs which is responsible for their inhibition on cholera toxin–stimulated fluid secretion and prevention in rotaviral diarrhea in previous studies [32,33]. In the present study, we identified another resveratrol oligomer, named trans-δ-viniferin (TVN) as a CaCC inhibitor that improved diarrhea symptoms of irritable bowel syndrome and rotavirus infection.

Section snippets

Animals, cells and chemicals

C57BL/6 mice aged 8 to 10 weeks were used in measurements of short-circuit current in colonic epithelium and determination of smooth muscle contraction in ileum. Mice with irritable bowel syndrome were established using two-day-old C57BL/6 pups. The feeding and management of all mice were in accordance with the “Guide for the Care and Use of Laboratory Animals of the National Institutes of Health”, and the experimental protocol was approved by Liaoning Normal University Committee on Animal

Inhibition of Clˉ current by TVN in TMEM16A expressed FRT cells

The direct inhibitory effect of TVN on TMEM16A activity was measured in TMEM16A expressed FRT cells. As shown in Fig. 1A and B, dehydrodimer of resveratrol TVN totally inhibited the TMEM16A-mediated Cl- current in a dose-dependent manner with IC50 of 19.7 μM which was higher than that of the TMEM16A selective inhibitor T16Ainh-A01. Since the activation of TMEM16A requires a certain concentration of Ca2+, and either Ca2+ released from calcium stores or extracellular Ca2+ influx can activate

Discussion

Viniferin is one of stilbenes found in many genera which is synthesized by trans-resveratrol through dehydrogenation polymerization, and when plants are infected by pathogens, chemical pollutants or stimulated by adversity stress, it is produced in large quantities and acts as a plant protectin [35,36]. The most commonly studied groups of viniferin in the field of pharmacy are dimers including trans-ε-viniferin, TVN, and the cis-and trans-isomers of trimer α-viniferin which were firstly

Conclusion

In summary, the present study identified TVN as a TMEM16A and intestinal epithelial CaCC inhibitor, which added a new pharmacological target of TVN. Antidiarrheal activity of TVN determined in rotavirus-infected mice and IBS-D model mice suggested that TVN might have potential as a precursor compound for the development of antisecretory and antimotility agents.

Funding

This work was supported by Scientific Research Fund of Liaoning Provincial Education Department (grants No. L201783646), Natural Science Foundation Project of Liaoning Province (grants No. 2019-BS-155), National Natural Science Foundation of China (grants Nos. 81973380 and 31471099) and National Science and Technology Major Project in major new drug discovery (grants Nos. 2017ZX09201-002-007).

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

References (58)

  • H. Seo et al.

    In vitro activity of alpha-viniferin isolated from the roots of Carex humilis against Mycobacterium tuberculosis

    Pulm. Pharmacol. Ther.

    (2017)
  • M.G. Dilshara et al.

    Anti-inflammatory mechanism of alpha-viniferin regulates lipopolysaccharide-induced release of proinflammatory mediators in BV2 microglial cells

    Cell. Immunol.

    (2014)
  • M. Yanez et al.

    (−)-trans-epsilon-viniferin, a polyphenol present in wines, is an inhibitor of noradrenaline and 5-hydroxytryptamine uptake and of monoamine oxidase activity

    Eur. J. Pharmacol.

    (2006)
  • P. Sasikumar et al.

    Isolation and characterization of resveratrol oligomers from the stem bark of Hopea ponga (Dennst.) Mabb. And their antidiabetic effect by modulation of digestive enzymes, protein glycation and glucose uptake in L6 myocytes

    J. Ethnopharmacol.

    (2019)
  • C. Hartzell et al.

    Calcium-activated chloride channels

    Annu. Rev. Physiol.

    (2005)
  • K. Kunzelmann et al.

    Anoctamins

    Arch. Eur. J. Physiol.

    (2011)
  • C.R. Bader et al.

    Calcium-activated chloride current in cultured sensory and parasympathetic quail neurones

    J. Physiol.

    (1987)
  • K.P. Currie et al.

    Activation of Ca(2+)-dependent Cl- currents in cultured rat sensory neurones by flash photolysis of DM-nitrophen

    J. Physiol.

    (1995)
  • W.A. Large et al.

    Characteristics and physiological role of the Ca(2+)-activated Cl- conductance in smooth muscle

    Am. J. Phys.

    (1996)
  • B. Nilius et al.

    Ion channels and their functional role in vascular endothelium

    Physiol. Rev.

    (2001)
  • A. Caputo et al.

    TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity

    Science.

    (2008)
  • Y.D. Yang et al.

    TMEM16A confers receptor-activated calcium-dependent chloride conductance

    Nature

    (2008)
  • C. Duran et al.

    Physiological roles and diseases of Tmem16/Anoctamin proteins: are they all chloride channels?

    Acta Pharmacol. Sin.

    (2011)
  • K. Kunzelmann et al.

    Role of the Ca2+ −activated Cl- channels bestrophin and anoctamin in epithelial cells

    Biol. Chem.

    (2011)
  • F. Huang et al.

    Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction

    Proc. Natl. Acad. Sci. U. S. A.

    (2012)
  • B. Manoury et al.

    TMEM16A/anoctamin 1 protein mediates calcium-activated chloride currents in pulmonary arterial smooth muscle cells

    J. Physiol.

    (2010)
  • C. Thomas-Gatewood et al.

    TMEM16A channels generate Ca(2)(+)-activated Cl(−) currents in cerebral artery smooth muscle cells

    Am. J. Physiol. Heart Circ. Physiol.

    (2011)
  • B. Liu et al.

    The acute nociceptive signals induced by bradykinin in rat sensory neurons are mediated by inhibition of M-type K+ channels and activation of Ca2+-activated Cl- channels

    J. Clin. Invest.

    (2010)
  • F. Huang et al.

    Studies on expression and function of the TMEM16A calcium-activated chloride channel

    Proc. Natl. Acad. Sci. U. S. A.

    (2009)
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