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Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel

Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel The volume-sensitive outwardly rectifying anion channel (VSOR) is a key component of volume regulation system critical for cell survival in non-isosmotic conditions. The aim of the present study was to test the effects of four tannin extracts with defined compositions on cell volume regulation and VSOR. Preparation I (98% of hydrolysable tannins isolated from leaves of sumac Rhus typhina L.) and Preparation II (100% of hydrolysable tannins isolated from leaves of broadleaf plantain Plantago major L) completely and irreversibly abolished swelling-activated VSOR currents in HCT116 cells. Both preparations profoundly suppressed the volume regulation in thymocytes with half-maximal effects of 40.9 µg/ml and 12.3 µg/ml, respectively. The inhibition was more efficient at lower concentrations but reverted at higher doses due to possible non-specific membrane- permeabilizing activity. Preparations III and IV (54,7% and 54.3% of hydrolysable tannins isolated, respectively, from roots and aboveground parts of Fergana spurge Euphorbia ferganensis B.Fedtch) inhibited VSOR activity in a partially reversible manner and suppressed the volume regulation with substantially higher half-maximal doses of 270 and 278 µg/ml, respectively, with no secondary reversion at higher doses. Hydrolysable tannins represent a novel class of VSOR channel inhibitors with the capacity to suppress the cell volume regulation machinery. Keywords Tannins – plant polyphenols – thymocytes – cell volume regulation – volume-sensitive anion channel This work was supported by the Ministry of Innovative component of the cellular volume regulation system and of Development of the Republic of Uzbekistan (under the grants some other physiological and pathophysiological processes FА-А11-Т060 and PZ2017092049). including cell proliferation, migration and apoptosis (Akita & Okada, 2014; Hoffmann et al., 2014; Okada et al., 2009). VSOR INTRODUCTION is the main pathway for the efflux of anions from swollen cells and, in cooperation with the Ca-activated potassium In order to survive in constantly changing osmotic conditions channels, provides a reduction of the intracellular osmotic caused by intensive physiological processes (breathing, food pressure during the active phase of volume regulation intake, fluid secretion and absorption, filtration and urine upon the hypoosmotic stress called the Regulatory Volume formation, etc.) and pathologies (inflammation, edema, Decrease (RVD) (Akita et al., 2011; Akita & Okada, 2014; Delpire trauma, ischemia and hypoxia), living cells developed & Gagnon, 2018; Hoffmann et al., 2014; Okada et al., 2006; elaborate volume regulatory mechanisms. The volume- Pedersen et al., 2016). Recent studies have demonstrated sensitive outwardly rectifying anion channel (VSOR) is a key that the LRRC8 family proteins constitute the molecular * E-mail: zairovich@gmail.com © European Pharmaceutical Journal OR 37 Eur. Pharm. J. 2019, 66(2), 37-44 Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel Tsiferova N.A. et al. basis of VSOR (Qiu et al., 2014; Voss et al., 2014), structurally and their composition was determined by a combination of organized into a hexameric pore (Deneka et al., 2018; Kasuya preparative column chromatography (silica gel, polyamide), et al., 2018; Kefauver et al., 2018). However, pharmacology quantitative paper chromatography and spectral methods as of this biologically important ion channel remains poorly described elsewhere (Abdulladzhanova et al., 2001). explored. Thus far, a number of structurally divers compounds Preparation I was obtained from the leaves of sumac (Rhus including stilbene derivatives, etacrynic acid analogs and typhina L.) and contained: 3,6-bis-О-di-O-galloyl-1,2,4-tri- flavonoids have been shown to suppress the activity of VSOR О-galloyl-β-D-glucose (74%); 1,2,3,4,6-penta-О-galloyl-β-D- in a voltage-dependent and independent manner (Okada et glucose (10%); 1,4,6-tri-О-galloyl-β-D-glucose (5%); 2,3-di- al., 2019; Xue et al., 2018). О-galloyl-β-D-glucose (2%); 2-О-galloyl-β-D-glucose (2%); Tannins are structurally heterogenous polyphenols, 3-О-galloyl-β-D-glucose (2%); 6-О-galloyl-β-D-glucose (2%); which bind to proteins and can trigger their precipitation gallic acid (1%); rutin (1%); quercetin (0.5%); kaempferol (Mavlyanov et al., 2001). Tannins are secondary metabolites (0.5%). and constitute a part of the plants’ defense system against Preparation II was obtained from the leaves of broadleaf pathogens and insect’s invasion. In addition, these substances plantain (Plantago major L.) and contained: diester exhibit a wide spectrum of biological activities such as of hexahydroxydiphenoyl-1-(О-2-О-galloyl-β-D- antimicrobial (Scalbert, 1991); antioxidant, (Rice-Evans et al., glucopyranosido)-1-(О-β-D-xylopyranoside (30.1%); diester 1995, 1996); anti-inflammatory (Terra et al., 2007; Xue et al., of hexahydroxydiphenoyl-1-(О-β-D-glucopyranosido)-2-(О- 2018); neuroprotective (Behravan et al., 2014). Tannins, along 4-О-galloyl-β-D-glucopyranoside) (27.9%); quercetin-3-О-(2”, with other polyphenolic compounds, have been considered 6”di- О- galloyl-3”- О- p- coumaroyl)-β -D - glucopyranoside to be responsible for the health benefits of red wine and (25.4%); kaempferol-3-O-(2’’,3’’-di-O-galloyl-6’’-О- coumaroyl- green tea, possibly by inhibiting the activity of the Ca- β-D-glucopyranoside (16.6%). activated chloride channels (CaCCs) (Namkung et al., 2010). Preparation III was obtained from the roots of Fergana spurge Penta-m-digalloyl-glucose, a hydrolysable tannin extracted (Euphorbia ferganensis B.Fedtch.) and contained: 1-О-galloyl- from the Chinese gallnut, was demonstrated to inhibit 2,4- v aloneoyl-4,6-he xahydrox ydiphenoyl-β -D - glucose the cystic fibrosis transmembrane conductance regulator (48%); gallic acid (15%); digallic acid (10%); ellagic acid (9%); protein (CFTR), a chloride channel activated by intracellular terchebin (8%); 2,3-digalloyl-β-D-glucose (6.7%); quercetin- cAMP (Wongsamitkul et al., 2010). Tannic acid was shown to 3-О-rutinoside (2.3%); myricetin (0.8%); iso-myricitrin (0.2%). inhibit CaCCs formed by TMEM16A and B (Cruz-Rangel et al., Preparation IV was obtained from the aboveground part of 2015; Namkung et al., 2010, 2011) and TMEM16F (Szteyn et Fergana spurge (Euphorbia humifusa Willd.) and contained: al., 2012), which is consistent with an antidiarrhoeic activity 1-О-galloyl-4,6-hexahydroxydiphenoyl-β-D-glucose (35%); of tannin-containing extracts reported earlier (Galvez et al., quercetin (17%); ellagic acid (10.8%); 3-О-galloyl-4,6- 1991). Tannic acid also blocks L-type Ca-channels (Zhu et hexahydroxydiphenoyl-β-D-glucose (8.3%); 1,2,3-tri-O- al., 2016) and the maxi-anion channel (Woll et al., 1987). The galloyl-β-D-glucose (7%); gallic acid (7%); geraniin (2.5%); latter is known to be swelling-activated (Okada et al., 2018, quercetin-3-О-rhamnoside (4%); quercetin-3-О-galactoside 2019; Sabirov & Merzlyak, 2012; Sabirov et al., 2016), although (3.2%); kaempferol-3-О-glucoside (2.7%); 1-O-galloyl-6-O-bis- it operates mostly when cells are metabolically deprived, galloyl-2,4-valoneoyl-β-D-glucose (1.5%); kaempferol (1%). whereas VSOR is the major contributor to the swelling- The tannin preparations were added from concentrated stock activated plasmalemmal conductance at normal intracellular solutions in dimethylsulfoxide (DMSO). Final concentration ATP levels. Tannins were never considered as modulators of DMSO did not exceed 0.1%, and at this concentration, the of the cell volume regulation system and its constituent solvent did not significantly affect the records. components. Here, we tested four tannin preparations of plant origin on their effects on the cell volume regulation Solutions in rat thymocytes and the VSOR channel activity in HCT116 human colon cancer cells. The normal Ringer solution contained (mM): 135 NaCl, 5 KCl, 2 CaCl , 1 MgCl , 11 HEPES, 5 glucose (рН 7.4, adjusted with 2 2 MATERIALS AND METHODS NaOH, 290 mOsm/kg-H O). The H-buffer contained (mM): 5 KCl, 2 CaCl , 1 MgCl , 10 HEPES, 5 glucose (рН 7.4, adjusted 2 2 Substances with NaOH, 40 mOsm/kg-H O). Hypotonic solutions were prepared by mixing the Ringer solutions with H-buffer in The plants were collected from the Tashkent environs a ratio of 3:4 (vol/vol). The pipette solution for whole-cell during the flowering stage and taxonomically identified experiments contained (in mM): 125 CsCl, 2 CaCl , 1 MgCl , 3 2 2 by Dr. Gnatchenko E.V. of the Institute of Botany of the Na ATP, 5 HEPES (pH 7.4 adjusted with CsOH), 10 EGTA, and 50 Academy of Sciences of Uzbekistan. Preparation of tannin mannitol (pCa 7.65; 320 mOsm/kg-H O). extracts was performed essentially as described previously (Islambekov et al., 1994; Olchowik-Grabarek et al., 2017) 38 39 Eur. Pharm. J. 2019, 66(2), 37-44 Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel Tsiferova N.A. et al. Cells Electrophysiology Human colon tumor cell line, HCT116, was cultured in DMEM Patch electrodes were fabricated from borosilicate glass supplemented with 10% of fetal bovine serum and antibiotics capillaries using a micropipette puller (PP-830, Narishige, (100 U/ml penicillin plus 100 mg/ml streptomycin) at 37 C Japan) and had a tip resistance of 3–5 MΩ when filled with and 5% CO . For patch-clamping, the cells were cultured in pipette solution. Fast and slow capacitive transients were suspension under mild stirring during 3–5 h. routinely compensated for. For whole-cell recordings, the All animal experiments were conducted in accordance access resistance did not exceed 10 MΩ and was always with the ARRIVE guidelines and approved by the Bioethics compensated for by 80%. Membrane currents were measured Committee of the Institute of Biophysics and Biochemistry. The with an EPC-9 patch-clamp system (Heka-Electronics, isolation of thymic lymphocytes (thymocytes) was performed Lambrecht/Pfalz, Germany). The membrane potential as described previously (Kurbannazarova et al., 2003, 2008, was controlled by shifting the pipette potential (Vp) and 2011; Sabirov et al., 2013). Briefly, the 6–8 weeks old rats, is reported as Vp for whole-cell recordings. Currents were kept in vivarium on an average diet, were anaesthetized filtered at 1 kHz and sampled at 5–10 kHz. Data acquisition and with halothane or diethyl ether and painlessly euthanized by analysis were done using Pulse + PulseFit (Heka-Electronics). cervical dislocation. The thymi were dissected and carefully Liquid junction potentials were calculated using pCLAMP washed with an ice-cold normal Ringer solution. The thymi 8.1 (Molecular Devices, Sunnyvale, CA) algorithms and were were then minced using fine forceps and passed through a corrected off-line when appropriate. All experiments were 100 µm-nylon mesh. The suspension was centrifuged at 1000 performed at room temperature (23–25 °C). g for 5 min; the pellet was washed two times with the normal Ringer solution and resuspended in this medium at a cell Data analysis density of 100 x 10 cells/ml. The cell suspension was kept on ice for ≤ 5 h and contained no more than 5% of damaged cells The dose-response data were approximated using a Hill as assayed by trypan blue exclusion. equation of the following form: Cell Volume Measurements RVD = RVD + (RVD – RVD )/(1 + (C/IC ) ) (2) min max min 50 Cell volume changes under non-isosmotic conditions were Here: RVD and RVD are the minimal and maximal values min max recorded by light transmittance measurement as described of RVD, C – concentration of the substance (μg/ml), IC – previously (Kurbannazarova et al., 2003, 2008, 2011). Briefly, concentration of the substance rendering a half-maximal 900 ml of the normal Ringer or hypotonic solutions was inhibitory effect (μg/ml), h – Hill coefficient. added to the 1.5 cm glass cuvette thermo stated with a water Data were analyzed using Origin 8 (OriginLab Corporation, jacket and equilibrated for 10 min. An aliquot (100 ml) of Northampton, MA, USA). Pooled data are given as means cell suspension was added to this medium to yield the final ± SEM of n observations. Comparisons between the two cell density of 10 x 10 cells/ml. The light transmittance was experimental groups were made using the unpaired two- measured at 610 nm (band-pass filter) using a photometer sample Student’s t-test. The data of Fig. 3d and 4d were MKMF-01 (Russia). The output signal was amplified by U5- analyzed using both unpaired two-sample (comparison 11 amplifier (Russia), digitized at 100 Hz using a USB sensor at different voltages) and one-sample (comparison with interface GO!Link and recorded by Logger Lite software control) t-test. Differences were considered to be statistically (Vernier, Beaverton, OR). significant at p < 0.05. The parameter RVD was calculated using the following RESULTS equation (1): RVD=( T -T )/( T -T )*100% (1) Tannin preparations inhibit thymocyte volume max 15 max 0 regulation under hypoosmotic stress where T and T are the initial and maximal light 0 max transmittances, and T is the light transmittance measured Blockers of VSOR channel are expected to suppress the 15 minutes after the onset of hypotonic stress. RVD = 100 regulatory volume decrease phase of cellular response to for complete recovery of the cell volume to the initial level, the hypoosmotic stress. In order to test this possibility, we and RVD = 0 when volume regulation is fully suppressed. employed the immature thymic lymphocytes which possess Under control conditions, RVD usually had values of 60– fully functional volume regulation machinery (Arrazola et al., 90% depending on the cells condition, osmotic gradient, 1993; Kurbannazarova et al., 2003; Soler et al., 1993). We have temperature and other experimental conditions. previously shown that thymocytes express the VSOR channels with the same biophysical and pharmacological profile as other cell types, and that VSOR blockers completely abolish 38 39 Eur. Pharm. J. 2019, 66(2), 37-44 Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel Tsiferova N.A. et al. RVD in these cells (Kurbannazarova et al., 2011; Sabirov et When preparations I and II were added to the flow chamber al., 2013). Therefore, we supposed that tannin preparations at a dose of 31 µg/ml and 41 µg/ml, respectively (at these might affect the volume regulation in thymocytes. doses, their effect on RVD was maximal, see Fig. 1c,d), we In our experiments, thymocytes, when challenged with observed almost instant suppression of the macroscopic hypoosmotic stress, first rapidly swelled (passive response) currents (Fig. 3a,b). The effect was essentially irreversible. The and then gradually restored their volume toward an initial ionic currents were suppressed at both positive and negative level (active response; Fig. 1a). The parameter RVD as defined potentials (Fig. 3c,d) suggesting that the channel blockage is by Equation (1) (see Experimental section) ranged from 66% voltage-independent. Voltage-independence together with to 93% and averaged at 79.6 ± 1.9% (n = 17). the irreversibility of blockage may indicate a strong, possibly When added to the hypoosmotic medium, tannin preparations covalent, interaction of the tannins with the channel protein. I and II exhibited a profound suppressive effect on the Preparations III and IV also exhibited suppressive effects on thymocyte volume regulation (Fig. 1a,b). For preparation I, the macroscopic swelling-induced conductance. However, an inhibition at lower doses was gradually lost as the amount in contrast to the preparations I and II, current inhibition by of the added preparation was increased above 30 µg/ml (Fig. preparations III and IV was slower (Fig. 4a,b) and partially 1a,c). Since the maximal swelling also declined at high doses, reversible. The currents in the presence of these preparations we supposed that high concentrations of preparation I were were decreased more efficiently at positive potentials (Fig. detrimental for cellular plasma membrane. A similar biphasic 4c,d) than at negative voltages. Reversibility of the inhibition action was also observed for preparation II (Fig. 1b,d). The together with voltage-dependency may suggest an open- half-maximal concentrations and Hill coefficients for the channel blockage mechanism: applied positive voltage drives inhibiting phase of the dose-response curves (solid circles the negatively charged polyphenolic compounds applied and solid lines in Fig. 1c,d) were as follows: preparation I (IC50 from the extracellular side into the channel lumen. = 40.9 ± 7.2 µg/ml; h = 0.96 ± 0.2) and preparation II (IC50 = DISCUSSION 12.3 ± 8.1 µg/ml; h = 0.59 ± 0.294). In contrast to the first two preparations, preparations III and IV did not affect the maximal swelling and did not display Thus, we have demonstrated that polyphenolic tannins of the secondary damaging phase on the dose-response curves plant origin represent a novel class of VSOR channel inhibitors. (Fig. 2). The half-maximal concentrations and Hill coefficients This activity may contribute to the well-documented for the inhibiting phase of the dose-response curves (solid beneficial health effects of polyphenol-rich food and drink circles and solid lines in Fig. 2c,d) were as follows: preparation products. III (IC50 = 270 ± 77 µg/ml; h = 0.63 ± 0.15) and preparation IV In our experiments, preparations (I and II) were more efficient (IC50 = 278 ± 43 µg/ml; h = 0.65 ± 0.07). inhibitors of VSOR and volume regulation system compared Comparison of the IC50 values suggested that preparations to the preparations III and IV. Since preparation II consisted I and II are more efficient inhibitors of the cell volume exclusively of hydrolysable tannins, and the content of regulation than preparations III and IV. hydrolysable tannins in preparation I reached 98%, we inferred that hydrolysable tannins represent a novel class of Tannin preparations block the swelling-induced VSOR channel inhibitors. Certainly, the tannin preparations anion conductance used in these experiments have rather complex composition, and thus, the individual tannins might be more effective and Tannins were never considered as volume-regulated anion selective modulators of the VSOR chloride channel. channel blockers. In order to test this possibility, we employed Preparations III and IV contained less overall tannin content direct electrophysiological assessment of the VSOR channel (54.7% and 54.3%, respectively) and were less effective. activity in human colorectal cancer HCT116 cells, which have Possibly, other types of polyphenols, which constituted a been used recently for molecular identification of the VSOR large part of these preparations may have weakened the channel proteins (Qiu et al., 2014; Voss et al., 2014). inhibitory effects of hydrolysable tannins of preparations III In our experiments, we filled the patch-pipettes with slightly and IV. hypertonic (by ~30 mOsm/kg-H O) solution to induce It should be noted that 3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl- cellular swelling as described previously (Kurbannazarova et β-D-glucose, which is the major component of Preparation al., 2011; Sabirov et al., 2013). Upon attaining the whole-cell I, was recently shown to form anion-selective channels in configuration, cells gradually swelled as could be observed lipid bilayers (Borisova et al., 2019). This effect may explain visually under phase-contrast microscopy. The cellular swelling the secondary rising phase on the dose-response curve was accompanied by a robust activation of the macroscopic on the Figure 1a,c, suggesting that these newly formed currents with outward rectification and inactivation at large pores functionally replace the VSOR channel by serving as a depolarizing positive potentials (Fig. 3a,b), a phenotypical pathway for anion efflux. Since Preparation II also displayed landmark of the VSOR anion channel (Okada, 1997). a biphasic effect of RVD, one may suppose that some of its 40 41 Eur. Pharm. J. 2019, 66(2), 37-44 Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel Tsiferova N.A. et al. Figure 1: Dose-dependent effects of Preparations I and II on the thymocyte volume regulation under hypoosmotic stress. (a, b) Representative recordings of light transmittance changes. (c, d) Dose-response curves; the solid lines are fits to the equation (2) with half-maximal concentrations and Hill coefficients given in the text. Figure 2. Dose-dependent effects of Preparations III and IV on the thymocyte volume regulation under hypoosmotic stress. (a, b) Representative recordings of light transmittance changes. (c, d) Dose-response curves; the solid lines are fits to the equation (2) with half-maximal concentrations and Hill coefficients given in the text. 40 41 Eur. Pharm. J. 2019, 66(2), 37-44 Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel Tsiferova N.A. et al. Figure 3: Inhibition of VSOR currents by Preparations I and II. (a, b) The time course of whole-cell current activation in response to cell swelling. Currents were elicited by application of alternating test-pulses from 0 to ±40 mV every 15 s. Arrowheads (▲) denote the time points where the step-pulses from ‒100 to +100 mV in 20 mV increments were applied to test the voltage-dependence of the macroscopic conductance. (c) Instantaneous current-to-voltage relationships measured at the beginning of test-pulses from recordings similar to those shown in (a) and (b); n = 5 for Preparation I and n = 4 for Preparation II. (d) Fractional currents measured at +40 mV (open bars) and ‒40 mV (hatched bars). *Significantly different from control values at p < 0.05. Figure 4: Inhibition of VSOR currents by Preparations III and IV. (a, b) The time course of whole-cell current activation in response to cell swelling. Currents were elicited by the application of alternating test-pulses from 0 to ±40 mV every 15 s. Arrowheads (▲) denote the time points where the step-pulses from ‒100 to +100 mV in 20 mV increments were applied to test the voltage- dependence of the macroscopic conductance. (c) Instantaneous current-to-voltage relationships measured at the beginning of test-pulses from recordings similar to those shown in (a) and (b); n = 5 for Preparation III and n = 4 for Preparation IV. (d) Fractional currents measured at +40 mV (open bars) and ‒40 mV (hatched bars). *Significantly different from control values at p < 0.05. Significantly different at p < 0.05 from values measured at negative voltages. 42 43 Eur. Pharm. J. 2019, 66(2), 37-44 Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel Tsiferova N.A. et al. components could also act as pore formers on lipid matrix of that tannins antagonizing activity of the VSOR anion channel the cellular plasma membrane. could be beneficial in protecting brain tissues, and possibly, What kind of pharmacological effects could be anticipated the heart, during ischemic/hypoxic injury. for VSOR-inhibitory hydrolysable tannins? It is known, that ACKNOWLEDGEMENTS the most effective and selective blocker of VSOR channel, 4-(2-but yl-6,7- dichloro -2- c yclopent yl-indan-1- on-5-yl) oxobutyric acid (DCPIB), exhibits a great beneficial effect This work was supported by the Ministry of Innovative in the reversible middle cerebral artery occlusion (rMCAO) Development of the Republic of Uzbekistan (under the grants model in adult rats (Han et al., 2014; Zhang et al., 2008) and FА-А11-Т060 and PZ2017092049). in neonatal mouse hypoxic-ischemic brain injury (Alibrahim CONFLICT OF INTEREST et al., 2013; Wong et al., 2018). The drug also protected cardiomyocytes from injury induced by hyperglycemia (Wang et al., 2017). Given the well-known beneficial effects The authors declare no conflict of interests. of the tannin-containing plant extracts in cerebral ischemia and stroke (Behravan et al., 2014), our results would suggest References [1] Abdulladzhanova NG, Mavlyanov SM, Dalimov DN. Phenolic cerebral ischemia. 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[37] Soler A, Rota R, Hannaert P, Cragoe EJ, Jr., Garay RP. Volume- dependent K+ and Cl- fluxes in rat thymocytes. J Physiol. 1993;465:387–401. [38] Szteyn K, Schmid E, Nurbaeva MK, et al. Expression and functional significance of the Ca(2+)-activated Cl(-) channel ANO6 in dendritic cells. Cell Physiol Biochem. 2012;30:1319–1332. [39] Terra X, Valls J, Vitrac X, et al. Grape-seed procyanidins act as antiinflammatory agents in endotoxin-stimulated RAW 264.7 macrophages by inhibiting NFkB signaling pathway. J AgricFood Chem. 2007;55:4357–4365. [40] Voss FK, Ullrich F, Munch J, et al. Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC. Science. 2014;344:634–638. 44 OR http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Facultatis Pharmaceuticae Universitatis Comenianae de Gruyter

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Abstract

The volume-sensitive outwardly rectifying anion channel (VSOR) is a key component of volume regulation system critical for cell survival in non-isosmotic conditions. The aim of the present study was to test the effects of four tannin extracts with defined compositions on cell volume regulation and VSOR. Preparation I (98% of hydrolysable tannins isolated from leaves of sumac Rhus typhina L.) and Preparation II (100% of hydrolysable tannins isolated from leaves of broadleaf plantain Plantago major L) completely and irreversibly abolished swelling-activated VSOR currents in HCT116 cells. Both preparations profoundly suppressed the volume regulation in thymocytes with half-maximal effects of 40.9 µg/ml and 12.3 µg/ml, respectively. The inhibition was more efficient at lower concentrations but reverted at higher doses due to possible non-specific membrane- permeabilizing activity. Preparations III and IV (54,7% and 54.3% of hydrolysable tannins isolated, respectively, from roots and aboveground parts of Fergana spurge Euphorbia ferganensis B.Fedtch) inhibited VSOR activity in a partially reversible manner and suppressed the volume regulation with substantially higher half-maximal doses of 270 and 278 µg/ml, respectively, with no secondary reversion at higher doses. Hydrolysable tannins represent a novel class of VSOR channel inhibitors with the capacity to suppress the cell volume regulation machinery. Keywords Tannins – plant polyphenols – thymocytes – cell volume regulation – volume-sensitive anion channel This work was supported by the Ministry of Innovative component of the cellular volume regulation system and of Development of the Republic of Uzbekistan (under the grants some other physiological and pathophysiological processes FА-А11-Т060 and PZ2017092049). including cell proliferation, migration and apoptosis (Akita & Okada, 2014; Hoffmann et al., 2014; Okada et al., 2009). VSOR INTRODUCTION is the main pathway for the efflux of anions from swollen cells and, in cooperation with the Ca-activated potassium In order to survive in constantly changing osmotic conditions channels, provides a reduction of the intracellular osmotic caused by intensive physiological processes (breathing, food pressure during the active phase of volume regulation intake, fluid secretion and absorption, filtration and urine upon the hypoosmotic stress called the Regulatory Volume formation, etc.) and pathologies (inflammation, edema, Decrease (RVD) (Akita et al., 2011; Akita & Okada, 2014; Delpire trauma, ischemia and hypoxia), living cells developed & Gagnon, 2018; Hoffmann et al., 2014; Okada et al., 2006; elaborate volume regulatory mechanisms. The volume- Pedersen et al., 2016). Recent studies have demonstrated sensitive outwardly rectifying anion channel (VSOR) is a key that the LRRC8 family proteins constitute the molecular * E-mail: zairovich@gmail.com © European Pharmaceutical Journal OR 37 Eur. Pharm. J. 2019, 66(2), 37-44 Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel Tsiferova N.A. et al. basis of VSOR (Qiu et al., 2014; Voss et al., 2014), structurally and their composition was determined by a combination of organized into a hexameric pore (Deneka et al., 2018; Kasuya preparative column chromatography (silica gel, polyamide), et al., 2018; Kefauver et al., 2018). However, pharmacology quantitative paper chromatography and spectral methods as of this biologically important ion channel remains poorly described elsewhere (Abdulladzhanova et al., 2001). explored. Thus far, a number of structurally divers compounds Preparation I was obtained from the leaves of sumac (Rhus including stilbene derivatives, etacrynic acid analogs and typhina L.) and contained: 3,6-bis-О-di-O-galloyl-1,2,4-tri- flavonoids have been shown to suppress the activity of VSOR О-galloyl-β-D-glucose (74%); 1,2,3,4,6-penta-О-galloyl-β-D- in a voltage-dependent and independent manner (Okada et glucose (10%); 1,4,6-tri-О-galloyl-β-D-glucose (5%); 2,3-di- al., 2019; Xue et al., 2018). О-galloyl-β-D-glucose (2%); 2-О-galloyl-β-D-glucose (2%); Tannins are structurally heterogenous polyphenols, 3-О-galloyl-β-D-glucose (2%); 6-О-galloyl-β-D-glucose (2%); which bind to proteins and can trigger their precipitation gallic acid (1%); rutin (1%); quercetin (0.5%); kaempferol (Mavlyanov et al., 2001). Tannins are secondary metabolites (0.5%). and constitute a part of the plants’ defense system against Preparation II was obtained from the leaves of broadleaf pathogens and insect’s invasion. In addition, these substances plantain (Plantago major L.) and contained: diester exhibit a wide spectrum of biological activities such as of hexahydroxydiphenoyl-1-(О-2-О-galloyl-β-D- antimicrobial (Scalbert, 1991); antioxidant, (Rice-Evans et al., glucopyranosido)-1-(О-β-D-xylopyranoside (30.1%); diester 1995, 1996); anti-inflammatory (Terra et al., 2007; Xue et al., of hexahydroxydiphenoyl-1-(О-β-D-glucopyranosido)-2-(О- 2018); neuroprotective (Behravan et al., 2014). Tannins, along 4-О-galloyl-β-D-glucopyranoside) (27.9%); quercetin-3-О-(2”, with other polyphenolic compounds, have been considered 6”di- О- galloyl-3”- О- p- coumaroyl)-β -D - glucopyranoside to be responsible for the health benefits of red wine and (25.4%); kaempferol-3-O-(2’’,3’’-di-O-galloyl-6’’-О- coumaroyl- green tea, possibly by inhibiting the activity of the Ca- β-D-glucopyranoside (16.6%). activated chloride channels (CaCCs) (Namkung et al., 2010). Preparation III was obtained from the roots of Fergana spurge Penta-m-digalloyl-glucose, a hydrolysable tannin extracted (Euphorbia ferganensis B.Fedtch.) and contained: 1-О-galloyl- from the Chinese gallnut, was demonstrated to inhibit 2,4- v aloneoyl-4,6-he xahydrox ydiphenoyl-β -D - glucose the cystic fibrosis transmembrane conductance regulator (48%); gallic acid (15%); digallic acid (10%); ellagic acid (9%); protein (CFTR), a chloride channel activated by intracellular terchebin (8%); 2,3-digalloyl-β-D-glucose (6.7%); quercetin- cAMP (Wongsamitkul et al., 2010). Tannic acid was shown to 3-О-rutinoside (2.3%); myricetin (0.8%); iso-myricitrin (0.2%). inhibit CaCCs formed by TMEM16A and B (Cruz-Rangel et al., Preparation IV was obtained from the aboveground part of 2015; Namkung et al., 2010, 2011) and TMEM16F (Szteyn et Fergana spurge (Euphorbia humifusa Willd.) and contained: al., 2012), which is consistent with an antidiarrhoeic activity 1-О-galloyl-4,6-hexahydroxydiphenoyl-β-D-glucose (35%); of tannin-containing extracts reported earlier (Galvez et al., quercetin (17%); ellagic acid (10.8%); 3-О-galloyl-4,6- 1991). Tannic acid also blocks L-type Ca-channels (Zhu et hexahydroxydiphenoyl-β-D-glucose (8.3%); 1,2,3-tri-O- al., 2016) and the maxi-anion channel (Woll et al., 1987). The galloyl-β-D-glucose (7%); gallic acid (7%); geraniin (2.5%); latter is known to be swelling-activated (Okada et al., 2018, quercetin-3-О-rhamnoside (4%); quercetin-3-О-galactoside 2019; Sabirov & Merzlyak, 2012; Sabirov et al., 2016), although (3.2%); kaempferol-3-О-glucoside (2.7%); 1-O-galloyl-6-O-bis- it operates mostly when cells are metabolically deprived, galloyl-2,4-valoneoyl-β-D-glucose (1.5%); kaempferol (1%). whereas VSOR is the major contributor to the swelling- The tannin preparations were added from concentrated stock activated plasmalemmal conductance at normal intracellular solutions in dimethylsulfoxide (DMSO). Final concentration ATP levels. Tannins were never considered as modulators of DMSO did not exceed 0.1%, and at this concentration, the of the cell volume regulation system and its constituent solvent did not significantly affect the records. components. Here, we tested four tannin preparations of plant origin on their effects on the cell volume regulation Solutions in rat thymocytes and the VSOR channel activity in HCT116 human colon cancer cells. The normal Ringer solution contained (mM): 135 NaCl, 5 KCl, 2 CaCl , 1 MgCl , 11 HEPES, 5 glucose (рН 7.4, adjusted with 2 2 MATERIALS AND METHODS NaOH, 290 mOsm/kg-H O). The H-buffer contained (mM): 5 KCl, 2 CaCl , 1 MgCl , 10 HEPES, 5 glucose (рН 7.4, adjusted 2 2 Substances with NaOH, 40 mOsm/kg-H O). Hypotonic solutions were prepared by mixing the Ringer solutions with H-buffer in The plants were collected from the Tashkent environs a ratio of 3:4 (vol/vol). The pipette solution for whole-cell during the flowering stage and taxonomically identified experiments contained (in mM): 125 CsCl, 2 CaCl , 1 MgCl , 3 2 2 by Dr. Gnatchenko E.V. of the Institute of Botany of the Na ATP, 5 HEPES (pH 7.4 adjusted with CsOH), 10 EGTA, and 50 Academy of Sciences of Uzbekistan. Preparation of tannin mannitol (pCa 7.65; 320 mOsm/kg-H O). extracts was performed essentially as described previously (Islambekov et al., 1994; Olchowik-Grabarek et al., 2017) 38 39 Eur. Pharm. J. 2019, 66(2), 37-44 Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel Tsiferova N.A. et al. Cells Electrophysiology Human colon tumor cell line, HCT116, was cultured in DMEM Patch electrodes were fabricated from borosilicate glass supplemented with 10% of fetal bovine serum and antibiotics capillaries using a micropipette puller (PP-830, Narishige, (100 U/ml penicillin plus 100 mg/ml streptomycin) at 37 C Japan) and had a tip resistance of 3–5 MΩ when filled with and 5% CO . For patch-clamping, the cells were cultured in pipette solution. Fast and slow capacitive transients were suspension under mild stirring during 3–5 h. routinely compensated for. For whole-cell recordings, the All animal experiments were conducted in accordance access resistance did not exceed 10 MΩ and was always with the ARRIVE guidelines and approved by the Bioethics compensated for by 80%. Membrane currents were measured Committee of the Institute of Biophysics and Biochemistry. The with an EPC-9 patch-clamp system (Heka-Electronics, isolation of thymic lymphocytes (thymocytes) was performed Lambrecht/Pfalz, Germany). The membrane potential as described previously (Kurbannazarova et al., 2003, 2008, was controlled by shifting the pipette potential (Vp) and 2011; Sabirov et al., 2013). Briefly, the 6–8 weeks old rats, is reported as Vp for whole-cell recordings. Currents were kept in vivarium on an average diet, were anaesthetized filtered at 1 kHz and sampled at 5–10 kHz. Data acquisition and with halothane or diethyl ether and painlessly euthanized by analysis were done using Pulse + PulseFit (Heka-Electronics). cervical dislocation. The thymi were dissected and carefully Liquid junction potentials were calculated using pCLAMP washed with an ice-cold normal Ringer solution. The thymi 8.1 (Molecular Devices, Sunnyvale, CA) algorithms and were were then minced using fine forceps and passed through a corrected off-line when appropriate. All experiments were 100 µm-nylon mesh. The suspension was centrifuged at 1000 performed at room temperature (23–25 °C). g for 5 min; the pellet was washed two times with the normal Ringer solution and resuspended in this medium at a cell Data analysis density of 100 x 10 cells/ml. The cell suspension was kept on ice for ≤ 5 h and contained no more than 5% of damaged cells The dose-response data were approximated using a Hill as assayed by trypan blue exclusion. equation of the following form: Cell Volume Measurements RVD = RVD + (RVD – RVD )/(1 + (C/IC ) ) (2) min max min 50 Cell volume changes under non-isosmotic conditions were Here: RVD and RVD are the minimal and maximal values min max recorded by light transmittance measurement as described of RVD, C – concentration of the substance (μg/ml), IC – previously (Kurbannazarova et al., 2003, 2008, 2011). Briefly, concentration of the substance rendering a half-maximal 900 ml of the normal Ringer or hypotonic solutions was inhibitory effect (μg/ml), h – Hill coefficient. added to the 1.5 cm glass cuvette thermo stated with a water Data were analyzed using Origin 8 (OriginLab Corporation, jacket and equilibrated for 10 min. An aliquot (100 ml) of Northampton, MA, USA). Pooled data are given as means cell suspension was added to this medium to yield the final ± SEM of n observations. Comparisons between the two cell density of 10 x 10 cells/ml. The light transmittance was experimental groups were made using the unpaired two- measured at 610 nm (band-pass filter) using a photometer sample Student’s t-test. The data of Fig. 3d and 4d were MKMF-01 (Russia). The output signal was amplified by U5- analyzed using both unpaired two-sample (comparison 11 amplifier (Russia), digitized at 100 Hz using a USB sensor at different voltages) and one-sample (comparison with interface GO!Link and recorded by Logger Lite software control) t-test. Differences were considered to be statistically (Vernier, Beaverton, OR). significant at p < 0.05. The parameter RVD was calculated using the following RESULTS equation (1): RVD=( T -T )/( T -T )*100% (1) Tannin preparations inhibit thymocyte volume max 15 max 0 regulation under hypoosmotic stress where T and T are the initial and maximal light 0 max transmittances, and T is the light transmittance measured Blockers of VSOR channel are expected to suppress the 15 minutes after the onset of hypotonic stress. RVD = 100 regulatory volume decrease phase of cellular response to for complete recovery of the cell volume to the initial level, the hypoosmotic stress. In order to test this possibility, we and RVD = 0 when volume regulation is fully suppressed. employed the immature thymic lymphocytes which possess Under control conditions, RVD usually had values of 60– fully functional volume regulation machinery (Arrazola et al., 90% depending on the cells condition, osmotic gradient, 1993; Kurbannazarova et al., 2003; Soler et al., 1993). We have temperature and other experimental conditions. previously shown that thymocytes express the VSOR channels with the same biophysical and pharmacological profile as other cell types, and that VSOR blockers completely abolish 38 39 Eur. Pharm. J. 2019, 66(2), 37-44 Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel Tsiferova N.A. et al. RVD in these cells (Kurbannazarova et al., 2011; Sabirov et When preparations I and II were added to the flow chamber al., 2013). Therefore, we supposed that tannin preparations at a dose of 31 µg/ml and 41 µg/ml, respectively (at these might affect the volume regulation in thymocytes. doses, their effect on RVD was maximal, see Fig. 1c,d), we In our experiments, thymocytes, when challenged with observed almost instant suppression of the macroscopic hypoosmotic stress, first rapidly swelled (passive response) currents (Fig. 3a,b). The effect was essentially irreversible. The and then gradually restored their volume toward an initial ionic currents were suppressed at both positive and negative level (active response; Fig. 1a). The parameter RVD as defined potentials (Fig. 3c,d) suggesting that the channel blockage is by Equation (1) (see Experimental section) ranged from 66% voltage-independent. Voltage-independence together with to 93% and averaged at 79.6 ± 1.9% (n = 17). the irreversibility of blockage may indicate a strong, possibly When added to the hypoosmotic medium, tannin preparations covalent, interaction of the tannins with the channel protein. I and II exhibited a profound suppressive effect on the Preparations III and IV also exhibited suppressive effects on thymocyte volume regulation (Fig. 1a,b). For preparation I, the macroscopic swelling-induced conductance. However, an inhibition at lower doses was gradually lost as the amount in contrast to the preparations I and II, current inhibition by of the added preparation was increased above 30 µg/ml (Fig. preparations III and IV was slower (Fig. 4a,b) and partially 1a,c). Since the maximal swelling also declined at high doses, reversible. The currents in the presence of these preparations we supposed that high concentrations of preparation I were were decreased more efficiently at positive potentials (Fig. detrimental for cellular plasma membrane. A similar biphasic 4c,d) than at negative voltages. Reversibility of the inhibition action was also observed for preparation II (Fig. 1b,d). The together with voltage-dependency may suggest an open- half-maximal concentrations and Hill coefficients for the channel blockage mechanism: applied positive voltage drives inhibiting phase of the dose-response curves (solid circles the negatively charged polyphenolic compounds applied and solid lines in Fig. 1c,d) were as follows: preparation I (IC50 from the extracellular side into the channel lumen. = 40.9 ± 7.2 µg/ml; h = 0.96 ± 0.2) and preparation II (IC50 = DISCUSSION 12.3 ± 8.1 µg/ml; h = 0.59 ± 0.294). In contrast to the first two preparations, preparations III and IV did not affect the maximal swelling and did not display Thus, we have demonstrated that polyphenolic tannins of the secondary damaging phase on the dose-response curves plant origin represent a novel class of VSOR channel inhibitors. (Fig. 2). The half-maximal concentrations and Hill coefficients This activity may contribute to the well-documented for the inhibiting phase of the dose-response curves (solid beneficial health effects of polyphenol-rich food and drink circles and solid lines in Fig. 2c,d) were as follows: preparation products. III (IC50 = 270 ± 77 µg/ml; h = 0.63 ± 0.15) and preparation IV In our experiments, preparations (I and II) were more efficient (IC50 = 278 ± 43 µg/ml; h = 0.65 ± 0.07). inhibitors of VSOR and volume regulation system compared Comparison of the IC50 values suggested that preparations to the preparations III and IV. Since preparation II consisted I and II are more efficient inhibitors of the cell volume exclusively of hydrolysable tannins, and the content of regulation than preparations III and IV. hydrolysable tannins in preparation I reached 98%, we inferred that hydrolysable tannins represent a novel class of Tannin preparations block the swelling-induced VSOR channel inhibitors. Certainly, the tannin preparations anion conductance used in these experiments have rather complex composition, and thus, the individual tannins might be more effective and Tannins were never considered as volume-regulated anion selective modulators of the VSOR chloride channel. channel blockers. In order to test this possibility, we employed Preparations III and IV contained less overall tannin content direct electrophysiological assessment of the VSOR channel (54.7% and 54.3%, respectively) and were less effective. activity in human colorectal cancer HCT116 cells, which have Possibly, other types of polyphenols, which constituted a been used recently for molecular identification of the VSOR large part of these preparations may have weakened the channel proteins (Qiu et al., 2014; Voss et al., 2014). inhibitory effects of hydrolysable tannins of preparations III In our experiments, we filled the patch-pipettes with slightly and IV. hypertonic (by ~30 mOsm/kg-H O) solution to induce It should be noted that 3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl- cellular swelling as described previously (Kurbannazarova et β-D-glucose, which is the major component of Preparation al., 2011; Sabirov et al., 2013). Upon attaining the whole-cell I, was recently shown to form anion-selective channels in configuration, cells gradually swelled as could be observed lipid bilayers (Borisova et al., 2019). This effect may explain visually under phase-contrast microscopy. The cellular swelling the secondary rising phase on the dose-response curve was accompanied by a robust activation of the macroscopic on the Figure 1a,c, suggesting that these newly formed currents with outward rectification and inactivation at large pores functionally replace the VSOR channel by serving as a depolarizing positive potentials (Fig. 3a,b), a phenotypical pathway for anion efflux. Since Preparation II also displayed landmark of the VSOR anion channel (Okada, 1997). a biphasic effect of RVD, one may suppose that some of its 40 41 Eur. Pharm. J. 2019, 66(2), 37-44 Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel Tsiferova N.A. et al. Figure 1: Dose-dependent effects of Preparations I and II on the thymocyte volume regulation under hypoosmotic stress. (a, b) Representative recordings of light transmittance changes. (c, d) Dose-response curves; the solid lines are fits to the equation (2) with half-maximal concentrations and Hill coefficients given in the text. Figure 2. Dose-dependent effects of Preparations III and IV on the thymocyte volume regulation under hypoosmotic stress. (a, b) Representative recordings of light transmittance changes. (c, d) Dose-response curves; the solid lines are fits to the equation (2) with half-maximal concentrations and Hill coefficients given in the text. 40 41 Eur. Pharm. J. 2019, 66(2), 37-44 Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel Tsiferova N.A. et al. Figure 3: Inhibition of VSOR currents by Preparations I and II. (a, b) The time course of whole-cell current activation in response to cell swelling. Currents were elicited by application of alternating test-pulses from 0 to ±40 mV every 15 s. Arrowheads (▲) denote the time points where the step-pulses from ‒100 to +100 mV in 20 mV increments were applied to test the voltage-dependence of the macroscopic conductance. (c) Instantaneous current-to-voltage relationships measured at the beginning of test-pulses from recordings similar to those shown in (a) and (b); n = 5 for Preparation I and n = 4 for Preparation II. (d) Fractional currents measured at +40 mV (open bars) and ‒40 mV (hatched bars). *Significantly different from control values at p < 0.05. Figure 4: Inhibition of VSOR currents by Preparations III and IV. (a, b) The time course of whole-cell current activation in response to cell swelling. Currents were elicited by the application of alternating test-pulses from 0 to ±40 mV every 15 s. Arrowheads (▲) denote the time points where the step-pulses from ‒100 to +100 mV in 20 mV increments were applied to test the voltage- dependence of the macroscopic conductance. (c) Instantaneous current-to-voltage relationships measured at the beginning of test-pulses from recordings similar to those shown in (a) and (b); n = 5 for Preparation III and n = 4 for Preparation IV. (d) Fractional currents measured at +40 mV (open bars) and ‒40 mV (hatched bars). *Significantly different from control values at p < 0.05. Significantly different at p < 0.05 from values measured at negative voltages. 42 43 Eur. Pharm. J. 2019, 66(2), 37-44 Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel Tsiferova N.A. et al. components could also act as pore formers on lipid matrix of that tannins antagonizing activity of the VSOR anion channel the cellular plasma membrane. could be beneficial in protecting brain tissues, and possibly, What kind of pharmacological effects could be anticipated the heart, during ischemic/hypoxic injury. for VSOR-inhibitory hydrolysable tannins? It is known, that ACKNOWLEDGEMENTS the most effective and selective blocker of VSOR channel, 4-(2-but yl-6,7- dichloro -2- c yclopent yl-indan-1- on-5-yl) oxobutyric acid (DCPIB), exhibits a great beneficial effect This work was supported by the Ministry of Innovative in the reversible middle cerebral artery occlusion (rMCAO) Development of the Republic of Uzbekistan (under the grants model in adult rats (Han et al., 2014; Zhang et al., 2008) and FА-А11-Т060 and PZ2017092049). in neonatal mouse hypoxic-ischemic brain injury (Alibrahim CONFLICT OF INTEREST et al., 2013; Wong et al., 2018). The drug also protected cardiomyocytes from injury induced by hyperglycemia (Wang et al., 2017). Given the well-known beneficial effects The authors declare no conflict of interests. of the tannin-containing plant extracts in cerebral ischemia and stroke (Behravan et al., 2014), our results would suggest References [1] Abdulladzhanova NG, Mavlyanov SM, Dalimov DN. Phenolic cerebral ischemia. 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Journal

Acta Facultatis Pharmaceuticae Universitatis Comenianaede Gruyter

Published: Nov 1, 2019

Keywords: Tannins; plant polyphenols; thymocytes; cell volume regulation; volume-sensitive anion channel

References