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Cellular mechanisms underlying the laxative effect of flavonol naringenin on rat constipation model.

Yang ZH, Yu HJ, Pan A, Du JY, Ruan YC, Ko WH, Chan HC, Zhou WL - PLoS ONE (2008)

Bottom Line: Naringenin could increase intracellular cAMP content and PKA activity, consisted with that MDL-12330A (N-(Cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride) pretreatment reduced the naringenin-induced I(SC).In addition, significant inhibition of the naringenin-induced I(SC) by quinidine indicated that basolateral K+ channels were involved in maintaining this cAMP-dependent Cl- secretion.Taken together, our data suggest that naringenin could stimulate Cl- secretion in colonic epithelium via a signaling pathway involving cAMP and PKA, hence provide an osmotic force for subsequent colonic fluid secretion by which the laxative effect observed in the rat constipation model.

View Article: PubMed Central - PubMed

Affiliation: The School of Life Science, Sun Yat-sen University, Guangzhou, China.

ABSTRACT

Background & aims: Symptoms of constipation are extremely common, especially in the elderly. The present study aim to identify an efficacious treatment strategy for constipation by evaluating the secretion-promoting and laxative effect of a herbal compound, naringenin, on intestinal epithelial anion secretion and a rat constipation model, respectively.

Methods/principal findings: In isolated rat colonic crypts, mucosal addition of naringenin (100 microM) elicited a concentration-dependent and sustained increase in the short-circuit current (I(SC)), which could be inhibited in Cl- free solution or by bumetanide and DPC (diphenylamine-2-carboxylic acid), but not by DIDS (4, 4'- diisothiocyanatostilbene-2, 2'-disulfonic acid). Naringenin could increase intracellular cAMP content and PKA activity, consisted with that MDL-12330A (N-(Cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride) pretreatment reduced the naringenin-induced I(SC). In addition, significant inhibition of the naringenin-induced I(SC) by quinidine indicated that basolateral K+ channels were involved in maintaining this cAMP-dependent Cl- secretion. Naringenin-evoked whole cell current which exhibited a linear I-V relationship and time-and voltage- independent characteristics was inhibited by DPC, indicating that the cAMP activated Cl- conductance most likely CFTR (cystic fibrosis transmembrane conductance regulator) was involved. In rat constipation model, administration of naringenin restored the level of fecal output, water content and mucus secretion compared to loperamide-administrated group.

Conclusions: Taken together, our data suggest that naringenin could stimulate Cl- secretion in colonic epithelium via a signaling pathway involving cAMP and PKA, hence provide an osmotic force for subsequent colonic fluid secretion by which the laxative effect observed in the rat constipation model. Naringenin appears to be a novel alternative treatment strategy for constipation.

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Characteristics of NAR-induced whole cell current in T84 cells.A, B, and C, typical whole cell currents recorded by holding the membrane potential at −30 mV and pulsing to voltages in a range of ±80 mV in 20-mV steps from (A) a control cell; (B) a cell applied with NAR (100 µM) to the bath solution; (C) a cell pre-exposed to 100 µM NAR applied with 1 mM DPC; (D) corresponding current-voltage relationship obtained from currents in A, B and C; (E) Whole-cell recordings obtained after NAR stimulation from a cell bathed in 70 mM NMDGCl with corresponding I–V relationships. Note that reversal potential was shifted from 0 to 17.3 mV, close to the new ECl = 18.7 mV.
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pone-0003348-g007: Characteristics of NAR-induced whole cell current in T84 cells.A, B, and C, typical whole cell currents recorded by holding the membrane potential at −30 mV and pulsing to voltages in a range of ±80 mV in 20-mV steps from (A) a control cell; (B) a cell applied with NAR (100 µM) to the bath solution; (C) a cell pre-exposed to 100 µM NAR applied with 1 mM DPC; (D) corresponding current-voltage relationship obtained from currents in A, B and C; (E) Whole-cell recordings obtained after NAR stimulation from a cell bathed in 70 mM NMDGCl with corresponding I–V relationships. Note that reversal potential was shifted from 0 to 17.3 mV, close to the new ECl = 18.7 mV.

Mentions: In the whole cell patch clamp experiments, addition of 100 µM NAR resulted in an inward current at holding potential −30 mV. The NAR-activated whole-cell current profile elicited by a series of voltages exhibited time- and voltage-independent characteristics with a linear I–V relationship (Figure 7A–C).The I–V relationships were shown in Figure 6D. The reversal potential of the NAR-induced currents in symmetrical Cl_ solutions was close to the Cl− equilibrium, 0 mV. In order to further identify whether the NAR-activated whole-cell currents were mediated by Cl−, and not through any nonselective conductance, Cl− concentration in the bath was changed from 140 mM to 70 mM while a pipette containing 140 mM NMDG (N-Methyl-D-Glucamine) -Cl was used. As shown in Figure 6, the reversal potential was shifted to a value 17.3 mV, close to the new theoretic equilibrium value for Cl− 18.7 mV (Figure 7E). The results suggested that currents activated by extracellular naringenin were mediated by Cl−. Chloride channel blocker DPC blocked the NAR-activated Cl− current when the membrane potential was held at negative potentials (Figure 7C). This voltage-dependent blockade by DPC was in line with the characteristic of CFTR previously reported in other epithelial cells [22], [23].


Cellular mechanisms underlying the laxative effect of flavonol naringenin on rat constipation model.

Yang ZH, Yu HJ, Pan A, Du JY, Ruan YC, Ko WH, Chan HC, Zhou WL - PLoS ONE (2008)

Characteristics of NAR-induced whole cell current in T84 cells.A, B, and C, typical whole cell currents recorded by holding the membrane potential at −30 mV and pulsing to voltages in a range of ±80 mV in 20-mV steps from (A) a control cell; (B) a cell applied with NAR (100 µM) to the bath solution; (C) a cell pre-exposed to 100 µM NAR applied with 1 mM DPC; (D) corresponding current-voltage relationship obtained from currents in A, B and C; (E) Whole-cell recordings obtained after NAR stimulation from a cell bathed in 70 mM NMDGCl with corresponding I–V relationships. Note that reversal potential was shifted from 0 to 17.3 mV, close to the new ECl = 18.7 mV.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2553183&req=5

pone-0003348-g007: Characteristics of NAR-induced whole cell current in T84 cells.A, B, and C, typical whole cell currents recorded by holding the membrane potential at −30 mV and pulsing to voltages in a range of ±80 mV in 20-mV steps from (A) a control cell; (B) a cell applied with NAR (100 µM) to the bath solution; (C) a cell pre-exposed to 100 µM NAR applied with 1 mM DPC; (D) corresponding current-voltage relationship obtained from currents in A, B and C; (E) Whole-cell recordings obtained after NAR stimulation from a cell bathed in 70 mM NMDGCl with corresponding I–V relationships. Note that reversal potential was shifted from 0 to 17.3 mV, close to the new ECl = 18.7 mV.
Mentions: In the whole cell patch clamp experiments, addition of 100 µM NAR resulted in an inward current at holding potential −30 mV. The NAR-activated whole-cell current profile elicited by a series of voltages exhibited time- and voltage-independent characteristics with a linear I–V relationship (Figure 7A–C).The I–V relationships were shown in Figure 6D. The reversal potential of the NAR-induced currents in symmetrical Cl_ solutions was close to the Cl− equilibrium, 0 mV. In order to further identify whether the NAR-activated whole-cell currents were mediated by Cl−, and not through any nonselective conductance, Cl− concentration in the bath was changed from 140 mM to 70 mM while a pipette containing 140 mM NMDG (N-Methyl-D-Glucamine) -Cl was used. As shown in Figure 6, the reversal potential was shifted to a value 17.3 mV, close to the new theoretic equilibrium value for Cl− 18.7 mV (Figure 7E). The results suggested that currents activated by extracellular naringenin were mediated by Cl−. Chloride channel blocker DPC blocked the NAR-activated Cl− current when the membrane potential was held at negative potentials (Figure 7C). This voltage-dependent blockade by DPC was in line with the characteristic of CFTR previously reported in other epithelial cells [22], [23].

Bottom Line: Naringenin could increase intracellular cAMP content and PKA activity, consisted with that MDL-12330A (N-(Cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride) pretreatment reduced the naringenin-induced I(SC).In addition, significant inhibition of the naringenin-induced I(SC) by quinidine indicated that basolateral K+ channels were involved in maintaining this cAMP-dependent Cl- secretion.Taken together, our data suggest that naringenin could stimulate Cl- secretion in colonic epithelium via a signaling pathway involving cAMP and PKA, hence provide an osmotic force for subsequent colonic fluid secretion by which the laxative effect observed in the rat constipation model.

View Article: PubMed Central - PubMed

Affiliation: The School of Life Science, Sun Yat-sen University, Guangzhou, China.

ABSTRACT

Background & aims: Symptoms of constipation are extremely common, especially in the elderly. The present study aim to identify an efficacious treatment strategy for constipation by evaluating the secretion-promoting and laxative effect of a herbal compound, naringenin, on intestinal epithelial anion secretion and a rat constipation model, respectively.

Methods/principal findings: In isolated rat colonic crypts, mucosal addition of naringenin (100 microM) elicited a concentration-dependent and sustained increase in the short-circuit current (I(SC)), which could be inhibited in Cl- free solution or by bumetanide and DPC (diphenylamine-2-carboxylic acid), but not by DIDS (4, 4'- diisothiocyanatostilbene-2, 2'-disulfonic acid). Naringenin could increase intracellular cAMP content and PKA activity, consisted with that MDL-12330A (N-(Cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride) pretreatment reduced the naringenin-induced I(SC). In addition, significant inhibition of the naringenin-induced I(SC) by quinidine indicated that basolateral K+ channels were involved in maintaining this cAMP-dependent Cl- secretion. Naringenin-evoked whole cell current which exhibited a linear I-V relationship and time-and voltage- independent characteristics was inhibited by DPC, indicating that the cAMP activated Cl- conductance most likely CFTR (cystic fibrosis transmembrane conductance regulator) was involved. In rat constipation model, administration of naringenin restored the level of fecal output, water content and mucus secretion compared to loperamide-administrated group.

Conclusions: Taken together, our data suggest that naringenin could stimulate Cl- secretion in colonic epithelium via a signaling pathway involving cAMP and PKA, hence provide an osmotic force for subsequent colonic fluid secretion by which the laxative effect observed in the rat constipation model. Naringenin appears to be a novel alternative treatment strategy for constipation.

Show MeSH
Related in: MedlinePlus