Abstract
Flavonoids belong to a large group of plant polyphenols that are consumed daily in large amounts. Our previous findings have shown that baicalein, a major flavonoid derived from the medicinal herb Scutellariae radix, induces Cl(-) secretion across rat colonic mucosa. The current study examines the effect of baicalein on Cl(-) secretion in human colonic epithelial (T84) cells and its interaction with Ca(2+)- and cAMP-dependent secretagogues. We have employed a technique that allows concurrent monitoring of short-circuit current (I(SC)) and [Ca(2+)](i) in polarized epithelium. Basolateral application of baicalein induced a concentration-dependent increase in I(SC). The increase in I(SC) was because of Cl(-) secretion and was not accompanied by any discernible increase in [Ca(2+)](i). Baicalein acted synergistically with Ca(2+)- but not cAMP-dependent secretagogues. In the presence of baicalein, the carbachol and histamine induced increases in I(SC) that were markedly potentiated while increases in [Ca(2+)](i) were not significantly enhanced. Baicalein treatment uncoupled Cl(-) secretion from inhibitory effects normally generated by muscarinic activation. Baicalein treatment also resulted in increased cAMP content and activated PKA activity. Nystatin permeabilization studies revealed that baicalein stimulated an apical Cl(-) current but did not activate any basolateral K(+) current. These data suggest that baicalein potentiates Ca(2+)-mediated Cl(-) secretion through a signaling pathway involving cAMP and protein kinase A, most likely through the cystic fibrosis transmembrane conductance regulator in the apical membrane.
Highlights
In the intestinal tract, flavonoids have been demonstrated to affect gastrointestinal motility both in vivo and in vitro [2]
Our previous findings have shown that baicalein, a major flavonoid derived from the medicinal herb Scutellariae radix, induces Cl؊ secretion across rat colonic mucosa
The current study examines the effect of baicalein on Cl؊ secretion in human colonic epithelial (T84) cells and its interaction with Ca2؉- and cAMP-dependent secretagogues
Summary
Culture of Cells—Experiments were performed using the human intestinal T84 cell line obtained from American Type Culture Collection. For simultaneous measurements of [Ca2ϩ]i and ISC, cells were seeded onto Transwell-col membranes (Costar, Cambridge, MA) with 0.4-m pore diameter (culture area 0.1 cm2) as previously described [18]. Cells grown on a Transwell-col membrane were loaded with Fura-2 by incubation (45 min, 37 °C) in bicarbonate-buffered Krebs-Henseleit solution containing 3 M Fura-2-AM and 1.6 M pluronic F127. Apical membrane ClϪ currents, defined as ICl(ap), were measured in cells permeabilized basolaterally with 360 g/ml nystatin in the presence of asymmetrical buffers that imposed an apical to basolateral ClϪ gradient [25]. Basolateral membrane Kϩ currents, defined as IK(bl), were measured in cells permeabilized apically with 360 g/ml nystatin for 30 min in the presence of asymmetrical buffers that imposed an apical-to-basolateral Kϩ gradient. Data were analyzed by Student’s t test for paired or unpaired variants and analysis of variance where appropriate, with p Ͻ 0.05 considered significant
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