Traditional Chinese formula, lubricating gut pill, stimulates cAMP-dependent CI(−) secretion across rat distal colonic mucosa.
ABSTRACT Lubricating gut pill (LGP), a traditional Chinese formula, had been conformed to improve the loperamide-induced rat constipation by stimulation of Cl(-) secretion, but its mechanism has not been fully explored. Thus, the purpose of this study was to identify the action sites of LGP-stimulated Cl(-) secretion across rat distal colonic mucosa.
Rat distal colonic mucosa was mounted in Ussing chambers and short circuit current (I(SC)), apical Cl(-) current and basolateral K(+) current were recorded. Intracellular cyclic adenosine monophosphate (cAMP) content and protein kinase A (PKA) activity were determined with ELISA kit and the non-radioactive PepTag test, respectively.
LGP at 800μg/ml elicited a sustained increase in Cl(-) secretory response, which was inhibited by CFTR(inh)172, a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor. Permeabilizing apical membrane with nystatin revealed that LGP-stimulated basolateral K(+) current was significantly inhibited by KCNQ1 K(+) channel inhibitor chromanol 293B. LGP-stimulated I(SC) was markedly reduced by pretreatment with cis-N-[2-phenylcyclopentyl]-azacyclotridec-1-en-2amine (MDL-12,330A) and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), but not with inhibitors of Ca(2+)-dependent signaling pathway. Treatment of tissue with LGP resulted in an increase in intracellular cAMP level and the activation in protein kinase A. The E-prostanoid(4) (EP)(4) receptor antagonist L-161,982 completely eliminated LGP-induced response.
The results showed that LGP enhances Cl(-) and fluid secretion via prostanoid receptor signaling and also cAMP and protein kinase A pathway, subsequently triggering the activation of apical Cl(-) channels mostly CFTR and basolateral cAMP-dependent K(+) channel.
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ABSTRACT: A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.Analytical Biochemistry 06/1976; 72:248-54. · 2.58 Impact Factor
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ABSTRACT: The cystic fibrosis transmembrane regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A. A cAMP-independent activation has been recently shown for the protein tyrosine kinase inhibitor genistein in CFTR-transfected NIH/3T3 fibroblasts. We further studied the role of genistein on Cl- secretion in HT-29/B6 and T84 colonic epithelial cells, which express native CFTR in their apical membranes. Transepithelial Cl- secretion was more effectively stimulated in T84 cells when compared with HT-29/B6 cells by mucosal perfusion with 50 microM genistein. Genistein, like the cAMP agonist forskolin, stimulated CFTR activity in cell-attached patches of single cells with similar slope conductances of 8.5 +/- 0.5 and 9.2 +/- 0.3 pS, respectively. Monolayers in Ussing chambers were basolaterally permeabilized with the pore former alpha-toxin, and gradient-driven Cl- current across the apical membrane (ICl) was measured. ICl was stimulated by serosal (i.e., cytosolic) cAMP (half-maximal stimulatory concentration = 9.8 +/- 1.9 microM). In the presence of cAMP (> 5 microM), subsequent mucosal, but not serosal, addition of genistein further increased Icl by approximately 16%; in the absence of cytosolic cAMP, genistein had no effect on ICl. The inactive analogue daidzein had no effect. When cAMP agonists were removed in the continued presence of genistein, ICl remained elevated in both permeabilized and intact monolayers as well as in cell-attached patches of single cells. In addition, genistein blocked K- currents across the basolateral membrane in apically amphotericin B-permeabilized monolayers (half maximal inhibitory concentration = 44.2 +/- 8.1 microM). Therefore, in intact epithelia, the overall secretory response to genistein is composed of stimulatory effects on the apical CFTR and inhibitory effects on the basolateral K+ conductance. We propose that genistein blocks a phosphatase, which regulates CFTR during cAMP-dependent stimulation.The American journal of physiology 01/1996; 270(1 Pt 1):C265-75. · 3.28 Impact Factor
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ABSTRACT: The protein tyrosine kinase inhibitor, genistein, caused an increase of short-circuit current (Isc) across the rat distal colon in forskolin-pretreated tissues, suggesting a synergistic interaction of the drug with cAMP-dependent secretion. In the absence of forskolin, genistein had a dual effect on Isc, it increased Isc in tissues with a low baseline, but decreased Isc in tissues with a high baseline Isc. The secretory effect of genistein was dependent on the presence of Cl- and was blocked by inhibitors of Cl- secretion like bumetanide, an inhibitor of the Na(+)-K(+)-Cl- cotransporter, or 5-nitro-2-(3- phenylpropylamino)-benzoate (NPPB), a Cl- channel blocker. Unidirectional flux measurements revealed that genistein inhibited Na+ and Cl- absorption and induced net Cl- secretion. The protein tyrosine phosphatase inhibitor vanadate suppressed the secretory effect of genistein. In contrast, genistein caused an inhibition of carbachol-induced, i.e. Ca(2+)-mediated secretion. Whole-cell patch-clamp experiments confirmed the synergistic effect of genistein on cAMP-induced Cl- currents. In the presence of forskolin, genistein caused a depolarization concomitant with an increase in membrane inward current. In addition, genistein caused an inhibition of a basal K+ conductance and inhibited the Ca(2+)-dependent K+ conductance stimulated by carbachol. These results suggest a complex role of the protein tyrosine kinase pathway in the control of colonic Cl- secretion, an antagonistic action on the cAMP pathway and a synergistic action on the Ca2+ pathway as revealed by the opposing effects of genistein. The physiological importance of this regulation remains to be clarified.European Journal of Pharmacology 04/1996; 299(1-3):161-70. · 2.59 Impact Factor