Differential CI^-and HCO_3^- mediated anion secretion by different colonic cell types in response to tetromethylpyrazine

AIM: Colonic epithelium is known to secrete both CI- and HCO3, but the secretory mechanisms of different colonic cell types are not fully understood. The present study aimed to investigate the differential activation of Cl-and HCO3 secretion by tetramethylpyrazine (TMP) in human crypt-like cell line, T84, and villus-like cell line, Caco-2, in comparison to the TMP-induced secretory response in freshly isolated rat colonic mucosa. METHODS: Colonic epithelial anion secretion was studied by using the short circuit current (Isc) technique. RT-PCR was used to examine the expression of Na^+-HCO3-cotranspoter in different epithelial cell types. RESULTS: TMP produced a concentration-dependent Isc which was increase in both T84 and Caco-2 cells. When extracellular CI was removed, TMP-induced Isc was abolished by 76.6% in T84 cells, but not in Caco-2 cells. However, after both CI and HCO3- were removed, TMP-induced Isc in Caco-2 cells was reduced to 10%. Bumetanide, an inhibitor of Na^+-K^+-Cl-cotranspoter, inhibited the TMP-induced Isc by 96.7% in T84 cells, but only 47.9% in Caco-2 cells. In the presence of bumetanide and 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid, an inhibitor of Na^+-HCO3- cotransporter, inhibited the TMP-induced current in Caco-2 cells by 93.3% .In freshly isolated rat colonic mucosa, TMP stimulated distinct Isc responses similar to that observed in T84 and Caco-2 cells depending on the concentration used. RT-PCR revealed that the expression of Na^+-HCO3- cotransporter in Caco-2 cells was 4-fold more greater than that in T84 cells. CONCLUSION: TMP exerts concentration-dependent differential effects on different colonic cell types with stimulation of predominant Cl- secretion by crypt cells at a lower concentration, but predominant HCO3- secretion by villus cells at a higher concentration, suggesting different roles of these cells in colonic Cl and HCO3 secretion.

Improvement of barrier function and stimulation of colonic epithelial anion secretion by Menoease Pills

AIM: Menoease Pills(MP)1 a Chinese medicine-based new formula for postmenopausal women, has been shown to modulate the endocrine and immune systems. The present study investigated the effects of MP and one of its active ingredients, ligustrazine, on epithelial barrier and ion transport function in a human colonic cell line, T84.METHODS: Colonic transepithelial electrophysiological characteristics and colonic anion secretion were studied using the short circuit current (Isc) technique. RT-PCR was used to examine the expression of cytoplasmic proteins associated with the tight junctions, ZO-l(zonula occludens-1) and ZO-2 (zonula occludens-2).RESULTS: Pretreatment of T84 cells with MP (15 μg/mL) for 72 h significantly increased basal potential difference,transepithelial resistance and basal ISC. RT-PCR results showed that the expressions of ZO-land ZO-2 were significantly increased after MP treatment, consistent with improved epithelial barrier function. Results of acute stimulation showed that apical addition of MP produced a concentrationdependent (10-5 000 μg/mL, EC50 = 293.9 μg/mL) increase in ISC. MP-induced ISC was inhibited by basolateral treatment with bumetanide (100 μmol/L), an inhibitor of the Na^+-K^+-2Cl^- cotransporter, apical addition of Cl^- channel blockers, diphenylamine-2, 2'-dicarboxylic acid (1mmol/L) or glibenclamide (1 retool/L), but not 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid or epithelial Na+ channel blocker,amiloride. The effect of MP on ZOo1 and ZO-2was mimicked by Ligustrazine and the ligustrazine-induced ISC was also blocked by basolateral application of bumetanide and apical addition of diphenylamine-2, 2'-dicarboxylic acid or glibenclamide, and reduced by a removal of extracellular Cl^-.CONCLUSION: The results of the present study suggest that MP and lligustrazine may improve epithelial barrier function and exert a stimulatory effect on colonic anion secretion, indicating the potential use of MP and its active ingredients for improvement of GI tract host defense and alleviation of constipation often seen in the elderly.

Effect of tetramethylpyrazine on exocrine pancreatic and bile secretion

AIM: To investigate the effect of tetramethylpyrazine (ligustrazine, TMP) on the secretion of exocrine pancreas (and biliary).METHODS: In in vivo study, we investigated the effect of TMP on the secretion of pancreatic-bile juice (PBJ) in rats.Using human pancreatic duct cell line, CAPAN-1, combined with the short-circuit current (ISC) technique we further studied the effect of TMP on the pancreatic anion secretion.RESULTS: Administration of TMP (80 mg/kg, ip) significantly increased the secretion of PBJ (P<0.05), but the pH of PBJ and the secretion of pancreatic protein were not significantly affected. Basolateral addition of TMP produced a dosedependent increase in ISC(EC50=1.56 mmol/L), which contained a fast transient ISC response followed by a slow decay. Apical application of Cl- channel blockers, DPC (1 mmol/L),decreased the response by about 67.1% (P<0.001), whereas amiloride (100 μmol/L), a epithelial sodium channel blockers,had no effect. Removal of extracellular HCO3- abolished TMP-induced increase in ISC by about 74.4 % (P<0.001),but the removal of external Cl- did not. Pretreatment with phosphodiesterase inhibitor, TBMX(0.5 mmol/L), decreased the TMP-induced ISC by 91% (P<0.001).CONCLUSION: TMP could stimulate the secretion of PBJ,especially pancreatic ductal HCO3- secretion via cAvlp or cGMP-dependent pathway. It need further study to investigate the roles of cAMP or cGMP in the effect of TMP on the secretion of exocrine pancreas.