Activation of apical CFTR and basolateral Ca(2+)-activated K+ channels by tetramethylpyrazine in Caco-2 cell line.
We have previously demonstrated that tetramethylpyrazine (TMP) could stimulate colonic and pancreatic anion secretion. The present study investigated the signaling pathways and cellular mechanisms underlying the effect of TMP using human colonic Caco-2 cells, with permeabilized apical or basolateral membranes, in conjunction with Ussing chamber technique, intracellular cAMP and Ca2+ measurements as well as competitive RT-PCR for mRNA expression of cystic fibrosis transmembrane conductance regulator (CFTR) and Ca(2+)-dependent Cl- channels (CACC). Basolateral addition of TMP induced a short circuit current (I(SC)) response, which could be mimicked by forskolin and 3-isobutyl-1-methylxanthine (IBMX). Adenylate cyclase inhibitor, MDL12330A, and intracellular Ca2+ chelator, BAPTA-AM, significantly inhibited the TMP-induced I(SC). In basolateral membrane-permeabilized cells, TMP, as well as forskolin and IBMX, induced an I(SC) response, which was sensitive to MDL-12330A, H89, and specific channel blocker CFTR(inh-172), but insensitive to apical application of 4-4'-didsothiocyanostilbene-2, 2'-disulfonic acid (DIDS) and basolateral pretreatment with BAPTA-AM. In apical membrane-permeabilized cells, TMP, similar to forskolin and IBMX, produced a very small current increase, which was sensitive to K+ channel blockers, BaCl2 and tetraethylammonium (TEA), but not Chromanol 293B and charybdotoxin (ChTX), alone or combined. However, in intact Caco-2 monolayers, the TMP-induced I(SC) could be partially inhibited by ChTX. TMP (5 mM) could stimulate intracellular cAMP production. Intracellular Ca2+ was also increased by TMP (5 mM) in both Ca(2+)-containing and Ca(2+)-free bathing solutions. RT-PCR showed that the expression of CFTR in Caco-2 cells was 5.2 fold higher than that of Ca(2+)-activated Cl- channel (CACC). In conclusion, TMP stimulates Cl- secretion by activating cAMP and [Ca2+]i signaling pathways leading to subsequent activation of apical CFTR and basolateral K+ channels.
Zhu
Jin Xia
JX
Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, PR China.
Zhang
Gui Hong
GH
Yang
Ning
N
Rowlands
Dewi Kenneth
DK
Wong
Hau Yan Connie
HY
Tsang
Lai Ling
LL
Chung
Yiu Wa
YW
Chan
Hsiao Chang
HC
D001483:Base Sequence; D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism; D003106:Colon / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism*; D011719:Pyrazines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
C505032:CFTR protein, human; C492109:CLCA1 protein, human; D018118:Chloride Channels; D024681:Potassium Channels, Calcium-Activated; D011719:Pyrazines; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA; D000242:Cyclic AMP; D002118:Calcium; C017953:tetramethylpyrazine