Activation of apical CFTR and basolateral Ca2+-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 Ca2+-dependent Cl− channels (CACC). Basolateral addition of TMP induced a short circuit current (ISC) 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 ISC. In basolateral membrane-permeabilized cells, TMP, as well as forskolin and IBMX, induced an ISC response, which was sensitive to MDL-12330A, H89, and specific channel blocker CFTRinh-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 ISC 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 Ca2+-containing and Ca2+-free bathing solutions. RT-PCR showed that the expression of CFTR in Caco-2 cells was 5.2 fold higher than that of Ca2+-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.