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Abstract: Our previous study demonstrated that NYD-SP27 is a novel inhibitory PLC isoform expressed endogenously in human pancreas and upregulated in CFPAC-1 cells. The present study investigated the effect of NYD-SP27 down-regulation on the ATP-stimulated and Ca2+-dpendent pancreatic anion secretion by CFPAC-1 cell line using short-circuit current (I SC) recording. NYD-SP27 antisense-transfected CFPAC-1 (AT-CF) cells exhibited a significantly higher basal transmembrane potential difference and current than those of empty vector-transfected CFPAC-1 (VT-CF) cells. Cl− channel blocker, DPC or Glibenclamide (1mM), and inhibitor of Na+-K+-Cl− cotransporter, bumetanide (100μM) significantly inhibited the basal current in AT-CF cells. The inhibitor of adenylate cyclase, MDL12330A (20μM), and Ca2+-dependent Cl− channel (CaCC) blocker, DIDS (100μM) also significantly reduced the basal current in AT-CF. Apical application of ATP (10μM) stimulated a fast transient I SC increase in VT-CF cells, but a more sustained rise with slower decline in AT-CF cells. Pretreatment with BAPTA-AM (50μM) reduced the ATP-induced I SC response in AT-CF cells by 77.9%. PMA (1μM), a PKC activator, inhibited the ATP-stimulated current increase (the transient peak) in VT-CF cells, but had no effect on the AT-CF cells. However, PKC inhibitor, staurosporine (40μM) could inhibit the ATP-induced I SC response in AT-CF cells. The present results confirm the previously proposed inhibitory role of NYD-SP27 in the PLC pathway and demonstrate that the suppression of its expression could result in an enhancement of ATP-stimulated Ca2+ dependent pancreatic anion secretion. [Copyright &y& Elsevier]

Abstract: We have previously obtained monoclonal bone marrow stem cells from adult rats (rMSCs) and induced them into phenotypic neurons. In the present study, we aimed to induce rMSCs into epithelial cells by culturing them onto compartmentalized permeable supports, which have been used for growing a variety of polarized epithelia in culture. Hematoxylin staining showed that after 4 days grown on permeable supports, rMSCs formed an epithelial-like monolayer. Immunofluorescence of the permeably-supported monolayers, but not the rMSCs grown in culture flasks, showed positive signals for epithelial markers, cytokeratin 5 & 8. RT-PCR results also showed the mRNA expression of epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) as well as tight junction protein ZO-1 in the rMSC-derived monolayers grown on permeable supports but absent from those grown in culture flasks. However, western blot only detected protein expression of ZO-1 but not ENaC nor CFTR. The short-circuit current measurements showed that the rMSC-derived monolayers grown on permeable supports exhibited a trans-monolayer resistance of 30–50Ωcm2; however, the monolayers did not respond to activators or blockers of CFTR or ENaC. The results suggest that compartmentalized or polarized culture conditions provide a suitable environment for rMSCs to differentiate into epithelial progenitor cells with tight junction formation; however, this condition is not sufficient for functional expression of epithelial ion channels associated with well-differentiated epithelia. [Copyright &y& Elsevier]

Abstract: In the present study we investigated the role of Ca2+ in tetramethylpyrazine (TMP)-induced anion secretion in the human colonic epithelial cell line, Caco-2, using the short-circuit current (I SC) technique in conjunction with intracellular Ca2+ measurements. The results showed that TMP-induced I SC response was significantly reduced by 58.8% and 38.3% after inhibiting Ca2+ ATPase of endoplasmic reticulum (ER) with thapsigargin and mobilizing ER stored Ca2+ release with ATP, respectively. Conversely, thapsigargin- and ATP-evoked I SC responses were also significantly reduced by pretreatment with TMP by 43.2% and 38.5%, respectively. Conversely, removal of extracellular Ca2+, apical but not basolateral, or the presence of the Ca2+ chelator (EGTA) significantly increased TMP-induced I SC by 47.1% and 37.8%, respectively. Similar to TMP, thapsigargin-induced current increase was also enhanced by chelating extracellular Ca2+ or in Ca2+ free solution; however, removal of extracellular Ca2+ did not significantly affect 3-isobutyl-1-methylxanthine (IBMX)- and forskolin-induced transepithelial current. Measurement of the intracellular concentration of free Ca2+ ([Ca2+]i) with fura-2/AM showed that TMP could induce an increase in [Ca2+]i but pretreatment with TMP significantly reduced thapsigargin-evoked, but not ATP-induced, [Ca2+]i increase. These results suggest that the effect of TMP on colonic anion secretion is partly mediated by TMP-increased [Ca2+]i by acting on a target similar to thapsigargin. The observed inhibitory effect of extracellular Ca2+ on Ca2+-dependent anion secretion represents a novel mechanism by which Ca2+-dependent regulation of epithelial electrolyte transport may be fine-tuned by extracellular Ca2+ in the apical domain. [Copyright &y& Elsevier]

The present study examined the effect of Bak Foong Pills (BFP), an over-the-counter traditional Chinese medicine (China registration no. Z980035), on anion secretion and the underlying signaling pathways in normal and cystic fibrosis pancreatic duct cell lines, CAPAN-1 and CFPAC-1, respectively, using the short-circuit current technique. Apical addition of BFP ethanol extract (600 μg/ml) induced a fast transient ISC peak that was followed by a slower but more sustained increase in ISC in CAPAN-1 cells. However, the response to BFP in CFPAC-1 was predominantly the first transient peak. Apical addition of DIDS (200 μM) inhibited the first peak by more than 60% in both cell lines without significantly affecting the second ISC rise. More than 85% of the BFP-induced first transient in both cell lines was inhibited when extra and intracellular Ca2+ was chelated or emptied by pre-treatment with BAPTA (100 μM) and thapsigargin (10 μM), respectively. Acute addition of PMA (1 μM), a PKC activator, blocked more than 95% of the BFP-induced first peak in both cell lines, consistent with previously reported PKC modulation of Ca2+-dependent pancreatic anion secretion. The BFP-induced second ISC rise in CAPAN-1 could be inhibited by 73.6% and 71.13% by pretreatment of the cells with MDL-12330A (20 μM), an adenylate cyclase inhibitor and Rp-cAMP (200 μM), a cyclic AMP antagonist, respectively. However, less than 25% of the ISC was inhibited by combined treatment with BAPTA and thapsigargin. The second rise was also completely blocked by DPC (2 mM) or Glibenclamide (1 mM). The results indicate that BFP ethanol extract stimulates pancreatic duct anion secretion in normal and CF cells via different signaling pathways involving both Ca2+ and cAMP. [Copyright &y& Elsevier]