Involvement of intracellular and extracellular Ca2+ in tetramethylpyrazine-induced colonic anion secretion

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 (ISC) technique in conjunction with intracellular Ca2+ measurements. The results showed that TMP-induced ISC 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 ISC 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 ISC 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.