Popis: |
Whole-cell patch-clamp techniques and fluorescence measurements of intracellular Ca2+ concentration, (Ca2+)i, were used to investigate the mechanism of taurodeoxycholate (TDC) stimulation of Cl- secretion in the T84 colonic cell line. During perforated whole-cell recordings, the cell membrane voltage was alternately clamped to EK and ECl. Initially, TDC (0.75 mM) stimulated inward nonselective cation currents that were composed of discrete large conductance single-channel events. This initial response was followed by activation of K+ and Cl- currents with peak values of 385 +/- 41 pA and 98 +/- 28 pA, respectively (n = 12). The K+ and Cl- currents oscillated while TDC was present and returned to baseline levels upon its removal. The threshold for activation of the oscillatory currents was 0.1 mM TDC. Taurocholate, a bile acid that does not stimulate colonic Cl- secretion, induced no current response. The TDC-induced currents could be activated in Ca(2+)-free bathing solutions. Preincubation of cells with the Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethy)-ester (20 microM), (BAPTA-AM), eliminated the K+ and Cl- current responses, although the nonselective cation channel events were still present. Replacement of bath Na+ with NMDG+ inhibited the TDC-induced nonselective cation current but did not affect the K+ or Cl- currents. TDC induced a transient (Ca2+)i rise of 575 +/- 70 nM from a baseline of 71 +/- 5 nM (n = 15); thereafter, (Ca2+)i either plateaued or oscillated. TDC-induced (Ca2+)i oscillations were observed in the absence of bath Ca2+; however, removal of bath Ca2+ during the TDC response caused (Ca2+)i to return to near baseline values. Simultaneous K+ current and (Ca2+)i measurements confirmed that the initial nonselective cation current was independent of (Ca2+)i, while K+ current oscillations were in phase with the (Ca2+)i oscillations. TDC induced inositol monophosphate (IP) accumulation, reflecting production of inositol 1,4,5-trisphosphate (IP3) during TDC stimulation. The response to TDC during standard whole-cell patch-clamp was similar to that observed with perforated whole-cell recordings, except the nonselective cation current was prolonged. When heparin (1 mg/ml) was added to the pipette under these conditions, the Ca(2+)-activated currents were inhibited, but the nonselective cation currents were unaffected. These data suggest that TDC induces a Ca(2+)-independent nonselective cation conductance, perhaps by directly permeabilizing the plasma membrane. TDC stimulates Cl- secretion by activating K+ and Cl- conductances via an IP3-mediated release of Ca2+ from intracellular stores. |