Taurodeoxycholate activates potassium and chloride conductances via an IP3-mediated release of calcium from intracellular stores in a colonic cell line (T84)
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.
Devor
D C
DC
Department of Physiology, State University of New York at Buffalo 14214.
Sekar
M C
MC
Frizzell
R A
RA
Duffey
M E
ME
D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000187:drug effects; D002794:Choline / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism*; D004553:Electric Conductivity / Q000187:drug effects; D005470:Fluorometry; D016257:Fura-2; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007473:Ion Channels / Q000187:drug effects*; D008839:Microelectrodes; D029363:Organic Anion Transporters, Sodium-Dependent; D010716:Phosphatidylinositols / Q000378:metabolism; D015221:Potassium Channels / Q000187:drug effects; D015398:Signal Transduction; D012964:Sodium / Q000378:metabolism; D027981:Symporters; D013657:Taurodeoxycholic Acid / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D002412:Cations; D018118:Chloride Channels; D007473:Ion Channels; D029363:Organic Anion Transporters, Sodium-Dependent; D010716:Phosphatidylinositols; D015221:Potassium Channels; D027981:Symporters; C071908:sodium-bile acid cotransporter; D013657:Taurodeoxycholic Acid; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D012964:Sodium; D002794:Choline; D002118:Calcium; D016257:Fura-2