ISSN:
1432-2013
Keywords:
Ca2+ channel
;
Stimulation-secretion coupling
;
Exocrine secretion
;
Colon
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Abstract Cl− secretion in HT29 cells is regulated by agonists such as carbachol, neurotensin and adenosine 5′-triphosphate (ATP). These agonists induce Ca2+ store release as well as Ca2+ influx from the extracellular space. The increase in cytosolic Ca2+ enhances the Cl− and K+ conductances of these cells. Removal of extracellular Ca2+ strongly attenuates the secretory response to the above-mentioned agonists. The present study utilises patch-clamp methods to characterise the Ca2+ influx pathway. Inhibitors which have been shown previously to inhibit non-selective cation channels, such as flufenamate (0.1 mmol·l−1, n=6) and Gd3+ (10 μmol·l−1, n=6) inhibited ATP (0.1 mmol·l−1) induced increases in whole-cell conductance (G m). When Cl− and K+ currents were inhibited by the presence of Cs2SO4 in the patch pipette and gluconate in the bath, ATP (0.1 mmol·l−1) still induced a significant increase in G m from 1.2±0.3 nS to 4.7±1 nS (n=24). This suggests that ATP induces a cation influx with a conductance of approximately 3–4 nS. This cation influx was inhibited by flufenamate (0.1 mmol·l−1, n=6) and Gd3+ (10 μmol·l−1, n=9). When Ba2+ (5 mmol·l−1) and 4,4′-diisothiocyanatostilbene-2-2′-disulphonic acid (DIDS, 0.1 mmol·l−1) were added to the KCl/K-gluconate pipette solution to inhibit K+ and Cl− currents and the cells were clamped to depolarised voltages, ATP (0.1 mmol·l−1) reduced the membrane current (I m) significantly from 86±14 pA to 54±11 pA (n=13), unmasking a cation inward current. In another series, the cation inward current was activated by dialysing the cell with a KCl/K-gluconate solution containing 5–10 mmol·l−1 1,2-bis-(2-aminoethoxy)ethane-N,N,N′,N′-tetraacetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The zero-current membrane voltage (V m) and I m (at a clamp voltage of +10 mV) were monitored as a function of time. A new steady-state was reached 30–120 s after membrane rupture. V m depolarised significantly from −33±2 mV to −12±1 mV, and I m fell significantly from 17±2 pA to 8.9±1.0 pA (n=71). This negative current, representing a cation inward current, was activated when Ca2+ stores were emptied and was reduced significantly (ΔI m) when Ca2+ and/or Na+ were removed from the bathing solution: removal of Ca2+ in the absence of Na+ caused a ΔI m of 5.0±1.2 pA (n=12); removal of Na+ in the absence of Ca2+ caused a ΔI m of 12.8±3.5 pA (n=4). The cation inward current was also reduced significantly by La3+, Gd3+, and flufenamate. We conclude that store depletion induces a Ca2+/Na+ influx current in these cells. With 145 mmol·l−1 Na+ and 1 mmol·l−1 Ca2+, both ions contribute to this cation inward current. This current is an important component in the agonist-regulated secretory response.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00386159
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