Ca²⁺ influx induced by store release and cytosolic Ca²⁺ chelation in HT₂₉ colonic carcinoma cells

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.