CA2-CARCINOMA CELLS( INFLUX INDUCED BY STORE RELEASE AND CYTOSOLIC CA2+ CHELATION IN HT29 COLONIC)

Cl- secretion in HT29 cells is regulated by agonists such as carbachol , neurotensin and adenosine 5'-triphosphate (ATP). These agonists indu ce Ca2+ store release as well as Ca2+ influx from the extracellular sp ace. The increase in cytosolic Ca2+ enhances the Cl- and K+ conductanc es of these cells. Removal of extracellular Ca2+ strongly attenuates t he secretory response to the above-mentioned agonists. The present stu dy utilises patch-clamp methods to characterise the Ca2+ influx pathwa y. Inhibitors which have been shown previously to inhibit non-selectiv e cation channels, such as flufenamate (0.1 mmol . l(-1), n = 6) and G d3+ (10 mu mol . l(-1), M = 6) inhibited ATP (0.1 mmol . l(-1)) induce d increases in whole-cell conductance (G(m)). When Cl- and K+ currents were inhibited by the presence of Cs2SO4 in the patch pipette and glu conate 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 (it = 24). This suggests that ATP induces a cation influx with a conductance of approx imately 3-4 nS. This cation influx was inhibited by flufenamate (0.1 m mol l(-1), n = 6) and Gd3+ (10 mu mol . l(-1), n = 9). When Ba2+(5 mmo l . l(-1)) and 4,4'-diisothiocyanatostilbene-2-2'-disulphonic acid (DI DS, 0.1 mmol . l(-1)) were added to the KCl/K-gluconate pipette soluti on to inhibit K+ and Cl- currents and the cells were clamped to depola rised 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 w as activated by dialysing the cell with a KCl/K-gluconate solution con taining 5-10 mmol . l(-1) ,2-bis-(2-aminoethoxy)ethane-N,N,N',N'-tetra acetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane- N,N,N',N'-tetra acetic 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,depola rised significantly from -33 +/- 2 mV to -12 +/- mV, and I-m fell sign ificantly from 17 +/- 2 pA to 8.9 +/- 1.0 pA (n = 71). This negative c urrent, representing a cation inward current, was activated when Ca2stores were emptied and was reduced significantly Delta I-m) when Ca2 and/or Na+ were removed from the bathing solution: removal of Ca2+ in the absence of Na+ caused a Delta I-m of 5.0 +/- 1.2 pA (n = 12); rem oval of Na+ in the absence of Ca2+ caused a Delta I-m of 12.8 +/- 3.5 pA (n = 4). The cation inward current was also reduced significantly b y La3+, Gd3+, and flufenamate. We conclude that store depletion induce s a Ca2+/Na+ influx current in these cells. With 145 mmol . l(-1) Naand 1 mmol . l(-1) Ca2+, both ions contribute to this cation inward cu rrent. This current is an important component in the agonist-regulated secretory response.