Evidence for agonist-induced export of intracellular Ca²⁺ in epithelial cells

Abstract

There is increasing evidence that some agonists not only induce intracellular Ca²⁺ increases, due to store release and transmembranous influx, but also that they stimulate Ca²⁺ efflux. We have investigated the agonist-stimulated response on the intracellular Ca²⁺ activity ([Ca²⁺]_i) in the presence of thapsigargin (10⁻⁸ mol/l, TG) in HT₂₉ and CFPAC-1 cells. For CFPAC-1 the agonists ATP (10⁻⁷–10⁻³ mol/l, n=9), carbachol (10⁻⁶–10⁻³ mol/l, n=5) and neurotensin (10⁻¹⁰–10⁻⁷ mol/l, n=6) all induced a concentration-dependent decrease in [Ca²⁺]_i in the presence of TG. Similar results were obtained with HT₂₉ cells. This decrease of [Ca²⁺]_i could be caused by a reduced Ca²⁺ influx, either due to a reduced driving force for Ca²⁺ in the presence of depolarizing agonists or due to agonist-regulated decrease in Ca²⁺ permeability. Using the fura-2 Mn²⁺ quenching technique we demonstrated that ATP did not slow the TG-induced Mn²⁺ quench. This indicates that the agonist-induced [Ca²⁺]_i decrease in the presence of TG was not due to a reduced influx of Ca²⁺ into the cell, but rather due to stimulation of Ca²⁺ export. We used the cell attached nystatin patch clamp technique in CFPAC-1 cells to examine whether, in the presence of TG, the above agonists still led to the previously described electrical changes. The cells had a mean membrane voltage of −49±3.6 mV (n=9). Within the first 3 min ATP was still able to induce a depolarization which could be attributed to an increase in Cl⁻ conductance. This was expected, since at this time after TG stimulation all Ca²⁺ agonists still liberated some [Ca²⁺]_i. When TG incubation was prolonged, agonist application led to strongly attenuated or to no electrical responses. Therefore, the agonist-stimulated [Ca²⁺]_i decrease cannot be explained by the reduction of the driving force for Ca²⁺ into the cell. In the same cells hypotonic swelling (160 mosmol/l, n=15) still induced a further [Ca²⁺]_i increase in the presence of TG and concomitantly induced Cl⁻ and K⁺ conductances. We conclude that the agonist-induced decrease of [Ca²⁺]_i in the presence of TG probably unmasks a stimulation of [Ca²⁺]_i export.