FLUFENAMATE AND GD3-CELL LINE CFPAC-1( INHIBIT STIMULATED CA2+ INFLUXIN THE EPITHELIAL)

The relevant influx pathway for stimulated Ca2+ entry into epithelial cells is largely unknown. Using flufenamate (Flu) and Gd3+, both known pharmacological blockers of non-selective cation currents in other ep ithelial preparations, we tested whether the stimulated Ca2+ entry in CFPAC-1 cells was inhibited by these agents. Transmembraneous Ca2+ inf lux into CFPAC-1 cells was stimulated by either ATP (10(-4) and 10(-5) mol/l), carbachol (CCH, 10(-4) mol/l) or thapsigargin (TG, 10(-8) mol /l). Three different experimental approaches were used. (1) Because th e plateau phase of an agonist-induced [Ca2+](i) transient reflects Ca2 + influx into these cells, we investigated the influence of Flu and Gd 3+ on the level of the stimulated [Ca2+](i) plateau. (2) The fura-2 Mn 2+-quenching technique was used to visualise divalent cation entry and monitor its inhibition. (3) During the ''refilling period'' after ago nist-induced discharge of the intracellular pools the putative influx inhibitors Flu and Gd3+ were given and subsequently the filling state of the agonist-sensitive intracellular stores tested. The results from the first experimental approach showed that both Flu and Gd3+ were po tent inhibitors of the stimulated Ca2+ entry in CFPAC-1 cells. Flu rev ersibly decreased the ATP-induced [Ca2+](i) plateau in a concentration dependent manner, with an IC50 Value of 33 mu mol/l (n = 6). Similar results were obtained for the CCH- (n = 5) and the TG-induced (n = 5) [Ca2+](i) plateau. Gd3+ concentration dependently inhibited the stimul ated Ca2+ plateau. A complete block of the ATP-induced [Ca2+](i) plate au was seen at 0.5 mu mol/l (ATP 10(-5) mol/l, n = 8). The second appr oach showed that Flu (10(-4) mol/l) completely inhibited the ATP- (10( -5) mol/l, n = 3), CCH- (10(-4) mol/l, n = 4) and TG- (10(-8) mol/l, n = 3)-induced fura-2 Mn2+ quench. Gd3+ also inhibited the fura-2 Mn2+- quenching rate (n = 9). The third approach showed that Flu (n = 6) and Gd3+ (n = 8) inhibited the refilling of the ATP-sensitive intracellul ar Ca2+ store. These results show that inhibitors of non-selective cat ion currents in other epithelial preparations are potent inhibitors of stimulated Ca2+ influx in CFPAC-1 cells. Whether this inhibitory effe ct concerns a non-selective cation channel remains to be established.