Flufenamate and Gd3+ inhibit stimulated Ca2+ influx in the epithelial cell line CFPAC-1.
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 epithelial preparations, we tested whether the stimulated Ca2+ entry in CFPAC-1 cells was inhibited by these agents. Transmembraneous Ca2+ influx 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 the plateau phase of an agonist-induced [Ca2+]i transient reflects Ca2+ influx into these cells, we investigated the influence of Flu and Gd3+ 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 agonist-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 potent inhibitors of the stimulated Ca2+ entry in CFPAC-1 cells. Flu reversibly decreased the ATP-induced [Ca2+]i plateau in a concentration dependent manner, with an IC50 value of 33 mumol/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 stimulated Ca2+ plateau. A complete block of the ATP-induced [Ca2+]i plateau was seen at 0.5 mumol/l (ATP 10(-5) mol/l, n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)
Physiologisches Institut, Albert-Ludwigs-Universität, Freiburg, Germany.
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S
Schumann
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R
Greger
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J
Leipziger
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D004848:Epithelium / Q000378:metabolism; D005439:Flufenamic Acid / Q000494:pharmacology*; D016257:Fura-2; D005682:Gadolinium / Q000494:pharmacology*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D013729:Terpenes; D005439:Flufenamic Acid; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D002217:Carbachol; D005682:Gadolinium; D002118:Calcium; D016257:Fura-2