calcimycin and 3--5-dichlorodiphenylamine-2-carboxylic-acid

The mechanism of regulated Cl- secretion was evaluated in the mucin-secreting cell line HT29-Cl.16E by transepithelial electrophysiology and fura 2 measurements of cytosolic Ca2+. Carbachol by itself was a weak secretagogue, but augmented adenosine 3',5'-cyclic monophosphate (cAMP)-mediated secretion more than twofold, consistent with activation of a rate-limiting K+ conductance. To characterize this conductance, monolayers were apically permeabilized with amphotericin B. At least two types of K+ conductances were identified. One type was activated by elevated cytosolic cAMP levels and inhibited by Ba2+ (inhibitor constant 0.3 mM) in the basolateral solution but was not affected by quinidine or elevated cytosolic Ca2+. The other type was activated by carbachol via cytosolic Ca2+ and was partially inhibited by quinidine (60% inhibition by 2.5 mM quinidine) but was not affected by Ba2+ up to 1 mM. Both conductances appear to be involved in active, transepithelial Cl- secretion in intact monolayers but under different conditions because 1) the cAMP-stimulated short-circuit current (Isc) can be partially inhibited by 1 mM Ba2+ (50%) but not quinidine, 2) the Ba2+ inhibition does not affect the carbachol-induced increase in Isc in cells with elevated cAMP levels, and 3) the carbachol-dependent Isc can be inhibited by quinidine. Therefore, the contribution of the cAMP-dependent K+ conductance appears important for maintaining the membrane potential and therewith Cl- secretion when cAMP is the only messenger for secretion signals, whereas the Ca(2+)-dependent K+ conductance is responsible for the carbachol-stimulated increase in Isc.

Topics: Amphotericin B; Barium; Bumetanide; Calcimycin; Calcium; Carbachol; Cell Line; Cell Membrane Permeability; Colforsin; Cyclic AMP; Digitonin; Diphenylamine; Electric Conductivity; Epithelial Cells; Epithelium; Fura-2; Humans; Intestinal Mucosa; Membrane Potentials; Models, Biological; Ouabain; Potassium; Potassium Channels; Quinidine; Tetradecanoylphorbol Acetate; Thionucleotides; Vasoactive Intestinal Peptide

Fenamates, a subgroup of nonsteroidal anti-inflammatory drugs, inhibit several functions of human polymorphonuclear leukocytes (PMNs) in vitro, by a thus far unknown mechanism. To determine the mechanism behind this action, we studied the effects of two fenamates (flufenamic and tolfenamic acids) on Ca2+ metabolism in human PMNs. The two fenamates inhibited the increases in intracellular free calcium concentration induced by either the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine or the calcium ionophore A23187 in fura-2-labeled PMNs. This inhibition was concluded to be due to blocking of the cation influx, as evidenced by measurement of Mn2+ influx and the influx of radioactive calcium. In addition, the actions of flufenamic and tolfenamic acids were similar to those of an experimental blocker of nonselective cation channels (SK&F 96365). The two other control compounds, an antagonist of voltage-dependent calcium channels (nifedipine) and an inhibitor of prostanoid synthesis (ketoprofen), were ineffective. In conclusion, inhibition of calcium influx in PMNs is introduced as a novel prostanoid-independent mode of action of two nonsteroidal anti-inflammatory drugs with fenamate structure, flufenamic and tolfenamic acids, which could explain their earlier documented inhibitory effects on PMN functions.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Calcimycin; Calcium; Calcium Channel Blockers; Diphenylamine; Flufenamic Acid; Humans; Imidazoles; In Vitro Techniques; Intracellular Fluid; Ion Transport; Ketoprofen; Manganese; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nifedipine; ortho-Aminobenzoates