Inhibition of human neutrophil function by tolfenamic acid involves inhibition of Ca2+ influx

The present work was designed to study the pharmacological control of the receptor-mediated activation of human neutrophils by tolfenamic acid (2-[(3-chloro-2-methylphenyl)-amino]benzoic acid). Tolfenamic acid inhibited in a concentration-dependent manner the degranulation response and Ca²⁺ influx in neutrophils activated either by the chemotactic peptide fMLP (N-formyl-methionyl-leucylphenylalanine) or Ca²⁺ ionophore A23187 (calcimycin). When fMLP was used to activate neutrophils, tolfenamic acid (30 μM) reduced Ca²⁺ influx by 50% and degranulation by 20%. A23187-triggered Ca²⁺ influx and degranulation were inhibited by 60% and 40%, respectively, by 30 μM tolfenamic acid. Tolfenamic acid did not inhibit the release of Ca²⁺ from intracellular stores induced either by fMLP or A23187. To confirm the inhibition of receptor-mediated cation influx by tolfenamic acid, the agonist induced Mn²⁺ influx was studied in Ca²⁺ free medium. Tolfenamic acid (10–30 μM) reduced fMLP-stimulated Mn²⁺ influx in neutrophils in a concentration-dependent manner. The simultaneous Ca²⁺ release from intracellular stores was not affected. Protein kinase C activity in sonicated human neutrophils and the purified enzyme from rat brain were inhibited by the protein kinase inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) but not by tolfenamic acid. Both failed to inhibit neutrophil degranulation induced by phorbol myristate acetate, a protein kinase C activator. Tolfenamic acid (100 μM) increased the cellular cAMP levels up to 1.3-fold in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. No effects on cellular cGMP levels were found. In conclusion, tolfenamic acid inhibits both fMLP-and Ca²⁺-ionophore induced Ca²⁺ influx in human neutrophils concomitantly with its inhibitory action on degranulation. This suggests that the inhibitory action of tolfenamic acid on human neutrophil functions is mediated by a mechanism involving inhibition of Ca²⁺ influx. Neither protein kinase C nor cyclic nucleotides seem to have a major role in the inhibitory action of tolfenamic acid on human neutrophil functions.