Cromolyn inhibits assembly of the NADPH oxidase and superoxide anion generation by human neutrophils.

Early and late phase reactions have been observed in asthma; the late phase reaction is characterized by accumulation of inflammatory cells such as neutrophils. Activated neutrophils degranulate and assemble an active NADPH oxidase, which generates superoxide anion (O2-), reactions that have been implicated in lung tissue damage. Preincubation of neutrophils with the asthma drug cromolyn sodium selectively inhibited FMLP (10(-7) M) and PMA (0.1 microgram/ml) elicited O2- generation but not degranulation. To further characterize the mechanism of this inhibition we examined the effect of cromolyn on the NADPH oxidase complex and the signaling pathways for its assembly. Ca2+ mobilization and activation of protein kinase C have been implicated as signals for activation of the NADPH oxidase. Ca2+ mobilization triggered by FMLP was significantly decreased by 21.2% in cromolyn-treated cells. In contrast, cromolyn did not interfere with translocation or activity of protein kinase C. Membranes prepared from neutrophils stimulated with 0.5 microgram/ml PMA generated O2-, indicating assembly of an active NADPH oxidase; cromolyn did not inhibit this membrane-associated, preassembled oxidase. In contrast, preincubation of neutrophils with 100 microM cromolyn before addition of PMA decreased the capacity of the membranes to generate O2- by 57.3%. These results indicate that cromolyn inhibited the assembly of an active NADPH oxidase. The efficacy of cromolyn may be associated with inhibition of assembly of an active NADPH oxidase in the neutrophil and prevention of oxygen radical-induced tissue damage.

Long chain acyl coenzyme A and signaling in neutrophils. An inhibitor of acyl coenzyme A synthetase, triacsin C, inhibits superoxide anion generation and degranulation by human neutrophils.

Ligand-initiated activation of neutrophils triggers O2- generation, degranulation, phospholipid remodeling, and release of fatty acids such as arachidonate, oleate, and palmitate. Long chain acyl-CoA synthetase converts free fatty acids to acyl-CoA esters; a role for acyl-CoA esters as positive modulators of neutrophil functions is proposed. Physiologically relevant concentrations (1-10 microM) of acyl-CoA esters such as palmitoyl-CoA, enhanced O2- generation triggered by fMet-Leu-Phe or guanosine 5'-O-(thiotriphosphate) (GTP gamma S) but did not act as a trigger per se. Triacsin C, an inhibitor of acyl-CoA synthetase, inhibited fMet-Leu-Phe-elicited O2- generation and degranulation in a concentration-dependent manner. Triacsin C inhibited O2- generation elicited by fMet-Leu-Phe and GTP gamma S in electroporated neutrophils, indicating that acyl-CoA acted downstream from the receptor. Palmitoyl-CoA reversed the Triacsin C-induced inhibition of O2- generation. fMet-Leu-Phe elicited a prompt increase in total long chain acyl-CoA esters. Arachidonoyl-CoA and oleoyl-CoA were elevated 5 s after addition of fMet-Leu-Phe, while palmitoyl-CoA was not elevated until 60 s. Triacsin C inhibited fMet-Leu-Phe-initiated increases in arachidonoyl-CoA, oleoyl-CoA, and palmitoyl-CoA. These results suggest a role for acyl-CoA esters in regulating activation of O2- generation and degranulation at the G protein or subsequent step(s).

Protein kinase C isotypes and signal-transduction in human neutrophils: selective substrate specificity of calcium-dependent beta-PKC and novel calcium-independent nPKC.

Neutrophils possess at least two phospholipid-dependent forms of protein kinase C, a classical Ca/PS/DG-dependent beta-isotype of protein kinase C and a Ca-independent but PS/DG-dependent novel protein kinase C (nPKC) which we now demonstrate to have different substrate specificities. Activation of human neutrophils triggers assembly of an NADPH oxidase in the membrane and generation of O2-. A role for the major Ca-dependent isotype beta-PKC in neutrophils is proposed in stimulus-induced phosphorylation and association of a cytosolic 47 kDa protein (p47-phox) with the membrane NADPH oxidase. In this study we demonstrate that purified beta-PKC and nPKC have very different substrate specificities; beta-PKC but not nPKC phosphorylated both endogenous and recombinant p47-phox. In addition, beta-PKC but not nPKC phosphorylated [ser25]PKC(19-31), the substrate peptide based on a sequence in the Ca-dependent alpha, beta and gamma-isotypes. Pseudosubstrate(19-36), derived from the C-terminus of Ca-dependent PKC isotypes, inhibited beta-PKC but not nPKC activity using either Histone IIIS or peptide(19-31) as substrate. Pseudosubstrate(19-36) also inhibited beta-PKC catalyzed phosphorylation of endogenous and recombinant p47-phox. Pseudosubstrate(19-36) also inhibited the O2- generation triggered by GTP gamma S in electroporated neutrophils by 50%. 32P-Labelled neutrophils electroporated in the presence of GTP gamma S showed phosphorylation of multiple cytosolic proteins including a 47 kDa band, and phosphorylation of membrane-associated 34 kDa, 47 kDa and 54 kDa proteins. Pseudosubstrate(19-36) inhibited phosphorylation of p47-phox in the membrane but not in the cytosol. These findings suggest translocatable, Ca-dependent isotypes of PKC such as beta-PKC may play a role in the phosphorylation of membrane associated p47-phox and the assembly or maintenance of an active NADPH oxidase.