Nitric Oxide Production by the Vacuolar-Type (H⁺)-ATPase Inhibitors Bafilomycin A1 and Concanamycin A and Its Possible Role in Apoptosis in RAW 264.7 Cells

Abstract

In the mouse leukemic monocyte cell line RAW 264.7, the vacuolar-type (H ϩ )-ATPase (V-ATPase) inhibitors bafilomycin A1 and concanamycin A induced nitric oxide (NO) production through the expression of inducible nitric-oxide synthase mRNA and its protein and decreased cell growth and survival as determined by 3-(4,5-dimethyl(thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Bafilomycin A1 and concanamycin A activated nuclear factor (NF)-B and activator protein-1 and decreased the level of IB-␣ and increased that of phosphorylated c-Jun N-terminal kinase (JNK). NO production induced by these V-ATPase inhibitors was suppressed by the NF-B inhibitor Bay 11-7082 [(E)3-[(4-methylphenyl)sulfonyl])-2-propenenitrile] and the JNK inhibitor SP600125 [anthra[1,9-cd]pyrazol-6(2H)-one] in parallel with the partial alleviation of the V-ATPase inhibitor-induced decrease in MTT response. The Na ϩ ,K ϩ -ATPase inhibitor dibucaine and the F-ATPase inhibitor oligomycin did not induce NO production at which concentrations the MTT response was decreased. The NO donor Snitroso-N-acetyl-DL-penicillamine further lowered the V-ATPase inhibitor-induced decrease in the MTT response, and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt (carboxy-PTIO) alleviated it partially. Mitochondrial depolarization, an index of apoptosis, was induced by bafilomycin A1 and concanamycin A. On treatment with the nitric-oxide synthase inhibitor N G -monomethyl-L-arginine acetate, the disruption of mitochondrial membrane potential induced by bafilomycin A1 and concanamycin A was alleviated partially in parallel with the decrease in NO production. Carboxy-PTIO also alleviated it partially. Our findings suggest that the V-ATPase inhibitors bafilomycin A1 and concanamycin A similarly induce NO production and the newly produced NO participates partially in the V-ATPase inhibitorinduced apoptosis in RAW 264.7 cells.

Genetic and pharmacologic manipulation of vacuolar ATPase: Effects on zymogen activation in pancreatic acini

“…2 Inhibition of vATPase with the classic vATPase inhibitors bafilomycin-A1 and concanamycin-A not only inhibits CER-stimulated zymogen activation, 2 but also blocks the enhancing effect of an extracellular acid load in vitro. 23 Because the classic vATPase inhibitors bafilomycin and concanamycin have been shown to have nonspecific effects, [6][7][8][9] the current study was undertaken to confirm our previous results utilizing the novel class of vATPase inhibitors, the benzolactone enamides, which have a different mode of inhibition, as well as genetic manipulation using siRNA.…”

“…Bafilomycin causes the degradation of IκB and phosphorylation of JNK resulting in the activation of nuclear factor-kappaB (NF-κB) and ultimately an increase in iNOS mRNA and protein. 7 The method by which bafilomycin causes IκB degradation and JNK phosphorylation are unclear, but raise the possibility that these effects might not be related to vATPase inhibition. Bafilomycin 8 and concanamycin 9 have been shown to block chloroquine induced apoptosis in cultured cerebellar granule neurons at concentrations which do not inhibit vATPase activity (1 nM).…”

Vacuolar ATPase as a potential therapeutic target and mediator of treatment resistance in cancer

“…V‐ATPase inhibition has been shown to result in increased reactive oxygen species (ROS) in cancer cells75, 76, 77 and HIF1α upregulation 74. Furthermore, V‐ATPase inhibition induces caspase‐dependent apoptosis in invasive tumor cells via the mitochondrial pathways 74, 78.…”

Omeprazole Antagonizes Virulence and Inflammation in Salmonella enterica -Infected RAW264.7 Cells

“…Proton pump inhibitors have been reported to affect inflammatory signaling (17) and the expression of inducible nitric oxide synthase (iNOS) in macrophages and macrophage-like cells (18). The expression of iNOS also can be induced in murine monocytic cells through infection with S. Typhimurium, whereupon subsequent NO production inhibits the intracellular replication of S. Typhimurium (4,42).…”