Received for publication June 14, 2006.
Revised August 6, 2006.
Accepted for publication August 7, 2006.

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⁺)-ATPase (V-ATPase) inhibitors bafilomycin A₁ and concanamycin A induced nitric oxide (NO) production through the expression of inducible nitric oxide synthase (iNOS) 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-kappa B (NF-B) and activator protein-1 (AP-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 and the JNK inhibitor SP600125 in parallel with the partial alleviation of the V-ATPase inhibitor-induced decrease in MTT response. The Na⁺,K⁺-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 S-nitroso-N-acetyl-DL-penicillamine (SNAP) 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 NOS inhibitor N^G-monomethyl-L-arginine acetate (L-NMMA), 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 inhibitor-induced apoptosis in RAW 264.7 cells.

Key words: MTT response, RAW 264.7 Cells, V-ATPase inhibitor, bafilomycin A1, concanamycin A, nitric oxide