Pepstatin a induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines

Mitsuko Okada, Shin Irie, Makoto Sawada, Ryuji Urae, Akinori Urae, Nakao Iwata, Norio Ozaki, Kohei Akazawa, Hiroshi Nakanishi

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)


The extrusion of protons is considered a very general parameter of the activation of many kinds of membrane or intracellular molecules, such as receptors, ion channels, and enzymes. We found that pepstatin A caused a reproducible, concentration-related increase in the extracellular acidification rate in two microglial cell lines, Ra2 and 6-3. Washing abolished pepstatin A-induced acidification immediately. However, pepstatin A did not cause the extracellular acidification in other cell types, such as CHO, C6 glioma, and NIH3T3 cells. These observations strongly suggest that pepstatin A interacts with certain membrane proteins specific to both Ra2 and 6-3 cells from outside. N-methylmaleimide and N-dicyclohexylcarbodiimide, N,N′-dicyclohexylcarbodiimide, inhibitors of H+-ATPase, were found to reduce pepstatin A-induced response strongly, while bafilomycin A1, a vacuolar H+-ATPase inhibitor, vanadate, a P-type H+-ATPase inhibitor, and NaN3, an F1 ATPase inhibitor, virtually did not. 5-(N-ethyl-N-isopropyl) amiloride, an inhibitor of Na+/H+ exchanger isoform 1, greatly enhanced pepstatin-induced response, while amiloride did not. Zn2+, a voltage-dependent proton channel blocker, did not affect pepstatin-induced response neither. Staurosporine, a nonspecific inhibitor of protein kinase C, inhibited pepstatin A-induced response, while chelerythrine, more selective inhibitor of protein kinase C, greatly enhanced it. H-7 and H-8 did not affected the response. These findings suggest that pepstatin A induces extracellular acidification in microglia cell lines, Ra2 and 6-3, through an N-methylmaleimide- and N,N′-dicyclohexylcarbodiimide-sensitive, but bafilomycin A1-insensitive, ATPase, which seems to be distinct from protein kinase C-dependent process.

Original languageEnglish
Pages (from-to)167-174
Number of pages8
Issue number2
Publication statusPublished - Aug 1 2003

All Science Journal Classification (ASJC) codes

  • Neurology
  • Cellular and Molecular Neuroscience


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