title: Pepstatin A induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines.
abstract: 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,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. Zn(2+), 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.
authors.affiliation: Laboratory of Molecular and Cellular Neurogenetics, LTA Medical Corporation, Fukuoka, Japan. mokada@med.osaka-cu.ac.jp
authors.forename: Mitsuko
authors.initials: M
authors.lastname: Okada
authors.forename: Shin
authors.initials: S
authors.lastname: Irie
authors.forename: Makoto
authors.initials: M
authors.lastname: Sawada
authors.forename: Ryuji
authors.initials: R
authors.lastname: Urae
authors.forename: Akinori
authors.initials: A
authors.lastname: Urae
authors.forename: Nakao
authors.initials: N
authors.lastname: Iwata
authors.forename: Norio
authors.initials: N
authors.lastname: Ozaki
authors.forename: Kohei
authors.initials: K
authors.lastname: Akazawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakanishi
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors*; D002478:Cells, Cultured; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005110:Extracellular Space / Q000187:drug effects* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration / Q000187:drug effects*; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D010436:Pepstatins / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats
chemical_list: D010436:Pepstatins; D011480:Protease Inhibitors; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin