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Pepstatin A induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines
Author(s) -
Okada Mitsuko,
Irie Shin,
Sawada Makoto,
Urae Ryuji,
Urae Akinori,
Iwata Nakao,
Ozaki Norio,
Akazawa Kohei,
Nakanishi Hiroshi
Publication year - 2003
Publication title -
glia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.954
H-Index - 164
eISSN - 1098-1136
pISSN - 0894-1491
DOI - 10.1002/glia.10237
Subject(s) - pepstatin , extracellular , bafilomycin , biology , biochemistry , protein kinase a , staurosporine , leupeptin , chelerythrine , microbiology and biotechnology , protein kinase c , kinase , enzyme , protease , apoptosis , autophagy
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 NaN 3 , 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. © 2003 Wiley‐Liss, Inc.