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

研究成果: Article

6 引用 (Scopus)

抄録

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.

元の言語English
ページ(範囲)167-174
ページ数8
ジャーナルGLIA
43
発行部数2
DOI
出版物ステータスPublished - 01-08-2003

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Peptide Hydrolases
Cell Line
Dicyclohexylcarbodiimide
Proton-Translocating ATPases
Protein Kinase C
Protons
Vacuolar Proton-Translocating ATPases
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
pepstatin
Sodium Azide
Intracellular Membranes
Sodium-Hydrogen Antiporter
Vanadates
Staurosporine
Amiloride
Microglia
Ion Channels
Glioma
Adenosine Triphosphatases
Protein Isoforms

All Science Journal Classification (ASJC) codes

  • Neurology
  • Cellular and Molecular Neuroscience

これを引用

Okada, Mitsuko ; Irie, Shin ; Sawada, Makoto ; Urae, Ryuji ; Urae, Akinori ; Iwata, Nakao ; Ozaki, Norio ; Akazawa, Kohei ; Nakanishi, Hiroshi. / Pepstatin a induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines. :: GLIA. 2003 ; 巻 43, 番号 2. pp. 167-174.
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Okada, M, Irie, S, Sawada, M, Urae, R, Urae, A, Iwata, N, Ozaki, N, Akazawa, K & Nakanishi, H 2003, 'Pepstatin a induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines', GLIA, 巻. 43, 番号 2, pp. 167-174. https://doi.org/10.1002/glia.10237

Pepstatin a induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines. / Okada, Mitsuko; Irie, Shin; Sawada, Makoto; Urae, Ryuji; Urae, Akinori; Iwata, Nakao; Ozaki, Norio; Akazawa, Kohei; Nakanishi, Hiroshi.

:: GLIA, 巻 43, 番号 2, 01.08.2003, p. 167-174.

研究成果: Article

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T1 - Pepstatin a induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines

AU - Okada, Mitsuko

AU - Irie, Shin

AU - Sawada, Makoto

AU - Urae, Ryuji

AU - Urae, Akinori

AU - Iwata, Nakao

AU - Ozaki, Norio

AU - Akazawa, Kohei

AU - Nakanishi, Hiroshi

PY - 2003/8/1

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N2 - 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.

AB - 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.

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