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.

Myxoma and vaccinia viruses exploit different mechanisms to enter and infect human cancer cells

“…These results were somewhat unexpected,since it is well known that pepstatin A inhibits the proteolysis of aspartic acid proteases that in turn inhibit endosomal acidification (Zaidi et al, 2007), which should affect significantly VACV entry via the endocytic route. However, Okada and co-workers reported that pepstatin A also displays an alternative role inducing extracellular acidification, which is unrelated to its inhibition of aspartic proteases (Okada et al, 2003). We conlcude that the external acidification produced via pepstatin does not significantly affect MYXV and VACV entry into these cancer cells.…”

A simple and high-yield method for preparation of rat microglial cultures utilizing Aclar plastic film

“…Microglia are known to consist of various subpopulations 23–26 . It is of note that the SMK cell line was cloned from a single cell by the limiting dilution method and does not necessarily represent the subpopulation of microglia that can be isolated by the current method.…”

Basic Science of PET Imaging for Inflammatory Diseases

“…It has been reported that production of IL-12 [151] and histamine [152] are different in the subtypes of microglia. Furthermore, the subtypes of microglia show distinct acidification profiles by treatment with pepstatin A [153] and selective clearance of oligomeric beta-amyloid peptide [154]. The distinct phenotypes in activated forms of microglia might result from this heterogeneity.…”