Evidence against a role for aspartyl proteases in intracellular processing of big endothelin.
CPAE endothelial cells were cultured in the presence of pepstatin, NH4Cl, or chloroquine in order to assess their effects on the secretion of endothelin-1 (ET-1). The first of these is an inhibitor of aspartyl proteases and the last two are known to neutralize acidic intracellular compartments. The pepstatin was encapsulated into liposomes to aid in its uptake, and uptake was confirmed by measuring the residual aspartyl protease activity in washed, lysed cells. Pepstatin had no effect (less than 5%) on the secretion of ET-1, 25 mM NH4Cl decreased secretion by 30-47%, and 25 microM chloroquine increased secretion by 37-79%. In contrast, each of these reagents is known to inhibit lysosomal degradation of intracellular proteins by 75-90%. Additionally, big ET was shown to be a very poor substrate, in terms of kcat/Km values, for aspartyl proteases. The rate constants were less than 10(4) M-1 s-1, which is approximately 1% of the value for the best substrates. The data, therefore, do not support a role for aspartyl proteases in the formation of ET-1. Similar to chloroquine, 0.5 microM monensin increased the secretion of ET-1 by 40-60%. Both of these reagents have previously been shown to increase the rate of constitutive secretion of peptides by affecting their partitioning between packaging into storage granules and constitutive secretion. The results would therefore provide supportive evidence for the existence of a storage form of ET-1 in endothelial cells.
Nichols
J S
JS
Glaxo Research Laboratories, Research Triangle Park, North Carolina 27709.
Berman
J
J
Wypij
D M
DM
Wiseman
J S
JS
D000643:Ammonium Chloride; D001224:Aspartic Acid / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002478:Cells, Cultured; D002738:Chloroquine; D002851:Chromatography, High Pressure Liquid; D019332:Endothelin-1; D016232:Endothelins / Q000378:metabolism*; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D008985:Monensin; D010436:Pepstatins / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*
D019332:Endothelin-1; D016232:Endothelins; D007202:Indicators and Reagents; D010436:Pepstatins; D011498:Protein Precursors; D000643:Ammonium Chloride; C014195:Streptomyces pepsin inhibitor; D001224:Aspartic Acid; D002738:Chloroquine; D008985:Monensin; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin