Hyperosmotic cellular volume regulation in the ribbed mussel Geukensia demissa: Inhibition by lysosomal and proteinase inhibitors

Abstract

AbstractThe microbial proteinase inhibitors bestatin, leupeptin, chymostatin, antipain, and pepstatin (all at 10−4 M) inhibit the proteolytic activity of gill homogenates of the ribbed mussel Geukensia demissa by 20–30%. In combination, these inhibitors reduced the hydrolysis of casein by 50%.Isolated gills from G. demissa acclimated to 500 mOsm seawater (SW) were incubated in 1000 mOsm SW for 8 h in the presence of the following proteinase and lysosome inhibitors: bestatin, pepstatin, leupeptin, antipain, chymostatin, NH4Cl, imidazole, and chloroquine. In control tissues the amino acid pool increased by 155 μmol/g dry wt. Imidazole (10 mM), NH4Cl (10 mM), and chloroquine (10−4 M) inhibited this increase by 70, 60, and 50%, respectively. Pepstatin, bestatin, and leupeptin (all 10−4 M) also inhibited the accumulation of amino acids in gills exposed to higher ambient salinity by 30–40%. In combination, these three microbial proteinase inhibitors reduced the enlargement of the amino acid pool by 60%. Chymostatin and antipain had no effect on the increase in the amino acid content of the tissues.The effects of these inhibitors on the amino acid levels of gill tissues incubated in isomotic seawater were minor. None of the inhibitors had significant effects on the oxygen uptake of isolated gills.The results from gills exposed to the amines and chloroquine suggest that lysosome‐mediated proteolysis is involved in the accumulation of alanine and glycine during cellular adjustment to hyperosmotic stress in marine bivalves. The effectiveness of pepstatin, leupeptin, and bestatin in reducing this accumulation implicates aminopeptidase I and cathepsins B and D as the specific enzymes involved in the proteolysis associated with adjustment to hyperosmotic stress.