Hypotonic activation of insulin-sensitive phosphodiesterase in rat fat cells.
The effect of hypotonic treatment on the low Km membrane-bound cyclic AMP phosphodiesterase was investigated. Isolated fat cells obtained from Sprague-Dawley rats were incubated at 37 degrees C with an without 2 nM insulin. A crude microsomal fraction prepared by differential centrifugation was suspended in a hypotonic buffer at 4 degrees C, with and without protease inhibitors. Following solubilization from the particulate fraction, hypotonic treatment stimulated the phosphodiesterase in a time-dependent manner. Among the protease inhibitors, E-64, leupeptin and antipain were effective in preventing hypotonic activation of the enzyme. The release of the enzyme from the particulate fraction was partially inhibited by antipain. Kinetic analysis of the enzyme from hypotonic activation was much the same as that of the enzyme from the isotonic buffer. These results suggest that hypotonic activation of the phosphodiesterase may be the result of stimulation of an endogenous thiol protease of lysosomal origin.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000273:Adipose Tissue / Q000201:enzymology*; D000818:Animals; D000976:Antipain / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007038:Hypotonic Solutions / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D008861:Microsomes / Q000201:enzymology; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D007038:Hypotonic Solutions; D007328:Insulin; D007976:Leupeptins; D011480:Protease Inhibitors; D000976:Antipain; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D007930:Leucine; C024974:E 64