A new method for simultaneous measurements of mast cell proteases in human vascular tissue.
1. Human mast cells contain carboxypeptidase A, chymase and tryptase. In the present study, in order to analyse the mast cell proteases simultaneously, we investigated a method for the measurement of carboxypeptidase A, chymase and tryptase in human vascular tissues. 2. Human vascular tissues were homogenized in 10 mmol/L phosphate buffer containing 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 or 1.8 mol/L KCl and 0.1% non-idet P-40 and samples were then extracted. Because carboxypeptidase A and chymase convert angiotensin (Ang)I to Ang-(1-9) and AngII, respectively, the extracts were incubated with AngI in the presence of an angiotensin-converting enzyme (ACE) inhibitor. The extract prepared in buffer with over 0.8 mol/L KCl converted AngI to Ang-(1-9) and AngII. Formation of Ang-(1-9) and AngII plateaued in extracts with 1.0 and 1.2 mol/L KCl, respectively. 3. The formation of Ang-(1-9) and AngII in the extract with 1.2 mol/L KCl was inhibited by inhibitors of carboxypeptidase A and chymase, respectively, suggesting that Ang-(1-9) and AngII were generated from AngI by carboxypeptidase A and chymase, respectively. 4. Using a specific tryptase substrate, tryptase activity was detected in extract in buffer with over 0.8 mol/L KCl and reached a plateau at concentrations of KCl over 1.0 mol/L. 5. These findings show that the maximum activity of carboxypeptidase A, chymase and tryptase was detected in extracts of human homogenized vascular tissues in buffer at 1.2 mol/L KCl. The present study demonstrates a new method for the simultaneous measurement of proteases derived from mast cells in humans.
Takai
S
S
Department of Pharmacology, Osaka Medical College, Takatsuki, Japan. pha010@art.osaka-med.ac.jp
Sakaguchi
M
M
Jin
D
D
Miyazaki
M
M
D000803:Angiotensin I / Q000378:metabolism; D000804:Angiotensin II / Q000096:biosynthesis; D001158:Arteries / Q000201:enzymology*; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D002851:Chromatography, High Pressure Liquid; D053818:Chymases; D004730:Endothelium, Vascular / Q000201:enzymology; D005260:Female; D006801:Humans; D008297:Male; D008407:Mast Cells / Q000201:enzymology*; D008875:Middle Aged; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D014020:Tissue Extracts / Q000032:analysis; D053802:Tryptases
D010446:Peptide Fragments; D014020:Tissue Extracts; D000804:Angiotensin II; D000803:Angiotensin I; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases