Role of Ca2+-dependent metalloprotease-2 in stimulating Ca2+ ATPase activity under peroxynitrite treatment in bovine pulmonary artery smooth muscle membrane.
We have determined effect of the oxidant peroxynitrite (ONOO-) on Ca2+-dependent matrix metalloprotease-2 (MMP-2) activity and the role of the protease on Ca2+ ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane under ONOO- -triggered conditions. The smooth muscle plasma membrane possesses a 72-kDa protease activity in a gelatin-containing zymogram. The 72-kDa protease activity has been found to be inhibited by tissue inhibitor of metalloprotease-2 (TIMP-2), indicating that the protease is the matrix metalloprotease-2 (MMP-2). Treatment of the membrane suspension with ONOO- caused stimulation of the MMP-2 activity (as evidenced by 14C-gelatin degradation) and also increased Ca2+ ATPase activity. The ONOO- -triggered protease activity and the Ca2+ ATPase activity were found to be inhibited by the antioxidants: vitamin E, thiourea, and mannitol. Pretreatment with catalase and superoxide dismutase did not significantly alter ONOO- -stimulated MMP-2 activity and Ca2+ATPase activity, indicating that peroxide and superoxide are not present in appreciable amount in ONOO-. Under both basal and ONOO- triggered conditions, the MMP-2 activity and the Ca2+ ATPase activity were also inhibited by EGTA, 1:10-phenanthroline, and TIMP-2. However, the ONOO- -stimulated MMP-2 activity and the Ca2+ ATPase activity were found to be insensitive to phenylmethylsulfonylfluoride, Bowman-Birk inhibitor, chymostatin, leupeptin, antipain, N-ethylmaleimide, and pepstatin. These results suggest that ONOO- caused stimulation of MMP-2 activity and that the increased MMP-2 activity subsequently played a pivotal role in stimulating Ca2+ ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane.
Chakraborti
Tapati
T
Department of Biochemistry and Biophysics, University of Kalyani, West Bengal, India. s_chakraborti@hotmail.com
Das
Sudip
S
Mandal
Malay
M
Mandal
Amritlal
A
Chakraborti
Sajal
S
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002374:Catalase / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011651:Pulmonary Artery / Q000201:enzymology*; D013482:Superoxide Dismutase / Q000494:pharmacology; D013890:Thiourea / Q000494:pharmacology; D014810:Vitamin E / Q000494:pharmacology
D000975:Antioxidants; D007527:Isoenzymes; D011480:Protease Inhibitors; D014810:Vitamin E; D030421:Peroxynitrous Acid; D002374:Catalase; D013482:Superoxide Dismutase; D020778:Matrix Metalloproteinase 2; D000252:Calcium-Transporting ATPases; D013890:Thiourea; D002118:Calcium