Role of MMP-2 in oxidant-mediated regulation of Ca2+ uptake in microsomes of bovine pulmonary artery smooth muscle.

Treatment of bovine pulmonary artery smooth muscle microsomes with tert-butylhydroperoxide (t-buOOH) (300 microM) markedly stimulated matrix metalloproteinase-2 (MMP-2) activity and enhanced Ca(2+)-ATPase activity and ATP-dependent Ca2+ uptake. Pre-treatment with vit. E (1 mM) and tissue inhibitor of metalloproteinase-2 (TIMP-2) (50 microg/ml) prevented t-buOOH-induced stimulation of MMP-2 activity, Ca(2+)-ATPase activity and ATP-dependent Ca2+ uptake. In contrast, Na(+)-dependent Ca2+ uptake was inhibited by t-buOOH and the inhibition was reversed by vit. E (1 mM) and TIMP-2 (50 microg/ml). However, t-buOOH-triggered changes in MMP-2 activity, and ATP- and Na(+)-dependent Ca2+ uptake were not reversed upon pre-treatment of the microsomes with a low concentration of 5 microg/ml of TIMP-2, which on the contrary reversed MMP-2 (1 microg/ml)-mediated alteration on these parameters. The inhibition of Na(+)-dependent Ca2+ uptake by MMP-2 under t-buOOH treatment overpowered the stimulation of ATP-dependent Ca2+ uptake in the microsomes. Combined treatment of the microsomes with low doses of MMP-2 (0.5 microg/ml) and t-buOOH (100 microM) augmented Ca(2+)-ATPase activity and ATP-dependent Ca2+ uptake, but inhibited Na(+)-dependent Ca2+ uptake, compared to that elicited by either MMP-2 (0.5 microg/ml) or t-buOOH (100 microM). Pre-treatment with TIMP-2 (50 microg/ml) reversed the effects of MMP-2 (0.5 microg/ml) and/or t-buOOH (100 microM). Although pre-treatment with 5 microg/ml of TIMP-2 reversed the effects produced by MMP-2 (0.5 microg/ml), but it did not inhibit the responses elicited by t-buOOH (300 microM) or t-buOOH (100 microM) plus MMP-2 (0.5 microg/ml) in the microsomes. Treatment with TIMP-2 (5 microg/ml) inhibited MMP-2 (1 microg/ml) activity (assessed by [14C]-gelatin degradation), whereas treatment of t-buOOH (300 microM) with TIMP-2 (5 microg/ml) abolished the inhibitory effect of TIMP-2 (5 microg/ml) on MMP-2 (1 microg/ml) activity (assessed by [14C]-gelatin degradation). Overall, these results suggested that t-buOOH inactivated TIMP-2, the ambient inhibitor of MMP-2, leading to activation of the ambient proteinase, MMP-2 which subsequently stimulated Ca(2+)-ATPase activity and ATP-dependent Ca2+ uptake, but inhibited Na(+)-dependent Ca2+ uptake, resulting in a marked decrease in Ca2+ uptake in the microsomes.