Mandal Amritlal, Chakraborti Tapati, Choudhury Rajdeep, Ghosh Biswarup, Chakraborti Sajal
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741 235, West Bengal, India.
Indian J Biochem Biophys. 2005 Feb;42(1):19-27.
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.
用叔丁基过氧化氢（t-buOOH）（300微摩尔）处理牛肺动脉平滑肌微粒体，显著刺激基质金属蛋白酶-2（MMP-2）活性，并增强Ca(2+)-ATP酶活性和ATP依赖的Ca2+摄取。用维生素E（1毫摩尔）和金属蛋白酶组织抑制剂-2（TIMP-2）（50微克/毫升）预处理可防止t-buOOH诱导的MMP-2活性、Ca(2+)-ATP酶活性和ATP依赖的Ca2+摄取的刺激。相反，t-buOOH抑制Na(+)-依赖的Ca2+摄取，而维生素E（1毫摩尔）和TIMP-2（50微克/毫升）可逆转这种抑制作用。然而，用低浓度5微克/毫升的TIMP-2预处理微粒体后，t-buOOH触发的MMP-2活性以及ATP和Na(+)-依赖的Ca2+摄取的变化并未逆转，相反，5微克/毫升的TIMP-2可逆转MMP-2（1微克/毫升）介导的这些参数的改变。在t-buOOH处理下，MMP-2对Na(+)-依赖的Ca2+摄取的抑制作用超过了微粒体中ATP依赖的Ca2+摄取的刺激作用。与单独用MMP-2（0.5微克/毫升）或t-buOOH（100微摩尔）相比，用低剂量的MMP-2（0.5微克/毫升）和t-buOOH（100微摩尔）联合处理微粒体可增强Ca(2+)-ATP酶活性和ATP依赖的Ca2+摄取，但抑制Na(+)-依赖的Ca2+摄取。用TIMP-2（50微克/毫升）预处理可逆转MMP-2（0.5微克/毫升）和/或t-buOOH（100微摩尔）的作用。虽然用5微克/毫升的TIMP-2预处理可逆转MMP-2（0.5微克/毫升）产生的作用，但它并未抑制微粒体中t-buOOH（300微摩尔）或t-buOOH（100微摩尔）加MMP-2（0.5微克/毫升）引起的反应。用TIMP-2（5微克/毫升）处理可抑制MMP-2（1微克/毫升）的活性（通过[14C]-明胶降解评估），而用TIMP-2（5微克/毫升）处理t-buOOH（300微摩尔）可消除TIMP-2（5微克/毫升）对MMP-2（1微克/毫升）活性的抑制作用（通过[14C]-明胶降解评估）。总体而言，这些结果表明，t-buOOH使MMP-2的内源性抑制剂TIMP-2失活，导致内源性蛋白酶MMP-2活化，随后MMP-2刺激Ca(2+)-ATP酶活性和ATP依赖的Ca2+摄取，但抑制Na(+)-依赖的Ca2+摄取，导致微粒体中Ca2+摄取显著减少。
