Mannaioni Guido, Orr Anna G, Hamill Cecily E, Yuan Hongjie, Pedone Katherine H, McCoy Kelly L, Berlinguer Palmini Rolando, Junge Candice E, Lee C Justin, Yepes Manuel, Hepler John R, Traynelis Stephen F
Dipartimento di Farmacologia, Università degli Studi di Firenze, Firenze, Italy.
J Biol Chem. 2008 Jul 18;283(29):20600-11. doi: 10.1074/jbc.M803015200. Epub 2008 May 12.
Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration (Ca(2+)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or Ca(2+) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase Ca(2+) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.
蛋白酶激活受体-1（PAR1）可被多种丝氨酸蛋白酶激活，包括纤溶酶。PAR1和纤溶酶原（纤溶酶的前体）均在中枢神经系统中表达。在本研究中，我们检测了纤溶酶对星形胶质细胞培养物、神经元培养物以及海马脑片的影响。我们发现，纤溶酶可引起皮质培养的小鼠星形胶质细胞中磷酸肌醇水解增加（半数有效浓度[EC(50)]为64纳米）以及Fura-2/AM荧光增强（比基线高195±6.7%，EC(50)为65纳米）。纤溶酶还可激活培养的星形胶质细胞内的细胞外信号调节激酶（ERK1/2）。在PAR1基因敲除（PAR1(-/-)）的星形胶质细胞中，纤溶酶诱导的细胞内钙离子浓度（[Ca(2+)]i）升高以及磷酸化ERK1/2水平增加均减弱，并被选择性PAR1拮抗剂1微摩尔BMS-200261阻断。然而，纤溶酶对原代培养的海马神经元或海马脑片中的CA1锥体细胞的ERK1/2或[Ca(2+)]i信号传导没有可检测到的影响。施加纤溶酶（100 - 200纳米）可增强从CA1锥体细胞记录到的微小兴奋性突触后电流中N-甲基-D-天冬氨酸（NMDA）受体依赖性成分，但对α-氨基-3-羟基-5-甲基-4-异恶唑丙酸或γ-氨基丁酸受体介导的突触电流没有影响。纤溶酶还增加了从CA1锥体细胞记录到的NMDA诱导的全细胞受体电流（比对照增强2.5±0.3倍）。这种效应被BMS-200261（1微摩尔；比对照增强1.02±0.09倍）阻断。这些数据表明，纤溶酶可能作为一种内源性PAR1激活剂，可增加星形胶质细胞中的[Ca(2+)]i，并增强CA1锥体细胞中的NMDA受体突触电流。
