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Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons.

Robinson SD, Lee TW, Christie DL, Birch NP - Front Cell Neurosci (2015)

Bottom Line: NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location.Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity.Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and receptor-associated protein (RAP), a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences and Centre for Brain Research, University of Auckland Auckland, New Zealand.

ABSTRACT
NMDA receptors (NMDARs) play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM) but not high (50 μM) concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-aminopyridine (4-AP). Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and receptor-associated protein (RAP), a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs.

No MeSH data available.


Related in: MedlinePlus

tPA inhibition of NMDA-induced calcium influx is independent of plasmin and lipoprotein receptor-related protein 1 (LRP-1). (A) Hippocampal cultures were preincubated with tPA (40 μg/ml; for 5 min) or α2-antiplasmin (140 nM; for 15 min) and tPA (for 5 min). Baseline Fluo-4 fluorescence was monitored for 15 s prior to the addition of NMDA (5 μM), at time = 0. Fluo-4 fluorescence was monitored for a further 45 s. Raw fluorescence values were converted to ΔF/F0, where F0 is the average fluorescence over the first 15 s of recording prior to addition of agonist (baseline) and ΔF is Fmax−F0. (B) The responses in A were quantitated by measuring the AUC and are presented relative to the AUC for 5 μM NMDA (100%). (C) Hippocampal cultures were preincubated with tPA (40 μg/ml) for 5 min or receptor-associated protein (RAP) (500 nM; for 15 min) and tPA (for 5 min). Baseline Fluo-4 fluorescence was monitored for 15 s prior to the addition of NMDA (5 μM), at time = 0. Fluo-4 fluorescence was monitored for a further 45 s. Raw fluorescence values were converted to ΔF/F0, where F0 is the average fluorescence over the first 15 s of recording prior to addition of agonist (baseline) and ΔF is Fmax−F0. (D) The responses in C were quantitated by measuring the AUC and are presented relative to the AUC for 5 μM NMDA (100%). RFU, Relative Fluorescent Units; n.s, not significant; ***p < 0.001. Error bar, SEM.
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Figure 5: tPA inhibition of NMDA-induced calcium influx is independent of plasmin and lipoprotein receptor-related protein 1 (LRP-1). (A) Hippocampal cultures were preincubated with tPA (40 μg/ml; for 5 min) or α2-antiplasmin (140 nM; for 15 min) and tPA (for 5 min). Baseline Fluo-4 fluorescence was monitored for 15 s prior to the addition of NMDA (5 μM), at time = 0. Fluo-4 fluorescence was monitored for a further 45 s. Raw fluorescence values were converted to ΔF/F0, where F0 is the average fluorescence over the first 15 s of recording prior to addition of agonist (baseline) and ΔF is Fmax−F0. (B) The responses in A were quantitated by measuring the AUC and are presented relative to the AUC for 5 μM NMDA (100%). (C) Hippocampal cultures were preincubated with tPA (40 μg/ml) for 5 min or receptor-associated protein (RAP) (500 nM; for 15 min) and tPA (for 5 min). Baseline Fluo-4 fluorescence was monitored for 15 s prior to the addition of NMDA (5 μM), at time = 0. Fluo-4 fluorescence was monitored for a further 45 s. Raw fluorescence values were converted to ΔF/F0, where F0 is the average fluorescence over the first 15 s of recording prior to addition of agonist (baseline) and ΔF is Fmax−F0. (D) The responses in C were quantitated by measuring the AUC and are presented relative to the AUC for 5 μM NMDA (100%). RFU, Relative Fluorescent Units; n.s, not significant; ***p < 0.001. Error bar, SEM.

Mentions: To investigate if tPA’s effects on intracellular calcium levels involved tPA-mediated activation of plasmin, cultures were preincubated with α2-antiplasmin. α2-antiplasmin (140 nM) alone did not affect 5 μM NMDA-induced calcium flux and did not block the effect on tPA on 5 μM NMDA-induced calcium flux (Figures 5A,B). This suggests that plasmin was not responsible for the observed tPA response. It does not rule out the possibility that tPA may be converting plasminogen to plasmin in our cultures, only that this conversion is not necessary for the observed effect of tPA on 5 μM NMDA-induced changes in calcium levels. As tPA has also been proposed to modulate NMDA-mediated calcium flux through interaction with LRP as a complex with a specific tPA inhibitor (Martin et al., 2008; Samson et al., 2008a) experiments were undertaken with the competitive LRP-1 receptor antagonist RAP. RAP alone had no effect on NMDA-mediated calcium flux (Figures 5C,D). RAP also failed to block the inhibitory effect of tPA on NMDA-mediated calcium flux (Figures 5C,D) suggesting that the tPA-mediated inhibition of 5 μM NMDA-induced calcium flux does not involve an interaction with LRP-1 or a similar RAP-sensitive receptor.


Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons.

Robinson SD, Lee TW, Christie DL, Birch NP - Front Cell Neurosci (2015)

tPA inhibition of NMDA-induced calcium influx is independent of plasmin and lipoprotein receptor-related protein 1 (LRP-1). (A) Hippocampal cultures were preincubated with tPA (40 μg/ml; for 5 min) or α2-antiplasmin (140 nM; for 15 min) and tPA (for 5 min). Baseline Fluo-4 fluorescence was monitored for 15 s prior to the addition of NMDA (5 μM), at time = 0. Fluo-4 fluorescence was monitored for a further 45 s. Raw fluorescence values were converted to ΔF/F0, where F0 is the average fluorescence over the first 15 s of recording prior to addition of agonist (baseline) and ΔF is Fmax−F0. (B) The responses in A were quantitated by measuring the AUC and are presented relative to the AUC for 5 μM NMDA (100%). (C) Hippocampal cultures were preincubated with tPA (40 μg/ml) for 5 min or receptor-associated protein (RAP) (500 nM; for 15 min) and tPA (for 5 min). Baseline Fluo-4 fluorescence was monitored for 15 s prior to the addition of NMDA (5 μM), at time = 0. Fluo-4 fluorescence was monitored for a further 45 s. Raw fluorescence values were converted to ΔF/F0, where F0 is the average fluorescence over the first 15 s of recording prior to addition of agonist (baseline) and ΔF is Fmax−F0. (D) The responses in C were quantitated by measuring the AUC and are presented relative to the AUC for 5 μM NMDA (100%). RFU, Relative Fluorescent Units; n.s, not significant; ***p < 0.001. Error bar, SEM.
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Figure 5: tPA inhibition of NMDA-induced calcium influx is independent of plasmin and lipoprotein receptor-related protein 1 (LRP-1). (A) Hippocampal cultures were preincubated with tPA (40 μg/ml; for 5 min) or α2-antiplasmin (140 nM; for 15 min) and tPA (for 5 min). Baseline Fluo-4 fluorescence was monitored for 15 s prior to the addition of NMDA (5 μM), at time = 0. Fluo-4 fluorescence was monitored for a further 45 s. Raw fluorescence values were converted to ΔF/F0, where F0 is the average fluorescence over the first 15 s of recording prior to addition of agonist (baseline) and ΔF is Fmax−F0. (B) The responses in A were quantitated by measuring the AUC and are presented relative to the AUC for 5 μM NMDA (100%). (C) Hippocampal cultures were preincubated with tPA (40 μg/ml) for 5 min or receptor-associated protein (RAP) (500 nM; for 15 min) and tPA (for 5 min). Baseline Fluo-4 fluorescence was monitored for 15 s prior to the addition of NMDA (5 μM), at time = 0. Fluo-4 fluorescence was monitored for a further 45 s. Raw fluorescence values were converted to ΔF/F0, where F0 is the average fluorescence over the first 15 s of recording prior to addition of agonist (baseline) and ΔF is Fmax−F0. (D) The responses in C were quantitated by measuring the AUC and are presented relative to the AUC for 5 μM NMDA (100%). RFU, Relative Fluorescent Units; n.s, not significant; ***p < 0.001. Error bar, SEM.
Mentions: To investigate if tPA’s effects on intracellular calcium levels involved tPA-mediated activation of plasmin, cultures were preincubated with α2-antiplasmin. α2-antiplasmin (140 nM) alone did not affect 5 μM NMDA-induced calcium flux and did not block the effect on tPA on 5 μM NMDA-induced calcium flux (Figures 5A,B). This suggests that plasmin was not responsible for the observed tPA response. It does not rule out the possibility that tPA may be converting plasminogen to plasmin in our cultures, only that this conversion is not necessary for the observed effect of tPA on 5 μM NMDA-induced changes in calcium levels. As tPA has also been proposed to modulate NMDA-mediated calcium flux through interaction with LRP as a complex with a specific tPA inhibitor (Martin et al., 2008; Samson et al., 2008a) experiments were undertaken with the competitive LRP-1 receptor antagonist RAP. RAP alone had no effect on NMDA-mediated calcium flux (Figures 5C,D). RAP also failed to block the inhibitory effect of tPA on NMDA-mediated calcium flux (Figures 5C,D) suggesting that the tPA-mediated inhibition of 5 μM NMDA-induced calcium flux does not involve an interaction with LRP-1 or a similar RAP-sensitive receptor.

Bottom Line: NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location.Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity.Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and receptor-associated protein (RAP), a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences and Centre for Brain Research, University of Auckland Auckland, New Zealand.

ABSTRACT
NMDA receptors (NMDARs) play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM) but not high (50 μM) concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-aminopyridine (4-AP). Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and receptor-associated protein (RAP), a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs.

No MeSH data available.


Related in: MedlinePlus