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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

The proteolytic activity of tPA is required for inhibition of NMDA-induced calcium influx. (A) Hippocampal cultures were preincubated with tPA or the enzymatically inactive tPA mutant tPAS478A (40 μg/ml) 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%). RFU, Relative Fluorescent Units; n.s, not significant; ***p < 0.001. Error bar, SEM.
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Figure 4: The proteolytic activity of tPA is required for inhibition of NMDA-induced calcium influx. (A) Hippocampal cultures were preincubated with tPA or the enzymatically inactive tPA mutant tPAS478A (40 μg/ml) 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%). RFU, Relative Fluorescent Units; n.s, not significant; ***p < 0.001. Error bar, SEM.

Mentions: As proteolytic and non-proteolytic mechanisms have been reported to modulate NMDA-mediated calcium levels, we tested the effects of tPA-STOP, a reversible competitive inhibitor of trypsin-like serine proteases, as well as an enzymatically inactive tPA mutant. Unexpectedly, preincubation of cultures with 1 μM tPA-STOP alone almost completely inhibited intracellular calcium changes activated by 5 μM NMDA (Supplementary Figure 2). This result suggested an off-target effect of tPA-STOP and so it was not used in any further experiments. In a second approach we tested the effect of an enzymatically-inactive tPA mutant, with the active site Ser478 residue mutated to alanine. In contrast to tPA, the enzymatically-inactive tPAS478A had no effect on 5 μM NMDA-mediated changes in calcium levels (Figures 4A,B).


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)

The proteolytic activity of tPA is required for inhibition of NMDA-induced calcium influx. (A) Hippocampal cultures were preincubated with tPA or the enzymatically inactive tPA mutant tPAS478A (40 μg/ml) 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%). RFU, Relative Fluorescent Units; n.s, not significant; ***p < 0.001. Error bar, SEM.
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Figure 4: The proteolytic activity of tPA is required for inhibition of NMDA-induced calcium influx. (A) Hippocampal cultures were preincubated with tPA or the enzymatically inactive tPA mutant tPAS478A (40 μg/ml) 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%). RFU, Relative Fluorescent Units; n.s, not significant; ***p < 0.001. Error bar, SEM.
Mentions: As proteolytic and non-proteolytic mechanisms have been reported to modulate NMDA-mediated calcium levels, we tested the effects of tPA-STOP, a reversible competitive inhibitor of trypsin-like serine proteases, as well as an enzymatically inactive tPA mutant. Unexpectedly, preincubation of cultures with 1 μM tPA-STOP alone almost completely inhibited intracellular calcium changes activated by 5 μM NMDA (Supplementary Figure 2). This result suggested an off-target effect of tPA-STOP and so it was not used in any further experiments. In a second approach we tested the effect of an enzymatically-inactive tPA mutant, with the active site Ser478 residue mutated to alanine. In contrast to tPA, the enzymatically-inactive tPAS478A had no effect on 5 μM NMDA-mediated changes in calcium levels (Figures 4A,B).

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