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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

Tissue-type plasminogen activator (tPA) inhibits increases in intracellular calcium in cultured rat hippocampal neurons stimulated with low (5 μM) but not high (50 μM) concentrations of NMDA. (A) Baseline Fluo-4 fluorescence was monitored in hippocampal cultures for 15 s prior to the addition of NMDA, at time = 0, to final concentrations ranging between 5 and 100 μM. 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 area under the curve (AUC) and are presented relative to the AUC for 5 μM NMDA (100%). Results are from three independent experiments. Error bar, SEM. (C,D) Hippocampal cultures were preincubated with tPA (40 μg/ml) for 5 min and Fluo-4 fluorescence monitored before and after addition of NMDA to a final concentration of 50 μM. Data was collected as in A and quantitated as in B. n.s, not significant. (E) Hippocampal cultures were preincubated with tPA (40 μg/ml) for 5 min and Fluo-4 fluorescence monitored before and after addition of NMDA to a final concentration of 5 μM. (F) Quantitation of responses to tPA (Actilyse, sourced from Boehringer Ingelheim), rtPA (rtPA sourced from Biopur) and dtPA (dialysed Actilyse). RFU, Relative Fluorescent Units, ***p < 0.001. Error bar, SEM.
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Figure 1: Tissue-type plasminogen activator (tPA) inhibits increases in intracellular calcium in cultured rat hippocampal neurons stimulated with low (5 μM) but not high (50 μM) concentrations of NMDA. (A) Baseline Fluo-4 fluorescence was monitored in hippocampal cultures for 15 s prior to the addition of NMDA, at time = 0, to final concentrations ranging between 5 and 100 μM. 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 area under the curve (AUC) and are presented relative to the AUC for 5 μM NMDA (100%). Results are from three independent experiments. Error bar, SEM. (C,D) Hippocampal cultures were preincubated with tPA (40 μg/ml) for 5 min and Fluo-4 fluorescence monitored before and after addition of NMDA to a final concentration of 50 μM. Data was collected as in A and quantitated as in B. n.s, not significant. (E) Hippocampal cultures were preincubated with tPA (40 μg/ml) for 5 min and Fluo-4 fluorescence monitored before and after addition of NMDA to a final concentration of 5 μM. (F) Quantitation of responses to tPA (Actilyse, sourced from Boehringer Ingelheim), rtPA (rtPA sourced from Biopur) and dtPA (dialysed Actilyse). RFU, Relative Fluorescent Units, ***p < 0.001. Error bar, SEM.

Mentions: To study the effect of recombinant tPA on NMDA-mediated calcium flux, intracellular calcium levels were monitored in embryonic hippocampal neurons cultured between 14 and 17 DIV (days in vitro), using a Fluo-4-based calcium assay and a high speed fluorometric plate reader. Treatment with the NMDAR agonist NMDA alone (5–100 μM) resulted in a rapid and concentration-dependent increase in intracellular calcium levels (Figures 1A,B). The fluorescent profiles indicated differences in handling between lower (<10 μM) and higher (>10 μM) concentrations of NMDA. Both responses were characterized by a rapid influx of calcium (the amplitude of which was concentration-dependent) which in the former gradually returned towards baseline, but in the latter plateaued, resulting in sustained calcium levels. In some instances small oscillations were observed at the lower NMDA concentrations. To investigate the effect of tPA on the calcium response, Fluo-4-loaded neurons were treated with tPA. Addition of tPA alone (40 μg/ml) did not produce any detectable change in Fluo-4 fluorescence (Supplementary Figure 1) and the calcium response to 50 μM NMDA was unaffected by pre-incubation with tPA (Figures 1C,D). However, the calcium response to 5 μM NMDA was significantly reduced to 38 ± 8% of control (Figures 1E,F). The tPA used in these experiments (Actilyse®) contains a number of excipients, including L-arginine, which has been suggested to be toxic to neurons (Oh et al., 2005; Samson et al., 2008b). To examine for any effects of the excipients in the Actilyse® tPA on the calcium response we repeated the experiments with a dialysed preparation of Actilyse® tPA (dtPA) and a second commercial recombinant tPA, Biopur tPA, which does not contain excipients. Both produced similar inhibition of the 5 μM NMDA-mediated calcium response (Figure 1F), supporting the designation of tPA as the active agent. For all subsequent experiments Actilyse® tPA, which will be referred to from now simply as tPA, was used.


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)

Tissue-type plasminogen activator (tPA) inhibits increases in intracellular calcium in cultured rat hippocampal neurons stimulated with low (5 μM) but not high (50 μM) concentrations of NMDA. (A) Baseline Fluo-4 fluorescence was monitored in hippocampal cultures for 15 s prior to the addition of NMDA, at time = 0, to final concentrations ranging between 5 and 100 μM. 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 area under the curve (AUC) and are presented relative to the AUC for 5 μM NMDA (100%). Results are from three independent experiments. Error bar, SEM. (C,D) Hippocampal cultures were preincubated with tPA (40 μg/ml) for 5 min and Fluo-4 fluorescence monitored before and after addition of NMDA to a final concentration of 50 μM. Data was collected as in A and quantitated as in B. n.s, not significant. (E) Hippocampal cultures were preincubated with tPA (40 μg/ml) for 5 min and Fluo-4 fluorescence monitored before and after addition of NMDA to a final concentration of 5 μM. (F) Quantitation of responses to tPA (Actilyse, sourced from Boehringer Ingelheim), rtPA (rtPA sourced from Biopur) and dtPA (dialysed Actilyse). RFU, Relative Fluorescent Units, ***p < 0.001. Error bar, SEM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4598481&req=5

Figure 1: Tissue-type plasminogen activator (tPA) inhibits increases in intracellular calcium in cultured rat hippocampal neurons stimulated with low (5 μM) but not high (50 μM) concentrations of NMDA. (A) Baseline Fluo-4 fluorescence was monitored in hippocampal cultures for 15 s prior to the addition of NMDA, at time = 0, to final concentrations ranging between 5 and 100 μM. 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 area under the curve (AUC) and are presented relative to the AUC for 5 μM NMDA (100%). Results are from three independent experiments. Error bar, SEM. (C,D) Hippocampal cultures were preincubated with tPA (40 μg/ml) for 5 min and Fluo-4 fluorescence monitored before and after addition of NMDA to a final concentration of 50 μM. Data was collected as in A and quantitated as in B. n.s, not significant. (E) Hippocampal cultures were preincubated with tPA (40 μg/ml) for 5 min and Fluo-4 fluorescence monitored before and after addition of NMDA to a final concentration of 5 μM. (F) Quantitation of responses to tPA (Actilyse, sourced from Boehringer Ingelheim), rtPA (rtPA sourced from Biopur) and dtPA (dialysed Actilyse). RFU, Relative Fluorescent Units, ***p < 0.001. Error bar, SEM.
Mentions: To study the effect of recombinant tPA on NMDA-mediated calcium flux, intracellular calcium levels were monitored in embryonic hippocampal neurons cultured between 14 and 17 DIV (days in vitro), using a Fluo-4-based calcium assay and a high speed fluorometric plate reader. Treatment with the NMDAR agonist NMDA alone (5–100 μM) resulted in a rapid and concentration-dependent increase in intracellular calcium levels (Figures 1A,B). The fluorescent profiles indicated differences in handling between lower (<10 μM) and higher (>10 μM) concentrations of NMDA. Both responses were characterized by a rapid influx of calcium (the amplitude of which was concentration-dependent) which in the former gradually returned towards baseline, but in the latter plateaued, resulting in sustained calcium levels. In some instances small oscillations were observed at the lower NMDA concentrations. To investigate the effect of tPA on the calcium response, Fluo-4-loaded neurons were treated with tPA. Addition of tPA alone (40 μg/ml) did not produce any detectable change in Fluo-4 fluorescence (Supplementary Figure 1) and the calcium response to 50 μM NMDA was unaffected by pre-incubation with tPA (Figures 1C,D). However, the calcium response to 5 μM NMDA was significantly reduced to 38 ± 8% of control (Figures 1E,F). The tPA used in these experiments (Actilyse®) contains a number of excipients, including L-arginine, which has been suggested to be toxic to neurons (Oh et al., 2005; Samson et al., 2008b). To examine for any effects of the excipients in the Actilyse® tPA on the calcium response we repeated the experiments with a dialysed preparation of Actilyse® tPA (dtPA) and a second commercial recombinant tPA, Biopur tPA, which does not contain excipients. Both produced similar inhibition of the 5 μM NMDA-mediated calcium response (Figure 1F), supporting the designation of tPA as the active agent. For all subsequent experiments Actilyse® tPA, which will be referred to from now simply as tPA, was used.

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