Limits...
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

Calcium responses of cultured hippocampal neurons to high (50 μM) and low (5 μM) concentrations of NMDA are pharmacologically distinct. Hippocampal cultures were preincubated with antagonists for 15 min before recording calcium responses. Baseline Fluo-4 fluorescence was monitored for 15 s prior to the addition of NMDA (A–C, 50 μM NMDA; E–G, 5 μM NMDA) at time = 0, then 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. Antagonists tested were amino-5-phosphonovalerate (APV) (A,E; 50 μM), MK-801 (B,F; 10 μM); nimodipine (C,G; 10 μM). (D,H) Responses were quantitated by measuring the AUC and are presented relative to the AUC for 5 μM NMDA (100%). RFU, Relative Fluorescent Units; **p < 0.005, ***p < 0.001. Error bar, SEM.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4598481&req=5

Figure 2: Calcium responses of cultured hippocampal neurons to high (50 μM) and low (5 μM) concentrations of NMDA are pharmacologically distinct. Hippocampal cultures were preincubated with antagonists for 15 min before recording calcium responses. Baseline Fluo-4 fluorescence was monitored for 15 s prior to the addition of NMDA (A–C, 50 μM NMDA; E–G, 5 μM NMDA) at time = 0, then 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. Antagonists tested were amino-5-phosphonovalerate (APV) (A,E; 50 μM), MK-801 (B,F; 10 μM); nimodipine (C,G; 10 μM). (D,H) Responses were quantitated by measuring the AUC and are presented relative to the AUC for 5 μM NMDA (100%). RFU, Relative Fluorescent Units; **p < 0.005, ***p < 0.001. Error bar, SEM.

Mentions: To investigate the NMDAR responses in more detail we assessed the effects of glutamate and calcium channel antagonists on tPA-sensitive calcium flux. The selective competitive NMDAR antagonist APV (50 μM) and the use-dependent NMDAR open channel blocker MK-801 (10 μM) both significantly inhibited the calcium response of neurons treated with 50 μM NMDA while the L-type voltage-gated calcium channel blocker nimodipine (10 μM) had no significant effect on calcium levels (Figures 2A–D). The calcium response of hippocampal neurons to 5 μM NMDA was also blocked by APV and MK-801. However, in contrast to stimulation with 50 μM NMDA, nimodipine also inhibited the response (Figures 2E–H). These results are consistent with previously published data (Jensen and Wang, 1996; Soriano et al., 2006) and support trans-synaptic activation of the postsynaptic cell by glutamate at 5 μM NMDA, and direct stimulation of postsynaptic NMDARs at 50 μM NMDA. We further investigated an effect of tPA on the calcium response stimulated by presynaptic release of glutamate using the GABAA receptor antagonist bicuculline (50 μM) and the potassium channel blocker 4-AP (250 μM; Hardingham et al., 2001b). Treatment resulted in synchronous spontaneous calcium oscillations suggestive of synaptic coupling between neurons (Figures 3A–C). These oscillations were markedly reduced by MK-801 and nimodipine (Figures 3A–C,D). Importantly, the calcium oscillations were also markedly inhibited by tPA (Figure 3D).


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)

Calcium responses of cultured hippocampal neurons to high (50 μM) and low (5 μM) concentrations of NMDA are pharmacologically distinct. Hippocampal cultures were preincubated with antagonists for 15 min before recording calcium responses. Baseline Fluo-4 fluorescence was monitored for 15 s prior to the addition of NMDA (A–C, 50 μM NMDA; E–G, 5 μM NMDA) at time = 0, then 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. Antagonists tested were amino-5-phosphonovalerate (APV) (A,E; 50 μM), MK-801 (B,F; 10 μM); nimodipine (C,G; 10 μM). (D,H) Responses were quantitated by measuring the AUC and are presented relative to the AUC for 5 μM NMDA (100%). RFU, Relative Fluorescent Units; **p < 0.005, ***p < 0.001. Error bar, SEM.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Calcium responses of cultured hippocampal neurons to high (50 μM) and low (5 μM) concentrations of NMDA are pharmacologically distinct. Hippocampal cultures were preincubated with antagonists for 15 min before recording calcium responses. Baseline Fluo-4 fluorescence was monitored for 15 s prior to the addition of NMDA (A–C, 50 μM NMDA; E–G, 5 μM NMDA) at time = 0, then 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. Antagonists tested were amino-5-phosphonovalerate (APV) (A,E; 50 μM), MK-801 (B,F; 10 μM); nimodipine (C,G; 10 μM). (D,H) Responses were quantitated by measuring the AUC and are presented relative to the AUC for 5 μM NMDA (100%). RFU, Relative Fluorescent Units; **p < 0.005, ***p < 0.001. Error bar, SEM.
Mentions: To investigate the NMDAR responses in more detail we assessed the effects of glutamate and calcium channel antagonists on tPA-sensitive calcium flux. The selective competitive NMDAR antagonist APV (50 μM) and the use-dependent NMDAR open channel blocker MK-801 (10 μM) both significantly inhibited the calcium response of neurons treated with 50 μM NMDA while the L-type voltage-gated calcium channel blocker nimodipine (10 μM) had no significant effect on calcium levels (Figures 2A–D). The calcium response of hippocampal neurons to 5 μM NMDA was also blocked by APV and MK-801. However, in contrast to stimulation with 50 μM NMDA, nimodipine also inhibited the response (Figures 2E–H). These results are consistent with previously published data (Jensen and Wang, 1996; Soriano et al., 2006) and support trans-synaptic activation of the postsynaptic cell by glutamate at 5 μM NMDA, and direct stimulation of postsynaptic NMDARs at 50 μM NMDA. We further investigated an effect of tPA on the calcium response stimulated by presynaptic release of glutamate using the GABAA receptor antagonist bicuculline (50 μM) and the potassium channel blocker 4-AP (250 μM; Hardingham et al., 2001b). Treatment resulted in synchronous spontaneous calcium oscillations suggestive of synaptic coupling between neurons (Figures 3A–C). These oscillations were markedly reduced by MK-801 and nimodipine (Figures 3A–C,D). Importantly, the calcium oscillations were also markedly inhibited by tPA (Figure 3D).

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