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. 2017 Jan 3:7:39610.
doi: 10.1038/srep39610.

Mechanical stress activates NMDA receptors in the absence of agonists

Mohammad Mehdi Maneshi  1   2 , Bruce Maki  3 , Radhakrishnan Gnanasambandam  1 , Sophie Belin  3 , Gabriela K Popescu  3 , Frederick Sachs  1 , Susan Z Hua  1   2
Affiliations

Mechanical stress activates NMDA receptors in the absence of agonists

Mohammad Mehdi Maneshi et al. Sci Rep. .

Abstract

While studying the physiological response of primary rat astrocytes to fluid shear stress in a model of traumatic brain injury (TBI), we found that shear stress induced Ca2+ entry. The influx was inhibited by MK-801, a specific pore blocker of N-Methyl-D-aspartic acid receptor (NMDAR) channels, and this occurred in the absence of agonists. Other NMDA open channel blockers ketamine and memantine showed a similar effect. The competitive glutamate antagonists AP5 and GluN2B-selective inhibitor ifenprodil reduced NMDA-activated currents, but had no effect on the mechanically induced Ca2+ influx. Extracellular Mg2+ at 2 mM did not significantly affect the shear induced Ca2+ influx, but at 10 mM it produced significant inhibition. Patch clamp experiments showed mechanical activation of NMDAR and inhibition by MK-801. The mechanical sensitivity of NMDARs may play a role in the normal physiology of fluid flow in the glymphatic system and it has obvious relevance to TBI.

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Figures

Figure 1
Figure 1. Sources of shear-activated Ca2+ increase in astrocytes using pharmacology.
(a) Fluorescence images of Fluo-4 loaded astrocytes before the stimulus pulse (23 dyn/cm2, 10 ms) and 6 s later at the peak of the response under control conditions and with treatments of various inhibitors. Yellow arrows indicate the responding cells. (b) Time dependent changes in Ca2+ in controls and with a mixture of MK-801 (10 μM) and Ruthenium red (30 μM). Each trace was measured from selected single cell. The shear stimulus was applied at the time indicated by the arrow. The data show that the mixture completely blocked the Ca2+ response. (c) The Ca2+ response in cells treated with MK-801 (dark blue traces, from cells in the image of panel a, section MK-801), memantine (light blue traces), and ketamine (green traces), compared with control cells (red traces). (d) The Ca2+ response in cells treated with 0, 2 and 10 mM Mg2+, showing NMDAR sensitivity to low dose Mg2+ (2 mM) was reduced by shear stress. (e) Ca2+ response to agonists, glutamate (1 mM) and glycine (1 mM) (green curves). MK-801 (50 μM) blocked the agonist activated Ca2+ response (blue curves). (f,g) Summary of peak Ca2+ responses (f) and the number of responding cells (g) in cultures treated with different drugs. The means are from N = 200 cells from >4 experiments under each condition. Cells treated with various drugs were compared with control cells with the same stimuli (*p < 0.05). Error bars indicate s.e.m.
Figure 2
Figure 2. Pharmacological inhibition of NMDA-evoked currents in cultured astrocytes.
(a) Representative whole-cell currents evoked by co-application of NMDA (100 μM) and glycine (100 μM); drugs were applied for 5 s to reach steady-state (Iss). Vertical scale bars represent 100 pA. (b) Summary of current blockage by IFN (10 μm, N = 4), AP5 (100 μM, N = 5), MK-801 (50 μM, N = 6), and NMB-1 (10 μM, N = 4); *Indicates significant differences relative to currents in the absence of drugs (p < 0.05).
Figure 3
Figure 3. Role of free glutamate and glutamate binding sites on shear induced Ca2+ response in astrocytes.
(a) Ca2+ response to shear pulse stimuli (23 dyn/cm2, 10 ms) in control (red), AP5 (50 μM, Gray), IFN (10 μm, orange), and MK-801 (50 μM, Blue) showing that blocking glutamate binding sites did not affect the Ca2+ response to shear. (b) Ca2+ response to agonists (1 mM glutamate and 1 mM glycine) in control (green), AP5 (200 μM), and MK-801 (50 μM). Each curve was an average of 200 cells from 4 experiments (*p < 0.05). (c) Peak Ca2+ intensity with and without glutamate inhibitors. Error bars indicate s.e.m.
Figure 4
Figure 4. Ca2+ response to fluid shear stimuli in GluN1,2A and 2B expressing CHO cells.
(a) The Ca2+ response to a shear pulse (23 dyn/cm2, 10 ms) in control cells (wild type, gray), with MSC inhibitor GsMTx4 (blue), and GluN1,2A (red) and GluN1,2B (orange) expressing cells in the present of GxMTx4, showing recombinant NMDA channels can be activated by shear stress without agonists. (b) Ca2+ response to shear pulse and glutamate-glycine (green) in GluN1,2A expressing cells, showing that glutamate antagonists do not inhibit shear responses. Results for the GluN1,2A expressing cells (red) are replotted for comparison. (c) Ca2+ response to shear pulse and glutamate-glycine (green) in GluN1,2B expressing cells, showing glutamate biding sites play a role on N2B subunit. Each curve is an average over 200 cells from >4 experiments under each condition. Error bars indicate s.e.m. (d–f) Peak Ca2+ response in each conditions corresponding to (a–c), respectively. Cells treated with various drugs were compared with control cells with corresponding stimuli (*p < 0.05). Error bars indicate s.e.m.
Figure 5
Figure 5. Mechanically-activated NMDAR current in outside-out patches.
Patches that showed channel activation in response to NMDA were used for further analysis. (a) Pulse train showing mechanically-activated unitary current from NMDARs and inhibited by 50 μM MK-801. (b) Block of NMDA (200 μM) activated current by MK-801 (100 μM) with patch at rest. (c) Representative traces showing MK-801 inhibition of a pressure pulse response (~60 mmHg) (n = 4, independent patches form different culture dishes).
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