Nitric oxide-induced calcium release via ryanodine receptors regulates neuronal function

Abstract

Mobilization of intracellular Ca(2+) stores regulates a multitude of cellular functions, but the role of intracellular Ca(2+) release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca(2+) release from intracellular stores of central neurons, and thereby promote prolonged Ca(2+) signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca(2+) release. NO-induced Ca(2+) release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.

Figure 1

NO induces intracellular Ca²⁺ release via RyR1 in cerebellar PCs. (A) Confocal Ca²⁺ imaging of PC dendrites (left) and pseudocolour Ca²⁺ imaging of cells (right) 120 s following NOC7 (300 μM) treatment. (B) NOC7-induced Ca²⁺ increase with or without thapsigargin (TG) (2 μM). (C) NOC7-induced Ca²⁺ increase at 36°C with or without dantrolene (30 μM). (D) Effect of various perturbations (i.e. removal of extracellular Ca²⁺ with 0.5 mM EGTA (0Ca), 2 μM TG, 4 mg/ml heparin applied through a patch pipette (Hep), and 30 μM dantrolene (Dan)) on the peak magnitude of the NO-induced Ca²⁺ response. (E) Confocal Ca²⁺ imaging in the soma of neonatal PCs 120 s after NOC7 (300 μM) treatment in control and Ryr1^−/− mice. (F) NOC7-induced Ca²⁺ increase in PCs treated with or without TG (2 μM) and in Ryr1^−/− PCs. (G) Summary of the effect of perturbations on the magnitude of NO-induced Ca²⁺ increase. In all graphs, error bars indicate s.e.m. (n=5–11). ^*P<0.01, ^**P<0.001, t-test compared with control.

Figure 2

NICR in HEK293 cells expressing RyR1. (A) Effect of various perturbations (i.e. without tetracycline induction, control, removal of extracellular Ca²⁺ with 1 mM EGTA (0Ca), 2 μM thapsigargin (TG), 1 μM ODQ, control at 35°C and 30 μM dantrolene) on the peak magnitude of NOC7 (300 μM)-induced Ca²⁺ increase; n=50–145. (B) RyR subtype dependence of the NO-induced Ca²⁺ response. Please refer to Supplementary Figure S2G for more information. (C, D) Ca²⁺ response to NOC7 and caffeine in HEK293 cells expressing RyR1 (left) or C3635A-RyR1 (right); n=121–245. (E) Time course of NOC7 (1 mM)-induced S-nitrosylation of wild-type and C3635A-RyR1 cells, as determined by the biotin-switch method. (F) Summary of the time course of S-nitrosylation; n=3–4. In all graphs, error bars indicate s.e.m. ^*P<0.02, ^**P<0.001, t-test compared with control.

Figure 3

Nerve activity induces NICR in PCs. (A) Confocal Ca²⁺ imaging of PC dendrites (left) and pseudocolour Ca²⁺ imaging of cells (right) 60 s after BS (five pulses at 50 Hz repeated 60 times every 1 s). (B) Spatial distribution of BS-induced Ca²⁺ release; n=5. (C) BS-induced Ca²⁺ increase in wild-type and Nos1^−/− mice with or without thapsigargin (2 μM); n=5–6. (D) BS-induced Ca²⁺ increase in wild-type mice with or without dantrolene (30 μM) at 36°C; n=5–6. (E) Effect of various perturbations on the magnitude of BS-induced Ca²⁺ increase. Control, thapsigargin, heparin (4 mg/ml) applied through a patch pipette, ODQ (1 μM), cocktail of glutamate receptor antagonists (αGluR: 20 μM NBQX and 100 μM CPCCOEt), L-NAME (100 μM), Nos1^−/−, control at 36°C, and dantrolene, n=5–7. In all graphs, error bars indicate s.e.m. ^*P<0.01, ^**P<0.001, t-test compared with control.

Figure 4

Involvement of NICR in synaptic plasticity. (A) LTP of excitatory postsynaptic currents (EPSCs) at the PF-PC synapse in wild-type and Nos1^−/− mice; n=5. (B) Dantrolene (30 μM) blocked PF-LTP at 36°C; n=5–6. (C) Effect of various perturbations on the magnitude of LTP (normalized EPSC averaged between 21 and 30 min after BS): control, thapsigargin (2 μM), heparin (4 mg/ml) applied through a patch pipette, CPCCOEt (100 μM, αmGluR), BAPTA (30 mM) applied through a patch pipette, control at 36°C, dantrolene (30 μM), and Nos1^−/−; n=4–6. ^**P<0.001, t-test compared with control.

Figure 5

Possible involvement of NICR in ischaemic brain damage. (A) Representative TTC-stained brain slices 24 h after transient MCAO. (B) Infarct volume 24 h after transient MCAO determined using the Leach correction; n=6 for each condition. A two-way ANOVA revealed a significant interaction (P=0.026), and subsequent analyses with Tukey's multiple comparison test indicated that Nos1^+/+ mice treated with dantrolene had a significantly smaller infarct volume compared with Nos1^−/− mice. Data are the mean±s.e.m. Physiological data of the animals are summarized in Supplementary Table S1.

Figure 6

Involvement of NICR in NO-induced neuronal cell death. (A) NOC12 (500 μM)-induced intracellular Ca²⁺ increase in cultured cerebral neurons of Ryr1^+/+ (left, n=30 cells) or Ryr1^−/− (right, n=28 cells) mice. Ca²⁺ measurements were made in the absence of extracellular Ca²⁺. (B) NOC12-induced Ca²⁺ release is blocked by dantrolene (10 μM) at 35°C. Control, n=22 cells; dantrolene, n=21 cells. (C) Neuronal cell death assayed 16 h after treatment with 500 μM NOC12 without or with 10 μM dantrolene in cultured cerebral neurons of Ryr1^+/+ (left) or Ryr1^−/− (right) mice. (D) The extent of cell death was expressed as a ratio of the number of PI-positive cells to that of Hoechst-positive cells. Numbers in parentheses (15–22) indicate the number of determinations in each condition using different cultures. In each determination, >120 cells were analysed. Data are expressed as mean±s.e.m. Statistical significance was determined via an ANOVA followed by a Tukey post-hoc test. ^***P<0.0001.