A novel hydrogen sulfide-releasing N-methyl-D-aspartate receptor antagonist prevents ischemic neuronal death - PubMed (original) (raw)

A novel hydrogen sulfide-releasing N-methyl-D-aspartate receptor antagonist prevents ischemic neuronal death

Eizo Marutani et al. J Biol Chem. 2012.

Abstract

Physiological levels of H(2)S exert neuroprotective effects, whereas high concentrations of H(2)S may cause neurotoxicity in part via activation of NMDAR. To characterize the neuroprotective effects of combination of exogenous H(2)S and NMDAR antagonism, we synthesized a novel H(2)S-releasing NMDAR antagonist N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzamide (S-memantine) and examined its effects in vitro and in vivo. S-memantine was synthesized by chemically combining a slow releasing H(2)S donor 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid (ACS48) with a NMDAR antagonist memantine. S-memantine increased intracellular sulfide levels in human neuroblastoma cells (SH-SY5Y) 10-fold as high as that was achieved by ACS48. Incubation with S-memantine after reoxygenation following oxygen and glucose deprivation (OGD) protected SH-SY5Y cells and murine primary cortical neurons more markedly than did ACS48 or memantine. Glutamate-induced intracellular calcium accumulation in primary cortical neurons were aggravated by sodium sulfide (Na(2)S) or ACS48, but suppressed by memantine and S-memantine. S-memantine prevented glutamate-induced glutathione depletion in SH-SY5Y cells more markedly than did Na(2)S or ACS48. Administration of S-memantine after global cerebral ischemia and reperfusion more robustly decreased cerebral infarct volume and improved survival and neurological function of mice than did ACS48 or memantine. These results suggest that an H(2)S-releasing NMDAR antagonist derivative S-memantine prevents ischemic neuronal death, providing a novel therapeutic strategy for ischemic brain injury.

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Figures

FIGURE 1.

Synthesis of ACS48 (A) and S-memantine (B).

FIGURE 2.

Protocols of OGD for SH-SY5Y cells and murine primary cortical neurons.

FIGURE 3.

A, sulfide levels (μ

) released from 20 μ

Na2S (no symbol), ACS48 (▾), S-memantine (▴), and medium alone (●) in DMEM/F12 supplemented with 10% FBS. Medium was incubated at 37 °C for 0 min, 1 min (only Na2S), 30 min, 1.5 h, 4 h, 8 h, or 26 h after addition of H2S donor, n = 3 each. Values of the medium-alone group differ significantly from all other groups at all time points except for Na2S at 8 and 26 h (p < 0.05). ACS48 differs significantly from S-memantine at all time points (p < 0.01), *, p < 0.01 versus Na2S by two-way ANOVA with Bonferroni post-test. B, intracellular sulfide levels of SH-SY5Y treated with 20 μ

Na2S (no symbol), ACS48 (▾), S-memantine (▴), and medium alone (●) in DMEM/F12 with 1% DMSO. Cells were incubated at 37 °C for 0 min, 30 min, 1.5 h, 4 h, 8 h, or 26 h after addition of H2S donor. n = 3 each. *, p < 0.001 versus all other groups by two-way ANOVA with Bonferroni post-test. C, intracellular sulfide levels of SH-SY5Y treated with S-memantine (green line with no symbol, 20 μ

), ACS48 (■, 20 μ

), ACS48 + memantine (□, 20 μ

+ 20 μ

), memantine (○, 20 μ

), and medium alone (●) in DMEM/F12 with 1% DMSO. Cells were incubated at 37 °C for 0 min, 30 min, 1.5 h, 4 h, or 8 h after addition of H2S donor. n = 3 each. No significant difference was found between “with memantine” and “without memantine.”

FIGURE 4.

Effects of Na2S on LDH-release from SH-SY5Y after OGD. Na2S at 10 or 50 μ

, or vehicle was added at pre-OGD or 30 min, 2 h, 5 h, or 8 h after the end of OGD. LDH released in the culture medium was measured 24 h after the end of OGD, n = 5 - 8 each. No significant difference was found between vehicle and Na2S.

FIGURE 5.

A, time dependence of the effects of H2S donors on LDH-release from SH-SY5Y (or murine primary cortical neurons) after OGD. ACS48, memantine, S-memantine at 10 μ

, or vehicle was added at 5 min before the initiation of OGD (pre-OGD) or 30 min, 2 h, 5 h, or 8 h (or 30 min, 2 h, or 5 h for primary neurons) after the end of OGD. LDH released in the culture medium was measured 24 h (or 21 h for primary neurons) after the end of OGD, n = 5 or 6 each. *, p < 0.001 versus vehicle, #, p < 0.01 versus ACS48 added at same time point. †, p < 0.05 versus memantine added at same time point. B, dose dependence of the effects of H2S donors on LDH release from SH-SY5Y (or murine primary cortical neurons) after OGD. ACS48, memantine, S-memantine at 10 or 50 μ

or vehicle was added 8 h (or 30 min for primary neurons) after the end of OGD. LDH released in the culture medium was measured 24 h (or 21 h for primary neurons) after the end of OGD, n = 5 or 6 each. *, p < 0.01 versus vehicle; #, p < 0.05 versus ACS48 at the same dose. †, p < 0.01 versus memantine at same dose.

FIGURE 6.

Effects of H2S donors and memantine on cell viability after OGD. A–D, ACS48, memantine, S-memantine at 50 μ

or vehicle was added 8 h after the end of OGD. LDH released in the culture medium was measured 24 h after the end of OGD. A, LDH release from SH-SY5Y after OGD, n = 5 or 6 each. *, p < 0.001 versus vehicle; #, p < 0.05. B, MTT assay, n = 5 or 6 each. No-OGD control (control) differs significantly from all other groups (p < 0.001). *, p < 0.01 versus vehicle; #, p < 0.05. C, CV assay and D, photographs of wells containing SH-SY5Y stained with CV after OGD, n = 5 each No OGD control (control) differs significantly from all other groups (p < 0.001). *, p < 0.001 versus vehicle, #, p < 0.05. E, LDH released from murine primary cortical neurons measured 2.5 h after the end of OGD. ACS48, memantine, S-memantine at 50 μ

, or vehicle was added 0.5 h after the end of OGD. LDH released in the culture medium was measured 21 h after the end of OGD, n = 5 or 6 each. *, p < 0.001 versus vehicle, #, p < 0.001 versus ACS48 and memantine.

FIGURE 7.

Cytotoxicity of Na2S, ACS48, and S-memantine. LDH in the culture medium released from murine primary cortical neurons were measured 24 h after treatment with Na2S, ACS48 or S-memantine at 25, 50 or 100 μ

with or without 0, 25, 50, or 100 μ

memantine, n = 5 or 6 each. *, p < 0.001 versus vehicle without memantine, #, p < 0.001 versus same dose of the same H2S donor without memantine, †, p < 0.05.

FIGURE 8.

A, intracellular calcium levels [Ca2+]i in murine primary cortical neurons incubated with 20 μ

Na2S, ACS48, memantine, or S-memantine with or without 0. 1 m

glutamate (Glu). Values are shown as fura-2 fluorescence intensity ratio (λex 340 nm/λex 380 nm). n = 5 each. *, p < 0.001 versus vehicle with the same dose of Glu, #, p < 0.01 versus Na2S with the same dose of Glu. †, p < 0.05 versus ACS48 with the same dose of Glu. B, intracellular GSH levels [GSH]i of SH-SY5Y was measured 8 h after incubation with 50 μ

ACS48, memantine, or S-memantine w/wo 2 m

of glutamate. Values were normalized with protein concentration in lysate and reported as a ratio to the values of vehicle-treated group without Glu, n = 4 each. *, p < 0.05 versus vehicle w/o Glu, #, p < 0.01 versus vehicle with Glu, †, p < 0.05 versus ACS48 and memantine w/o Glu, ‡, p < 0.01 versus Na2S, ACS48 and memantine with Glu.

FIGURE 9.

Representative immunoblot and densitometric analysis of cleaved caspase-3 (A), phosphorylated Akt (p-Akt) (B), phosphorylated extracellular-signal regulated kinase 1/2 (p-ERK) (C) protein expression in SH-SY5Y after 15 h of OGD and 24 h of reoxygenation with or without the addition of S-memantine or memantine at 50 μm at 8 h after the end of OGD. Relative intensity was normalized to total caspase-3, total Akt, or total ERK, respectively. n = 3 or 4 each. **, p < 0.01, or ***, p < 0.001 versus control. #, p < 0.05, ##, p < 0.01.

FIGURE 10.

Plasma (A) and cerebral sulfide (B) levels of mice, measured 90 min after intraperitoneal administration of Na2S, ACS48, or S-memantine at 25 μmol/kg. n = 4 each. *, p < 0.05; **, p < 0.01.

FIGURE 11.

A, percent survival of mice subjected to 40 min of bilateral carotid artery occlusion (BCAO) and reperfusion and treated with S-memantine, memantine, ACS48, Na2S, or vehicle 1 min after reperfusion. n = 12 each. *, p < 0.05 versus Na2S, ACS48, memantine and vehicle by log-rank test. B, neurological score after BCAO. Vehicle and ACS48 (n = 12, 6, 3, 3, 3 on day 0, 1, 2, 3, 4, respectively), Na2S (n = 12, 8, 4, 4, 4), memantine (n = 12, 8, 3, 3, 3), S-memantine (n = 12, 11, 8, 7, 7), Sham (n = 5). *, **, or ***, p < 0.05, 0.01, or 0.001 versus vehicle, respectively. C, cerebral infarct volume and representative photographs of TTC-stained brain of mice subjected to sham operation or BCAO and treated with vehicle or S-memantine. n = 5 each. ***, p < 0.001 versus vehicle by unpaired t test.

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A novel hydrogen sulfide-releasing N-methyl-D-aspartate receptor antagonist prevents ischemic neuronal death - PubMed (original) (raw)