Calcium Export from Neurons and Multi-Kinase Signaling Cascades Contribute to Ouabain Neuroprotection in Hyperhomocysteinemia

Abstract

Pathological homocysteine (HCY) accumulation in the human plasma, known as hyperhomocysteinemia, exacerbates neurodegenerative diseases because, in the brain, this amino acid acts as a persistent N-methyl-d-aspartate receptor agonist. We studied the effects of 0.1-1 nM ouabain on intracellular Ca²⁺ signaling, mitochondrial inner membrane voltage (φ_mit), and cell viability in primary cultures of rat cortical neurons in glutamate and HCY neurotoxic insults. In addition, apoptosis-related protein expression and the involvement of some kinases in ouabain-mediated effects were evaluated. In short insults, HCY was less potent than glutamate as a neurotoxic agent and induced a 20% loss of φ_mit, whereas glutamate caused a 70% decrease of this value. Subnanomolar ouabain exhibited immediate and postponed neuroprotective effects on neurons. (1) Ouabain rapidly reduced the Ca²⁺ overload of neurons and loss of φ_mit evoked by glutamate and HCY that rescued neurons in short insults. (2) In prolonged 24 h excitotoxic insults, ouabain prevented neuronal apoptosis, triggering proteinkinase A and proteinkinase C dependent intracellular neuroprotective cascades for HCY, but not for glutamate. We, therefore, demonstrated here the role of PKC and PKA involving pathways in neuronal survival caused by ouabain in hyperhomocysteinemia, which suggests existence of different appropriate pharmacological treatment for hyperhomocysteinemia and glutamate excitotoxicity.

Keywords: NMDA receptors; calcium; cortical neurons; glutamate; homocysteine; ouabain.

Figure 1

Neuroprotective effect of ouabain (Oua) against neurotoxicity induced by 4 h excitotoxic insults in rat cortical neurons. (A–C) Confocal images represent an overlay of those recorded in green and red spectral regions of rat cortical neurons after 4 h treatment with the bathing solution (control, panel A), and 100 μM glutamate + 30 μM glycine (Glu, panel B) and Glu with 1 nM ouabain (Oua, C) obtained with the fluorescent viability assay. Scale bar, 100 µm. The correlation plots shown on the right of corresponding images allow estimation of the cell viability (percentages of live, necrotic and apoptotic neurons). (D) Histogram quantitatively compares the data obtained in control (n = 8) and in the presence Glu (n = 7) along and with 0.1 nM (n = 8) or 1 nM (n = 9) ouabain (Oua). Experimental conditions are indicated below the plots. Green columns represent percentages of live, red—of necrotic and orange—of apoptotic neurons. Mean values ± SEM are plotted. Experimental conditions are indicated below the plots. ** (p = 0.002), *** (p = 0.0001)—data are significantly different from control by one-way ANOVA with Tukey’s post-hoc test. (E) Histogram quantitatively compares the data obtained in control (n = 8) and in the presence of 100 μM homocysteine + 30 μM glycine (HCY, n = 6) and 0.1 (n = 7) nM or 1 nM (n = 8) ouabain (Oua). Mean values ± SEM are plotted. Experimental conditions are indicated below the plots. Green columns represent percentages of live, red of necrotic, and orange of apoptotic neurons. *** (p = 0.0002)—data are significantly different from control by one-way ANOVA with Tukey’s post-hoc test, n = 7.

Figure 2

Ouabain prevents the expression of pro-apoptotic proteins induced by 4 h glutamate (Glu) treatment of cortical neurons. Glutamate inhibits expression of Bcl-2 and enhances expression of AIF, Cas-3, p53, and BAX. Co-application of Glu with 1 nM ouabain (Oua) prevents pro-apoptotic protein expression so that the profile of protein expression remains similar to that of control values. Representative images of Western blots showing the protein expression in control, after 4 h treatment with 100 μM glutamate + 30 μM glycine and with 100 μM glutamate + 30 μM glycine + 1 nM ouabain. For quantitative analysis, the blots were scanned, and the intensities of bands after normalizing to β-actin were plotted as means ± SEM (n = 6 for each bar). One-way ANOVA with Tukey’s post-hoc test was utilized to reveal the significance of difference from control (* p = 0.03; ** p = 0.005; *** p = 0.0002).

Figure 3

Ouabain prevents the intracellular Ca²⁺ overload and the loss of mitochondrial inner membrane potential of cortical neurons caused by glutamate. (A) Fluorescent Ca²⁺ responses of neurons loaded with Fluo-8 evoked by an application of 100 μM glutamate + 30 μM glycine (Glu) obtained from the same experiment and normalized to the fluorescence intensity recorded without Glu. Gray lines—responses of single neurons. Red line—an average response. (B) Ca²⁺ responses when 1 nM Oua was added on the top of Glu responses. (C) Example of Glu responses revealing generation of Ca²⁺ transients by neurons. (D) Average cumulative curves for Ca²⁺ overload evoked Glu (red line) and with an addition of Oua (blue line), which represent an integral of fluorescent Ca²⁺ responses shown in panels (A,B). Mean value ± SEM for each point is plotted (n = 7). (E) Fluorescent responses of neurons loaded with rhodamine-123 evoked by an application of 100 μM glutamate + 30 μM glycine (Glu) that reflect the loss of mitochondrial inner membrane voltage (φ_mit) obtained from the same experiment and normalized to the fluorescence intensity recorded in the presence of 4 µM CCCP. Gray lines—responses of single neurons. Red line—an average response. (F) Changes of φ_mit when 1 nM Oua was added to Glu. (G) Histograms compare average values of φ_mit obtained with Glu and combined application of Glu and Oua in relation to full loss of φ_mit in the presence of CCCP (on the left, n = 7) and Ca²⁺ response amplitudes obtained under control and with Glu and combined application of Glu and Oua (on the right, n = 7). ***—Data are significantly different (p = 0.0006, ANOVA with Tukey’s post-hoc test).

Figure 4

Ouabain prevents the intracellular Ca²⁺ overload and the loss of mitochondrial inner membrane potential of cortical neurons caused by homocystein. (A) Fluorescent Ca²⁺ responses of neurons loaded with Fluo-8 evoked by an application of 100 μM homocystein + 30 μM glycine (HCY) obtained from the same experiment and normalized to the fluorescence intensity recorded without HCY. Gray lines—responses of single neurons. Red line—an average response. (B) Ca²⁺ responses when 1 nM Oua was added on top of HCY responses. (C) Average cumulative curves for Ca²⁺ overload evoked HCY (red line) and with an addition of Oua (blue line), which represent an integral of fluorescent Ca²⁺ responses shown in panels (A,B). Mean value ± SEM for each point is plotted (n = 4). (D) Fluorescent responses of neurons loaded with rhodamine-123 evoked by an application of 100 μM homocystein + 30 μM glycine (HCY), which reflect the loss of mitochondrial inner membrane voltage (φ_mit) obtained from the same experiment and normalized to the fluorescence intensity recorded in the presence of 4 µM CCCP. Gray lines—responses of single neurons. Red line—an average response. (E) Changes of φ_mit when 1 nM Oua was added to HCY. (F) Histograms compare average values of φ_mit obtained with HCY and combined application of HCY and Oua in relation to full loss of φ_mit in the presence of CCCP (on the left, n = 5) and Ca²⁺ response amplitudes ([Ca²⁺]) obtained under control and with Glu and combined application of HCY and Oua (on the right, n = 5). ***—Data are significantly different (p = 0.0004, ANOVA with Tukey’s post-hoc test).

Figure 5

Ouabain prevents the neurotoxicity induced by 24 h excitotoxic insult in rat cortical neurons. (A) Confocal image after 24 h treatment with 100 μM Glu + 30 μM glycine (Glu) and (B) with 100 μM HCY + 30 μM glycine (HCY); (C,D) addition of 1 nM ouabain. Scale bar, 100 μm. Data obtained from the control and that after treated with 100 μM Glu or 100 μM HCY and either 0.1 nM or 1 nM Oua. The correlation plots shown on the right of corresponding images allow estimation of the cell viability (percentages of live, necrotic, and apoptotic neurons). (E) Data obtained from the control and that after treatment with 100 μM Glu or 100 μM HCY and either 0.1 nM or 1 nM Oua. Green columns: percentages of live. Red: necrotic. Orange: apoptotic neurons. Data are expressed as mean ± SEM. * p = 0.03; ** p = 0.002; *** p = 0.0008, respectively, are significantly different from the control, one-way ANOVA with Tukey’s post-hoc test).

Figure 6

Effects of inhibitors of different protein kinases in ouabain-induced neuroprotection. The histograms quantitatively compare the data obtained in the presence of 100 μM glutamate (+30 μM glycine, Glu) or homocysteine (+30 μM glycine, HCY) with 1 nM ouabain (Oua), and in combination with 0.6 μM PKA inhibitor (PKA_i), 1 μM PKC inhibitor chelerythrine (Chel), or 3 μM CaMKII inhibitor (KN93) or during excitotoxic insults without Oua. Mean values ± SEM are plotted. Experimental conditions are indicated below the plot. Green columns represent percentages of live, red of necrotic, and orange of apoptotic neurons. Data were obtained in 4 h (A) and in 24 h (B) excitotoxic insults. *, **, ***—Data are significantly different from the values obtained in HCY with Oua (* p = 0.03; ** p = 0.009; *** p = 0.0001; ANOVA with Tukey’s post-hoc test). The number of experiments (n) for each group is indicated above the columns.

Figure 7

Schematics of data interpretation of antagonism between hyperhomocysteinemia-induced neuronal death and ouabain triggered NKA signaling. The 4 h ouabain effects are kinase-independent and include (A) acceleration of Ca²⁺ removal from the cell by NCX, and (B) prevention of Bax translocation to mitochondria. Both (A) and (B) prevent severe ionic imbalance in neurons and augment downstream necrotic and apoptotic neuronal death. The 24 h ouabain effects additionally involve PKC- (C) and PKA-dependent (D) inhibition of HCY-specific ERK/p38 MAPK apoptotic pathways. [Ca²⁺]—free intracellular calcium concentration; AIF—apoptosis inducing factor; ATP—adenosine-3-phosphate; CaMKII—Ca²⁺/calmodulin-dependent protein kinase II; Cas-3 and Cas-9—caspases 3 and 9, respectively; Cyt-C—cytochrome C; GluN2A—subunit of N-methyl-d-aspartate receptors; HCY—homocysteine; MAPK—mitogen-activated protein kinase; MTX—mitochondria; NCX—sodium–calcium exchanger; NKA—Na/K-ATPase; Oua—ouabain; PKA—protein kinase A; PKC—protein kinase C; PTP—mitochondrial permeability transition pore; φMTX—mitochondrial inner membrane potential, V_m—cell membrane potential.