Nitric oxide signaling participates in norepinephrine-induced activity of neuronal intracellular survival pathways
Introduction
Nitric oxide (NO) is a volatile gas that has been attributed with the ability to promote both cell survival and neuronal death, depending upon the levels of NO present, as well as the specific cellular system involved (Araujo and Carvalho, 2005). Although high levels of NO can lead to tissue damage via the rapid formation of free radical species (Maher and Schubert, 2000), this molecule can also play a prominent role in neural plasticity (Contestabile, 2000, Garthwaite and Boulton, 1995, Holscher, 1997) through highly specific signaling mechanisms (Gross and Wolin, 1995, Bredt, 1999) that are capable of directing cellular activities via the activation of protein kinases (Holscher, 1997). This highly important biological action of NO involves its binding to the heme moiety in the heterodimeric enzyme, soluble guanylate cyclase. Activation of this enzyme by NO results in the production of the second messenger molecule, cGMP, which can regulate numerous physiological events such as vasodilatation and neurotransmission (Krumenacker et al., 2004). As NO is closely associated with the NMDA receptor complex (Contestabile, 2000), via this interaction, glutamatergic activation following sensory stimulation, learning or heightened neuronal activity activates the synthesis of NO, which in turn activates cGMP (Garthwaite and Boulton, 1995). As noted above, via the activation of protein kinases, cGMP activation may lead to growth or regenerative activities within the cell (Hindley et al., 1997). Therefore, NO signaling could be one means by which environmental enrichment, learning and/or physical activity lead to enhanced neuronal survival or growth.
In recent years, it has been observed that chronic treatment with antidepressant medications enhances the expression of a key neurotrophin, brain-derived neurotrophic factor (BDNF), in the hippocampus and neocortex, and that increase in BDNF may be an important aspect of the therapeutic response (Duman et al., 1997). General physical activity (exercise, voluntary wheel running) also increases BDNF (Neeper et al., 1996). This effect is more rapid than antidepressants (enhanced BDNF transcription is evident within hours rather than weeks), and voluntary wheel running combined with antidepressant treatment potentiates BDNF mRNA expression in an additive manner (Oliff et al., 1998, Russo-Neustadt et al., 1999). Other evidence exists that antidepressant-induced regulation and activity-induced changes in BDNF transcription may occur through convergent intracellular mechanisms (Russo-Neustadt et al., 2000).
Both antidepressant treatment and voluntary exercise enhance BDNF transcription through increased activation of monoaminergic neurotransmitter signaling, and evidence is particularly strong that exercise-induced changes in BDNF are dependent upon norepinephrine (NE) activation (Garcia et al., 2003, Ivy et al., 2003, Russo-Neustadt and Chen, 2005). This exercise-induced NE release activates the protein kinase A/cAMP pathway, just as increased intrasynaptic NE and/or 5-HT following antidepressant treatment is thought to do (Duman et al., 2001, Russo-Neustadt and Chen, 2005) leading to CREB activation and enhanced BDNF synthesis. In addition to this mechanism, one means by which exercise can rapidly and robustly increase BDNF is via glutamatergic activation and NO signaling. General physical activity enhances NE content (Dishman et al., 2000) and metabolism (Dunn et al., 1996) and glutamate (Leung et al., 2006, Richter-Levin et al., 1998) release, as well as NMDA receptor levels (Farmer et al., 2004, Molteni et al., 2002) within the hippocampus. These stimuli activate NO synthesis (Fedele et al., 2001), which is capable of enhancing NE release through increased glutamate release and rapid amplification of the signal (Lonart et al., 1992, Stout and Woodward, 1994, Stout and Woodward, 1995). In addition, NO is a mediator of calcium-dependent activation of the G-protein p21 Ras (Ras) through NMDA receptor stimulation (Deora et al., 1998, Koo et al., 2005). By this means, the pro-survival phosphatidylinositol 3′ kinase (PI-3K) pathway is activated, leading to the subsequent transcription of many pro-survival genes (Kang et al., 2004).
The hypothesis that exercise-induced increases in BDNF may be dependent upon NO synthesis was recently tested by treating animals chronically with the NO synthase inhibitor, l-NAME (Chen et al., 2006). The results supported this hypothesis, as exercising l-NAME treated animals did not show increased hippocampal BDNF mRNA, unlike their vehicle-treated counterparts. On the other hand, increases in BDNF transcription resulting from an exercise/antidepressant combination were not prevented with concurrent l-NAME treatment (Chen et al., 2006). It is possible that only the exercise-induced increases in BDNF, and not those stimulated by antidepressant treatment, are NO-dependent.
In both the whole animal and in cell culture models, exercise or NE application respectively increase cell survival signaling via activation of the PI-3K pathway (Chen and Russo-Neustadt, 2005, Chen et al., 2007). In the current study, we wished to assess whether the phenomenon of enhanced BDNF expression following the application of NE is also dependent upon NO signaling. In addition, we have investigated whether cGMP activation is necessary and vital for the observed increase in BDNF and/or PI-3K within hippocampal neurons.
Section snippets
Antibodies
Antibodies were purchased from the following sources: anti-BDNF (Santa Cruz Biotech, Santa Cruz, CA); anti-phospho-T308-Akt and anti-Akt (Cell Signaling, Beverly, MA); anti-phospho-ERK1/2, anti-ERK1/2, anti-PI-3K, and anti-α-tubulin (Upstate Biotech. Inc., Charlottesville, VA).
Hippocampal dissection at embryonic day 18 (E18) and tissue culture
Hippocampi of each embryo were excised and cultured according to the method of Banker and Cowan (1977). Briefly, on their 18th day of pregnancy, female rats (Sprague–Dawley) were sacrificed using an overdose of
NE increases BDNF, PI-3K, P-Akt and P-ERK1 immunoreactivity
Application of NE to E18 hippocampal neurons led to a time-and concentration-dependent increase in BDNF, PI-3K and P-ERK. Previous studies revealed that exposure to 100 nM NE for 2 h led to peak levels of BDNF immunoreactivity in these cultures (Chen et al. 2007). These conditions were therefore used for our subsequent experiments. Under these conditions, NE exposure led to a robust increase in BDNF expression [F(15,32) = 16.46, p < .0001] (Fig. 1). Similarly, NE also increased the expression of
Discussion
Our experimental evidence suggests that the BDNF-enhancing effects of NE, as applied to primary hippocampal neurons, are dependent upon NO signaling. Increased NE neurotransmission is thought to be an important component of the BDNF-enhancing response to exercise or antidepressant treatment in vivo. (Duman, 1998, Ivy et al., 2003). Our results support the hypothesis that NO signaling is an important part of this NE-mediated mechanism in vitro. The results of our current study are consistent
Conclusions
In conclusion, in isolated hippocampal neurons, we have shown that BDNF and PI-3K immunoreactivity can be increased by application of NE (or an NO donor), and that these changes are NO-dependent. Similar to earlier results gleaned in vivo (Chen et al., 2006), NOS inhibition decreases BDNF and PI-3K immunoreactivity. Because BDNF and PI-3K are survival-associated proteins (Brunet et al., 1999, Huang and Reichardt, 2003, Rossler et al., 2004, Segal, 2003), it is logical to infer that the presence
Acknowledgements
Funded by PHS Grant MH 59776 to ARN.
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2011, Brain ResearchCitation Excerpt :Physical activity in rats and humans showed a positive effect on depression, anxiety disorders and mental activity in the elderly (Dik et al., 2003; Kramer et al., 2006; Lytle et al., 2004). Many researchers reported that exercise increases gene expression of neurotrophic factors (Chen et al., 2006) involved in development, growth and survival of certain neuronal types (Anderson et al., 2000; Bimonte et al., 2003; Chen and Russo-Neustadt, 2007; Estévez et al., 2006) in rodents. Nitric oxide (NO), a gaseous signaling molecule, is involved in the regulation of the cardiovascular, nervous and immune systems and neurotoxicity (Alderton et al., 2001; Zhou and Zhu, 2009).