The present findings demonstrate that BDNF secretion from neuronally differentiated PC12 cells is markedly increased by exposure to hypoxic stimuli. Thus, our data support the hypothesis that oxidative stress can increase BDNF availability by stimulating BDNF release. Mechanisms underlying this release, including the requirement for (i) sodium influx through TTX sensitive channels, (ii) Ca2+ influx through voltage-gated channels and (iii) Ca2+ release from IP3- and ryanodine-sensitive stores, closely resemble those previously described for activity-dependent BDNF release from neurons (Balkowiec and Katz 2000, 2002). However, an unexpected finding was that most of the BDNF release induced by IH was blocked by dopamine receptor antagonists or inhibition of dopamine synthesis. These data indicate that the effect of IH on BDNF release is largely indirect and requires autocrine or paracrine signaling by endogenous dopamine.
Role of voltage-gated sodium and Ca2+ channels
The dependence of IH-induced BDNF secretion on voltage-gated sodium and Ca2+ channels is similar to that of activity-dependent neuronal BDNF release (Balkowiec and Katz 2000, 2002). These findings are consistent with the fact that acute hypoxic stimulation of PC12 cells results in membrane depolarization by inhibition of the Kv1.2 O2 sensitive potassium current (Zhu et al. 1996; Conforti et al. 2000) and with previous observations that PC12 cells express a full complement of voltage-gated Ca2+ channels (UsowicZ et al. 1990; Liu et al. 1996). Moreover, Ca2+ influx through voltage-gated Ca2+ channels has previously been shown to regulate catecholamine release from PC12 cells in response to acute hypoxic stimuli (Kumar et al. 1998; Taylor and Peers 1998; Kim et al. 2004).
Ca2+ mobilization from internal stores
In addition to influx of extracellular Ca2+, IH-induced BDNF release requires Ca2+ mobilization from internal stores through ryanodine receptors and IP3 receptors. This accords well with previous reports from our laboratory demonstrating that ryanodine receptors are required for activity-dependent BDNF release from hippocampal neurons (Balkowiec and Katz 2002). Moreover, IP3 receptors are required for hypoxia-induced catecholamine release from PC12 cells (Kim et al. 2004). The fact that regulated secretion of BDNF from PC12 cells and hippocampal neurons requires activation of both Ca2+ influx and Ca2+ mobilization from internal stores suggests a role for Ca2+-induced Ca2+ release, a mechanism by which cytoplasmic Ca2+ levels can be amplified and prolonged by Ca2+ release from internal stores (Albrecht et al. 2002). In some cell types, the effect of ROS on Ca2+ homeostasis can be attributed, in part, to Ca2+ release from internal stores (Kourie 1998). Thus, it is possible that decreased Ca2+ mobilization may contribute to the inhibition of IH-induced BDNF release that we observed following treatment of cells with NAC, a ROS scavenger.
Dopaminergic regulation of brain-derived neurotrophic factor release
Dopamine receptors, particularly the D2 subtype, have a well defined role in modulating dopamine synthesis and release in neurons (Goldstein et al. 1990) and PC12 cells (Courtney et al. 1991; Pothos et al. 1998). In addition, multiple reciprocal interactions have been found between dopamine and BDNF signaling. For example, BDNF can stimulate dopamine release from cultured mesencephalic (Blochl and Sirrenberg 1996) and striatal (Goggi et al. 2003) neurons and retinal amacrine cells (Neal et al. 2003). In addition, BDNF is required for expression of dopamine D3 receptors in the nucleus accumbens and striatum (Guillin et al. 2001; Sokoloff et al. 2002; Guillin et al. 2003) and can also up-regulate dopamine D5 receptors in cultured striatal astrocytes (Brito et al. 2004). On the other hand, BDNF expression in cultured striatal neurons (Kuppers and Beyer 2001), NT2/N cells (Fang et al. 2003) and transfected NG108-15 cells (Takeuchi et al. 2002) can be up-regulated by exogenous dopamine and D1 or D2 dopamine receptor agonists. Similarly, treatment of mice with the dopamine precursor levodopa acutely increases BDNF mRNA in mouse striatum (Okazawa et al. 1992). In NT2/N cells, BDNF up-regulation by activation of D1 receptors is associated with protection against oxygen–glucose deprivation induced cell death that is dependent on TrkB, indicating that dopamine signaling can trigger BDNF release from these cells (Fang et al. 2003). Moreover, the non-selective dopamine agonist apomorphine has been reported to stimulate BDNF secretion from embryonic ventral mesencephalic neurons (Guo et al.2002).
Our data indicate that IH is largely ineffective at stimulating BDNF release from PC12 cells when either dopamine synthesis or dopamine receptors are blocked. These data suggest that ROS generation, which is also required for IH-induced BDNF secretion, acts upstream of dopamine receptor activation. This possibility is consistent with previous observations that ROS generation can potentiate dopamine release from PC12 cells (Kim et al. 2004). Moreover, the fact that D2 antagonists, as well as AMPT, blocked IH-induced BDNF release argues against the possibility that dopamine stimulates BDNF release by acting intracellularly to promote ROS formation.
The importance of dopamine receptor activation for most IH-induced BDNF release may be related in part to the ability of dopamine to stimulate Ca2+ release from endoplasmic reticulum stores (Hernandez-Lopez et al. 2000; Takeuchi et al. 2002). Ca2+ mobilization from internal stores is a key step in regulation of neurotrophin release in general and a focus of diverse signals that can either enhance or suppress BDNF secretion (Balkowiec and Katz 2002; Canossa et al. 2002). Indeed, the present findings demonstrate that Ca2+ mobilization from the endoplasmic reticulum, as well as Ca2+ influx, are required for IH-induced BDNF release. However, increased Ca2+ release from internal stores occurs rapidly following dopamine receptor activation (Hernandez-Lopez et al. 2000; Takeuchi et al. 2002), whereas 90 or more cycles of IH are required to elicit a significant increase in BDNF release. Thus, it seems unlikely that an acute effect of dopamine receptor activation on Ca2+ mobilization alone can explain IH-induced BDNF release. Therefore, our data indicate a role for other, relatively slow, Ca2+-dependent processes mediated by dopamine. In particular, the fact that multiple cycles of IH increased the percentage of total BDNF content that is released, as well as BDNF expression, suggests that prolonged dopamine signaling up-regulates the secretory pathway used by BDNF in these cells. This could occur, for example, through transcriptional activation of genes involved in peptide packaging and/or release. This possibility is consistent with the fact that D2 agonists can activate the mitogen-associated protein kinase and protein kinase A transcriptional cascades (Yan et al. 1999; Bonci and Hopf 2005), phosphorylate the cyclic AMP response element binding protein (Yan et al. 1999) and up-regulate neuronal gene expression (Berke et al. 1998).
Nonetheless, our finding that IH-induced BDNF release was blocked by D2 antagonists seems paradoxical in view of the fact that D2 activation normally inhibits neurosecretion. However, recent studies indicate that prolonged exposure to dopamine or D2 agonists, as in our study, targets D2 receptors for internalization and degradation, whereas excitatory D1 receptors are recycled to the plasma membrane (Bartlett et al. 2005). The net result is a relative increase in dopamine mediated excitation at the expense of inhibition. Perhaps, therefore, the increases in BDNF release that we observed in response to multiple cycles of IH, or prolonged exposure to pergolide, result from a predominance of D1 mediated excitation that is normally masked by the presence of D2 receptors (Bartlett et al. 2005). Finally, the fact that BDNF can stimulate dopamine secretion (Blochl and Sirrenberg 1996; Goggi et al. 2003; Neal et al. 2003) and vice versa (present study) raises the possibility that BDNF and dopamine may mutually enhance each other's release.
Dopamine receptor blockade does not completely inhibit IH-induced BDNF release (Fig. 7b). It is possible, therefore, that the dopamine antagonists we used did not completely inhibit dopamine signaling in our cultures. Alternatively, these data may indicate that non-dopaminergic mechanisms, such as direct effects of ROS, may also be involved in IH-induced BDNF release. This possibility is supported by the fact that the BDNF release induced by short-term stimulation with H2O2 (Fig. 3) is not blocked by pretreatment of cells with AMPT (data not shown), indicating that dopamine signaling is not required for this acute effect. Perhaps therefore the small component of IH-induced BDNF release that is not blocked by dopamine antagonists results from a direct effect of ROS, possibly on Ca2+ mobilization (see above). This would be consistent with previous reports demonstrating that ROS can directly stimulate secretion of classical transmitters, such as acetylcholine, from PC12 cells (Kim et al. 2004).
Although the bulk of our studies focused on IH as a model of oxidative stress, we also found that BDNF release is weakly stimulated by exposure to 1 h of SH. Several factors may account for the fact that IH is a more potent secretagogue than SH, despite the fact that the cumulative exposure to hypoxia was the same in both paradigms. One possibility is that the repetitive reoxygenation that occurs with multiple cycles of IH, as well as the hypoxic episodes themselves, augments release by increasing the production of ROS (Yuan et al. 2004). Alternatively, or, in addition, it is possible that the onset of each hypoxic cycle, rather than the cumulative time in hypoxia, is the key stimulus for BDNF release. For example, it is well known that the peak secretory response of PC12 cells to sustained depolarization is transient, even in the presence of sustained elevations in intracellular Ca2+ (Di Virgilio et al. 1987). If this is also true for hypoxic stimuli, then the efficacy of repeated cycles of IH may in part reflect the cumulative effect of multiple peak responses at the onset of each hypoxic event.
In vivo, dopamine and BDNF are colocalized within several neuronal populations, including subsets of neurons in the substantia nigra and ventral tegmental area (Seroogy et al. 1994) and the petrosal ganglion of the glossopharyngeal nerve (Brady et al. 1999). Therefore, to the degree that our data can be extrapolated to primary dopaminergic neurons, our results suggest that oxidative stress may stimulate autocrine/paracrine signaling by dopamine, and BDNF release, in these populations. In light of evidence that BDNF can attenuate oxyradical damage (Mattson et al. 1995; Petersen et al. 2001), these findings raise the possibility that coexpression of BDNF may confer increased resistance to oxidative stress within subsets of dopaminergic neurons.