The role of NHE proteins in DA neurotransmission is essentially unknown. We have explored the effects of the NHE inhibitor HOE-642 on striatal DA neurotransmission using in vivo microdialysis. In addition, we have examined mRNA expression of NHE1–5 as well as the location of NHE1 protein in the striatum and midbrain. Intrastriatal delivery of HOE-642 caused an increase followed by a decrease in DA overflow with a concomitant decline in striatal DA content. HOE-642 delivery also attenuated DA overflow induced by the mitochondrial inhibitor MAL without modifying the subsequent DAergic terminal damage. Expression of all five NHE isoforms was detected in the striatum and midbrain. Moreover, NHE1 protein was found in striatal synaptosomes but was not directly co-located with nigrostriatal DAergic neurons. The apparent absence of NHE1 protein on DAergic neurons suggests that the effects of HOE-642 on striatal DA overflow are either mediated via NHE1 located on other cell types or that HOE-642 is acting through NHE isoforms other than NHE1. The latter is a likely possibility because the HOE-642 related effects were seen at concentrations that might inhibit multiple NHE isoforms.
Effects of HOE-642 on striatal DA neurotransmission under normal conditions
The NHE inhibitor HOE-642 belongs to the family of benzoylguanidines that exhibit higher affinities for NHE1, NHE2, and NHE5 (IC50 values of 0.05, 3, and 9.1 μM, respectively), and lower affinities for NHE4 and NHE3 (IC50 values of 250 μM and 1 mM, respectively) (Scholz et al. 1995; Szabo et al. 2000; Luo et al. 2005). Expression of the five NHE isoforms was found in the striatum and ventral midbrain (present study). Based on diffusion characteristics in brain tissue and drug recovery rates in vitro (Nicholson and Rice 1986; Lindefors et al. 1989; Westerink and De Vries 2001), we estimate that reverse microdialysis of HOE-642 (0.3–3 nmol/min) would achieve concentrations of 15–150 μM in the striatum that fall well within the range to block multiple NHE isoforms.
Prolonged HOE-642 delivery caused an early increase followed by a progressive reduction in DA overflow with a concomitant increase in DA turnover and reduced striatal DA content. These findings, which are similar to those seen with the administration of VMAT2 inhibitors such as tetrabenazine (TBZ) or Ro4-1284, indicate a disruption in vesicular DA storage. The inhibition of vesicular DA uptake causes transient accumulation of DA in the cytosol which leads to DAT-mediated DA release, increased intraneuronal DA metabolism and a decrease in pre-synaptic DA stores (Colzi et al. 1993; Moy et al. 2000; Staal and Sonsalla 2000; Andersson et al. 2006). While TBZ and Ro4-1284 directly inhibit the VMAT2 transporter, we propose that HOE-642 disrupts transvesicular ion gradients and indirectly affects vesicular DA storage. This might occur via two non-exclusive mechanisms. First, inhibition of plasma membrane NHE activity on DAergic terminals would cause cytosolic acidification that could reduce the transvesicular pH gradient necessary for adequate DA transport into vesicles (Johnson et al. 1982; Drapeau and Nachshen 1988; Camacho et al. 2006). Secondly, inhibition of any NHE located on synaptic vesicles (Szaszi et al. 2002) could also play a role in the regulation of vesicular volume (via Na+ or K+ influx) as is seen with the organelle NHE isoforms 6–9 (Orlowski and Grinstein 2004; Nakamura et al. 2005). Reduction of vesicular volume is thought to reduce vesicular DA storage (Pothos 2002). While both mechanisms may be in effect, the slower reduction in DA overflow after onset of reverse microdialysis of HOE-642 (> 2 h) compared with the onset of reverse microdialysis of TBZ (30 min, see Andersson et al. 2006) may reflect the need for a sufficient build-up of cytosolic H+ to disrupt vesicular DA uptake.
Although an effect of HOE-642 on vesicular disruption might contribute to the observed changes in DA neurotransmission, additional factors are likely to account for the initial large increase in DA overflow. This notion is underscored by the findings that delivery of HOE-642 causes an eightfold increase in DA overflow compared with the maximal effect of TBZ, which elicits only a twofold increase in DA overflow (Andersson et al. 2006). Several potential mechanisms might account for this observation. First, as DA overflow elicited by Ro4 administration is much greater during MAO-A inhibition because of reduced DA degradation (Colzi et al. 1993), inhibition of MAO after HOE-642 delivery could similarly contribute to the increase in DA overflow. However, while amiloride and its analogs inhibit MAO activity (Zubieta et al. 1988), HOE-642 appeared to be a poor inhibitor of either MAO-A or -B in vitro (present findings). Moreover, the significant increases in the overflow of DOPAC and HVA, and DA turnover elicited by HOE-642 indicate a lack of MAO inhibition in vivo.
Another possible mechanism by which HOE-642 could produce elevations in striatal DA overflow is by blockade of DAT and consequent inhibition of extracellular DA re-uptake. This is an important consideration in light of an earlier report that amiloride analogs block striatal synaptosomal 3H-DA uptake (Callahan et al. 2001). However, the present data do not support this hypothesis. HOE-642 did not inhibit 3H-DA uptake into synaptosomes up to 100 μM in contrast to the amiloride analog EIPA that completely blocked 3H-DA uptake at 30 μM (Table 2). In addition, prolonged reverse microdialysis of DAT inhibitors, such as cocaine or nomifensine, causes sustained increases in striatal DA overflow (Tolliver et al. 1999; Westerink and De Vries 2001) in contrast to the HOE-642 mediated bi-phasic effect. Moreover, DAT inhibition protects against MAL-induced damage (Moy et al. 2007), whereas HOE-642 did not provide protection. Hence it is unlikely that HOE-642 modified striatal DA overflow via blockade of extracellular DA re-uptake.
A third possibility is that HOE-642 induces exocytotic DA release in addition to the DAT-mediated DA release, which is typically associated with disruption of vesicular DA stores. This is in accordance with brain synaptosome studies suggesting that NHE inhibition causes exocytotic DA release. Amiloride analogs stimulate synaptosomal 3H-DA efflux that is abolished in Ca2+-free media and is unchanged by DAT inhibition (Cannizzaro et al. 2003), and cytosolic acidification enhances basal DA release in the presence of a DAT inhibitor (Drapeau and Nachshen 1988). Thus, striatal NHE inhibition might trigger exocytotic DA release accompanied by alterations in vesicular DA storage. Such effects invoke the presence of one or more NHE isoforms on the DAergic neurons. Although NHE1 protein was not co-localized on DAergic neurons (Fig. 6), we found that NHE2–5 were also expressed in the striatum and therefore could mediate the effects of HOE-642 on DA release (Fig. 7).
Finally, it is possible that the changes in DA neurotransmission seen with HOE-642 also occur via inhibition of NHE activity in non-DAergic cell types in the striatum. For example, NHE activity contributes to H+ extrusion in cultured astrocytes (Kimelberg et al. 1979; Shrode and Putnam 1994; Pizzonia et al. 1996), which actively modulate neurotransmission (Rothstein et al. 1996; Kang et al. 1998). In particular, astrocytes have been implicated in regulating interstitial pH in the brain by secreting acid equivalents (Chesler and Kraig 1989; Chesler 2003), which down-regulate neuronal activity in part via blockade of pre-synaptic voltage-dependent Ca2+ channels (Prod’hom et al. 1989). Although an acid secretory function of glia has been mostly attributed to the Na+-HCO3− cotransporter in other brain regions (Grichtchenko and Chesler 1994; Newman 1996), it is tempting to speculate that NHE inhibition might reduce the ability of astrocytes to secrete H+ surrounding DAergic varicosities, thereby enhancing activity-dependent DA release. The impact of NHE inhibition on corticostriatal projections and striatal interneurons might also affect local release of other transmitters such as glutamate and GABA (Trudeau et al. 1999; Jang et al. 2006) which could indirectly modify nigrostriatal DA activity. While future studies are needed to characterize the cellular mechanisms involved in the HOE-642 induced DA release, our data is consistent with the notion that striatal NHE inhibition triggers a combination of events that contribute to modify DA neurotransmission in vivo.
Effects of HOE-642 on malonate-induced DA overflow and DAergic terminal damage
Earlier studies demonstrated that depletion of striatal DA content (Globus et al. 1987; Ferger et al. 1999; Moy et al. 2000) or administration of DAT inhibitors (Xia et al. 2001; Moy et al. 2007) prior to a metabolic stress attenuate the associated increase in extracellular DA levels and striatal tissue damage. Conversely, disruption of vesicular DA stores during a metabolic stress enhances MAL-induced damage (Albers et al. 1996; Burrows et al. 2000; Nixdorf et al. 2001). Such evidence implies a direct correlation between the extent of DA release during metabolic stress and striatal tissue damage. In the present study, we tested the hypothesis that HOE-642 might protect DAergic terminals against metabolic stress. To eliminate the confounding of HOE-642-induced DA release with MAL-induced toxicity, the mitochondrial inhibitor was infused only after DA overflow had stabilized at or below pre-drug levels. This approach revealed that the MAL-induced DA overflow was attenuated by HOE-642 pre-treatment likely because of the reduced striatal DA pools (Table 1). However, HOE-642 pre-treatment did not modify the MAL-induced DAergic terminal damage, nor did it alter striatal lactate overflow, suggesting that the changes in DA overflow were not because of an attenuation of the metabolic stress per se. While it is not clear how DA itself might contribute to neuronal damage caused by mitochondrial inhibition, one view is that mishandling of striatal DA exacerbates oxidative stress (Hastings et al. 1996; Ferger et al. 1999; Xia et al. 2001). However, the lack of striatal DA terminal protection in mice pre-treated with HOE-642 suggests that the magnitude of DA release during metabolic stress is not predictive of neuronal damage.
The finding that striatal HOE-642 delivery did not modify MAL-induced DA terminal damage is in accordance with previously reported data that systemic EIPA administration does not prevent loss of DAergic terminals caused by the neurotoxin MPTP in mice (Callahan et al. 2001). Together these data are in contrast to studies of animal models of brain ischemia reporting neuroprotection with systemic pre-treatment of HOE-642 or EIPA (Phillis et al. 1999; Castella et al. 2005; Luo et al. 2005). Although such different outcomes might be because of the variation between the experimental models utilized, another possibility is that NHE inhibition does not attenuate DAergic terminal damage because these neurons do not express NHE1.
NHE1 localization and expression of other NHE isoforms in the striatum and midbrain
Na+/H+ exchanger isoform 1 is the most abundant NHE isoform in the brain yet it is differentially expressed across brain regions and neuronal subpopulations (Ma and Haddad 1997; Douglas et al. 2001). The present findings are the first to demonstrate NHE1 protein in the striatum and substantia nigra as well as its localization to striatal synaptosomes. These data indicate that NHE1 is located at the synaptic level, which is in line with earlier demonstrations of amiloride-sensitive NHE activity in whole brain synaptosomes (Sauvaigo et al. 1984; Jean et al. 1985). Together these observations bring further support to the notion that NHE inhibition modifies neurotransmission in general (Trudeau et al. 1999; Jang et al. 2006).
Consistent with our western blot results, immunofluorescence confocal imaging revealed widespread NHE1 labeling in the striatal neuropil. Surprisingly, double-label immunofluorescence revealed that NHE1 signal was predominantly found in TH-negative cell processes. These data indicate that nigrostriatal DAergic neurons express low levels of NHE1 protein, if any, in relation to neighboring subpopulations of cells. These findings do not exclude the possibility that NHE1 protein is found in nigrostriatal DAergic fibers at levels below the limits of our detection method. However, the apparent lack of co-location of NHE1 on nigrostriatal DAergic neurons is consistent with the observation that striatal HOE-642 delivery does not protect DAergic terminals against metabolic stress as it does against ischemic damage to other neuronal populations (Luo et al. 2005).
The differential localization of NHE1 between DAergic and non-DAergic cells in the adult mouse brain is in agreement with a comprehensive demonstration of uneven NHE1 expression in other rat brain regions such as the cerebellum, where NHE1 mRNA signal was found concentrated in the granule and Purkinje cells compared with very weak signals in the cells of the molecular layer (Ma and Haddad 1997). Absence of NHE1 immunoreactivity in epithelial cells of the human choroid plexus has also been reported (Praetorius and Nielsen, 2006). Hence, evidence from our study and others underscores the differential expression of NHE1 protein across subpopulations of cells throughout the brain. Interestingly, both NHE1 activity and gene expression are, respectively, reduced by reactive oxygen species and H2O2 (Mulkey et al. 2004; Kumar et al. 2007). Given the high level of oxidative stress in nigrostriatal DAergic neurons, it is possible that NHE1 expression or activity might be reduced in these cells.
The presence of NHE activity in many other neuronal subtypes (Chesler 2003) in contrast to the apparent absence of NHE1 location on DAergic neurons in our studies would suggest that DAergic neurons express alternative NHE isoforms. In addition to NHE1, we have documented mRNA expression of NHE2–5 in the striatum and ventral midbrain. Future studies are needed to characterize which NHE isoforms are found on nigrostriatal DAergic neurons. A likely candidate seems to be the brain specific NHE5 as it has been localized in hippocampal neuronal processes and speculated to be involved in modulating neurotransmission (Baird et al. 1999; Szaszi et al. 2002). Localization studies of NHE isoforms in non-DAergic cells in the striatum and substantia nigra will also be important to elucidate the role of NHE proteins in DA neurotransmission under physiological and pathological conditions.