Light- and electron microscopy
An earlier mapping study [
7], reported that in the rat brain, NTPDase3 is present exclusively in neurons, mostly in neuronal processes. In the present study, we confirmed those previous results, showing IR cell bodies localized in the LHN and AN, while in the rest of the hypothalamus only cell processes were observed, frequently in close apposition to the wall of hypothalamic vessels. The latter observations raise the possibility that at least part of NTPDase3-IR hypothalamic cells might be involved in the mediation of peripheral humoral signals by either the release of neuronal end-products into the blood circulation, and/or, intracellular events of such neurons could be affected by peripheral humoral factors through a yet unidentified NTPDase3-mediated intracellular signaling system.
Our light- and correlated electron microscopic studies showed that NTPDase3-IR is present at certain well-demarked segments of the plasma membrane. This finding is consonant with the generally accepted view of NTPDases being transmembrane proteins and hydrolyzing phosphorylated nucleotides outside of the cell. With respect to the cytoplasmic immunoreactivity, part of the cytoplasmic IR particles was associated with ribosomes, some of which were free cytoplasmic, but the majority of which were linked to the endoplasmic reticulum. This finding is not surprising in light of the fact that the amino acid sequence used for the generation of the antibody is part of the core protein; therefore, it is immunohistochemically detectable readily after translation. Another source of immunoreactive cytoplasmic particles appeared to be linked to mitochondria.
The detection of NTPDase3-IR in the mitochondrial matrix or closely associated with the inner mitochondrial membrane is probably the most intriguing finding of this study. Since immunolabeled mitochondria were typically found near asymmetric (putative excitatory) synaptic membrane specializations, it is reasonable to assume that mitochondrial NTPDase3 activity is functionally linked to excitatory, rather than inhibitory neuronal functions.
Immunolabeling for NTPDase3 and GAD
NTPDase3-IR was found in the vicinity of asymmetric (therefore presumably excitatory), but not symmetric, synaptic membrane specializations (presynaptic axon terminals, dendrites and dendritic spines). Additionally, by immunolabeling for GAD and NTPDase3 and applying the well-known mirror-technique, we found that none of the GAD-IR neurons examined contained NTPDase3. Although we have not examined all hypothalamic synapses and GAD-IR neurons for the localization of NTPDase3, and some other, non-GABAergic neurons (e.g., dopaminergic) that are inhibitory in function may also express NTPDase3, our present findings still suggest that the vast majority of hypothalamic NTPDase3 is expressed in excitatory neurons.
In dendrites, free cytosolic (a possible soluble form), ribosome-associated, as well as mitochondrial labelings were detected. In contrast, in myelinated axons and axon terminals only mitochondrial immunoreactivity was observed. While the versatility in the appearance of immunoreactive material in dendrites may refer to the cellular processes of NTPDase3-metabolism (biosynthesis, protein maturation, explantation and organelle-linked function), the mitochondrial presence of this enzyme in axons and axon terminals suggests that the modulation of mitochondrial ATP-levels required to fuel neuronal output may be, at least to some extent, regulated by NTPDase3 activity.
Estrogen effects on hypothalamic NTPDase3 expression
As mentioned earlier, multiple NTPDase3-IR bands were detected in western blot studies using rat hypothalamus homogenates. This phenomenon may indicate that there is "incomplete" processing of the enzyme, however, it is more likely that the distinct bands observed represent different maturational forms of the enzyme. In this study, we detected NTPDase3-IR linked to multiple subcellular structures (plasma membrane, ribosomes, endoplasmic reticulum) including neuronal mitochondria. Additionally, we provided evidence for NTPDase-activity in synaptosomal preparations. Therefore, it is also possible that one or more of the protein-forms detected on western blots represent functional forms of the protein adapted or adjusted to the microenvironment or functional attributes of the cell organelles.
Since the neuroendocrine hypothalamus is highly E
2-responsive, it was reasonable to assume that E
2 may influence the expression level of NTPDase3 within this brain area. Therefore, in a pilot study we investigated whether E
2-treatment of ovx animals affects NTPDase3-levels in tissue blocks containing the entire hypothalamus. That study showed that a single subcutaneous injection of E
2 results in significantly increased levels of NTPDase3 (unpublished observation). Those results prompted us to examine and analyze the temporal changes in NTPDase3-expression in lateral- and medial hypothalamic tissue samples as described above. The present findings indicate that in response to a single subcutaneous injection of E
2, NTPDase3 expression increases in just a few hours after E
2-treatment in both hypothalamic areas, however, the pattern of temporal changes in the medial hypothalamus differs from that observed in the lateral part of this brain area. Since the mediobasal hypothalamus, including the AN, is a major player in the biphasic (positive- and negative feedback) regulation of the gonadotrophin secretion and release, it is reasonable to speculate that in the medial part of the hypothalamus, NTPDase3 may be involved in the estrogenic control of gonadotrophins. The idea of a causal coincidence between the two peaks in NTPDase3 levels and the positive/negative gonadotrophin feedbacks raises several questions. For example, a number of data suggest that NTPDase3 may be involved in the hypothalamic regulation of gonadotrophin release. We have previously described that during the E
2-induced gonadotrophin surge, an E
2-dependent synaptic reorganization on hypothalamic neurons occurs. This phenomenon we termed "phased synaptic remodeling", that shows specific changes in the ratio of inhibitory/excitatory synapses during the two (positive- and negative-) states of the gonadotrophin feedback control [
21]. A sharp rise in the number of excitatory synapses was observed at the time of the E
2-surge, and the formation of new synapses may very well include ones equipped with NTPDase3-containing mitochondria. This hypothesis is consonant with our observation that inhibition of NTPDase activity decreases state 3 mitochondrial respiration and the total mitochondrial respiratory capacity, ergo an increased amount of mitochondrial NTPDase would well serve the energy-needs of a transient intensification in excitatory neuronal activity.
Both medial and lateral hypothalamic functions are known to involve mechanisms mediated by various purinoceptors, such as A
1, P
2X, and the activity of NTPDase3-containing hypocretin-orexin neurons in the LHN is directly influenced by such receptor actions [
22‐
29]. Since here we found neuronal membrane-linked NTPDase3-IR in the AN/LHN, and E
2 induced a transient increase in NTPDase3-levels in both hypothalamic sites, it is also possible that E
2 increases the amount of membrane-incorporated NTPDase3 to transiently intensify purinergic interneuronal signaling. Further studies are underway to clarify this issue.
In lateral hypothalamic samples, NTPDase3 levels peaked at 4 hrs after E
2-treatment followed by a gradual decrease, and reached ovx levels by 26 hrs. It has been demonstrated by Belcher et al. [
7] that LHN NTPDase3-containing cells are nearly all (96–97%) hypocretin-orexin-containing neurons. These neurons are known to be direct modulators of the midbrain raphe serotonergic neurons [
30] to influence sleep-wake states. It is also known that E
2 influences arousal mechanisms in many ways [
31]. Thus, it is possible that the mechanism through which E
2 facilitates wakefulness involves increased NTPDase3-activity. On the other hand, LHN hypocretin-orexin (plus NTPDase3-IR) neurons are not only targets of E
2, but also that of the gastric hormone ghrelin; at the same time, these neurons also represent the major excitatory input of AN neuropeptide Y/Agouti-related protein-containing cells whose activity is responsible for the initiation of food intake. Changes in the functional intensity of this circuit also involve synaptic remodeling [
32]. It is therefore possible that in response to E
2-treatment, LHN NTPDase3-IR hypocretin-orexin-containing neurons intensify their action on AN neuropeptide Y/Agouti-related protein-containing cells, thereby leading to increased NTPDase3 levels in both (medial and lateral) parts of the hypothalamus. If this was the case, one could speculate that the orexigenic effect of E
2 may in some way involve the action of NTPDase3.
Mitochondrial respiration measurements
It has been shown that interneuronal signaling is a highly ATP-dependent, energy-demanding process [
33]. To supply the energy needs of neurotransmission, ATP is produced and maintained in neuronal mitochondria in a regulated fashion. We have previously proposed [
34] that one potential mechanism down-regulating mitochondrial ATP production may involve uncoupling proteins (UCPs), specifically UCP2, which was only found in inhibitory neurons of the hypothalamus. However, the specific mechanism involved in the regulation of mitochondrial ATP levels in excitatory hypothalamic neurons is currently unknown. Therefore, the identification of NTPDase3 in mitochondria in synaptic or perikaryal sites of excitatory hypothalamic neurons might be the most novel and intriguing finding of this study, and warrants further experiments to elucidate the exact functional role of mitochondrial NTPDase3 in neurotransmission.
To confirm our morphological findings, we isolated synaptosomes from hypothalamic tissue homogenates and examined mitochondrial respiration in control versus suramin (an NTPDase inhibitor) treated samples. Suramin blockade of NTPDases reduced mitochondrial oxygen consumption in state 3 mitochondrial respiration by 30%, and also decreased the total mitochondrial respiratory capacity by 34%. These findings imply that induction of NTPDase activity in the mitochondria by hydrolyzing ATP to ADP increases mitochondrial state 3 respiration and the total mitochondrial respiration capacity. It should be noted that the polyoxometalate suramin is not a fully selective NTPDase3-inhibitor, as such inhibitors, to the best of our knowledge, for the 8 known NTPDases have not yet been found. However, suramin has been shown to be a potent inhibitor of NTPDase3 and other NTPDases [
16], therefore the observed reduction in oxygen consumption in hypothalamic synaptosomes can be, at least in part, attributed to the inhibition of NTPDase3. This idea is supported by previous results on synaptosome fractions isolated from rat brain cortex and striatum [
35], and from hippocampal synaptosomes [
36]. These studies report a transient accumulation of ADP after addition of ATP followed by the subsequent metabolization of ADP to AMP and adenosine. As a result, the aforementioned studies argue against a considerable contribution by NTPDase1 and/or NTPDase2 and suggest that the observations would rather be compatible with a neuronal expression of NTPDase3.
Based on the present findings, it is tempting to speculate that an increase in the activity level of NTPDases (NTPDase3?) may result in the exhaustion of mitochondria (and the parent cell), whereas partial inhibition of NTPDases may be neuroprotective. Ongoing studies in our laboratory test this hypothesis. The reported pharmacological effects of polyoxometalates, such as suramin, seem to support this idea. For example, some data show that NTPDase inhibition is also antidiabetic [
37], although the exact mechanisms through which the beneficial effects of NTPDase inhibitors act are unknown. Therefore, the present results rise the possibility that in the pancreas, inhibition of NTPDases (NTPDase3) may protect the insulin-producing beta cells from overt ATP consumption and consequential exhaustion.