Background: Based on its distribution in the brain, ecto-nucleoside triphosphate
diphosphohydrolase 3 (NTPDase3) may play a role in the hypothalamic regulation of
homeostatic systems, including feeding, sleep-wake behavior and reproduction. To further
characterize the morphological attributes of NTPDase3-immunoreactive (IR) hypothalamic
structures in the rat brain, here we investigated: 1.) The cellular and subcellular
NTPDase3; 2.) The effects of 17-estradiol on the expression level of hypothalamic
NTPDase3; and 3.) The effects of NTPDase inhibition in hypothalamic synaptosomal
Methods: Combined light- and electron microscopic analyses were carried out to characterize
the cellular and subcellular localization of NTPDase3-immunoreactivity. The effects
estrogen on hypothalamic NTPDase3 expression was studied by western blot technique.
Finally, the effects of NTPDase inhibition on mitochondrial respiration were investigated
using a Clark-type oxygen electrode.
Results: Combined light- and electron microscopic analysis of immunostained hypothalamic
slices revealed that NTPDase3-IR is linked to ribosomes and mitochondria, is predominantly
present in excitatory axon terminals and in distinct segments of the perikaryal plasma
membrane. Immunohistochemical labeling of NTPDase3 and glutamic acid decarboxylase
(GAD) indicated that -amino-butyric-acid- (GABA) ergic hypothalamic neurons do not
express NTPDase3, further suggesting that in the hypothalamus, NTPDase3 is predominantly
present in excitatory neurons. We also investigated whether estrogen influences the
level of NTPDase3 in the ventrobasal and lateral hypothalamus. A single subcutaneous
injection of estrogen differentially increased NTPDase3 expression in the medial and
parts of the hypothalamus, indicating that this enzyme likely plays region-specific
estrogen-dependent hypothalamic regulatory mechanisms. Determination of mitochondrial
respiration rates with and without the inhibition of NTPDases confirmed the presence
NTPDases, including NTPDase3 in neuronal mitochondria and showed that blockade of
mitochondrial NTPDase functions decreases state 3 mitochondrial respiration rate and
mitochondrial respiratory capacity.
Conclusions: Altogether, these results suggest the possibility that NTPDases, among
NTPDase3, may play an estrogen-dependent modulatory role in the regulation of intracellular
availability of ATP needed for excitatory neuronal functions including neurotransmission.