Ecto-nucleoside triphosphate diphosphohydrolase 3 in the ventral and lateral hypothalamic area of female rats: morphological characterization and functional implications.
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 localization of NTPDase3; 2.) The effects of 17beta-estradiol on the expression level of hypothalamic NTPDase3; and 3.) The effects of NTPDase inhibition in hypothalamic synaptosomal preparations.


METHODS
Combined light- and electron microscopic analyses were carried out to characterize the cellular and subcellular localization of NTPDase3-immunoreactivity. The effects of 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 gamma-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 expression level of NTPDase3 in the ventrobasal and lateral hypothalamus. A single subcutaneous injection of estrogen differentially increased NTPDase3 expression in the medial and lateral parts of the hypothalamus, indicating that this enzyme likely plays region-specific roles in estrogen-dependent hypothalamic regulatory mechanisms. Determination of mitochondrial respiration rates with and without the inhibition of NTPDases confirmed the presence of NTPDases, including NTPDase3 in neuronal mitochondria and showed that blockade of mitochondrial NTPDase functions decreases state 3 mitochondrial respiration rate and total mitochondrial respiratory capacity.


CONCLUSIONS
Altogether, these results suggest the possibility that NTPDases, among them NTPDase3, may play an estrogen-dependent modulatory role in the regulation of intracellular availability of ATP needed for excitatory neuronal functions including neurotransmission.
Kiss
David S
DS
Department of Physiology & Biochemistry, Szent Istvan University Faculty of Veterinary Science, Budapest, Hungary. kiss.david@aotk.szie.hu
Zsarnovszky
Attila
A
Horvath
Krisztina
K
Gyorffy
Andrea
A
Bartha
Tibor
T
Hazai
Diana
D
Sotonyi
Peter
P
Somogyi
Virag
V
Frenyo
Laszlo V
LV
Diano
Sabrina
S
D000818:Animals; D015153:Blotting, Western; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D005968:Glutamate Decarboxylase / Q000032:analysis / Q000378:metabolism; D006706:Homeostasis / Q000502:physiology; D007026:Hypothalamic Area, Lateral / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D010100:Oxygen / Q000378:metabolism; D011755:Pyrophosphatases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012270:Ribosomes / Q000378:metabolism
D004958:Estradiol; D011755:Pyrophosphatases; C439652:nucleoside-triphosphate diphosphohydrolase 3; D005968:Glutamate Decarboxylase; D010100:Oxygen