NTPDases in the neuroendocrine hypothalamus: possible energy regulators of the positive gonadotrophin feedback

Abstract

Background: Brain-derived ectonucleoside triphosphate diphosphohydrolases (NTPDases) have been known as plasma membrane-incorporated enzymes with their ATP-hydrolyzing domain outside of the cell. As such, these enzymes are thought to regulate purinergic intercellular signaling by hydrolyzing ATP to ADP-AMP, thus regulating the availability of specific ligands for various P2X and P2Y purinergic receptors. The role of NTPDases in the central nervous system is little understood. The two major reasons are the insufficient knowledge of the precise localization of these enzymes in neural structures, and the lack of specific inhibitors for the various NTPDases. To fill these gaps, we recently studied the presence of neuron-specific NTPDase3 in the mitochondria of hypothalamic excitatory neurons by morphological and functional methods. Results from those studies suggested that intramitochondrial regulation of ATP levels may play a permissive role in the neural regulation of physiological functions by tuning the level of ATP-carried energy that is needed for neuronal functions, such as neurotransmission and/or intracellular signaling.

Presentation of the hypothesis: In the lack of highly specific inhibitors, the determination of the precise function and role of NTPDases is hardly feasable. Yet, here we attempt to find an approach to investigate a possible role for hypothalamic NTPDase3 in the initiation of the midcycle luteinizing hormone (LH) surge, as such a biological role was implied by our recent findings. Here we hypothesize that NTPDase-activity in neurons of the AN may play a permissive role in the regulation of the estrogen-induced pituitary LH-surge.

Testing the hypothesis: We propose to test our hypothesis on ovariectomized rats, by stereotaxically injecting 17beta-estradiol and/or an NTPDase-inhibitor into the arcuate nucleus and determine the consequential levels of blood LH, mitochondrial respiration rates from arcuate nucleus synaptosomal preparations, NTPDase3-expression from arcuate nucleus tissue samples, all compared to sham and intact controls.

Implications of the hypothesis: Results from these studies may lead to the conclusion that estrogen may modulate the activity of mitochondrial, synapse-linked NTPDase3, and may show a correlation between mitochondrial NTPDase3-activity and the regulation of LH-release by estrogen.

Figure 1

Experimental design for testing the proposed hypothesis. Animals will be ovariectomized (ovx) 7 days prior to further experimentation. Seven days after ovariectomy, animals will receive intracerebral (i.c.) stereotaxic injections into the arcuate nucleus as follows: Group 1 will receive 17beta-estradiol (E₂) 20 minutes after initial injection of saline; Group 2 will receive E₂ 20 minutes after initial i.c. injection of suramin (NTPDase-inhibitor); Group 3 will receive two consecutive injections of physiological saline (sham control) with a timing presented above. An additional group of animals (Group 4) not subjected to i.c. injections will be investigated. Animals will be sacrificed 2-6-10-18 and 26 hours after the i.c. injections. Blood samples will be drawn from each individual and plasma LH will be determined. Brains will be quickly removed and the medial part of the hypothalami will be isolated. Tissue samples from left and right sides will be separately handled. Tissue samples will be homogenized and protein concentrations quickly determined. Based on sample protein concentrations, aliquotes will be prepared from tissue homogenates for further determination of NTPDase3-levels by Western blot analysis. Mitochondrial oxygen consumption (mitochondrial respiration rates, especially ADP-dependent state 3 respiration and total mitochondrial respiratory capacity) will be determined from synaptosomal preparations of tissue aliquotes without further inhibition (suramin) of the sample (i.e., the left-sided medial hypothalamus), or with further suramin-pretreatment of the contralateral (i.e., the right-sided) sample.