Metabolism of extracellular adenine nucleotides in rat hippocampal synaptosomes - sex differences and the roll of female sex steroids

Adenozin-5-trifosfat (ATP) u centralnom nervnom sistemu (CNS) ima ulogu brzog ekscitatornog neurotransmitera i kotransmitera ali i trofičkog faktora, gliotransmitera i signalnog molekula koji učestvuje u komunikaciji između ćelija CNS. Kada se oslobodi u vanćelijski prostor, npr. sinaptičku pukotinu i aktivira odgovarajuće receptore (P1 i P2), ATP se brzo degradira, posredstvom enzima ektonukleotidaza. Primarna uloga ovih enzima je sekvencijalna hidroliza adeninskih nukleotida, kao što su ATP, adenozin-5-difosfat (ADP) i adenozin-5-monofosfat (AMP) čime nastaje nukleozid adenozin, potentni neuromodulator i homeostatski regulator u CNS. S obzirom na to da koncentracije vanćelijskog ATP i adenozina direktno zavise od stepena aktivnosti ektonukleotidaza, svaka promena ekspresije i aktivnosti ovih enzima može biti relevantna u kontekstu fiziologije i patologije. Oskudni literaturni podaci ukazuju da su ektonukleotidaze deo složene molekulske mreže koja je pod uticajem polnih hormona. Kako ženski polni hormoni, pogotovo 17β-estradiol (E2), utiču na gustinu sinapsi, složenost dendritskih grananja i broja trnolikih izraštaja u hipokampusu, ostvarajući tako snažan uticaj na sinaptičku plastičnost, učenje i pamćenje kod pacova oba pola, u ovoj tezi ispitan je uticaj hormonskog statusa i mehanizam regulacije/modulacije sinaptičkih ektonukleotidaza, ektonukleozid 5'-trifosfat difosfohidrolaze 1-3 (NTPD-aza 1-3) i ekto-5'-nukleotidaze (eN) 17β- estradiolom u hipokampusu ženki i mužjaka pacova. Takođe, ispitan je uticaj ponovljenog (sedmodnevnog) tretmana 17-estradiolom (E), E2 i progesteronom (P4) na aktivnost i ekspresiju eN u totalnoj membranskoj frakciji hipokampusa oba pola. Rezultati biohemijskih analiza ukazuju da aktivnost ispitanih ektonukleotidaza diskretno fluktuira tokom estrusnog ciklusa, kao i da uklanjanje jajnika (OVX), primarnog izvora ženskih polnih hormona, smanjuje nivo hidrolize ATP, ADP, AMP u sinaptozomima hipokampusa ženki pacova. Ovi nalazi jasno ukazuju na to da su ektonukleotidaze (NTPDaza 1-3 i eN) pod regulatornom kontrolom endogenih hormona jajnika. Promena stepena hidrolize adeninskih nukleotida uočena je kod OVX ženki nakon jednokratnog tretmana E2, pri čemu je stepen hidrolize ATP i ADP povećan verovatno kao rezultat povećane ekspresije NTPD-aze 1 i 2, dok relativna zastupljenost NTPD-aze 3 ostaje nepromenjena. E2 takođe dovodi do porasta nivoa hidrolize AMP pri čemu reguliše/moduliše eN aktivacijom klasičnih unutarćelijskih estradiolskih receptora (ER i ERβ) koji različitim mehanizmima dovode do povećanja aktivnosti eN u sinaptozomima hipokampusa OVX ženki. E2 moduliše vanćelijski metabolizam adeninskih nukleotida i u sinaptozomima hipokampusa mužjaka pacova. E2 smanjuje proteinsku ekspresiju i/ili aktivnost NTPD-aza 1, 2 i eN, ali ne menja relativnu zastupljenost NTPD-aze 3 u sinaptozomima hipokampusa mužjaka...Adenosine-5´-triphosphate (ATP) is an important extracellular signaling molecule. It acts as neurotransmitter, co-transmitter, gliotransmitter and trophic factor in the central nervous system (CNS). Upon the release, ATP modulates synaptic transmission by acting at either ionotropic (P2X) or metabotropic (P2Y) receptors, and is sequentially catabolized by the action of ectonucleotidases. The first step of ATP inactivation is mediated by the family of ecto-nucleoside triphosphate diphosphohydrolase (NTPDase), which are able to hydrolyze ATP and adenosine-5´-diphosphate (ADP) to adenosine-5´-monophosphate (AMP). The last and the rate-limiting step in of the ATP conversion is catalyzed by ecto-5´- nucleotidase (eN), which hydrolases AMP to adenosine, potent neuromodulator and homeostatic regulator in the CNS. Since ectonucleotidases is crucial for maintaining of physiological levels of extracellular adenine nucleotides in the CNS, any alteration in their activity and expression can be relevant in context of physiology and pathology. Literature data indicate that ectonucleotidases may be part of complex molecular network which may be modulated by ovarian steroids. Based on this findings, and the fact that female sex steroids, particularly 17β-estradiol (E2) play an essential role in the modulation of hippocampal synaptic plasticity therefore influencing hippocampal dependent learning and memory, we hypothesized that steroid hormones, in particular E2 has potential to modulate the activity and expression of ectonucleotidases (NTPDase 1-3 and eN) in hippocampal synaptosomes of male and female rats. Also, the aim of this doctoral dissertation was to determine the impact of steroid hormones on eN after repeated administration of 17α- estradiol, E2 and progesterone (P4) for seven consecutive days in hippocampus of both sexes. It is shown that the activity of the examined ectonucleotidases fluctuates across the estrous cycle in the hippocampal synaptosomes of female rats, while significant reduction level of ATP, ADP and AMP hydrolysis is observed in hippocampal synaptosomes after bilateral ovariectomy. These results clearly indicate that examined ectonucleotidases are regulated/modulated by endogenous female sex steroids. Biochemical analysis indicate that acute E2 treatment in the OVX rats significantly increase the level of ATP and ADP hydrolysis, probably as a result of upregulated NTPDase 1 and 2, while E2 had no effect on NTPDase 3. The level of AMP hydrolysis was also augmented in hippocampal synaptosomes of OVX rats after E2 treatment. Our initial evaluation imply that regulatory E2 action at the activity and protein abundance of eN is mediated by both intracellular estradiol receptors (ERα and ERβ) through different mechanisms in hippocampal synaptosomes of OVX rats..