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 brzogekscitatornog neurotransmitera i kotransmitera ali i trofičkog faktora, gliotransmitera isignalnog molekula koji učestvuje u komunikaciji između ćelija CNS. Kada se oslobodi uvanćelijski prostor, npr. sinaptičku pukotinu i aktivira odgovarajuće receptore (P1 i P2),ATP se brzo degradira, posredstvom enzima ektonukleotidaza. Primarna uloga ovih enzimaje sekvencijalna hidroliza adeninskih nukleotida, kao što su ATP, adenozin-5-difosfat(ADP) i adenozin-5-monofosfat (AMP) čime nastaje nukleozid adenozin, potentnineuromodulator i homeostatski regulator u CNS. S obzirom na to da koncentracijevanćelijskog ATP i adenozina direktno zavise od stepena aktivnosti ektonukleotidaza,svaka promena ekspresije i aktivnosti ovih enzima može biti relevantna u kontekstufiziologije i patologije. Oskudni literaturni podaci ukazuju da su ektonukleotidaze deosložene molekulske mreže koja je pod uticajem polnih hormona. Kako ženski polnihormoni, pogotovo 17β-estradiol (E2), utiču na gustinu sinapsi, složenost dendritskihgrananja i broja trnolikih izraštaja u hipokampusu, ostvarajući tako snažan uticaj nasinaptičku plastičnost, učenje i pamćenje kod pacova oba pola, u ovoj tezi ispitan je uticajhormonskog statusa i mehanizam regulacije/modulacije sinaptičkih ektonukleotidaza, ektonukleozid5'-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 iekspresiju eN u totalnoj membranskoj frakciji hipokampusa oba pola.Rezultati biohemijskih analiza ukazuju da aktivnost ispitanih ektonukleotidazadiskretno fluktuira tokom estrusnog ciklusa, kao i da uklanjanje jajnika (OVX), primarnogizvora ženskih polnih hormona, smanjuje nivo hidrolize ATP, ADP, AMP u sinaptozomimahipokampusa ženki pacova. Ovi nalazi jasno ukazuju na to da su ektonukleotidaze (NTPDaza1-3 i eN) pod regulatornom kontrolom endogenih hormona jajnika. Promena stepenahidrolize adeninskih nukleotida uočena je kod OVX ženki nakon jednokratnog tretmana E2,pri čemu je stepen hidrolize ATP i ADP povećan verovatno kao rezultat povećaneekspresije 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 eNaktivacijom klasičnih unutarćelijskih estradiolskih receptora (ER i ERβ) koji različitimmehanizmima dovode do povećanja aktivnosti eN u sinaptozomima hipokampusa OVXženki.E2 moduliše vanćelijski metabolizam adeninskih nukleotida i u sinaptozomimahipokampusa mužjaka pacova. E2 smanjuje proteinsku ekspresiju i/ili aktivnost NTPD-aza1, 2 i eN, ali ne menja relativnu zastupljenost NTPD-aze 3 u sinaptozomima hipokampusamužjaka...Adenosine-5´-triphosphate (ATP) is an important extracellular signaling molecule. Itacts as neurotransmitter, co-transmitter, gliotransmitter and trophic factor in the centralnervous system (CNS). Upon the release, ATP modulates synaptic transmission by actingat either ionotropic (P2X) or metabotropic (P2Y) receptors, and is sequentially catabolizedby the action of ectonucleotidases. The first step of ATP inactivation is mediated by thefamily of ecto-nucleoside triphosphate diphosphohydrolase (NTPDase), which are able tohydrolyze 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 andhomeostatic regulator in the CNS. Since ectonucleotidases is crucial for maintaining ofphysiological levels of extracellular adenine nucleotides in the CNS, any alteration in theiractivity and expression can be relevant in context of physiology and pathology. Literaturedata indicate that ectonucleotidases may be part of complex molecular network which maybe modulated by ovarian steroids. Based on this findings, and the fact that female sexsteroids, particularly 17β-estradiol (E2) play an essential role in the modulation ofhippocampal synaptic plasticity therefore influencing hippocampal dependent learning andmemory, we hypothesized that steroid hormones, in particular E2 has potential to modulatethe activity and expression of ectonucleotidases (NTPDase 1-3 and eN) in hippocampalsynaptosomes of male and female rats. Also, the aim of this doctoral dissertation was todetermine the impact of steroid hormones on eN after repeated administration of 17α-estradiol, E2 and progesterone (P4) for seven consecutive days in hippocampus of bothsexes.It is shown that the activity of the examined ectonucleotidases fluctuates across theestrous cycle in the hippocampal synaptosomes of female rats, while significant reductionlevel of ATP, ADP and AMP hydrolysis is observed in hippocampal synaptosomes afterbilateral ovariectomy. These results clearly indicate that examined ectonucleotidases areregulated/modulated by endogenous female sex steroids. Biochemical analysis indicate thatacute E2 treatment in the OVX rats significantly increase the level of ATP and ADPhydrolysis, probably as a result of upregulated NTPDase 1 and 2, while E2 had no effect onNTPDase 3. The level of AMP hydrolysis was also augmented in hippocampalsynaptosomes of OVX rats after E2 treatment. Our initial evaluation imply that regulatoryE2 action at the activity and protein abundance of eN is mediated by both intracellularestradiol receptors (ERα and ERβ) through different mechanisms in hippocampalsynaptosomes of OVX rats..