Vanćelijski metabolizam adeninskih nukleotida u sinaptozomima hipokampusa pacova - polne specifičnosti i uloga ženskih polnih hormona
Metabolism of extracellular adenine nucleotides in rat hippocampal synaptosomes - sex differences and the roll of female sex steroids
Author
Mitrović, Nataša Lj.
Mentor
Grković, Ivana
Committee members
Nedeljković, Nadežda
Bjelobaba, Ivana

Vujović, Predrag
Adžić, Miroslav

Metadata
Show full item recordAbstract
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. Ka...ko 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 rel...evant 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...