Estradiol induces physical association of neuronal nitric oxide synthase with NMDA receptor and promotes nitric oxide formation via estrogen receptor activation in primary neuronal cultures

Abstract

Estrogens and nitric oxide (NO) exert wide-ranging effects on brain function. Recent evidence suggested that one important mechanism for the regulation of NO production may reside in the differential coupling of the calcium-activated neuronal NO synthase (nNOS) to glutamate NMDA receptor channels harboring NR2B subunits by the scaffolding protein post-synaptic density-95 (PSD-95), and that estrogens promote the formation of this ternary complex. Here, we demonstrate that 30-min estradiol-treatment triggers the production of NO by physically and functionally coupling NMDA receptors to nNOS in primary neurons of the rat preoptic region in vitro. The ability of estradiol to activate neuronal NO signaling in preoptic neurons and to promote changes in protein-protein interactions is blocked by ICI 182,780, an estrogen receptor antagonist. In addition, blockade of NMDA receptor NR2B subunit activity with ifenprodil or disruption of PSD-95 synthesis in preoptic neurons by treatment with an anti-sense oligodeoxynucleotide inhibited the estradiol-promoted stimulation of NO release in cultured preoptic neurons. Thus, estrogen receptor-mediated stimulation of the nNOS/PSD-95/NMDA receptor complex assembly is likely to be a critical component of the signaling process by which estradiol facilitates coupling of glutamatergic fluxes for NO production in neurons.

Figure 1

Neuronal NOS and NMDA receptor subunit NR2B are expressed and interact physically in a subset of hypothalamic neurons of the preoptic region in vitro. A, Confocal microscopy image illustrating the co-localization of NR2B (green) and nNOS (red) immunofluorescent stainings in cultured neurons derived from the preoptic region from new born rats. Note the expression of the NR2B immunoreactive material both at soma (arrow) and dendrites (arrowheads) in nNOS neurons in vitro. Scale bar: 20 μm. B–C, Neuronal NOS, NR2B and PSD-95 coimmunoprecipitate from primary cultures of preoptic neurons. Solubilized rat preoptic region primary culture homogenates were immunoprecipitated (IPP) with an antibody to nNOS (B) or PSD-95 (C). Starting material before IPP, immunoprecipitated proteins and depleted fraction after IPP were analyzed by immunoblotting (IB) for NR2B. Then immunoblots were stripped and reprobed with antibodies to nNOS and/or PSD-95. Beads alone incubated with proteins extracts were used as a negative control. D, Absence of detectable NR2A protein expression in primary cultures of preoptic neurons as assessed by straight Western blot analyses. Protein extracts from the adult preoptic region (POA) served as a positive control, and the effective amount of protein loaded is represented by NR2B blot. In B–D, all experiments were performed at least in tree independent cultures.

Figure 2

Estradiol promotes nNOS/NR2B complex formation in neurons of the preoptic region in vitro. Treatment of preoptic region neuronal cultures with 17-b-estradiol (E2) results in an increased physical association of nNOS with NR2B. Neurons were cultured in neuronal defined medium and were exposed to E2 1 nM for 30 min or 48 h or to ethanol for control cultures (CTL). Upper panel, 500 μg of proteins per condition were immunoprecipitated (IPP) with a specific nNOS antibody, electrophoresed and immunobloted (IB) for NR2B. Then the immunoblot was stripped and reprobed with antibodies to nNOS to ensure that equal amounts of nNOS protein had been immunoprecipitated and loaded on the gel. Lower panel, bar graph showing the quantitative analysis of the differential NR2B/nNOS association among E₂ treatment (n = 4 independent experiments; * p = 0.025 vs CTL). Error bars indicate SEM. Statistical differences were established using a one way ANOVA by the Student-Newman-Keuls multiple comparison test.

Figure 3

Estradiol stimulates NOS catalytic activity in hypothalamic preoptic neurons in vitro. Both estradiol-stimulated NO formation and nNOS/NR2B complex formation requires estrogen receptor activation in cultured neurons of the preoptic region. Neurons were treated with 17-b-estradiol (E2) 1 nM for 30 min and exposed to ICI 182,780 1 μM, a pure estrogen receptor antagonist, or to L-NAME 1 mM, a NOS inhibitor, for 30 min before this treatment. A, An example of fluorescent images of purified neurons loaded with the NO-sensitive dye DAF-FM (arrows) in control- (CTL) 17-b-estradiol- and 17-b-estradiol plus ICI 182,780-treated conditions (E2 + ICI). Scale bar, 40 μm. B, Summary graph showing that the pure estrogen receptor inhibitor ICI 182,780 and the NOS inhibitor L-NAME significantly reduced the amplitude of 17-b-estradiol-induced DAF fluorescent increase in neurons. ***, p < 0.001. Statistical differences were established using a one way ANOVA by the Student-Newman- Keuls multiple comparison test; n = 6–8 independent observations. C, ICI 182,780 Inhibits 17-b-estradiol-stimulated NR2B/nNOS complex formation in neurons of the rat preoptic region in culture. After treatment proteins were extracted and processed as indicated above in Figure 2. IPP, immunoprecipitation; IB, immunoblot.

Figure 4

Estradiol requires both NR2B-containing NMDA receptors activity and PSD-95 expression to mediate its stimulatory effect on NO production in hypothalamic preoptic neurons in vitro. Neurons were treated with 17-b-estradiol (E2) 1 nM for 30 min and exposed to ifenprodil (3 and 10 μM), a NR2B antagonist, or PSD-95 antisense ODNs 5 μM for 30 min or 7 d, respectively, before this treatment. Statistical differences were established using a one way ANOVA by the Student-Newman-Keuls multiple comparison test. **, p < 0.01, n = 4–6 independent observations.

Figure 5

Schematic representation of the possible estradiol-mediated changes in proteinprotein interactions involved in the control of nNOS activity in neurons of the preoptic region. Neuronal NOS activity is primarily regulated by increases in the local intracellular [Ca²⁺], which activates nNOS through calmoduling (CaM) binding (Bredt and Snyder 1990). Importantly, Ca²⁺ influx through the NMDA receptor (NMDA-R) but not other Ca²⁺ influx pathways efficiently promotes NO synthesis (Garthwaite et al. 1988; Bredt and Snyder 1990) One estrogen receptor (ER)-dependent mechanism used for the regulation of nNOS activity may reside in the alternative coupling and uncoupling of the enzyme to NR2B-containing NMDA receptors channels by the scaffolding protein PSD-95, in the presence or the absence of estrogens, respectively. PSD-95 acts as an adaptor protein, thereby physically and functionally coupling NMDA receptors to nNOS and thus enables to couple glutamatergic fluxes with NO production. NO is formed enzymatically from L-arginine (L-Arg) in equimolar amounts with L-citrulline (L-Cit) by nNOS. Functional glutamate NMDA receptors are composed of NR1 and NR2 subunits (Hollmann and Heinemann 1994). The estrogen receptor antagonist ICI 182,780 competitively inhibits binding of estradiol to the estrogen receptor and impairs receptor dimerisation (Osborne et al. 2004); Antisense oligodeoxynucleotides to PSD-95 (AS PSD-95) selectively decreases PSD-95 expression in primary cultures of preoptic neurons (d’Anglemont de Tassigny et al. 2007); infenprodil is a specific antagonist of NR2B-containing NMDA receptors (Williams 1993).