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Glycine receptor in rat hippocampal and spinal cord neurons as a molecular target for rapid actions of 17-beta-estradiol.

Jiang P, Kong Y, Zhang XB, Wang W, Liu CF, Xu TL - Mol Pain (2009)

Bottom Line: Here we show that, in cultured rat hippocampal (HIP) and spinal dorsal horn (SDH) neurons, 17-beta-estradiol (E2) rapidly and reversibly reduced the peak amplitude of whole-cell glycine-activated currents (IGly).Moreover, the E2 effect on IGly persisted in the presence of the calcium chelator BAPTA, the protein kinase inhibitor staurosporine, the classical ER (i.e. ERalpha and ERbeta) antagonist tamoxifen, or the G-protein modulators, favoring a direct action of E2 on GlyRs.In HEK293 cells expressing various combinations of GlyR subunits, E2 only affected the IGly in cells expressing alpha2, alpha2beta or alpha3beta subunits, suggesting that either alpha2-containing or alpha3beta-GlyRs mediate the E2 effect observed in neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neuroscience and State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, PR China.

ABSTRACT
Glycine receptors (GlyRs) play important roles in regulating hippocampal neural network activity and spinal nociception. Here we show that, in cultured rat hippocampal (HIP) and spinal dorsal horn (SDH) neurons, 17-beta-estradiol (E2) rapidly and reversibly reduced the peak amplitude of whole-cell glycine-activated currents (IGly). In outside-out membrane patches from HIP neurons devoid of nuclei, E2 similarly inhibited IGly, suggesting a non-genomic characteristic. Moreover, the E2 effect on IGly persisted in the presence of the calcium chelator BAPTA, the protein kinase inhibitor staurosporine, the classical ER (i.e. ERalpha and ERbeta) antagonist tamoxifen, or the G-protein modulators, favoring a direct action of E2 on GlyRs. In HEK293 cells expressing various combinations of GlyR subunits, E2 only affected the IGly in cells expressing alpha2, alpha2beta or alpha3beta subunits, suggesting that either alpha2-containing or alpha3beta-GlyRs mediate the E2 effect observed in neurons. Furthermore, E2 inhibited the GlyR-mediated tonic current in pyramidal neurons of HIP CA1 region, where abundant GlyR alpha2 subunit is expressed. We suggest that the neuronal GlyR is a novel molecular target of E2 which directly inhibits the function of GlyRs in the HIP and SDH regions. This finding may shed new light on premenstrual dysphoric disorder and the gender differences in pain sensation at the CNS level.

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E2-induced inhibition of IGly is independent of intracellular signaling pathways and classical estrogen receptors (ERs). (A) Sample traces illustrating the inhibitory effects of E2 on the peak IGly under the conditions of intracellular application 15 mM BAPTA (A1), 5 μM staurosporine (A2), 0.5 mM GTP-γ-S (A3) and 0.5 mM GTP-β-S (A4), respectively. A5, Effect of E2 on IGly after incubation of neurons with tamoxifen for 2 h. A6, Effect of 17-α-E2 on IGly. (B) Pooled data summarizing the effect of E2 on IGly under various conditions shown in A. Each column represents the average values from 4–6 neurons, ***P < 0.001, Paired Student's t-test, compared with control without adding E2 or 17-α-E2 (dashed line). NS indicates no significant difference in this and the following figures.
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Figure 2: E2-induced inhibition of IGly is independent of intracellular signaling pathways and classical estrogen receptors (ERs). (A) Sample traces illustrating the inhibitory effects of E2 on the peak IGly under the conditions of intracellular application 15 mM BAPTA (A1), 5 μM staurosporine (A2), 0.5 mM GTP-γ-S (A3) and 0.5 mM GTP-β-S (A4), respectively. A5, Effect of E2 on IGly after incubation of neurons with tamoxifen for 2 h. A6, Effect of 17-α-E2 on IGly. (B) Pooled data summarizing the effect of E2 on IGly under various conditions shown in A. Each column represents the average values from 4–6 neurons, ***P < 0.001, Paired Student's t-test, compared with control without adding E2 or 17-α-E2 (dashed line). NS indicates no significant difference in this and the following figures.

Mentions: Previous studies have indicated that the acute effect of E2 occurring within a time course of milliseconds to minutes are attributed to the activation of intracellular signaling pathways mediated by presumably membrane-bound classical ERs [7,8,34,35] or novel ERs [9,10]. Additionally, E2 can modulate calcium channels and affect the intracellular calcium level [36,37]. To explore the possible involvement of any intracellular pathways in mediating E2 inhibition of IGly, following experiments were conducted. We first examined the role of the intracellular Ca2+. When neurons were loaded with 15 mM BAPTA via the recording pipette, E2 reduced the peak IGly to 62.7 ± 2.0% of the control, which was not significantly different from that obtained in the absence of BAPTA (Figure 2A1 and 2B1, P > 0.05, Unpaired Student's t-test). In order to test the role of protein phosphorylation and dephosphorylation in E2 inhibition, we loaded the neurons with staurosporine (5 μM), a nonselective protein kinase inhibitor, to disrupt the balance between phosphorylation and dephosphorylation. Likewise, the inhibitory effect of E2 on IGly was not altered (Figure 2A2 and 2C1, P > 0.05, Unpaired Student's t-test). A previous study [38] demonstrated that the GlyR is a target of the G protein βγ dimer. To examine the role of G proteins, we loaded the neurons with GTP-γ-S (500 μM) or GDP-β-S (500 μM) to activate or block the G protein, respectively. Neither of these treatments affected the inhibition induced by E2 on IGly (Figure 2A3, A4, and 2C, P > 0.05, Unpaired Student's t-test). Thus, it is unlikely that E2 exerts its inhibition on IGly through intracellular signaling pathways.


Glycine receptor in rat hippocampal and spinal cord neurons as a molecular target for rapid actions of 17-beta-estradiol.

Jiang P, Kong Y, Zhang XB, Wang W, Liu CF, Xu TL - Mol Pain (2009)

E2-induced inhibition of IGly is independent of intracellular signaling pathways and classical estrogen receptors (ERs). (A) Sample traces illustrating the inhibitory effects of E2 on the peak IGly under the conditions of intracellular application 15 mM BAPTA (A1), 5 μM staurosporine (A2), 0.5 mM GTP-γ-S (A3) and 0.5 mM GTP-β-S (A4), respectively. A5, Effect of E2 on IGly after incubation of neurons with tamoxifen for 2 h. A6, Effect of 17-α-E2 on IGly. (B) Pooled data summarizing the effect of E2 on IGly under various conditions shown in A. Each column represents the average values from 4–6 neurons, ***P < 0.001, Paired Student's t-test, compared with control without adding E2 or 17-α-E2 (dashed line). NS indicates no significant difference in this and the following figures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: E2-induced inhibition of IGly is independent of intracellular signaling pathways and classical estrogen receptors (ERs). (A) Sample traces illustrating the inhibitory effects of E2 on the peak IGly under the conditions of intracellular application 15 mM BAPTA (A1), 5 μM staurosporine (A2), 0.5 mM GTP-γ-S (A3) and 0.5 mM GTP-β-S (A4), respectively. A5, Effect of E2 on IGly after incubation of neurons with tamoxifen for 2 h. A6, Effect of 17-α-E2 on IGly. (B) Pooled data summarizing the effect of E2 on IGly under various conditions shown in A. Each column represents the average values from 4–6 neurons, ***P < 0.001, Paired Student's t-test, compared with control without adding E2 or 17-α-E2 (dashed line). NS indicates no significant difference in this and the following figures.
Mentions: Previous studies have indicated that the acute effect of E2 occurring within a time course of milliseconds to minutes are attributed to the activation of intracellular signaling pathways mediated by presumably membrane-bound classical ERs [7,8,34,35] or novel ERs [9,10]. Additionally, E2 can modulate calcium channels and affect the intracellular calcium level [36,37]. To explore the possible involvement of any intracellular pathways in mediating E2 inhibition of IGly, following experiments were conducted. We first examined the role of the intracellular Ca2+. When neurons were loaded with 15 mM BAPTA via the recording pipette, E2 reduced the peak IGly to 62.7 ± 2.0% of the control, which was not significantly different from that obtained in the absence of BAPTA (Figure 2A1 and 2B1, P > 0.05, Unpaired Student's t-test). In order to test the role of protein phosphorylation and dephosphorylation in E2 inhibition, we loaded the neurons with staurosporine (5 μM), a nonselective protein kinase inhibitor, to disrupt the balance between phosphorylation and dephosphorylation. Likewise, the inhibitory effect of E2 on IGly was not altered (Figure 2A2 and 2C1, P > 0.05, Unpaired Student's t-test). A previous study [38] demonstrated that the GlyR is a target of the G protein βγ dimer. To examine the role of G proteins, we loaded the neurons with GTP-γ-S (500 μM) or GDP-β-S (500 μM) to activate or block the G protein, respectively. Neither of these treatments affected the inhibition induced by E2 on IGly (Figure 2A3, A4, and 2C, P > 0.05, Unpaired Student's t-test). Thus, it is unlikely that E2 exerts its inhibition on IGly through intracellular signaling pathways.

Bottom Line: Here we show that, in cultured rat hippocampal (HIP) and spinal dorsal horn (SDH) neurons, 17-beta-estradiol (E2) rapidly and reversibly reduced the peak amplitude of whole-cell glycine-activated currents (IGly).Moreover, the E2 effect on IGly persisted in the presence of the calcium chelator BAPTA, the protein kinase inhibitor staurosporine, the classical ER (i.e. ERalpha and ERbeta) antagonist tamoxifen, or the G-protein modulators, favoring a direct action of E2 on GlyRs.In HEK293 cells expressing various combinations of GlyR subunits, E2 only affected the IGly in cells expressing alpha2, alpha2beta or alpha3beta subunits, suggesting that either alpha2-containing or alpha3beta-GlyRs mediate the E2 effect observed in neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neuroscience and State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, PR China.

ABSTRACT
Glycine receptors (GlyRs) play important roles in regulating hippocampal neural network activity and spinal nociception. Here we show that, in cultured rat hippocampal (HIP) and spinal dorsal horn (SDH) neurons, 17-beta-estradiol (E2) rapidly and reversibly reduced the peak amplitude of whole-cell glycine-activated currents (IGly). In outside-out membrane patches from HIP neurons devoid of nuclei, E2 similarly inhibited IGly, suggesting a non-genomic characteristic. Moreover, the E2 effect on IGly persisted in the presence of the calcium chelator BAPTA, the protein kinase inhibitor staurosporine, the classical ER (i.e. ERalpha and ERbeta) antagonist tamoxifen, or the G-protein modulators, favoring a direct action of E2 on GlyRs. In HEK293 cells expressing various combinations of GlyR subunits, E2 only affected the IGly in cells expressing alpha2, alpha2beta or alpha3beta subunits, suggesting that either alpha2-containing or alpha3beta-GlyRs mediate the E2 effect observed in neurons. Furthermore, E2 inhibited the GlyR-mediated tonic current in pyramidal neurons of HIP CA1 region, where abundant GlyR alpha2 subunit is expressed. We suggest that the neuronal GlyR is a novel molecular target of E2 which directly inhibits the function of GlyRs in the HIP and SDH regions. This finding may shed new light on premenstrual dysphoric disorder and the gender differences in pain sensation at the CNS level.

Show MeSH
Related in: MedlinePlus