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Beneficial effects of estrogen in a mouse model of cerebrovascular insufficiency.

Kitamura N, Araya R, Kudoh M, Kishida H, Kimura T, Murayama M, Takashima A, Sakamaki Y, Hashikawa T, Ito S, Ohtsuki S, Terasaki T, Wess J, Yamada M - PLoS ONE (2009)

Bottom Line: Agonists of ERalpha, ERbeta, and GPR30 promoted ERK activation in this cell line.This phenotype was reversed by E2 treatment, similar to the observed deficits in dendrite morphology and the number of synapses.E2 may offer new therapeutic perspectives for the treatment of cerebrovascular insufficiency related memory dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Yamada Research Unit, RIKEN Brain Science Institute, Saitama, Japan.

ABSTRACT

Background: The M(5) muscarinic acetylcholine receptor is known to play a crucial role in mediating acetylcholine dependent dilation of cerebral blood vessels. Previously, we reported that male M(5) muscarinic acetylcholine knockout mice (M5R(-/-) mice) suffer from a constitutive constriction of cerebral arteries, reduced cerebral blood flow, dendritic atrophy, and short-term memory loss, without necrosis and/or inflammation in the brain.

Methodology/principal findings: We employed the Magnetic Resonance Angiography to study the area of the basilar artery in male and female M5R(-/-) mice. Here we show that female M5R(-/-) mice did not show the reduction in vascular area observed in male M5R(-/-) mice. However, ovariectomized female M5R(-/-) mice displayed phenotypic changes similar to male M5R(-/-) mice, strongly suggesting that estrogen plays a key role in the observed gender differences. We found that 17beta-estradiol (E2) induced nitric oxide release and ERK activation in a conditional immortalized mouse brain cerebrovascular endothelial cell line. Agonists of ERalpha, ERbeta, and GPR30 promoted ERK activation in this cell line. Moreover, in vivo magnetic resonance imaging studies showed that the cross section of the basilar artery was restored to normal in male M5R(-/-) mice treated with E2. Treatment with E2 also improved the performance of male M5R(-/-) mice in a cognitive test and reduced the atrophy of neural dendrites in the cerebral cortex and hippocampus. M5R(-/-) mice also showed astrocyte swelling in cortex and hippocampus using the three-dimensional reconstruction of electron microscope images. This phenotype was reversed by E2 treatment, similar to the observed deficits in dendrite morphology and the number of synapses.

Conclusions/significance: Our findings indicate that M5R(-/-) mice represent an excellent novel model system to study the beneficial effects of estrogen on cerebrovascular function and cognition. E2 may offer new therapeutic perspectives for the treatment of cerebrovascular insufficiency related memory dysfunction.

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Bethanechol and E2 induce ERK phosphorylation and NO production in TM-BBB cells in a time-dependent manner.(A) TM-BBB cells were stimulated with 100 µM of the muscarinic agonist, bethanechol (Bch), or 10 nM E2 for different periods of time (1–60 min), and cell lysates were analyzed by western blotting with anti-pERK or anti-total ERK antibodies. The cumulative results of Western blot analysis are reported from three independent experiments (10 min after treatment with Bch or E2). Values are means±SEM. *p<0.05 (vs vehicle control). (B) TM-BBB cells were stimulated with 100 µM Bch or 10 nM E2 for different time periods. Subsequently, nitrite levels were measured in the culture medium. Values are means±SEM. **p<0.001 (vs vehicle control).
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pone-0005159-g002: Bethanechol and E2 induce ERK phosphorylation and NO production in TM-BBB cells in a time-dependent manner.(A) TM-BBB cells were stimulated with 100 µM of the muscarinic agonist, bethanechol (Bch), or 10 nM E2 for different periods of time (1–60 min), and cell lysates were analyzed by western blotting with anti-pERK or anti-total ERK antibodies. The cumulative results of Western blot analysis are reported from three independent experiments (10 min after treatment with Bch or E2). Values are means±SEM. *p<0.05 (vs vehicle control). (B) TM-BBB cells were stimulated with 100 µM Bch or 10 nM E2 for different time periods. Subsequently, nitrite levels were measured in the culture medium. Values are means±SEM. **p<0.001 (vs vehicle control).

Mentions: 17β-estradiol (E2), the primary estrogen, has been reported to signal rapidly through ERK/MAP kinase [21]–[23] and PI3K/Akt [20], [24] to induce eNOS activity and NO generation in peripheral blood vessel derived cells [25], [26] and in neuroblastoma cells [27]. To examine the effect of estrogen on NO release in cerebral endothelial cells, we used the immortalized TM-BBB cell line, which is derived from mouse brain vascular endothelial cells [28] (See material and methods). TM-BBB cells were treated with bethanechol (Bch; 100 µM), a muscarinic receptor agonist, and E2 (10 nM) for 0–60 min. Bch and E2 induced the phosphorylation of ERK1/2 in a similar manner (Figure 2A,B). Moreover, both compounds stimulated NO production by ∼1.6-fold at the 180 min time point (Figure 2B). These results support the concept that both muscarinic and estrogen receptor signaling leads to enhanced NO production via activation of eNOS through ERK 1/2 pathways in TM-BBB cells [29]–[31].


Beneficial effects of estrogen in a mouse model of cerebrovascular insufficiency.

Kitamura N, Araya R, Kudoh M, Kishida H, Kimura T, Murayama M, Takashima A, Sakamaki Y, Hashikawa T, Ito S, Ohtsuki S, Terasaki T, Wess J, Yamada M - PLoS ONE (2009)

Bethanechol and E2 induce ERK phosphorylation and NO production in TM-BBB cells in a time-dependent manner.(A) TM-BBB cells were stimulated with 100 µM of the muscarinic agonist, bethanechol (Bch), or 10 nM E2 for different periods of time (1–60 min), and cell lysates were analyzed by western blotting with anti-pERK or anti-total ERK antibodies. The cumulative results of Western blot analysis are reported from three independent experiments (10 min after treatment with Bch or E2). Values are means±SEM. *p<0.05 (vs vehicle control). (B) TM-BBB cells were stimulated with 100 µM Bch or 10 nM E2 for different time periods. Subsequently, nitrite levels were measured in the culture medium. Values are means±SEM. **p<0.001 (vs vehicle control).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664330&req=5

pone-0005159-g002: Bethanechol and E2 induce ERK phosphorylation and NO production in TM-BBB cells in a time-dependent manner.(A) TM-BBB cells were stimulated with 100 µM of the muscarinic agonist, bethanechol (Bch), or 10 nM E2 for different periods of time (1–60 min), and cell lysates were analyzed by western blotting with anti-pERK or anti-total ERK antibodies. The cumulative results of Western blot analysis are reported from three independent experiments (10 min after treatment with Bch or E2). Values are means±SEM. *p<0.05 (vs vehicle control). (B) TM-BBB cells were stimulated with 100 µM Bch or 10 nM E2 for different time periods. Subsequently, nitrite levels were measured in the culture medium. Values are means±SEM. **p<0.001 (vs vehicle control).
Mentions: 17β-estradiol (E2), the primary estrogen, has been reported to signal rapidly through ERK/MAP kinase [21]–[23] and PI3K/Akt [20], [24] to induce eNOS activity and NO generation in peripheral blood vessel derived cells [25], [26] and in neuroblastoma cells [27]. To examine the effect of estrogen on NO release in cerebral endothelial cells, we used the immortalized TM-BBB cell line, which is derived from mouse brain vascular endothelial cells [28] (See material and methods). TM-BBB cells were treated with bethanechol (Bch; 100 µM), a muscarinic receptor agonist, and E2 (10 nM) for 0–60 min. Bch and E2 induced the phosphorylation of ERK1/2 in a similar manner (Figure 2A,B). Moreover, both compounds stimulated NO production by ∼1.6-fold at the 180 min time point (Figure 2B). These results support the concept that both muscarinic and estrogen receptor signaling leads to enhanced NO production via activation of eNOS through ERK 1/2 pathways in TM-BBB cells [29]–[31].

Bottom Line: Agonists of ERalpha, ERbeta, and GPR30 promoted ERK activation in this cell line.This phenotype was reversed by E2 treatment, similar to the observed deficits in dendrite morphology and the number of synapses.E2 may offer new therapeutic perspectives for the treatment of cerebrovascular insufficiency related memory dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Yamada Research Unit, RIKEN Brain Science Institute, Saitama, Japan.

ABSTRACT

Background: The M(5) muscarinic acetylcholine receptor is known to play a crucial role in mediating acetylcholine dependent dilation of cerebral blood vessels. Previously, we reported that male M(5) muscarinic acetylcholine knockout mice (M5R(-/-) mice) suffer from a constitutive constriction of cerebral arteries, reduced cerebral blood flow, dendritic atrophy, and short-term memory loss, without necrosis and/or inflammation in the brain.

Methodology/principal findings: We employed the Magnetic Resonance Angiography to study the area of the basilar artery in male and female M5R(-/-) mice. Here we show that female M5R(-/-) mice did not show the reduction in vascular area observed in male M5R(-/-) mice. However, ovariectomized female M5R(-/-) mice displayed phenotypic changes similar to male M5R(-/-) mice, strongly suggesting that estrogen plays a key role in the observed gender differences. We found that 17beta-estradiol (E2) induced nitric oxide release and ERK activation in a conditional immortalized mouse brain cerebrovascular endothelial cell line. Agonists of ERalpha, ERbeta, and GPR30 promoted ERK activation in this cell line. Moreover, in vivo magnetic resonance imaging studies showed that the cross section of the basilar artery was restored to normal in male M5R(-/-) mice treated with E2. Treatment with E2 also improved the performance of male M5R(-/-) mice in a cognitive test and reduced the atrophy of neural dendrites in the cerebral cortex and hippocampus. M5R(-/-) mice also showed astrocyte swelling in cortex and hippocampus using the three-dimensional reconstruction of electron microscope images. This phenotype was reversed by E2 treatment, similar to the observed deficits in dendrite morphology and the number of synapses.

Conclusions/significance: Our findings indicate that M5R(-/-) mice represent an excellent novel model system to study the beneficial effects of estrogen on cerebrovascular function and cognition. E2 may offer new therapeutic perspectives for the treatment of cerebrovascular insufficiency related memory dysfunction.

Show MeSH
Related in: MedlinePlus