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Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions?

Blohberger J, Kunz L, Einwang D, Berg U, Berg D, Ojeda SR, Dissen GA, Fröhlich T, Arnold GJ, Soreq H, Lara H, Mayerhofer A - Cell Death Dis (2015)

Bottom Line: AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo.The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death.Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis.

View Article: PubMed Central - PubMed

Affiliation: Anatomy III - Cell Biology, Ludwig-Maximilian-University (LMU), Munich, Germany.

ABSTRACT
Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel approaches to influence ovarian functions.

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Related in: MedlinePlus

Monitoring of the ACh system in human GCs and evidence for AChE in FF and cultured GCs. (a–c) Change in confluence (relative to control) of cultured human GCs after 6, 12 and 24 h of treatment with different combinations of ACh (10 μM), HupA (10 μM), atropine (1 μM) and nicotine (10 μM). ACh initially shows a trophic effect on GCs but is degraded during 24 h of stimulation. HupA blocks ACh-degradation and the trophic ACh-effect remains visible after 24 h. Atropine is able to block the ACh-mediated effect and also decreases basal confluence change after 24 h. Nicotine was used as a control at 6 h and 24 h and shows no significant effect. Values are the mean±S.E.M. of n=3 independent preparations of cells pooled from two to five patients each. For each stimulation, a parallel control experiment was performed. Different letters indicate significant differences (P<0.05; analysis of variance). (d) AChE and BChE activity in FF, shown by the Ellman assay. Values are the mean±S.E.M. of n=15 FFs of different patients (Total: 521±45 mU/ml; BChE: 251±23 mU/ml; AChE: 251±26 mU/ml). (e) Identification of AChE protein in FF and a preadsorption western blotting experiment. Arrow indicates expected mass of protein (82 kDa). (f) Lysates of cultured human GCs possess AChE activity and low BChE activity in the Ellman assay. Values are the mean±S.E.M. of n=8 independent preparations of cells from two to five patients each (Total: 10±3 mU/mg; BChE: 0.7±0.3 mU/mg; AChE: 9±2 mU/mg)
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fig1: Monitoring of the ACh system in human GCs and evidence for AChE in FF and cultured GCs. (a–c) Change in confluence (relative to control) of cultured human GCs after 6, 12 and 24 h of treatment with different combinations of ACh (10 μM), HupA (10 μM), atropine (1 μM) and nicotine (10 μM). ACh initially shows a trophic effect on GCs but is degraded during 24 h of stimulation. HupA blocks ACh-degradation and the trophic ACh-effect remains visible after 24 h. Atropine is able to block the ACh-mediated effect and also decreases basal confluence change after 24 h. Nicotine was used as a control at 6 h and 24 h and shows no significant effect. Values are the mean±S.E.M. of n=3 independent preparations of cells pooled from two to five patients each. For each stimulation, a parallel control experiment was performed. Different letters indicate significant differences (P<0.05; analysis of variance). (d) AChE and BChE activity in FF, shown by the Ellman assay. Values are the mean±S.E.M. of n=15 FFs of different patients (Total: 521±45 mU/ml; BChE: 251±23 mU/ml; AChE: 251±26 mU/ml). (e) Identification of AChE protein in FF and a preadsorption western blotting experiment. Arrow indicates expected mass of protein (82 kDa). (f) Lysates of cultured human GCs possess AChE activity and low BChE activity in the Ellman assay. Values are the mean±S.E.M. of n=8 independent preparations of cells from two to five patients each (Total: 10±3 mU/mg; BChE: 0.7±0.3 mU/mg; AChE: 9±2 mU/mg)

Mentions: To study functional components of the ovarian ACh system, we monitored cell confluence of cultured human GCs, that is, the major cell population of large human ovarian follicles. An increase in confluence due to cell spreading and/or cell number was regarded as a trophic influence. Several independent experiments showed a significant increase in confluence after addition of ACh (10 μM; Figures 1a and b). After 24 h, no difference between ACh-treated and the control group was observed (Figure 1c), possibly because ACh becomes degraded. Indeed, when the AChE inhibitor huperzine A (HupA; 10 μM) was added to ACh, a significant increase of confluence resulted after 12 and 24 h. This suggests the presence of an intrinsic ACh degradation system in the cultures, which can be blocked. AChE may be produced by GCs or be present in the medium, which was supplemented with fetal calf serum (FCS). Supporting the prediction, the addition of HupA alone also increased confluence, as seen after 12 h and 24 h and suggested that endogenous ACh production and action are amenable for manipulation. Blockage of the muscarinic ACh receptors of GCs by atropine (1 μM) decreased confluence after 12 and 24 h. Simultaneous addition of ACh, HupA and atropine resulted in unchanged confluence levels compared with controls after 12 and 24 h. This indicates that the trophic ACh effect relies on the activation of muscarinic receptors. Nicotine (10 μM) was not able to induce trophic effects in GCs. Thus ACh exerts trophic actions via muscarinic receptors in human GCs. The use of HupA revealed that AChE normally restricts trophic ACh actions. HupA imbalances the cholinergic system of production and breakdown events, and this action results in a net trophic action of ACh.


Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions?

Blohberger J, Kunz L, Einwang D, Berg U, Berg D, Ojeda SR, Dissen GA, Fröhlich T, Arnold GJ, Soreq H, Lara H, Mayerhofer A - Cell Death Dis (2015)

Monitoring of the ACh system in human GCs and evidence for AChE in FF and cultured GCs. (a–c) Change in confluence (relative to control) of cultured human GCs after 6, 12 and 24 h of treatment with different combinations of ACh (10 μM), HupA (10 μM), atropine (1 μM) and nicotine (10 μM). ACh initially shows a trophic effect on GCs but is degraded during 24 h of stimulation. HupA blocks ACh-degradation and the trophic ACh-effect remains visible after 24 h. Atropine is able to block the ACh-mediated effect and also decreases basal confluence change after 24 h. Nicotine was used as a control at 6 h and 24 h and shows no significant effect. Values are the mean±S.E.M. of n=3 independent preparations of cells pooled from two to five patients each. For each stimulation, a parallel control experiment was performed. Different letters indicate significant differences (P<0.05; analysis of variance). (d) AChE and BChE activity in FF, shown by the Ellman assay. Values are the mean±S.E.M. of n=15 FFs of different patients (Total: 521±45 mU/ml; BChE: 251±23 mU/ml; AChE: 251±26 mU/ml). (e) Identification of AChE protein in FF and a preadsorption western blotting experiment. Arrow indicates expected mass of protein (82 kDa). (f) Lysates of cultured human GCs possess AChE activity and low BChE activity in the Ellman assay. Values are the mean±S.E.M. of n=8 independent preparations of cells from two to five patients each (Total: 10±3 mU/mg; BChE: 0.7±0.3 mU/mg; AChE: 9±2 mU/mg)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4385929&req=5

fig1: Monitoring of the ACh system in human GCs and evidence for AChE in FF and cultured GCs. (a–c) Change in confluence (relative to control) of cultured human GCs after 6, 12 and 24 h of treatment with different combinations of ACh (10 μM), HupA (10 μM), atropine (1 μM) and nicotine (10 μM). ACh initially shows a trophic effect on GCs but is degraded during 24 h of stimulation. HupA blocks ACh-degradation and the trophic ACh-effect remains visible after 24 h. Atropine is able to block the ACh-mediated effect and also decreases basal confluence change after 24 h. Nicotine was used as a control at 6 h and 24 h and shows no significant effect. Values are the mean±S.E.M. of n=3 independent preparations of cells pooled from two to five patients each. For each stimulation, a parallel control experiment was performed. Different letters indicate significant differences (P<0.05; analysis of variance). (d) AChE and BChE activity in FF, shown by the Ellman assay. Values are the mean±S.E.M. of n=15 FFs of different patients (Total: 521±45 mU/ml; BChE: 251±23 mU/ml; AChE: 251±26 mU/ml). (e) Identification of AChE protein in FF and a preadsorption western blotting experiment. Arrow indicates expected mass of protein (82 kDa). (f) Lysates of cultured human GCs possess AChE activity and low BChE activity in the Ellman assay. Values are the mean±S.E.M. of n=8 independent preparations of cells from two to five patients each (Total: 10±3 mU/mg; BChE: 0.7±0.3 mU/mg; AChE: 9±2 mU/mg)
Mentions: To study functional components of the ovarian ACh system, we monitored cell confluence of cultured human GCs, that is, the major cell population of large human ovarian follicles. An increase in confluence due to cell spreading and/or cell number was regarded as a trophic influence. Several independent experiments showed a significant increase in confluence after addition of ACh (10 μM; Figures 1a and b). After 24 h, no difference between ACh-treated and the control group was observed (Figure 1c), possibly because ACh becomes degraded. Indeed, when the AChE inhibitor huperzine A (HupA; 10 μM) was added to ACh, a significant increase of confluence resulted after 12 and 24 h. This suggests the presence of an intrinsic ACh degradation system in the cultures, which can be blocked. AChE may be produced by GCs or be present in the medium, which was supplemented with fetal calf serum (FCS). Supporting the prediction, the addition of HupA alone also increased confluence, as seen after 12 h and 24 h and suggested that endogenous ACh production and action are amenable for manipulation. Blockage of the muscarinic ACh receptors of GCs by atropine (1 μM) decreased confluence after 12 and 24 h. Simultaneous addition of ACh, HupA and atropine resulted in unchanged confluence levels compared with controls after 12 and 24 h. This indicates that the trophic ACh effect relies on the activation of muscarinic receptors. Nicotine (10 μM) was not able to induce trophic effects in GCs. Thus ACh exerts trophic actions via muscarinic receptors in human GCs. The use of HupA revealed that AChE normally restricts trophic ACh actions. HupA imbalances the cholinergic system of production and breakdown events, and this action results in a net trophic action of ACh.

Bottom Line: AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo.The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death.Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis.

View Article: PubMed Central - PubMed

Affiliation: Anatomy III - Cell Biology, Ludwig-Maximilian-University (LMU), Munich, Germany.

ABSTRACT
Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel approaches to influence ovarian functions.

Show MeSH
Related in: MedlinePlus