Assay to measure sphingomyelinase and ceramidase activities efficiently and safely

Mühle, Christiane; Kornhuber, Johannes

doi:10.1016/j.chroma.2016.12.033

Cited by 32 publications

(23 citation statements)

References 37 publications

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“…Protein concentrations were determined using a bicinchoninic acid assay (Smith et al ; Wiechelman et al ). ASM activity assay was conducted as previously described (Reichel et al ; Mühle and Kornhuber ). Briefly, 1 μg of protein was incubated with 0.58 μM N‐(4,4‐difluoro‐5,7‐dimethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene‐3‐dodecanoyl)‐sphingosyl phosphocholine (BODIPY FL‐C12‐sphingomyelin) in 50 μL reaction buffer (50 mM sodium acetate pH 5.0, 0.3 M NaCl, 0.2% NP‐40) for 1 h at 37°C; after incubation, 3 μL of the reaction volume was spotted on a silica gel 60 Å plate, and ceramide and sphingomyelin were separated by thin layer chromatography using 99% ethyl acetate/1% acetic acid (v/v) as a solvent.…”

Section: Methodsmentioning

confidence: 99%

Acid sphingomyelinase – a regulator of canonical transient receptor potential channel 6 (TRPC6) activity

Zeitler

Lian

Andreyeva

et al. 2019

Journal of Neurochemistry

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Recent investigations propose the acid sphingomyelinase (ASM)/ceramide system as a novel target for antidepressant action. ASM catalyzes the breakdown of the abundant membrane lipid sphingomyelin to the lipid messenger ceramide. This ASM‐induced lipid modification induces a local shift in membrane properties, which influences receptor clustering and downstream signaling. Canonical transient receptor potential channels 6 (TRPC6) are non‐selective cation channels located in the cell membrane that play an important role in dendritic growth, synaptic plasticity and cognition in the brain. They can be activated by hyperforin, an ingredient of the herbal remedy St. John’s wort for treatment of depression disorders. Because of their role in the context of major depression, we investigated the crosstalk between the ASM/ceramide system and TRPC6 ion channels in a pheochromocytoma cell line 12 neuronal cell model (PC12 rat pheochromocytoma cell line). Ca2+ imaging experiments indicated that hyperforin‐induced Ca2+ influx through TRPC6 channels is modulated by ASM activity. While antidepressants, known as functional inhibitors of ASM activity, reduced TRPC6‐mediated Ca2+ influx, extracellular application of bacterial sphingomyelinase rebalanced TRPC6 activity in a concentration‐related way. This effect was confirmed in whole‐cell patch clamp electrophysiology recordings. Lipidomic analyses revealed a decrease in very long chain ceramide/sphingomyelin molar ratio after ASM inhibition, which was connected with changes in the abundance of TRPC6 channels in flotillin‐1–positive lipid rafts as visualized by western blotting. Our data provide evidence that the ASM/ceramide system regulates TRPC6 channels likely by controlling their recruitment to specific lipid subdomains and thereby fine‐tuning their physical properties.

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Section: Methodsmentioning

confidence: 99%

Acid sphingomyelinase – a regulator of canonical transient receptor potential channel 6 (TRPC6) activity

Zeitler

Lian

Andreyeva

et al. 2019

Journal of Neurochemistry

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“…The activity of S-ASM was quantified using the fluorescent substrate BODIPY-FL-C12-SM ( N -(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)sphingosyl phosphocholine, D-7711, Thermo Fisher Scientific, Waltham, MA, USA), as described previously [67]. Briefly, the reaction was performed in 96-well polystyrene plates with 58 pmol sphingomyelin in a reaction buffer totaling 50 µL in volume, in the following composition: 200 mM sodium acetate buffer (pH 5.0), 500 mM NaCl, 0.2% IGEPAL® CA-630 (NP 40), and 500 µM ZnCl 2 .…”

Section: Methodsmentioning

confidence: 99%

Peripheral Acid Sphingomyelinase Activity Is Associated with Biomarkers and Phenotypes of Alcohol Use and Dependence in Patients and Healthy Controls

Mühle

Weinland

Gulbins

et al. 2018

IJMS

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By catalyzing the hydrolysis of sphingomyelin into ceramide, acid sphingomyelinase (ASM) changes the local composition of the plasma membrane with effects on receptor-mediated signaling. Altered enzyme activities have been noted in common human diseases, including alcohol dependence. However, the underlying mechanisms remain largely unresolved. Blood samples were collected from early-abstinent alcohol-dependent in-patients (n[♂] = 113, n[♀] = 87) and matched healthy controls (n[♂] = 133, n[♀] = 107), and analyzed for routine blood parameters and serum ASM activity. We confirmed increased secretory ASM activities in alcohol-dependent patients compared to healthy control subjects, which decreased slightly during detoxification. ASM activity correlated positively with blood alcohol concentration, withdrawal severity, biomarkers of alcohol dependence (liver enzyme activities of gamma-glutamyl transferase, alanine aminotransferase, aspartate aminotransferase; homocysteine, carbohydrate-deficient transferrin; mean corpuscular volume, and creatine kinase). ASM activity correlated negatively with leukocyte and thrombocyte counts. ASM and gamma-glutamyl transferase were also associated in healthy subjects. Most effects were similar for males and females with different strengths. We describe previously unreported associations between ASM activity and markers of liver damage and myelosuppression. Further research should investigate whether this relationship is causal, or whether these parameters are part of a common pathway in order to gain insights into underlying mechanisms and develop clinical applications.

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“…The protein concentrations were determined using a bicinchoninic acid kit (Sigma, Darmstadt, Germany). For the determination of Asm activity, 1 µg of protein was incubated with 0.58 µM N-(4,4-difluoro-5,7-dimethyl-4bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-sphingosylphosphocholine (BODIPY®FL C 12 -sphingomyelin; D-7711; Life Technologies, Darmstadt, Germany) in a 50 µL reaction buffer (50 mM sodium acetate pH 5.0, 0.3 M NaCl, and 0.2% NP-40) for 2 h at 37 • C; after incubation, 3 µL of the reaction mixture was spotted on a silica gel 60 plate (Macherey-Nagel; Düren, Germany), and the spots of ceramide and sphingomyelin were separated by thin-layer chromatography using 99% ethyl acetate/1% acetic acid (v/v) as a solvent [25]. The intensities of the BODIPY-conjugated ceramide and sphingomyelin fractions were determined using a Typhoon Trio scanner (GE Healthcare, München, Germany) and quantified with QuantityOne software (BioRad, München, Germany).…”

Section: Determination Of Asm Activity In Vitromentioning

confidence: 99%

The Forebrain-Specific Overexpression of Acid Sphingomyelinase Induces Depressive-Like Symptoms in Mice

Zoicas

Schumacher

Kleuser

et al. 2020

Cells

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Human and murine studies identified the lysosomal enzyme acid sphingomyelinase (ASM) as a target for antidepressant therapy and revealed its role in the pathophysiology of major depression. In this study, we generated a mouse model with overexpression of Asm (Asm-tgfb) that is restricted to the forebrain to rule out any systemic effects of Asm overexpression on depressive-like symptoms. The increase in Asm activity was higher in male Asm-tgfb mice than in female Asm-tgfb mice due to the breeding strategy, which allows for the generation of wild-type littermates as appropriate controls. Asm overexpression in the forebrain of male mice resulted in a depressive-like phenotype, whereas in female mice, Asm overexpression resulted in a social anxiogenic-like phenotype. Ceramides in male Asm-tgfb mice were elevated specifically in the dorsal hippocampus. mRNA expression analyses indicated that the increase in Asm activity affected other ceramide-generating pathways, which might help to balance ceramide levels in cortical brain regions. This forebrain-specific mouse model offers a novel tool for dissecting the molecular mechanisms that play a role in the pathophysiology of major depression.

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Assay to measure sphingomyelinase and ceramidase activities efficiently and safely

Cited by 32 publications

References 37 publications

Acid sphingomyelinase – a regulator of canonical transient receptor potential channel 6 (TRPC6) activity

Acid sphingomyelinase – a regulator of canonical transient receptor potential channel 6 (TRPC6) activity

Peripheral Acid Sphingomyelinase Activity Is Associated with Biomarkers and Phenotypes of Alcohol Use and Dependence in Patients and Healthy Controls

The Forebrain-Specific Overexpression of Acid Sphingomyelinase Induces Depressive-Like Symptoms in Mice

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