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Distinct single cell signal transduction signatures in leukocyte subsets stimulated with khat extract, amphetamine-like cathinone, cathine or norephedrine.

Bredholt T, Ersvær E, Erikstein BS, Sulen A, Reikvam H, Aarstad HJ, Johannessen AC, Vintermyr OK, Bruserud Ø, Gjertsen BT - BMC Pharmacol Toxicol (2013)

Bottom Line: Cathinone, cathine and norephedrine resulted in unique signaling profiles, with B-lymphocytes and natural killer cells more responsive compared to T-lymphocytes and monocytes.Treatment with norephedrine resulted in significantly increased T-lymphocyte proliferation, whereas khat-extract reduced proliferation and induced cell death.This study suggests that protein modification-specific single-cell analysis of immune cells could unravel pharmacologic effects of amphetamines and amphetamine-like agents, and further could represent a valuable tool in elucidation of mechanism(s) of action of complex botanical extracts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Science, University of Bergen, Bergen, Norway.

ABSTRACT

Background: Amphetamine and amphetamine derivatives are suggested to induce an immunosuppressive effect. However, knowledge of how amphetamines modulate intracellular signaling pathways in cells of the immune system is limited. We have studied phosphorylation of signal transduction proteins (Akt, CREB, ERK1/2, NF-κB, c-Cbl, STAT1/3/5/6) and stress sensors (p38 MAPK, p53) in human leukocyte subsets following in vitro treatment with the natural amphetamine cathinone, the cathinone derivatives cathine and norephedrine, in comparison with a defined extract of the psychostimulating herb khat (Catha edulis Forsk.). Intracellular protein modifications in single cells were studied using immunostaining and flow cytometry, cell viability was determined by Annexin V-FITC/Propidium Iodide staining, and T-lymphocyte proliferation was measured by (3)H-thymidine incorporation.

Results: Cathinone, cathine and norephedrine generally reduced post-translational modifications of intracellular signal transducers in T-lymphocytes, B-lymphocytes, natural killer cells and monocytes, most prominently affecting c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), p38 MAPK (p-Thr180/p-Tyr182) and p53 (both total p53 protein and p-Ser15). In contrast, the botanical khat-extract induced protein phosphorylation of STAT1 (p-Tyr701), STAT6 (p-Tyr641), c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), NF-κB (p-Ser529), Akt (p-Ser473), p38 MAPK (p-Thr180/p-Tyr182), p53 (Ser15) as well as total p53 protein. Cathinone, cathine and norephedrine resulted in unique signaling profiles, with B-lymphocytes and natural killer cells more responsive compared to T-lymphocytes and monocytes. Treatment with norephedrine resulted in significantly increased T-lymphocyte proliferation, whereas khat-extract reduced proliferation and induced cell death.

Conclusions: Single-cell signal transduction analyses of leukocytes distinctively discriminated between stimulation with cathinone and the structurally similar derivatives cathine and norephedrine. Cathinone, cathine and norephedrine reduced phosphorylation of c-Cbl, ERK1/2, p38 MAPK and p53(Ser15), and norephedrine induced T-lymphocyte proliferation. Khat-extract induced protein phosphorylation of signal transducers, p38 MAPK and p53, followed by reduced cell proliferation and cell death. This study suggests that protein modification-specific single-cell analysis of immune cells could unravel pharmacologic effects of amphetamines and amphetamine-like agents, and further could represent a valuable tool in elucidation of mechanism(s) of action of complex botanical extracts.

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Signal transduction in B-lymphocytes. (A) Heatmap of statistical significant changes in protein-modification levels and total protein levels in B-lymphocytes. (B) Unsupervised hierarchical clustering analysis of the different stimuli and signal transduction proteins in B-lymphocytes following 15 minutes of stimulation.
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Figure 2: Signal transduction in B-lymphocytes. (A) Heatmap of statistical significant changes in protein-modification levels and total protein levels in B-lymphocytes. (B) Unsupervised hierarchical clustering analysis of the different stimuli and signal transduction proteins in B-lymphocytes following 15 minutes of stimulation.

Mentions: Fold change values based on median fluorescence intensity (MFI) measurements were used as basis for data analyses, and signal transduction signatures for the leukocyte subsets are displayed as heatmaps of statistical significant changes (Figure 1B, 2A, 3A, 4A), and as cluster analyses (Figure 1C, 2B, 3B, 4B). The levels of phosphorylated (p) signal transducer and activator of transcription (STAT) 5, p-CREB, and p53 acetylated (ac)-Lysine (Lys)382 were not significantly altered by any stimulus (Additional file 1: Table S1) and were excluded from the heatmaps (Figure 1B, 2A, 3A, 4A) but included in the cluster analyses of signaling responses (Figure 1C, 2B, 3B, 4B). Cathinone did not induce significant signaling responses at the concentration and exposure times tested.


Distinct single cell signal transduction signatures in leukocyte subsets stimulated with khat extract, amphetamine-like cathinone, cathine or norephedrine.

Bredholt T, Ersvær E, Erikstein BS, Sulen A, Reikvam H, Aarstad HJ, Johannessen AC, Vintermyr OK, Bruserud Ø, Gjertsen BT - BMC Pharmacol Toxicol (2013)

Signal transduction in B-lymphocytes. (A) Heatmap of statistical significant changes in protein-modification levels and total protein levels in B-lymphocytes. (B) Unsupervised hierarchical clustering analysis of the different stimuli and signal transduction proteins in B-lymphocytes following 15 minutes of stimulation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3733921&req=5

Figure 2: Signal transduction in B-lymphocytes. (A) Heatmap of statistical significant changes in protein-modification levels and total protein levels in B-lymphocytes. (B) Unsupervised hierarchical clustering analysis of the different stimuli and signal transduction proteins in B-lymphocytes following 15 minutes of stimulation.
Mentions: Fold change values based on median fluorescence intensity (MFI) measurements were used as basis for data analyses, and signal transduction signatures for the leukocyte subsets are displayed as heatmaps of statistical significant changes (Figure 1B, 2A, 3A, 4A), and as cluster analyses (Figure 1C, 2B, 3B, 4B). The levels of phosphorylated (p) signal transducer and activator of transcription (STAT) 5, p-CREB, and p53 acetylated (ac)-Lysine (Lys)382 were not significantly altered by any stimulus (Additional file 1: Table S1) and were excluded from the heatmaps (Figure 1B, 2A, 3A, 4A) but included in the cluster analyses of signaling responses (Figure 1C, 2B, 3B, 4B). Cathinone did not induce significant signaling responses at the concentration and exposure times tested.

Bottom Line: Cathinone, cathine and norephedrine resulted in unique signaling profiles, with B-lymphocytes and natural killer cells more responsive compared to T-lymphocytes and monocytes.Treatment with norephedrine resulted in significantly increased T-lymphocyte proliferation, whereas khat-extract reduced proliferation and induced cell death.This study suggests that protein modification-specific single-cell analysis of immune cells could unravel pharmacologic effects of amphetamines and amphetamine-like agents, and further could represent a valuable tool in elucidation of mechanism(s) of action of complex botanical extracts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Science, University of Bergen, Bergen, Norway.

ABSTRACT

Background: Amphetamine and amphetamine derivatives are suggested to induce an immunosuppressive effect. However, knowledge of how amphetamines modulate intracellular signaling pathways in cells of the immune system is limited. We have studied phosphorylation of signal transduction proteins (Akt, CREB, ERK1/2, NF-κB, c-Cbl, STAT1/3/5/6) and stress sensors (p38 MAPK, p53) in human leukocyte subsets following in vitro treatment with the natural amphetamine cathinone, the cathinone derivatives cathine and norephedrine, in comparison with a defined extract of the psychostimulating herb khat (Catha edulis Forsk.). Intracellular protein modifications in single cells were studied using immunostaining and flow cytometry, cell viability was determined by Annexin V-FITC/Propidium Iodide staining, and T-lymphocyte proliferation was measured by (3)H-thymidine incorporation.

Results: Cathinone, cathine and norephedrine generally reduced post-translational modifications of intracellular signal transducers in T-lymphocytes, B-lymphocytes, natural killer cells and monocytes, most prominently affecting c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), p38 MAPK (p-Thr180/p-Tyr182) and p53 (both total p53 protein and p-Ser15). In contrast, the botanical khat-extract induced protein phosphorylation of STAT1 (p-Tyr701), STAT6 (p-Tyr641), c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), NF-κB (p-Ser529), Akt (p-Ser473), p38 MAPK (p-Thr180/p-Tyr182), p53 (Ser15) as well as total p53 protein. Cathinone, cathine and norephedrine resulted in unique signaling profiles, with B-lymphocytes and natural killer cells more responsive compared to T-lymphocytes and monocytes. Treatment with norephedrine resulted in significantly increased T-lymphocyte proliferation, whereas khat-extract reduced proliferation and induced cell death.

Conclusions: Single-cell signal transduction analyses of leukocytes distinctively discriminated between stimulation with cathinone and the structurally similar derivatives cathine and norephedrine. Cathinone, cathine and norephedrine reduced phosphorylation of c-Cbl, ERK1/2, p38 MAPK and p53(Ser15), and norephedrine induced T-lymphocyte proliferation. Khat-extract induced protein phosphorylation of signal transducers, p38 MAPK and p53, followed by reduced cell proliferation and cell death. This study suggests that protein modification-specific single-cell analysis of immune cells could unravel pharmacologic effects of amphetamines and amphetamine-like agents, and further could represent a valuable tool in elucidation of mechanism(s) of action of complex botanical extracts.

Show MeSH
Related in: MedlinePlus