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Efficient entry of cell-penetrating peptide nona-arginine into adherent cells involves a transient increase in intracellular calcium.

Melikov K, Hara A, Yamoah K, Zaitseva E, Zaitsev E, Chernomordik LV - Biochem. J. (2015)

Bottom Line: We found that a similar entry pathway is induced at 1-2 μM concentrations of R9 if peptide application is accompanied by a rapid temperature drop to 15°C.Intriguingly, we found that R9 at 10-20 μM and 37°C induces repetitive spikes in intracellular Ca(2+) concentration.To conclude, we uncovered a novel mechanistic link between calcium signalling and entry of cationic peptides.

View Article: PubMed Central - PubMed

Affiliation: Section on Membrane Biology, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Building 10/Room 10D05, 10 Center Drive, Bethesda, MD 20892-1855, U.S.A. melikovk@mail.nih.gov.

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An increase in intracellular calcium concentration by flufenamic acid induces efficient entry of R9-TAMRA into the cytosol and nucleus at 37°C(A) Cells were transferred into 37°C buffer containing 2 μM R9-TAMRA. Flufenamic acid (100 μM) was added either together with the peptide (NEFA) or 5 min later (NEFA, 5 min) and drug was not added to control cells (control). In all cases, cells were incubated for 15 min in the presence of the peptide before washing and imaging. Results of all individual experiments are shown as dots. Means for experiments together with 95% confidence intervals are denoted by points with error bars. (B) Representative fluorescence signal time traces from individual cells pre-loaded with calcium indicator dye are shown. Traces are shifted vertically for clarity. Cells were imaged live at 37°C and 100 μM flufenamic acid was added approximately 30 s after the start of the imaging (shown by black arrows). R9-TAMRA (2 μM) was added together with the flufenamic acid (bottom trace) or 5 min before application of the drug.
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Figure 10: An increase in intracellular calcium concentration by flufenamic acid induces efficient entry of R9-TAMRA into the cytosol and nucleus at 37°C(A) Cells were transferred into 37°C buffer containing 2 μM R9-TAMRA. Flufenamic acid (100 μM) was added either together with the peptide (NEFA) or 5 min later (NEFA, 5 min) and drug was not added to control cells (control). In all cases, cells were incubated for 15 min in the presence of the peptide before washing and imaging. Results of all individual experiments are shown as dots. Means for experiments together with 95% confidence intervals are denoted by points with error bars. (B) Representative fluorescence signal time traces from individual cells pre-loaded with calcium indicator dye are shown. Traces are shifted vertically for clarity. Cells were imaged live at 37°C and 100 μM flufenamic acid was added approximately 30 s after the start of the imaging (shown by black arrows). R9-TAMRA (2 μM) was added together with the flufenamic acid (bottom trace) or 5 min before application of the drug.

Mentions: Mechanistic dependence of the R9 entry on calcium signalling suggested that drugs known to release calcium from intracellular stores may lower concentration of the R9-TAMRA required for its efficient entry at 37°C. We treated the cells with flufenamic acid, a member of fenamate class of non-steroidal anti-inflammatory drugs, which have been shown to release calcium form mitochondria [32,33]. Since, in our experiments, we consistently observed a several minute delay between addition of 10 μM R9-TAMRA and the onset of calcium spikes (Figure 5B; Supplementary Video S2), we applied flufenamic acid to cells either together with 2 μM R9-TAMRA or 5 min after the peptide addition. We observed no effect on the entry of R9-TAMRA when flufenamic acid was added together with the peptide (Figure 10A). In contrast, application of flufenamic acid 5 min after peptide addition resulted in efficient entry of R9-TAMRA into the cytosol and nucleus of many cells (Figure 10A). Interestingly, flufenamic acid applied either with or before the peptide induced an increase in intracellular calcium concentration (Figure 10B). However, whereas addition of drug 5 min after peptide application resulted in a sustained increase in calcium concentration (Figure 10B, top trace), in the case of simultaneous application of flufenamic acid and the peptide, the calcium level rapidly returned to background levels (Figure 10B, bottom trace). We conclude that efficient entry of R9-TAMRA can be promoted by a drug-induced increase in intracellular calcium concentration.


Efficient entry of cell-penetrating peptide nona-arginine into adherent cells involves a transient increase in intracellular calcium.

Melikov K, Hara A, Yamoah K, Zaitseva E, Zaitsev E, Chernomordik LV - Biochem. J. (2015)

An increase in intracellular calcium concentration by flufenamic acid induces efficient entry of R9-TAMRA into the cytosol and nucleus at 37°C(A) Cells were transferred into 37°C buffer containing 2 μM R9-TAMRA. Flufenamic acid (100 μM) was added either together with the peptide (NEFA) or 5 min later (NEFA, 5 min) and drug was not added to control cells (control). In all cases, cells were incubated for 15 min in the presence of the peptide before washing and imaging. Results of all individual experiments are shown as dots. Means for experiments together with 95% confidence intervals are denoted by points with error bars. (B) Representative fluorescence signal time traces from individual cells pre-loaded with calcium indicator dye are shown. Traces are shifted vertically for clarity. Cells were imaged live at 37°C and 100 μM flufenamic acid was added approximately 30 s after the start of the imaging (shown by black arrows). R9-TAMRA (2 μM) was added together with the flufenamic acid (bottom trace) or 5 min before application of the drug.
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Figure 10: An increase in intracellular calcium concentration by flufenamic acid induces efficient entry of R9-TAMRA into the cytosol and nucleus at 37°C(A) Cells were transferred into 37°C buffer containing 2 μM R9-TAMRA. Flufenamic acid (100 μM) was added either together with the peptide (NEFA) or 5 min later (NEFA, 5 min) and drug was not added to control cells (control). In all cases, cells were incubated for 15 min in the presence of the peptide before washing and imaging. Results of all individual experiments are shown as dots. Means for experiments together with 95% confidence intervals are denoted by points with error bars. (B) Representative fluorescence signal time traces from individual cells pre-loaded with calcium indicator dye are shown. Traces are shifted vertically for clarity. Cells were imaged live at 37°C and 100 μM flufenamic acid was added approximately 30 s after the start of the imaging (shown by black arrows). R9-TAMRA (2 μM) was added together with the flufenamic acid (bottom trace) or 5 min before application of the drug.
Mentions: Mechanistic dependence of the R9 entry on calcium signalling suggested that drugs known to release calcium from intracellular stores may lower concentration of the R9-TAMRA required for its efficient entry at 37°C. We treated the cells with flufenamic acid, a member of fenamate class of non-steroidal anti-inflammatory drugs, which have been shown to release calcium form mitochondria [32,33]. Since, in our experiments, we consistently observed a several minute delay between addition of 10 μM R9-TAMRA and the onset of calcium spikes (Figure 5B; Supplementary Video S2), we applied flufenamic acid to cells either together with 2 μM R9-TAMRA or 5 min after the peptide addition. We observed no effect on the entry of R9-TAMRA when flufenamic acid was added together with the peptide (Figure 10A). In contrast, application of flufenamic acid 5 min after peptide addition resulted in efficient entry of R9-TAMRA into the cytosol and nucleus of many cells (Figure 10A). Interestingly, flufenamic acid applied either with or before the peptide induced an increase in intracellular calcium concentration (Figure 10B). However, whereas addition of drug 5 min after peptide application resulted in a sustained increase in calcium concentration (Figure 10B, top trace), in the case of simultaneous application of flufenamic acid and the peptide, the calcium level rapidly returned to background levels (Figure 10B, bottom trace). We conclude that efficient entry of R9-TAMRA can be promoted by a drug-induced increase in intracellular calcium concentration.

Bottom Line: We found that a similar entry pathway is induced at 1-2 μM concentrations of R9 if peptide application is accompanied by a rapid temperature drop to 15°C.Intriguingly, we found that R9 at 10-20 μM and 37°C induces repetitive spikes in intracellular Ca(2+) concentration.To conclude, we uncovered a novel mechanistic link between calcium signalling and entry of cationic peptides.

View Article: PubMed Central - PubMed

Affiliation: Section on Membrane Biology, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Building 10/Room 10D05, 10 Center Drive, Bethesda, MD 20892-1855, U.S.A. melikovk@mail.nih.gov.

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Related in: MedlinePlus