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Measurement of shear stress-mediated intracellular calcium dynamics in human dermal lymphatic endothelial cells.

Jafarnejad M, Cromer WE, Kaunas RR, Zhang SL, Zawieja DC, Moore JE - Am. J. Physiol. Heart Circ. Physiol. (2015)

Bottom Line: Removal of the extracellular calcium from the buffer or pharmocological blockade of calcium release-activated calcium (CRAC) channels significantly reduced the peak [Ca(2+)]i, demonstrating a role of extracellular calcium entry.Inhibition of endoplasmic reticulum (ER) calcium pumps showed the importance of intracellular calcium stores in the initiation of this signal.In conclusion, we demonstrated that the shear-mediated calcium signal is dependent on the magnitude of the shear and involves ER store calcium release and extracellular calcium entry.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Imperial College, London, England;

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Effect of a calcium release-activated calcium (CRAC) channel blocker S66 on [Ca2+]i dynamics to shear. A: shear-mediated calcium transient under 10 dyn/cm2 using DMEM/F-12 is shown here (similar to Fig. 2C; means ± SE; n = 9). B: after the baseline was recorded for 2 min, HDLEC were perfused slowly (∼0.2 dyn/cm2) with 10 µM S66 in DMEM/F-12 for 8 min. Then a similar shear stress waveform to previous experiments was used to investigate the effect of blocking CRAC channels flow-induced calcium signals (means ± SE; n = 5). The 1st and 2nd peaks were both higher than their baselines. The 2nd peak was also significantly smaller than the 1st peak. C: blocking CRAC channels by S66 resulted in smaller 1st and 2nd peaks compared with control experiments with DMEM/F-12 under 10 dyn/cm2. The peaks for C are calculated in each experiment individually and then are averaged over the number of experiments (*P < 0.05).
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Figure 5: Effect of a calcium release-activated calcium (CRAC) channel blocker S66 on [Ca2+]i dynamics to shear. A: shear-mediated calcium transient under 10 dyn/cm2 using DMEM/F-12 is shown here (similar to Fig. 2C; means ± SE; n = 9). B: after the baseline was recorded for 2 min, HDLEC were perfused slowly (∼0.2 dyn/cm2) with 10 µM S66 in DMEM/F-12 for 8 min. Then a similar shear stress waveform to previous experiments was used to investigate the effect of blocking CRAC channels flow-induced calcium signals (means ± SE; n = 5). The 1st and 2nd peaks were both higher than their baselines. The 2nd peak was also significantly smaller than the 1st peak. C: blocking CRAC channels by S66 resulted in smaller 1st and 2nd peaks compared with control experiments with DMEM/F-12 under 10 dyn/cm2. The peaks for C are calculated in each experiment individually and then are averaged over the number of experiments (*P < 0.05).

Mentions: The specific role of calcium release-activated calcium (CRAC) channels in mediating Ca2+ entry in response to the ER calcium release was investigated by inhibiting these channels using Syntha66 (also known as S66, a specific CRAC channel blocker) (14, 29, 36), thereby revealing their contribution to the shear-mediated [Ca2+]i signal (Fig. 5, A and B). Blockade of CRAC channels decreased the height of both the first (1.02 ± 0.03) and second peaks (0.73 ± 0.01) relative to their untreated controls (Fig. 5C). Moreover, S66 treatment often produced a delay of about 2–5 min in the shear-induced calcium response. Specifically, S66 increased the time to reach the first peak to 5.2 ± 0.4 min (2- to 5-min delay + an increase with a rate of 0.165 ratio/min; Table 1) compared with 1.8 ± 0.1 min without CRAC channel blockade (Fig. 5, A and B).


Measurement of shear stress-mediated intracellular calcium dynamics in human dermal lymphatic endothelial cells.

Jafarnejad M, Cromer WE, Kaunas RR, Zhang SL, Zawieja DC, Moore JE - Am. J. Physiol. Heart Circ. Physiol. (2015)

Effect of a calcium release-activated calcium (CRAC) channel blocker S66 on [Ca2+]i dynamics to shear. A: shear-mediated calcium transient under 10 dyn/cm2 using DMEM/F-12 is shown here (similar to Fig. 2C; means ± SE; n = 9). B: after the baseline was recorded for 2 min, HDLEC were perfused slowly (∼0.2 dyn/cm2) with 10 µM S66 in DMEM/F-12 for 8 min. Then a similar shear stress waveform to previous experiments was used to investigate the effect of blocking CRAC channels flow-induced calcium signals (means ± SE; n = 5). The 1st and 2nd peaks were both higher than their baselines. The 2nd peak was also significantly smaller than the 1st peak. C: blocking CRAC channels by S66 resulted in smaller 1st and 2nd peaks compared with control experiments with DMEM/F-12 under 10 dyn/cm2. The peaks for C are calculated in each experiment individually and then are averaged over the number of experiments (*P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4385995&req=5

Figure 5: Effect of a calcium release-activated calcium (CRAC) channel blocker S66 on [Ca2+]i dynamics to shear. A: shear-mediated calcium transient under 10 dyn/cm2 using DMEM/F-12 is shown here (similar to Fig. 2C; means ± SE; n = 9). B: after the baseline was recorded for 2 min, HDLEC were perfused slowly (∼0.2 dyn/cm2) with 10 µM S66 in DMEM/F-12 for 8 min. Then a similar shear stress waveform to previous experiments was used to investigate the effect of blocking CRAC channels flow-induced calcium signals (means ± SE; n = 5). The 1st and 2nd peaks were both higher than their baselines. The 2nd peak was also significantly smaller than the 1st peak. C: blocking CRAC channels by S66 resulted in smaller 1st and 2nd peaks compared with control experiments with DMEM/F-12 under 10 dyn/cm2. The peaks for C are calculated in each experiment individually and then are averaged over the number of experiments (*P < 0.05).
Mentions: The specific role of calcium release-activated calcium (CRAC) channels in mediating Ca2+ entry in response to the ER calcium release was investigated by inhibiting these channels using Syntha66 (also known as S66, a specific CRAC channel blocker) (14, 29, 36), thereby revealing their contribution to the shear-mediated [Ca2+]i signal (Fig. 5, A and B). Blockade of CRAC channels decreased the height of both the first (1.02 ± 0.03) and second peaks (0.73 ± 0.01) relative to their untreated controls (Fig. 5C). Moreover, S66 treatment often produced a delay of about 2–5 min in the shear-induced calcium response. Specifically, S66 increased the time to reach the first peak to 5.2 ± 0.4 min (2- to 5-min delay + an increase with a rate of 0.165 ratio/min; Table 1) compared with 1.8 ± 0.1 min without CRAC channel blockade (Fig. 5, A and B).

Bottom Line: Removal of the extracellular calcium from the buffer or pharmocological blockade of calcium release-activated calcium (CRAC) channels significantly reduced the peak [Ca(2+)]i, demonstrating a role of extracellular calcium entry.Inhibition of endoplasmic reticulum (ER) calcium pumps showed the importance of intracellular calcium stores in the initiation of this signal.In conclusion, we demonstrated that the shear-mediated calcium signal is dependent on the magnitude of the shear and involves ER store calcium release and extracellular calcium entry.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Imperial College, London, England;

Show MeSH
Related in: MedlinePlus