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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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[Ca2+]i dynamics under different levels of shear stress. A–C: shear stimuli of 1 (A), 3 (B), or 10 (C) dyn/cm2 showed significant increase in averaged ratio measurements for both shear stimuli compared with respective baselines. However, only for shears higher than 1 dyn/cm2 was the 2nd peak significantly smaller than the 1st peak. The ratio signal is the trace with error bars on the primary axis and the shear stress is the square wave shown on the secondary axis. The measurements are reported as means ± SE (A: n = 9; B: n = 4; and C: n = 9). D: average peak response to the 1st shear stimulus shows that the 3 dyn/cm2 response is not significantly different from the 10 dyn/cm2 response meaning that [Ca2+]i response plateaus at ∼3 dyn/cm2. The peaks for D are calculated in each experiment individually and then are averaged over the number of experiments. E. When the no-shear period (recovery time) between the 2 stimuli was increased from 10 to 30 min, the 2nd peak significantly increased (means ± SE; n = 6). F: with 10-min recovery time, the 2nd peak height was only 30% of the initial peak height, however, when this time was increase to 30 min, the 2nd peak height significantly increase to 54% of the initial peak height (*P < 0.05; ns: nonsignificant).
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Figure 2: [Ca2+]i dynamics under different levels of shear stress. A–C: shear stimuli of 1 (A), 3 (B), or 10 (C) dyn/cm2 showed significant increase in averaged ratio measurements for both shear stimuli compared with respective baselines. However, only for shears higher than 1 dyn/cm2 was the 2nd peak significantly smaller than the 1st peak. The ratio signal is the trace with error bars on the primary axis and the shear stress is the square wave shown on the secondary axis. The measurements are reported as means ± SE (A: n = 9; B: n = 4; and C: n = 9). D: average peak response to the 1st shear stimulus shows that the 3 dyn/cm2 response is not significantly different from the 10 dyn/cm2 response meaning that [Ca2+]i response plateaus at ∼3 dyn/cm2. The peaks for D are calculated in each experiment individually and then are averaged over the number of experiments. E. When the no-shear period (recovery time) between the 2 stimuli was increased from 10 to 30 min, the 2nd peak significantly increased (means ± SE; n = 6). F: with 10-min recovery time, the 2nd peak height was only 30% of the initial peak height, however, when this time was increase to 30 min, the 2nd peak height significantly increase to 54% of the initial peak height (*P < 0.05; ns: nonsignificant).

Mentions: A step change in shear stress (10-min long) applied to the HDLEC resulted in an increase in [Ca2+]i that peaked and then generally decayed back towards basal levels (Fig. 2, A–C). The magnitudes of the peak rise and the decay were dependent on the shear stress applied. For example, upon initiating a shear stress of 10 dyn/cm2, the fluorescence ratio (the index of [Ca2+]i) increased significantly (Fig. 2C), within 1.75 min (on average for calcium-containing DMEM/F12) from a basal ratio of 0.74 ± 0.02 to a peak value of 1.11 ± 0.03. The calcium signal then decreased exponentially even though shear stress remained elevated (Fig. 2C). Indeed, at the end of the 10 min of constant shear (10 dyn/cm2), calcium had fallen to values below the original baseline. Shear stress was then returned back to zero for 10 min before a second step of similar shear stress was applied to evaluate the recovery capability of these cells. During the 10-min “resting” period when shear stress was back to zero, the calcium signal generally recovered back toward the initial baseline, often falling below the initial basal level (Fig. 2, A–C). When a second identical shear stimulus was applied after the resting period (Fig. 2C), the magnitude of the second peak (0.79 ± 0.02) was significantly lower than the first. Based on the calibration constants, a ratio of 1.11 ± 0.03 for the first peak indicates 126.2 ± 5.8 nM, and a ratio of 0.79 ± 0.02 for the second peak indicates 57.3 ± 4.5 nM.


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)

[Ca2+]i dynamics under different levels of shear stress. A–C: shear stimuli of 1 (A), 3 (B), or 10 (C) dyn/cm2 showed significant increase in averaged ratio measurements for both shear stimuli compared with respective baselines. However, only for shears higher than 1 dyn/cm2 was the 2nd peak significantly smaller than the 1st peak. The ratio signal is the trace with error bars on the primary axis and the shear stress is the square wave shown on the secondary axis. The measurements are reported as means ± SE (A: n = 9; B: n = 4; and C: n = 9). D: average peak response to the 1st shear stimulus shows that the 3 dyn/cm2 response is not significantly different from the 10 dyn/cm2 response meaning that [Ca2+]i response plateaus at ∼3 dyn/cm2. The peaks for D are calculated in each experiment individually and then are averaged over the number of experiments. E. When the no-shear period (recovery time) between the 2 stimuli was increased from 10 to 30 min, the 2nd peak significantly increased (means ± SE; n = 6). F: with 10-min recovery time, the 2nd peak height was only 30% of the initial peak height, however, when this time was increase to 30 min, the 2nd peak height significantly increase to 54% of the initial peak height (*P < 0.05; ns: nonsignificant).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: [Ca2+]i dynamics under different levels of shear stress. A–C: shear stimuli of 1 (A), 3 (B), or 10 (C) dyn/cm2 showed significant increase in averaged ratio measurements for both shear stimuli compared with respective baselines. However, only for shears higher than 1 dyn/cm2 was the 2nd peak significantly smaller than the 1st peak. The ratio signal is the trace with error bars on the primary axis and the shear stress is the square wave shown on the secondary axis. The measurements are reported as means ± SE (A: n = 9; B: n = 4; and C: n = 9). D: average peak response to the 1st shear stimulus shows that the 3 dyn/cm2 response is not significantly different from the 10 dyn/cm2 response meaning that [Ca2+]i response plateaus at ∼3 dyn/cm2. The peaks for D are calculated in each experiment individually and then are averaged over the number of experiments. E. When the no-shear period (recovery time) between the 2 stimuli was increased from 10 to 30 min, the 2nd peak significantly increased (means ± SE; n = 6). F: with 10-min recovery time, the 2nd peak height was only 30% of the initial peak height, however, when this time was increase to 30 min, the 2nd peak height significantly increase to 54% of the initial peak height (*P < 0.05; ns: nonsignificant).
Mentions: A step change in shear stress (10-min long) applied to the HDLEC resulted in an increase in [Ca2+]i that peaked and then generally decayed back towards basal levels (Fig. 2, A–C). The magnitudes of the peak rise and the decay were dependent on the shear stress applied. For example, upon initiating a shear stress of 10 dyn/cm2, the fluorescence ratio (the index of [Ca2+]i) increased significantly (Fig. 2C), within 1.75 min (on average for calcium-containing DMEM/F12) from a basal ratio of 0.74 ± 0.02 to a peak value of 1.11 ± 0.03. The calcium signal then decreased exponentially even though shear stress remained elevated (Fig. 2C). Indeed, at the end of the 10 min of constant shear (10 dyn/cm2), calcium had fallen to values below the original baseline. Shear stress was then returned back to zero for 10 min before a second step of similar shear stress was applied to evaluate the recovery capability of these cells. During the 10-min “resting” period when shear stress was back to zero, the calcium signal generally recovered back toward the initial baseline, often falling below the initial basal level (Fig. 2, A–C). When a second identical shear stimulus was applied after the resting period (Fig. 2C), the magnitude of the second peak (0.79 ± 0.02) was significantly lower than the first. Based on the calibration constants, a ratio of 1.11 ± 0.03 for the first peak indicates 126.2 ± 5.8 nM, and a ratio of 0.79 ± 0.02 for the second peak indicates 57.3 ± 4.5 nM.

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