PMC

Effect of IL-13 and inhibited mitochondrial transport on [Ca²⁺]_cyt and [Ca²⁺]_mito. A: compared with the vehicle control, IL-13 increased [Ca²⁺]_cyt responses to His. Inhibition of MCU with Ru360 (dotted line) further increased [Ca²⁺]_cyt responses in IL-13-exposed cells, whereas inhibition of mitochondrial NCX with CGP (dashed line) decreased [Ca²⁺]_cyt responses. B and C: effects of IL-13 and inhibited mitochondrial transport on [Ca²⁺]_mito responses. Compared with the vehicle control, IL-13 had no effect on [Ca²⁺]_mito responses to His measured from both mitochondria in the perinuclear (B) and distal (C) regions of cells. Ru360 significantly decreased [Ca²⁺]_mito in IL-13-exposed cells, whereas CGP increased [Ca²⁺]_mito responses measured from mitochondria in the perinuclear regions but not from mitochondria in the distal regions of cells.

Effect of inhibited mitochondrial transport on [Ca²⁺]_cyt and [Ca²⁺]_mito. ASM cells loaded with fluo-3 AM and rhod-2 AM were exposed to 10 μM His. A: compared with control cells (solid line), inhibition of MCU with Ru360 (dotted line) increased [Ca²⁺]_cyt induced by His (10 μM). A similar result was obtained when the mitochondrial Na⁺/Ca²⁺ exchanger (NCX) was inhibited with CGP (dashed line). B and C: compared with control cells (solid line), Ru360 (dotted line) decreased [Ca²⁺]_mito induced by His (10 μM) in the perinuclear regions (B) or distal regions (C) of the cells. CGP (dashed line) increased [Ca²⁺]_mito in the perinuclear regions (B) but not in the distal regions (C) of the cells.

A and B: summary of the effect of TNF-α and inhibited mitochondrial transport on the amplitude of [Ca²⁺]_cyt (A) and [Ca²⁺]_mito (B) responses. C: the rate of fall expressed as a percentage of the vehicle control and corresponding to the areas under the curves (starting at the peak until the return to the basal Ca²⁺ level). TNF-α increased the decay of [Ca²⁺]_cyt and decreased the decay of [Ca²⁺]_mito responses from the distal regions of the cells. Ru360 increased the decay of [Ca²⁺]_cyt responses but not [Ca²⁺]_mito responses. Compared with the vehicle control, CGP increased the decay of [Ca²⁺]_cyt and [Ca²⁺]_mito responses from the perinuclear regions of the cells. Values are means ± SE. *Significant effect (P < 0.05).

A and B: summary of the effects of IL-13 and inhibited mitochondrial transport on the amplitude of [Ca²⁺]_cyt (A) and [Ca²⁺]_mito (B) responses (expressed as a percentage of the vehicle control). Values are means ± SE. *Significant effect (P < 0.05). C: the rate of fall expressed as a percentage of the vehicle control and corresponding to the areas under the curves (starting at the peak until the return to the basal Ca²⁺ level). IL-13 increased the decay of [Ca²⁺]_cyt and [Ca²⁺]_mito responses from the distal regions of the cells, whereas it decreased the decay of [Ca²⁺]_mito responses from the perinuclear regions. Ru360 did not further increased the decay of [Ca²⁺]_cyt responses but decreased the decay of [Ca²⁺]_mito responses from both the perinuclear and distal regions of the cells. Compared with the vehicle control, CGP increased the decay of [Ca²⁺]_cyt and [Ca²⁺]_mito responses from the perinuclear regions of the cells. Values are means ± SE. *Significant effect (P < 0.05).

Effect of the proinflammatory cytokine TNF-α and inhibited mitochondrial transport on [Ca²⁺]_cyt and [Ca²⁺]_mito. ASM cells incubated for 48 h with TNF-α and then loaded with fluo-3 AM and rhod-2 AM were exposed to 10 μM His. A: compared with the vehicle control, TNF-α increased [Ca²⁺]_cyt responses induced by His (10 μM). A similar result was obtained when MCU was inhibited with Ru360 (dotted line). Inhibition of mitochondrial NCX with CGP (dashed line) decreased [Ca²⁺]_cyt in TNF-α-exposed cells. B and C: TNF-α decreased [Ca²⁺]_mito responses to His measured from mitochondria in the perinuclear regions (B) but not from mitochondria in the distal regions of cells (C). Ru360 did not further decrease [Ca²⁺]_mito in TNF-α-exposed cells. In contrast, CGP significantly increased [Ca²⁺]_mito measured from mitochondria in the perinuclear regions of cells but not from mitochondria in the distal regions.

A–C: summary of the effects of inhibited mitochondrial transport on the amplitude of the [Ca²⁺]_cyt response (expressed as a percentage of control; A), the amplitude of the [Ca²⁺]_mito amplitude (B), and the rate of fall (C; expressed as a percentage of control and corresponding to the areas under the curves, starting at the peak until the return to the basal Ca²⁺ level). Ru360 did not affect the decay of the [Ca²⁺]_cyt responses but decreased the associated rate of fall of [Ca²⁺]_mito for both the perinuclear and distal regions of the cells. CGP increased the rate of fall of [Ca²⁺]_cyt responses, which was accompanied by a decrease in the decay of [Ca²⁺]_mito responses. Values are means ± SE. *Significant effect (P < 0.05).

Comparison of [Ca²⁺]_cyt and [Ca²⁺]_mito in human ASM. A: basal [Ca²⁺]_cyt and [Ca²⁺]_mito from mitochondria in the distal versus perinuclear regions of cells and the His effect on the amplitude of [Ca²⁺] responses (peak amplitude, n = 31). Values are means ± SE. B: effect of inhibited mitochondrial transport on the delay (in s) between [Ca²⁺]_cyt and [Ca²⁺]_mito. When measured at the initiation of either response, the delay between [Ca²⁺]_cyt and [Ca²⁺]_mito was estimated at 11 ± 3 and 9 ± 3 s for the mitochondria localized in the perinuclear or distal regions of the cells, respectively. Inhibition of the mitochondrial uniporter (MCU) with Ru360 increased this delay for both mitochondria in the perinuclear regions (15 ± 2 s) and distal regions of the cells (16 ± 3 s). CGP-37157 (CGP) had no effect on the delays between [Ca²⁺]_cyt and [Ca²⁺]_mito. *Significant effect (P < 0.05).

Measurement of [Ca²⁺]_cyt and [Ca²⁺]_mito in human ASM cells. A: fluorescence image of human ASM cells loaded with fluo-3 AM with an example of the ROI used to measure [Ca²⁺]_cyt. B: 10 μM His first induced a rise in [Ca²⁺]_cyt. C: the same ROI was used to measure the change in [Ca²⁺]_mito with the same cells simultaneously loaded with rhod-2 AM. Examples of the ROIs used to measure [Ca²⁺]_mito in the perinuclear and distal regions are also shown. D: traces obtained from the ROIs in C showed that the changes in fluorescence only occur when the ROIs are placed within the mitochondria in either the distal (dotted line) or perinuclear (dashed line) region of the cells. E and F: an overlay of the two fluorescence images (E) along with a time-extended view of [Ca²⁺] responses (F) from the graphs in B and D showed a significant delay between [Ca²⁺]_cyt and [Ca²⁺]_mito responses. Histamine (His) was added at 30 s.

Transient increases in rhod-2 fluorescence are localized within mitochondria. A and B: fluorescence images of human ASM cells loaded with MitoTracker green (A) and rhod-2 AM (B). C: overlay of the two fluorescence images with examples of regions of interest (ROIs) chosen within the mitochondria (region 1), in the cytosol (region 2), and near mitochondria (region 3), where some background fluorescence could be detected. Bar = 10 μm. D: measurement of rhod-2 fluorescence expressed as [Ca²⁺] (in nM) in the ROIs shown in C. A transient increase in [Ca²⁺] in response to histamine (His; 10 μM) was observed for the ROI chosen within the mitochondria (region 1, solid line) and not for the ROIs placed in either the cytosol (region 2, dotted line) or near the mitochondria (region 3, dashed line). E and F: MitoTracker green-loaded cells excited at 488 nm showed significant specific fluorescence at 501 nm, corresponding to MitoTracker green (E), whereas excitation of the cells at 568 nm (rhod-2 AM excitation wavelength) did not show significant (above background) fluorescence at 590 nm (F). G and H: similarly, excitation of ASM cells loaded with 2.5 μM rhod-2 AM at 488 nm (G; Mitotracker green excitation wavelength) did not result in any nonspecific fluorescence at 501 nm (above background), whereas excitation at 568 nm resulted in specific fluorescence at 590 nm, corresponding to rhod-2 AM (H). Ex, excitation; Em, emission.

Dye loading of human airway smooth muscle (ASM) cells. A and B: fluorescence images of human ASM cells loaded with 2.5 μM fluo-3 AM (A) and 2.5 μM rhod-2 AM (B) to estimate cytosolic ([Ca²⁺]_cyt) and mitochondrial Ca²⁺ concentration ([Ca²⁺]_mito), respectively. C: relative distribution of fluo-3 AM and rhod-2 AM fluorescence with the merged image of the two fluorescence images. D and E: in a separate set of experiments, human ASM cells were loaded simultaneously with rhod-2 AM (D) and then with 500 nM MitoTracker green (E) for 5 min to visualize mitochondria and showed consistent and matching patterns of mitochondria in the perinuclear areas and more distal (potentially perimembranous) areas of cells. F: an overlay of the two fluorescence images confirmed that the pattern of rhod-2 staining was mainly within mitochondria. G and H: in additional experiments, ASM cells were loaded with 1 μM BODIPY-FL-thapsigargin (G; endoplasmic reticulum Ca²⁺ pumps) and then with 500 nM MitoTracker red CMXRos (H). I: overlays of the two fluorescence images along with image analysis of the mitochondrial network were used to define the perinuclear regions. N, nucleus. Bars = 10 μm.