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Antagonistic Regulation of Parvalbumin Expression and Mitochondrial Calcium Handling Capacity in Renal Epithelial Cells.

Henzi T, Schwaller B - PLoS ONE (2015)

Bottom Line: With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated.Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected.In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells.

View Article: PubMed Central - PubMed

Affiliation: Anatomy, Department of Medicine, University of Fribourg, Fribourg, Switzerland.

ABSTRACT
Parvalbumin (PV) is a cytosolic Ca2+-binding protein acting as a slow-onset Ca2+ buffer modulating the shape of Ca2+ transients in fast-twitch muscles and a subpopulation of neurons. PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca2+ shuttle facilitating transcellular Ca2+ resorption. In excitable cells, upregulation of mitochondria in "PV-ergic" cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit β was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca2+ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca2+ buffer PV in kidney cells was counterbalanced in vivo and in vitro by adjusting the relative mitochondrial volume and modifying the mitochondrial protein composition conceivably to increase their Ca2+-buffering/sequestration capacity.

No MeSH data available.


Related in: MedlinePlus

CCCP-induced changes in the mitochondrial membrane potential evaluated by two fluorescent lipophilic cationic dyes: Mitotracker Red (MTR)/Green (MTG) (A) and JC-1 (B).Treatment with the uncoupler CCCP decreased the mitochondrial membrane potential. The concentration dependent decrease was bigger in the PV-expressing MDCK PV15 cells compared to control MDCK cells (n = 3 independent experiments, mean ± sem, differences between control and PV15 were statistically significant at all CCCP concentrations (pairwise t-test; p<0.05 marked with (*) for all pairs in (A) and (B)). C) The cuvette measurements (A,B) were confirmed by qualitative fluorescence microscopy. Collapsing of the mitochondrial membrane potential by CCCP reduced MTR fluorescence emission in both control (upper row) and PV15 MDCK (lower row) cells; the decrease was more pronounced in the PV-expressing PV15 clone.
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pone.0142005.g007: CCCP-induced changes in the mitochondrial membrane potential evaluated by two fluorescent lipophilic cationic dyes: Mitotracker Red (MTR)/Green (MTG) (A) and JC-1 (B).Treatment with the uncoupler CCCP decreased the mitochondrial membrane potential. The concentration dependent decrease was bigger in the PV-expressing MDCK PV15 cells compared to control MDCK cells (n = 3 independent experiments, mean ± sem, differences between control and PV15 were statistically significant at all CCCP concentrations (pairwise t-test; p<0.05 marked with (*) for all pairs in (A) and (B)). C) The cuvette measurements (A,B) were confirmed by qualitative fluorescence microscopy. Collapsing of the mitochondrial membrane potential by CCCP reduced MTR fluorescence emission in both control (upper row) and PV15 MDCK (lower row) cells; the decrease was more pronounced in the PV-expressing PV15 clone.

Mentions: The precise mitochondrial protein composition with respect to expression levels of individual protein is assumed to be the result of tissue- or even cell-specific mitochondria function [6,28]. Based on this assumption, we hypothesized that mitochondria from PV-expressing and non-PV-expressing MDCK cells would differ in their capacity to withstand Ca2+ influx-induced membrane depolarization. For this we relied on fluorescent dyes suitable to measure changes in the mitochondrial membrane potential. Since intracellular fluorescence increases as the result of either a rise in ΔΨm or an increase in mitochondrial mass density, a method of controlling for changes in mitochondrial mass is required to monitor changes in ΔΨm [29]. We used two approaches, either the ratiometric dye (JC-1) or a combination of two dyes, Mitotracker Red (MTR) sensitive to ΔΨm and Mitotracker Green (MTG) that is insensitive to changes in ΔΨm. Normalized ratios for both approaches demonstrated that an increase in the concentration of the uncoupler CCCP in the range of 2.5–10 μM led to a minor decrease in ΔΨm in control cells (Fig 7A and 7B). In PV-expressing PV15 cells, CCCP induced a strong, concentration-dependent decrease of the red/green fluorescence ratio (Fig 7A) and JC-1 (Fig 7B) fluorescence. Representative images of MTR (red) fluorescence in MDCK cells without (control) or with PV expression (clone PV15) are depicted in Fig 7C. Qualitative staining was similar in control and PV15 MDCK cells in the absence of the uncoupler. The collapsing of the mitochondrial membrane potential by addition of CCCP caused a loss in red fluorescence in mitochondria, at 2.5 μM mostly in PV15 cells (Fig 7C). At the higher CCCP concentration (7.5 μM), mitochondrial staining completely disappeared in PV15 cells, while in control MDCK cells, mitochondria staining was clearly reduced, but few visibly stained mitochondria were still present. The results from both approaches (JC-1 and MTR/MTG) demonstrate that the expression of PV impairs/attenuates the capacity of mitochondria to maintain ΔΨm in the presence of CCCP. Next we investigated, whether these differences might be due, at least in part, to alterations in intracellular ATP levels in control MDCK and PV15 cells. Relative ATP levels were not different in PV15 and control MDCK cells maintained at basal cell culture conditions, i.e. during unperturbed cell proliferation in vitro (93.3 ± 12.1%, vs. 100 ± 3.9%, respectively; n.s., n = 4 independent experiments).


Antagonistic Regulation of Parvalbumin Expression and Mitochondrial Calcium Handling Capacity in Renal Epithelial Cells.

Henzi T, Schwaller B - PLoS ONE (2015)

CCCP-induced changes in the mitochondrial membrane potential evaluated by two fluorescent lipophilic cationic dyes: Mitotracker Red (MTR)/Green (MTG) (A) and JC-1 (B).Treatment with the uncoupler CCCP decreased the mitochondrial membrane potential. The concentration dependent decrease was bigger in the PV-expressing MDCK PV15 cells compared to control MDCK cells (n = 3 independent experiments, mean ± sem, differences between control and PV15 were statistically significant at all CCCP concentrations (pairwise t-test; p<0.05 marked with (*) for all pairs in (A) and (B)). C) The cuvette measurements (A,B) were confirmed by qualitative fluorescence microscopy. Collapsing of the mitochondrial membrane potential by CCCP reduced MTR fluorescence emission in both control (upper row) and PV15 MDCK (lower row) cells; the decrease was more pronounced in the PV-expressing PV15 clone.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4634853&req=5

pone.0142005.g007: CCCP-induced changes in the mitochondrial membrane potential evaluated by two fluorescent lipophilic cationic dyes: Mitotracker Red (MTR)/Green (MTG) (A) and JC-1 (B).Treatment with the uncoupler CCCP decreased the mitochondrial membrane potential. The concentration dependent decrease was bigger in the PV-expressing MDCK PV15 cells compared to control MDCK cells (n = 3 independent experiments, mean ± sem, differences between control and PV15 were statistically significant at all CCCP concentrations (pairwise t-test; p<0.05 marked with (*) for all pairs in (A) and (B)). C) The cuvette measurements (A,B) were confirmed by qualitative fluorescence microscopy. Collapsing of the mitochondrial membrane potential by CCCP reduced MTR fluorescence emission in both control (upper row) and PV15 MDCK (lower row) cells; the decrease was more pronounced in the PV-expressing PV15 clone.
Mentions: The precise mitochondrial protein composition with respect to expression levels of individual protein is assumed to be the result of tissue- or even cell-specific mitochondria function [6,28]. Based on this assumption, we hypothesized that mitochondria from PV-expressing and non-PV-expressing MDCK cells would differ in their capacity to withstand Ca2+ influx-induced membrane depolarization. For this we relied on fluorescent dyes suitable to measure changes in the mitochondrial membrane potential. Since intracellular fluorescence increases as the result of either a rise in ΔΨm or an increase in mitochondrial mass density, a method of controlling for changes in mitochondrial mass is required to monitor changes in ΔΨm [29]. We used two approaches, either the ratiometric dye (JC-1) or a combination of two dyes, Mitotracker Red (MTR) sensitive to ΔΨm and Mitotracker Green (MTG) that is insensitive to changes in ΔΨm. Normalized ratios for both approaches demonstrated that an increase in the concentration of the uncoupler CCCP in the range of 2.5–10 μM led to a minor decrease in ΔΨm in control cells (Fig 7A and 7B). In PV-expressing PV15 cells, CCCP induced a strong, concentration-dependent decrease of the red/green fluorescence ratio (Fig 7A) and JC-1 (Fig 7B) fluorescence. Representative images of MTR (red) fluorescence in MDCK cells without (control) or with PV expression (clone PV15) are depicted in Fig 7C. Qualitative staining was similar in control and PV15 MDCK cells in the absence of the uncoupler. The collapsing of the mitochondrial membrane potential by addition of CCCP caused a loss in red fluorescence in mitochondria, at 2.5 μM mostly in PV15 cells (Fig 7C). At the higher CCCP concentration (7.5 μM), mitochondrial staining completely disappeared in PV15 cells, while in control MDCK cells, mitochondria staining was clearly reduced, but few visibly stained mitochondria were still present. The results from both approaches (JC-1 and MTR/MTG) demonstrate that the expression of PV impairs/attenuates the capacity of mitochondria to maintain ΔΨm in the presence of CCCP. Next we investigated, whether these differences might be due, at least in part, to alterations in intracellular ATP levels in control MDCK and PV15 cells. Relative ATP levels were not different in PV15 and control MDCK cells maintained at basal cell culture conditions, i.e. during unperturbed cell proliferation in vitro (93.3 ± 12.1%, vs. 100 ± 3.9%, respectively; n.s., n = 4 independent experiments).

Bottom Line: With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated.Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected.In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells.

View Article: PubMed Central - PubMed

Affiliation: Anatomy, Department of Medicine, University of Fribourg, Fribourg, Switzerland.

ABSTRACT
Parvalbumin (PV) is a cytosolic Ca2+-binding protein acting as a slow-onset Ca2+ buffer modulating the shape of Ca2+ transients in fast-twitch muscles and a subpopulation of neurons. PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca2+ shuttle facilitating transcellular Ca2+ resorption. In excitable cells, upregulation of mitochondria in "PV-ergic" cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit β was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca2+ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca2+ buffer PV in kidney cells was counterbalanced in vivo and in vitro by adjusting the relative mitochondrial volume and modifying the mitochondrial protein composition conceivably to increase their Ca2+-buffering/sequestration capacity.

No MeSH data available.


Related in: MedlinePlus