Stadlmann 2006 Cell Biochem Biophys
Stadlmann S, Renner K, Pollheimer J, Moser PL, Zeimet AG, Offner FA, Gnaiger E (2006) Preserved coupling of oxydative phosphorylation but decreased mitochondrial respiratory capacity in IL-1Γ treated human peritoneal mesothelial cells. Cell Biochem Biophys 44:179-86. |
Β» PMID: 16456220,
Stadlmann S, Renner K, Pollheimer J, Moser PL, Zeimet AG, Offner FA, Gnaiger E (2006) Cell Biochem Biophys
Abstract: The peritoneal mesothelium acts as a regulator of serosal responses to injury, infection, and neoplastic diseases. After inflammation of the serosal surfaces, proinflammatory cytokines induce an βactivatedβ mesothelial cell phenotype, the mitochondrial aspect of which has not previously been studied. After incubation of cultured human peritoneal mesothelial cells with interleukin (IL)-1Ξ² for 48 h, respiratory activity of suspended cells was analyzed by high-resolution respirometry. Citrate synthase (CS) and lactate dehydrogenase (LDH) activities were determined by spectrophotometry. Treatment with IL-1Ξ² resulted in a significant decline of respiratory capacity (p < 0.05). Respiratory control ratios (i.e., uncoupled respiration at optimum carbonyl cyanide p-trifluoromethoxyphenylhydrazone concentration divided by oligomycin inhibited respiration measured in unpermeabilized cells) remained as high as 11, indicating well-coupled mitochondria and functional integrity of the inner mitochondrial membrane. Whereas respiratory capacities of the cells declined in proportion with decreased CS activity (p < 0.05), LDH activity increased (p < 0.05). Taken together, these results indicate that IL-1Ξ² exposure of peritoneal mesothelial cells does not lead to irreversible defects or inhibition of specific components of the respiratory chain, but is associated with a decrease of mitochondrial content of the cells that is correlated with an increase in LDH (and thus glycolytic) capacity. β’ Keywords: Peritoneal mesothelial cells, Interleukin-1Ξ², Mitochondria, Respiration, Citrate synthase, Lactate dehydrogenase, Cell viability
β’ O2k-Network Lab: AT Innsbruck Gnaiger E
Coupling control protocol
- Β» CCP(S)01
Step | <Symbol in 2006 publication> | Respiratory flow | Comment |
---|---|---|---|
1ce | < E > | ROUTINE, R; flow: 1ce=Rce=Rvce+ROXdce | Cells (ce=vce+dce); cells (ce) are viable (vce) or dead (dce); if mitochondria of dce are intact, respiration of dce is identical to digitonin-permeabilized cells (pce). dce without substrate are in a state of ROXdce. |
1SD | < S > | flow: 1SD=Rvce+SP,dce | ROUTINE respiration of vce is not affected by extracellular succinate and ADP (SD); dce (with intact mt) are in state SP,dce. Limitation: In the absence of rotenone, S-OXPHOS capacity may be underestimated in some types of mitochondria, whereas it is overestimated if anaplerotic pathways from malate are active, e.g. mt-malic enzyme would additionally activate the N-pathway. |
2Omy | < O > | LEAK, L; flow: 2Omy=Lvce+SL,dce | vce and dce are in the LEAK state. |
3U | < 3u > | ETS, E; flow: Evce+SE,dce | vce and dce are in the ETS state. |
4Rot | < R > | ROX; flow: 4ROX=ROXvce+SE,dce | vce are in the ROX state, since succinate is not generated intracellularly after blocking CI by Rot and thus interrjupting the TCA cycle; dce (with intact mt) are in the S-ETS state. |
5Ama | < A > | ROXAma | All ce are in the ROX state. |
Cell membrane permeability index from the CCP
- In many studies of respiration using 'intact cells', respiratory flow, IO2, is expressed per million cells. The total number of cells, Nce, is the sum of viable cells Nvce and dead cells Ndce. Cell viability is the ratio of viable cells in the total cell count, Nvce/Nce. Cell viability can be calculated from respirometric indices, based on the assumption that respiratory integrity of mitochondria is maintained in dce suspended in mitochondrial respiration medium MiR05.
- The labels of the axes show the respiratory states as steps (mark names), and respiratory states.
- The two indexes derived from (1) the succinate control step, and (2) the antimycin A control step are in close agreement (modified Fig. 2A from Stadlmann et al 2006).
Succinate control step
Step | <Symbol in 2006 publication> | Respiratory state | Comment |
---|---|---|---|
1SD-R | < S-E > | R+SP - R | Stimulation of respiration by succinate & ADP (SD) in dead cells (dce, with intact mt) over endogenous ROUTINE respiration (R) in all counted cells (ce). vce are not permeable to SD, and dce are depleted of substrates and adenylates. Hence SD stimulates mitochondrial respiration in dce only. |
3U β R | < 3u-E > | E+SE - R | Total capacity over endogenous ROUTINE respiration in ce=vce+dce (viable and dead
cells). All oligomycin-inhibited cells are stimulated by uncouplers (3U), because intact cell membranes are permeable for the uncoupler and respiration is supported by endogenous substrates in vce, whereas dce respire on succinate. |
- R+SP - R = SP (dce only)
- E-R is the excess E-R capacity, ExR (vce only)
- Normalization of the cell membrane permeability index
- SP / (ExR+SE)
- If all cells are viable (vce), then this index equals 0, since SP = 0.
- If all cells are permeable (dce), then this index equals 1, since SP and SE are practically identical in many cases, then SP/SE = 1.0.
Antimycin A control step
Step | <Symbol in 2006 publication> | Respiratory state | Comment |
---|---|---|---|
4Rot-5Ama | < R-A > | SE,mt (mt is ROX-corrected) | Succinate-supported respiration in dce (in the presence of the CI inhibitor rotenone, Rot) over antimycin Aβinhibited oxygen uptake (Ama) of all cells. This effect is specific for dce in the presence of succinate and uncoupler. |
3U-5Ama | < 3u-A > | (E+SE)mt (mt is ROX-corrected) | ETS capacity of ce plus SE of dce over antimycin Aβinhibited respiration (mt; ROX-corrected). |
- SE - ROX = SE,mt (dce only)
- (E+SE) - ROX = (E+SE)mt
- Normalization of the cell membrane permeability index
- SE,mt / (E+SE)mt
- If all cells are viable (vce), then this index equals 0, since SE,mt = 0.
- If all cells are permeable (dce), then this index equals 1, since in this case Emt = E-ROX = 0.
Labels: MiParea: Respiration, mt-Biogenesis;mt-density, Pharmacology;toxicology
Stress:Cell death Organism: Human Tissue;cell: Endothelial;epithelial;mesothelial cell Preparation: Intact cells
Regulation: Coupling efficiency;uncoupling, Substrate Coupling state: ROUTINE, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.
HRR: Oxygraph-2k