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-1ß treated human peritoneal mesothelial cells. Cell Biochem Biophys 44:179-86.

» 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 “activated” mesothelial cell phenotype, the mitochondrial aspect of which has not previously been studied. After incubation of cultured human peritoneal mesothelial cells with interleukin (IL)-1β 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-1β 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-1β 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. • Keywords: Peritoneal mesothelial cells, Interleukin-1β, Mitochondria, Respiration, Citrate synthase, Lactate dehydrogenase, Cell viability

• O2k-Network Lab: AT Innsbruck Gnaiger E

Coupling control protocol

» CCP(S)01

Step	<Symbol in 2006 publication>	Respiratory state	Pathway control	Comment
1ce	< E >	ROUTINE, R	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 ROX.
1SD	< S >	R+SP	ROUTINE (R) + S (SP)	vce are not stimulated by SD; dce (with intact mt) are in state SP.
2Omy	< O >	L+SL	LEAK (L) + S (SL)	vce and dce are in the LEAK state.
3U	< 3u >	E+SE	ETS (E) + S (SE)	vce and dce are in the ETS state.
4Rot	< R >	ROX+SE	ROX+S (SE)	vce are in the ROX state, dce (with intact mt) are in the ETS state.
5Ama	< A >	ROXAma	ROX	All ce are in the ROX state.

Cell membrane permeability index from the CCP

These respiratory indices are based on the assumption that respiratory integrity of mitochondria is maintained after cell membrane permeabilization using the 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 – 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+S_P - R = S_P (dce only)

E-R is the excess E-R capacity, ExR (vce only)

• Normalization of the cell membrane permeability index

S_P / (ExR+S_E)

• If all cells are viable (vce), then this index equals 0, since S_P = 0.
• If all cells are permeable (dce), then this index equals 1, since S_P and S_E are practically identical in many cases, then S_P/S_E = 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 A–inhibited 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 A–inhibited respiration (mt; ROX-corrected).

S_E - ROX = S_E,mt (dce only)

(E+S_E) - ROX = (E+S_E)_mt

• Normalization of the cell membrane permeability index

S_E,mt / (E+S_E)_mt

• If all cells are viable (vce), then this index equals 0, since S_E,mt = 0.
• If all cells are permeable (dce), then this index equals 1, since in this case E_mt = 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