Troglitazone induced cytosolic acidification via extracellular signal-response kinase activation and mitochondrial depolarization: complex I proton pumping regulates ammoniagenesis in proximal tubule-like LLC-PK1 cells.
OBJECTIVE
To determined the mechanism(s) through which troglitazone induces cytosolic acidification and glutamine-dependent ammoniagenesis in pig kidney derived LLC-PK1 cells.


METHODS
Acute experiments measured acid extrusion, acid production and simultaneous Extracellular Signal-Regulated Kinase activation. TRO-enhanced acid production was correlated with mitochondrial membrane potential and rotenone and 5-(N-ethyl-N-isopropyl) amiloride, were employed to test specifically the role of Complex I proton pumping. Chronic experiments correlated inhibitors of Complex I with prevention of TRO-increased ammoniagenesis and affects on glutamine metabolism.


RESULTS
Exposure to TRO acutely activated Extracellular Signal-Regulated Kinase in a dose dependent manner associated with a fall in spontaneous cytosolic pH. Cytosolic acidosis was associated with both an increase in acid production and inhibition of sodium/hydrogen ion exchanger -mediated acid extrusion. Preventing TRO-induced Extracellular Signal-Regulated Kinase activation with Mitogen Activated Protein Kinase Kinase inhibitors blocked the increase in acid production, restored sodium/hydrogen ion exchanger-activity and prevented cytosolic acidification. Mechanistically, increased acid production was associated with a rapid mitochondrial depolarization and Complex I proton pumping. Blocking Extracellular Signal-Regulated Kinase activation prevented both the fall in Psim and the increased acid production suggesting that the former underlies the accelerated mitochondrial 'acid production'. Mitochondrial Complex I inhibitors EIPA and rotenone prevented increased acid production despite Extracellular Response Kinase activation and reduced sodium/hydrogen ion activity. Inhibition of Complex I prevented TRO's effects on glutamine metabolism.


CONCLUSIONS
TRO induces cellular acidosis through Extracellular Signal-Regulated Kinase activation-associated acid production and impaired acid extrusion. Acutely, increased acid production reflects mitochondrial Complex I proton pumping into the cytosol while chronically Complex I activity appears coupled to mitochondrial glutamate uptake and oxidation to ammonium at the expense of cytosolic transamination and alanine formation in these proximal tubule-like cells.
Oliver
Robert
R
Department of Molecular and Cellular Physiology, LSUHSC, Shreveport, LA 71130, USA.
Friday
Ellen
E
Turturro
Francesco
F
Welbourne
Tomas
T
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002839:Chromans / Q000494:pharmacology*; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D042967:Electron Transport Complex I / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D017494:Proton Pumps / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013552:Swine; D045162:Thiazolidinediones / Q000494:pharmacology*; D000077288:Troglitazone
D002839:Chromans; D017494:Proton Pumps; D045162:Thiazolidinediones; D000641:Ammonia; D000584:Amiloride; D045304:Cytochromes c; D003576:Electron Transport Complex IV; D048049:Extracellular Signal-Regulated MAP Kinases; D042967:Electron Transport Complex I; D000077288:Troglitazone; C039614:ethylisopropylamiloride