Troglitazone's rapid and sustained activation of ERK1/2 induces cellular acidosis in LLC-PK1-F+ cells: physiological responses.
We studied the signal pathway through which troglitazone (TRO) acts in inducing cellular acidosis in LLC-PK1-F+ cells in relation to ammoniagenesis and DNA synthesis. Cells were grown to confluent monolayers in 30-mm chambers and monitored for intracellular pH (pHi) by the BCECF assay and activated ERK by phospo-ERK1/2 antibodies. TRO induces a severe cellular acidosis (pHi 6.68 +/- 0.10 vs. 7.28 +/- 0.07 time control at 4 min, P < 0.01), whereas phospho-ERK1/2 to total ERK1/2 ratio increases 3.4-fold (P < 0.01). To determine whether ERK1/2 was activated by cellular acidosis or TRO was acting via MEK1/2 to activate ERK1/2, cells were pretreated with specific inhibitors of MEK1/2 activity, PD-098059 and U-0126, followed by the addition of TRO or vehicle. With MEK1/2 activity inhibited, TRO treatment failed to activate ERK1/2. Preventing ERK1/2 activation abrogated the TRO-induced cellular acidosis and maintained the pHi within the low normal range (7.06 +/- 0.11). To determine whether blocking ERK activation prevents TRO's inhibitory effect on NHE activity, cells were acid-loaded and the recovery response was monitored as DeltapHi/t over a 4-min recovery period. TRO inhibited NHE activity by 85% (P < 0.01), whereas blocking ERK activation restored the response. We measured activated ERK levels and pHi after 3- and 18-h exposure to TRO or extracellular acidosis (pHe = 6.95) to determine whether ERK activation was sustained. Whereas both TRO and extracellular acidosis increased activated ERK and decreased pHi after 3 h, only TRO sustained this response at 18 h. Furthermore, both enhanced ammoniagenesis and decreased DNA synthesis reflected the effect of TRO to induce and sustain a cellular acidosis.
Oliver
Robert
R
Dept. of Molecular and Cellular Physiology, LSUHSC, Shreveport, LA 71130, USA.
Friday
Ellen
E
Turturro
Francesco
F
Lacy
Ashley
A
Welbourne
Tomas
T
D000136:Acid-Base Equilibrium / Q000187:drug effects; D000138:Acidosis / Q000139:chemically induced* / Q000378:metabolism; D000818:Animals; D002839:Chromans / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism; D018374:LLC-PK1 Cells; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D013552:Swine; D045162:Thiazolidinediones / Q000494:pharmacology*; D000077288:Troglitazone
D002839:Chromans; D007004:Hypoglycemic Agents; D045162:Thiazolidinediones; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D000077288:Troglitazone