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Troglitazone's rapid and sustained activation of ERK1/2 induces cellular acidosis in LLC-PK₁-F⁺ cells: physiological responses

¹Departments of Molecular and Cellular Physiology and ²Department of Medicine, Feist-Weiller Cancer Center and ³Gene Therapy Program, Louisiana State University Health Science Center, Shreveport, Louisiana

Submitted 2 June 2004 ; accepted in final form 21 January 2005

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 (pH_i) by the BCECF assay and activated ERK by phospo-ERK1/2 antibodies. TRO induces a severe cellular acidosis (pH_i 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 pH_i 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 pH_i/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 pH_i 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 pH_i 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.

peroxisome proliferator-activated receptor- independent; intracellular pH; P-ERK; sodium hydrogen exchanger; ammoniagenesis; nonvolatile acid production; DNA synthesis

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