Purpose: The purpose of this study was to assess whether troglitazone (TRO) would induce cellular acidosis by inhibiting Na ؉ /H ؉ exchanger (NHE) 1 in breast carcinoma-derived cell lines and, if so, whether cellular acidosis would be associated with a reduction in proliferation.Experimental Design: Intracellular pH (pH i ) and acid extrusion capacity after an exogenous acid load were assayed using (2, 7)-biscarboxyethyl-5(6)-carboxyfluorescein in MCF-7 and MDA-MB-231 cells treated with TRO. Radiolabeled thymidine incorporation was used to assess DNA synthesis. Peroxisome proliferator-activated receptor (PPAR) ␥ involvement was assessed using an antagonist and PPAR␥ ؊/؊ NIH3T3 cells. Results: TRO induced a prompt (<4 minute) and severe cellular acidosis in both MCF-7 (7.54 ؎ 0.23 to 6.77 ؎ 0.06; P < 0.001) and MDA-MB-231 cells (7.38 ؎ 0.18 to 6.89 ؎ 0.25; P < 0.05) after 12 minutes, without increasing acid production. Acid extrusion as assessed by the response to an exogenous acid load (NH 4 Cl pulse) was markedly blunted (MDA-MB-231, P < 0.01) or eliminated (MCF-7, P < 0.001). Chronic exposure to TRO resulted in NHE1 activity reduction (P < 0.05) and a dose-dependent decrease in DNA synthesis (<75% inhibition at 100 mol/L; P < 0.001 and P < 0.01 for MCF-7 and MDA-MB-231, respectively) associated with a decreased number of viable cells. TROmediated inhibition of proliferation was not reversed by the presence of the PPAR␥ inhibitor GW9662 and was demonstrable in PPAR␥ ؊/؊ NIH3T3 cells, consistent with a PPAR␥-independent mechanism.Conclusions: TRO induces marked cellular acidosis in MCF-7 and MDA-MD-231 cells. Sustained acidosis is consonant with decreased proliferation and growth that is not reversed by a PPAR␥ antagonist. Our results support a NHE-mediated action of TRO that exerts its effect independent of PPAR␥.

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Troglitazone was studied in pH-sensitive LLC-PK1-F+ cells to determine the effect on pHi and glutamine metabolism as well as the role of peroxisome proliferator-activated receptor (PPARgamma)-dependent and PPARgamma-independent signaling pathways. Troglitazone induces a dose-dependent cellular acidosis that occurs within 4 min and persists over 18 h as a result of inhibiting Na+/H+ exchanger-mediated acid extrusion. Cellular acidosis was associated with glutamine-dependent augmented [15N]ammonium production and decreased [15N]alanine formation from 15N-labeled glutamine. The shift in glutamine metabolism from alanine to ammoniagenesis appears within 3 h and is associated after 18 h with both a reduction in assayable alanine aminotransferase (ALT) activity as well as cellular acidosis. The relative contribution of troglitazone-induced cellular acidosis vs. the decrease in assayable ALT activity to alanine production could be demonstrated. The PPARgamma antagonist bisphenol A diglycide ether (BADGE) reversed both the troglitazone-induced cellular acidosis and ammoniagenesis but enhanced the troglitazone reduction of assayable ALT activity; BADGE also blocked troglitazone induction of peroxisome proliferator response element-driven firefly luciferase activity. The protein kinase C (PKC) inhibitor chelerythrine mimics troglitazone effects, whereas phorbol ester reverses the effects on ammoniagenesis consistent with troglitazone negatively regulating the DAG/PKC/ERK pathway. Although functional PPARgamma signaling occurs in this cell line, the major troglitazone-induced acid-base responses appear to be mediated by pathway(s) involving PKC/ERK.