Telmisartan, an Angiotensin II receptor blocker, abrogates troglitazone‐induced cell signaling coupled to ammoniagenesis in proximal tubule‐like LLC‐PK1 cells

Abstract

Troglitazone(TRO) induces a large increase in ammonium production from glutamine via the mitochondrial pathway in proximal tubule‐like LLC‐PK1 cells. To elucidate the upstream site of tro action we measured the acute effects on cell signaling in LLC‐PK1 cells grown in chambers designed for fluorometric ratiometric imagining (BCECF and Fura 2) and in 24 well plates for 18h metabolic studies. TRO (25uM) induces a large fall (p<0.01) in cytosolic pH (7.19±0.09 to 6.85±0.08) and a doubling of cell calcium occurring within 8minutes. Associated with these responses was a similarly rapid and large increase (p<0.05) in phospho‐ERK1/2(p‐ERK to total ERK ratio=1±0.2 for control vs 10.1±3 fold for TRO). Treating the cells with 30uM telmisartan (pre‐incubation for 4minutes and during 8minutes with TRO) markedly reduced both the rise in p‐ERK1/2(10.1±3 to 2.4±0.5) and acidity (6.85±0.08 to 7.11± 0.07) suggesting that telmisartan acutely blocks TRO's upstream site of action. At the downstream site, TRO (25uM) enhanced (p<0.01) mitochondrial ammoniagenesis (NH4+ production 1011±110 to 1678±84nmol/mg/18h) despite a normal extracellular bicarbonate concentration and pH; addition of telmisartan (30uM) to the TRO‐media prevented (p<0.05) the enhanced ammonium production (1193±142 vs 1678±84 nmol/mg/18h for TRO+telmisartan vs TRO). These findings are consonant with angiotensin II‐like receptor involvement in TRO's initial activation of cell signaling pathways underlying the ammoniagenic response in these model porcine proximal tubule‐like cells.