P‐ERK Activation by Extracellular or Intracellular Acidosis Slows NHE3‐Mediated Acid Extrusion And Activates Ammoniagenesis in LLC‐PK1‐F+ Cells

Abstract

We exposed proximal tubule-like F+ cells to extracellular acidosis pHo=6.8 or troglitazone (TRO)-induced intracellular acidosis pHi<6.8(pHo=7.4) for 4min and 3h in order to determine cell signaling and physiological responses (NHE3 activity and NH4+production). Exposure to pHo =6.8 for 4min and 3h reduced the pHi from 7.32 ±0.07 to 6.96±0.04 and 6.75±0.11 associated with a 2.3 and 2.7 fold rise in PERK/T-ERK ratio (both p<0.05). Exposure to TRO (20uM) reduced pHi from 7.32±0.07 to 6.60±0.08 and 6.86±0.15 associated with a 3.7 and 2.1 rise in P-ERK at 4min and 3h respectively. NHE3 activity measured after transient 6min exposure to pHo=6.8 was reduced by 32% (p<0.01) while NH4+ increased by 1.65 fold (p<0.05) at 3h; TRO reduced NHE3 activity by 82% and NH4+ 2.4 fold (both p<0.01). TRO-induced P-ERK activation and fall in pHi was prevented by inhibiting EGFR in contrast EGFR inhibition with pHo 6.8 did not decrease P-ERK activation or cellular acidosis. Pre-incubation with MEKK inihibitors PD98059 and UO126 prevented the acidosis-induced increase in P-ERK and ammoniagenesis. Our results are consonant with P-ERK activation associated with extracellular acidosis and intracellular acidosis (K+ deficiency?) playing a major role in regulating acute physiological responses.