Fig. 8.
AMPK inhibits the PKA effects on V-ATPase activity in cultured S3 cells. Cells were grown on 24-well plate for 5 days before incubation in 0 Na/0 Bicarbonate solution in the absence/presence of PKA and/or AMPK activators. The rate of extracellular acidification in each set of treatments was measured in a low buffering capacity solution before and after the addition of bafilomycin, a specific V-ATPase inhibitor. The mean (±SE) rate of extracellular acidification (−[final buffer pH − initial buffer pH]/Δt) was obtained in the presence and absence of bafilomycin for each treatment condition. There was negligible cell death after these experiments, as determined by a Trypan blue exclusion assay across conditions. Data were obtained from 4 separate experiments per condition (*P = 0.06 vs. 0 Na/0 Bicarbonate solution pH 7.4; **P < 0.05 vs. 0 Na/0 Bicarbonate solution at pH 7.4; ***P < 0.05 vs. 0 Na/0 Bicarbonate solution at pH 7.4 + AICAR and 0 Na/0 Bicarbonate solution at pH 7.4 + 6-MB-cAMP + IBMX; ANOVA followed by paired t-tests). B: slot blot using the PKA phosphorylation substrate-specific antibody (top) with β-actin antibody as loading control (bottom) of whole S3 cell lysates following treatment of polarized S3 cells with 0 Na/0 Bicarbonate solution at pH 7.4 in the absence/presence of the PKA-activating drug cocktail (6-MB-cAMP:IBMX; 1 mM:0.5 mM) for 20 min. C: PKA activator 6-MB-cAMP significantly increased the PKA phosphorylation substrate signal compared with the control 0 Na/0 Bicarbonate solution at pH 7.4. Values are means (±SE) of PKA phosphorylation substrate signal normalized to β-actin signal (*P < 0.05; n = 3). D: average rate of change in extracellular proton concentration as measured by extracellular pH (pHo) using the pH-sensitive dye SNARF-5F. These measurements were performed over a 25-min time period at room temperature on the apical solution of confluent S3 cell monolayers grown on filters. Prior to and during the pHo measurement period, the monolayers were treated with either vehicle or 2 mM AICAR apically and basolaterally. At the start of the measurement period, the media on the apical sides were replaced with 0 Na/0 Bicarbonate pH 7.4 containing the dye SNARF-5F in the presence or absence of 1 mM 6-MB-cAMP/0.5 mM IBMX and in the continued presence or absence of AICAR (gray bars). In addition for each of the 4 treatment conditions, the pHo measurements were repeated for filters treated with the V-ATPase inhibitor bafilomycin (50 nM; black bars; *P < 0.05 with respect to 0 Na/0 Bicarbonate pH 7.4 alone; **P < 0.05 respect to 0 Na/0 Bicarbonate pH 7.4 6-MB-cAMP/IBMX and with respect to 0 Na/0 Bicarbonate pH 7.4 plus bafilomycin; n = 6–12).