Figure 2.

Pathophysiologic pH_o changes also affects CaR-induced ERK activation, actin polymerization, and L-Phe responsiveness in CaR-HEK cells. (A) Representative immunoblot showing ERK phosphorylation (pERK) after treatment with 3.5 mM Ca²⁺_o for 10 minutes under different pH_o conditions (7.4±0.2). Mean changes in ERK phosphorylation were determined by densitometry and expressed on a bar graph in arbitrary units. n=9 dishes from two independent experiments. *P<0.05 and **P<0.01 versus pH_o 7.4 by one-way ANOVA (Dunnet). (B) Representative immunofluoresence images (i) showing phalloidin-TRITC staining following CaR-HEK treatment with 1.5 mM Ca²⁺_o for 3 hours in buffers of pH_o ranging from 7.2 to 7.6. Quantification presented on a bar graph as pixels (phalloidin-TRITC) per unit area in five randomly selected regions of the cells is shown below (iii). Also shown are bar graphs from separate experiments where the cells were exposed to 0.5 (ii) or 1.8 (iv) mM Ca²⁺_o. n≥5 coverslips. *P<0.05 and **P<0.01 versus pH_o 7.4 by one-way ANOVA (Dunnet). (C) Representative Ca²⁺_i trace (i) showing CaR-HEK cells stimulated first with 10 mM L-Phe (in 1.8 mM Ca²⁺_o) and then with 3 mM Ca²⁺_o under various pH_o conditions as shown (0.5 Ca²⁺_o, baseline, pH 7.4 unless otherwise stated). The traces include a typical response in a single cell (gray) as well as the total response in the cell cluster (>10 cells; “global”, black). Quantification (ii) is shown as a bar graph (percentage change in L-Phe responsiveness relative to pH_o 7.4 control). n=7 coverslips from two independent experiments. **P<0.01 by repeated-measures ANOVA (Bonferroni).