Fig. 4.

Carbonic anhydrase IX (CA IX) is critical to intracellular pH regulation in pulmonary microvascular endothelial cells (PMVECs). A and B: PMVECs were seeded at 2.0 × 10⁵ cells/well on 6-well plates with glass coverslips on bicarbonate-buffered media and grown to confluence for 3 days. Media was changed to HEPES-buffered pH 7.4 or 2-(N-morpholino)ethanesulfonic acid (MES)-buffered pH 6.8 media, and cells were treated with DMSO (0.15%), acetazolamide (AZ) (150 µM), or SLC-0111 (150 µM). 2 days after inhibitor treatment, intracellular (A) and extracellular (B) pH was measured by pH-sensitive fluorescent dye BCECF-AM and pH meter, respectively. C: wild-type and CA IX knockout (KO) PMVECs were grown to confluence for 3 days as described above, and media was changed to MES-buffered pH 6.2 media. 3 days after media change, intracellular pH was measured by BCECF-AM. Intracellular pH is reported as percentage of baseline [pH 7.4 DMSO (A) and wild-type (C)]. Either chemical or genetic CA IX inhibition decreased intracellular pH in PMVECs. Data represent means ± SD. 5 or more separate experiments were performed. 1-way ANOVA was used to compare between groups. *Significant difference (P < 0.05) from DMSO (A and B) or wild-type (C) groups. ^Significant difference (P < 0.05) from pH 7.4 SLC-0111 group.