Carbonic anhydrase IX unifies spatial pH i regulation in large cell‐clusters

Abstract

Carbonic anhydrase (CA) IX is a hypoxia-induced transmembrane enzyme that catalyses reversible CO2 hydration. Many tumours express CAIX, and CA inhibitors can limit their growth. We induced CAIX expression in RT112 cells (derived from human bladder cancer) and studied its effect on spatial pHi regulation. Cells were grown into multicellular clusters (300μm radius) and loaded with a pH-dye (carboxy SNARF-1). A pHi map was imaged confocally every 4s, horizontally through the cluster. CAIX-free clusters, when superfused with 5% CO2/22mM HCO3− buffer, displayed a core-to-periphery standing pHi gradient of 0.24±0.01(n=10) units (acidic core). In contrast, CAIX-expressing clusters showed a greatly diminished gradient of 0.09±0.01(n=9). This increased to 0.15±0.01(n=10) in the presence of 200nM 14v, a membrane-impermeant CA inhibitor. Superfusion with 20% CO2/88mM HCO3− induced a reversible fall of cluster pHi. In CAIX-free clusters, pHi changes in the core were 45% slower than in the periphery. This difference was attenuated to 16% in CAIX-expressing clusters, while it increased again to 26% when CAIX was inhibited with 14v. These results suggest that, in non-vascularized groups of cells, a key role of extracellular-oriented CAIX is to unify pHi spatially within the cluster, both under steady state conditions and during dynamic changes. The coordination of pHi may facilitate further growth and development. Supported by the BHF (UK) and CR-UK.