Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC189
The dual role of CO2/HCO3- buffer in the regulation of intracellular pH of three-dimensional, multi-cellular tumour growths
A. Hulikova1, R. D. Vaughan-Jones1, P. Swietach1
1. Physiology, Anatomy and Genetics, Oxford University, Oxford, United Kingdom.
Histograms show the pHi gradient from the spheroid core to its periphery measured after 12 minutes of recovery from an imposed acid-load (produced by a 6 minute 20 mM ammonium prepulse). Error bars denote SEM. Asterisks denote statistically significant differences compared with control (shaded bars) at the 5% level using unpaired t-tests [except for testing the effects of acetazolamide (ATZ), where paired t-tests were performed]. CO2/HCO3- serves a dual role in pHi regulation in tumours: as a substrate for HCO3--dependent acid-extruders and as a mobile buffer assisting H+ ion extruders by facilitating extracellular H+ ion diffusion.
Intracellular pH (pHi), a major modulator of cell function, is regulated by acid/base transport across membranes. Excess intracellular H+-ions (e.g. produced by respiration) are extruded by transporters such as Na+/H+ exchange, or neutralized by HCO3- taken-up by carriers such as Na+-HCO3- cotransport. Using fluorescence pHi-imaging, we show that cancer-derived cell-lines (colorectal HCT116 and HT29, breast MDA-MB-468, pancreatic MiaPaca2, cervical HeLa) extrude acid by H+-efflux and HCO3−-influx, largely sensitive to 30 μM dimethylamiloride (DMA) and 100 μM 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), respectively. The magnitude of HCO3--influx was comparable among the cell-lines, and may represent a constitutive element of tumor pHi regulation (3.0±0.4[SEM] to 4.3±0.4 mM/min at pHi=6.7). In contrast, H+-efflux varied considerably (MDA-MB-468>HCT116>HT29>MiaPaca2> HeLa; from 7.0±0.4[SEM] to 0.7±0.2[SEM] mM/min at pHi=6.7). When HCO3--flux was pharmacologically inhibited with DIDS, acid-extrusion in multi-cellular HT29 and HCT116 spheroids (radii 130-200 μm; ~10,000-50,000 cells) was highly non-uniform and produced low pHi at the core (see Figure). With depth, acid-extrusion became relative more DIDS-sensitive because the low extracellular pH at the spheroid core inhibits H+-flux more than HCO3--flux. HCO3--flux inhibition also decelerated HCT116 spheroid growth. In the absence of CO2/HCO3-, acid-extrusion by H+-flux in HCT116 and MDA-MB-468 spheroids became highly non-uniform and inadequate at the core (see Figure). This is because H+-transporters require extracellular mobile pH-buffers, like CO2/HCO3-, to overcome low H+-ion mobility and chaperone H+-ions away from cells. The effect of 5% CO2/22 mM HCO3- was mimicked by 30 mM Hepes (an artificial buffer system) but reduced when carbonic anhydrase activity (which normally keeps CO2/HCO3- at equilibrium) was inhibited with acetazolamide (100 μM; see Figure). CO2/HCO3- exerts a dual effect: as substrate for membrane-bound HCO3--transporters, and as a mobile buffer for facilitating extracellular diffusion of H+-ions extruded from cells. These processes can be augmented by carbonic anhydrase activity. We conclude that CO2/HCO3- is important for maintaining uniformly alkaline pHi in small, non-vascularized tumor-growths and may be important for cancer disease progression.
Where applicable, experiments conform with Society ethical requirements