title: Dual role of CO2/HCO3(-) buffer in the regulation of intracellular pH of three-dimensional tumor growths.
abstract: Intracellular pH (pH(i)), 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 HCO(3)(-) taken up by carriers such as Na(+)-HCO(3)(-) cotransport. Using fluorescence pH(i) imaging, we show that cancer-derived cell lines (colorectal HCT116 and HT29, breast MDA-MB-468, pancreatic MiaPaca2, and cervical HeLa) extrude acid by H(+) efflux and HCO(3)(-) influx, largely sensitive to dimethylamiloride and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), respectively. The magnitude of HCO(3)(-) influx was comparable among the cell lines and may represent a constitutive element of tumor pH(i) regulation. In contrast, H(+) efflux varied considerably (MDA-MB-468 > HCT116 > HT29 > MiaPaca2 > HeLa). When HCO(3)(-) flux was pharmacologically inhibited, acid extrusion in multicellular HT29 and HCT116 spheroids (∼10,000 cells) was highly non-uniform and produced low pH(i) at the core. With depth, acid extrusion became relatively more DIDS-sensitive because the low extracellular pH at the spheroid core inhibits H(+) flux more than HCO(3)(-) flux. HCO(3)(-) flux inhibition also decelerated HCT116 spheroid growth. In the absence of CO(2)/HCO(3)(-), acid extrusion by H(+) flux in HCT116 and MDA-MB-468 spheroids became highly non-uniform and inadequate at the core. This is because H(+) transporters require extracellular mobile pH buffers, such as CO(2)/HCO(3)(-), to overcome low H(+) ion mobility and chaperone H(+) ions away from cells. CO(2)/HCO(3)(-) exerts a dual effect: as substrate for membrane-bound HCO(3)(-) 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 CO(2)/HCO(3)(-) is important for maintaining uniformly alkaline pH(i) in small, non-vascularized tumor growths and may be important for cancer disease progression.
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, United Kingdom.
authors.forename: Alzbeta
authors.initials: A
authors.lastname: Hulikova
authors.forename: Richard D
authors.initials: RD
authors.lastname: Vaughan-Jones
authors.forename: Pawel
authors.initials: P
authors.lastname: Swietach
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D001639:Bicarbonates / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport; D002021:Buffers; D002245:Carbon Dioxide / Q000378:metabolism*; D045744:Cell Line, Tumor; D004058:Diffusion; D018450:Disease Progression; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D018613:Microscopy, Confocal / Q000379:methods; D009369:Neoplasms / Q000473:pathology*; D011522:Protons; D018874:Spheroids, Cellular / Q000473:pathology
chemical_list: D001639:Bicarbonates; D002021:Buffers; D007477:Ions; D011522:Protons; D002245:Carbon Dioxide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid