Intracellular pH regulation in colonocytes of rat proximal colon

AbstractThe regulation of intracellular pH (pHi) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pHi in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pHi=7.24±0.04 and removal of extracellular Na+ lowered pHi by 0.24 pH units. Acid-loaded colonocytes by an NH3/NH+4 prepulse exhibited a spontaneous recovery that was partially Na+-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na+-dependent recovery rate was enhanced by increasing the extracellular Na+ concentration and was further stimulated by aldosterone. In an Na+- and K+-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K+ and this K+-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pHi exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO−3/CO2-containing solution, pHi of the proximal colonocytes was 7.20±0.04. Removal of external Cl− caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO−3/CO2 from the medium was Cl−-dependent, Na+-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na+-free HCO−3/CO2-containing solution was accelerated by addition of Na+. Removal of Cl− inhibited the effect of Na+. In summary, the freshly isolated proximal colonocytes of rats express Na+/H+ exchanger, H+/K+ exchanger ((H+-K+)-ATPase) and Na+-dependent Cl−/HCO−3 exchanger that contribute to acid extrusion and Na+-independent Cl−/HCO−3 exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pHi in vivo. Proximal colonocytes are able to maintain a more stable pHi than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity