Intracellular pH regulation in colonocytes of rat proximal colon

Abstract

The regulation of intracellular pH (pH i) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH i in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH i=7.24±0.04 and removal of extracellular Na + lowered pH i by 0.24 pH units. Acid-loaded colonocytes by an NH 3/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 pH i 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/CO 2-containing solution, pH i 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/CO 2 from the medium was Cl −-dependent, Na +-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na +-free HCO − 3/CO 2-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 pH i in vivo. Proximal colonocytes are able to maintain a more stable pH i than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.