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 NH3/NH4+ 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 HCO3-/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 HCO3-/CO2 from the medium was Cl--dependent, Na+-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na+-free HCO3-/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-/HCO3- exchanger that contribute to acid extrusion and Na+-independent Cl-/HCO3- 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.