The glow of the colonic pH microclimate kindled by short- chain fatty acids, chloride and bicarbonate

Epithelial cells are interface engines. They sit at the boundary between two distinct compartments (e.g. the intestinal lumen and the body) and consume cellular energy to shuttle solutes and water between the two compartments. Theoretical and experimental evidence suggests that one consequence of this transepithelial transport is a microscopic environment directly adjacent to epithelial cells. This microenvironment can maintain a composition distinct from the bulk solutions because of (1) limited mixing near the cellular surface, (2) fixed surface charges on the membrane, and (3) robust transport across the cell membrane. The concept is important because this is the environment which controls activation of membrane transport to drive transepithelial transport and the uptake of many drugs. Such microenvironments have been proposed to have physiological roles in the stomach, airways and intestines.

In this issue of The Journal of Physiology Genz, Engelhardt & Busche explore the extracellular pH directly adjacent to the apical membrane of colonic epithelia (referred to as the pH microclimate). The investigators are not alone in this goal. Early studies with pH-sensitive microelectrodes discovered a relatively constant pH microclimate near the surface epithelium of mammalian colon (McNeil et al. 1987), but results were plagued with questions about the precise location of the electrodes and the spatial resolution of such methods. More recent studies with water-soluble pH-sensitive fluorescent dyes and new modes of microscopy (confocal and digital deconvolution) have successfully measured the dynamic changes of extracellular pH in mouse colonic crypts (Chu & Montrose, 1996) with submicrometre resolution. However, these measurements from the aqueous phase leave lingering doubts about the events directly at the membrane surface.

Enter the innovative contribution by Genz et al. who used a near-membrane pH probe composed of a pH-sensitive fluorescent dye coupled to an aliphatic (lipophilic) chain. This idea builds on a landmark paper by Dragsten et al. (1981) which shows that long saturated fatty acids stay in the outer leaflet of the membrane bilayer and hence can be restricted to either the apical or basolateral domain in epithelial cells. Genz et al. extended this observation to develop a method for measurement of pH with 5-N-hexadecanoyl-aminofluorescein (HAF), which inserts into the outer leaflet of the apical membrane of isolated guinea-pig colonic epithelium. The authors carefully documented the site of dye insertion, tissue viability, and the quality of fluorescence response to changes in pH in the range of pH 6-8. Thus, the authors established a method for the non-invasive measurement of pH directly at the outer surface of the apical membrane of the surface epithelium.

The authors applied the method to investigate the effects of chloride, bicarbonate and butyrate on the pH microclimate. An important contribution in the paper is a novel observation regarding chloride. Genz et al. show for the first time that a Cl⁻-HCO₃⁻ exchanger is probably a major contributor to surface pH microclimate. This is an important observation regarding the transporter widely believed to mediate colonic bicarbonate secretion. In contrast, there was little support for expectations that H⁺-K⁺-ATPase was the dominant apical transporter mediating proton efflux (and thereby supplying luminal protons to sustain SCFA absorption) in guinea-pig distal colon (Englelhardt et al. 1993). Genz et al. report that H⁺-K⁺-ATPase had only a slight influence on the surface pH.

The colonic lumen is a bacterial broth that metabolizes undigested protein and carbohydrate to produce short-chain fatty acids (SCFAs) such as acetate, propionate and butyrate. SCFAs hold fascination for investigators as the most abundant anions and osmolytes in the colonic lumen, as physiological stimulators of sodium absorption and bicarbonate secretion, and because they hold a tantalizing link to the health of the colon which goes awry in ulcerative colitis. Genz et al. found that a SCFA (butyrate) qualitatively affected surface pH in a similar way to HCO₃, which gives important corroboration for earlier observations by Chu & Montrose (1996) with a different method, a different species (mouse), and a different part of the epithelium (surface versus crypt). Results of Genz et al. hint that Cl⁻-SCFA exchange may explain part of the effects of luminal chloride on the pH microclimate, although evidence is also presented that other (Cl⁻ independent) routes of SCFA flux must also be important to microclimate regulation. However, it is exciting to have a putative physiological role for the Cl⁻-SCFA exchange reported in colonic membrane vesicles by Rajendran & Binder (1994).

The contribution of Genz et al. strengthens the belief that regulated extracellular microenvironments are part of the cellular milieu, at least for transporting epithelia. The existence of a microscopic space with its own behaviour is undeniably awkward for studies of cell and tissue response. However, it should be recognized that microenvironments also provide a new opportunity. Disturbances in microenvironment may contribute to disease states by causing changes in drug uptake, cell/tissue viability, or changing the activity of membrane transporters.

Optical approaches have become the preferred tools for non-invasively measuring microenvironments, and this fuels their ascent as powerful and essential tools for the study of ion transport by cells and tissues. We now have the opportunity for a clearer vision of the world from a cellular perspective.