Regulation of pH in individual pancreatic ß-cells as evaluated by fluorescence ratio microscopy

Abstract

Pancreatic ß-cells are known to maintain intracellular pH (pH i) at a value well above that predicted from the electrochemical gradient. The mechanisms for the active extrusion of protons were examined by continuously monitoring pH i in individual ß-cells from ob/ob mice using the fluorescent indicator 2′,7′-bis(car☐yethyl)-5(6)-car☐yfluorescein (BCECF). In a medium nominally devoid of bicarbonate, the steady-state pH i was 6.82±0.02 and the intracellular buffering capacity was equivalent to 79±3 mM/pH unit. pH i remained unaffected after raising the glucose concentration from 3 to 20 mM, it was lowered when depolarizing the ß-cells with tolbutamide and it increased in the presence of carbachol. After removal of Na + there was a significant drop of pH i and blockage of the pH i recovery following acid loading with the NH + 4 prepulse technique. Whereas addition of amiloride had a similar, but less pronounced effect, omission of Cl − resulted in moderate alkalinisation. After switching to a medium containing bicarbonate, minor acidification was followed by adjustment of pH i to a steady state higher than the initial one. The results indicate that the acid load arising from glucose metabolism in the ß-cells is effectively buffered and the protons extruded both by Na + H + and Cl − HCO − 3 exchangers.