Effects of nutrient and non-nutrient stimuli on cytosolic pH in cultured insulinoma (HIT-T15) cells

Abstract

Intracellular pH (pH i) was measured in the insulin-secreting HIT-T15 cell line using the pH-sensitive fluorescent dye, 2′,7′-bis(carboxyethyl)-5′(6′)-carboxyfluorescein (BCECF). It was observed that the addition of a weak acid (e.g., acetate or propionate) caused a rapid decrease in pH i, followed by a slower recovery to the resting pH value. Conversely the addition of N 4Cl caused an increase in pH i followed by recovery. The addition of amiloride caused a fall in pH i; however, in this case no recovery to basal pH levels was observed. Subsequent addition of a weak acid caused a further fall in pH i with no recovery. The addition of glucose caused a transient acidification followed by alkalinization. When glucose was added to cells which had been pretreated with amiloride, the initial acidification was not followed by recovery or alkalinization. Addition of glyceraldehyde, α-ketoisocaproate, lactate or pyruvate to HIT cells also resulted in intracellular acidification followed by recovery. Similarly, depolarisation of HIT cells by treatment with high K + or with Ba 2+ was associated with a pronounced fall in pH i, followed by a gradual recovery. Insulin secretion from HIT cell was stimulated by glucose, glyceraldehyde, α-ketoisocaproate, lactate, pyruvate and KCl, whilst amiloride and weak acids exerted only modest effects in the absence of glucose, but amiloride in particular markedly potentiated glucose-induced insulin release. Thus, HIT cells appear to have an amiloride-sensitive mechanism for the extrusion of protons, probably Na +-H + exchange. Whilst intracellular acidification appears to potentiate secretory responses to nutrient stimuli, it seems unlikely that the activation of HIT cells by these nutrients occurs as a result of intracellular acidification. The mechanisms by which various nutrient and non-nutrient stimuli might exert distinct effects on pH i are discussed.