Effects of Alanine on Insulin-secreting Cells: Patch-clamp and Single Cell Intracellular Ca2+ Measurements

The effects of alanine, glucose and tolbutamide on insulin-secreting cells (RINm5F) have been investigated using patch-clamp and single cell intracellular Ca2+ measurements. When directly challenged with the amino acid L-alanine (2-10 mM) the cells underwent a sharp depolarization, which led to the generation of Ca2+ spike potentials and an increase in [Ca2+]i. The L-alanine-induced depolarization was associated with a net inward membrane current but no measurable change in the resistance of the cell. The latter effect was found to be in contrast to the actions of glucose (5-10 mM) and tolbutamide (100 microM), both of which depolarized cells and raised [Ca2+]i by an increase in the input resistance of the cell membrane, due to the closure of ATP-sensitive potassium channels. In the complete absence of external Na+ (by replacement with 140 mM NMDG+), L-alanine had no effects on either the membrane potential or [Ca2+]i. Similarly, replacing Na+ with NMDG+ in the continued presence of the amino acid resulted in a repolarization of the membrane and an attenuation of the L-alanine-induced rise in [Ca2+]i. The Na+ channel blocker TTX (1-2 microM) had no effects on the alanine-evoked electrical activity. Exchange of the L-form of the amino acid with the D-stereoisomer had similar actions to those of removing external Na+, since D-alanine had no effects on the membrane potential or [Ca2+]i. The actions of L-alanine were also found to be mimicked by the N-methylated amino acid analogue methylamino isobutyric acid (MeAIB) (2-10 mM), suggesting that the A-type electrogenic amino acid cotransport system operates in the RINm5F insulin-secreting cell line.