title: Effects of alanine on insulin-secreting cells: patch-clamp and single cell intracellular Ca2+ measurements.
abstract: 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.
authors.affiliation: MRC Secretory Control Research Group, University of Liverpool, U.K.
authors.forename: M J
authors.initials: MJ
authors.lastname: Dunne
authors.forename: D I
authors.initials: DI
authors.lastname: Yule
authors.forename: D V
authors.initials: DV
authors.lastname: Gallacher
authors.forename: O H
authors.initials: OH
authors.lastname: Petersen
mesh_terms: D000409:Alanine / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D010190:Pancreatic Neoplasms; D051381:Rats; D014044:Tolbutamide / Q000494:pharmacology
chemical_list: D007328:Insulin; D014044:Tolbutamide; D005947:Glucose; D000409:Alanine; D002118:Calcium