Identification of α- and β-cells in intact isolated islets of Langerhans by their characteristic cytoplasmic Ca2+ concentration dynamics and immunocytochemical staining

Ratiometric images of cytoplasmic Ca2+ concentration ([Ca2+](c)) in individual cells were recorded simultaneously with a confocal ultraviolet-laser microscope in the Indo-1-loaded islets isolated from mice. After changes in [Ca2+](c) in response to glucose or amino acids were recorded, the islet was fixed, permeabilized, and stained by the indirect immunofluorescence method against insulin or glucagon in situ; the individual cells were then identified in the focal plain identical to that used for the [Ca2+](c) imaging. Almost all cells identified as insulin-positive (beta-cells) by their distinct immunofluorescence responded to the increase in glucose concentration from 3 to 11 mmol/l with an increase in [Ca2+](c). Major populations of cells (similar to 65%) identified as glucagon-positive (alpha-cells) responded to the addition of arginine (5-10 mmol/l to 3 mmol/l glucose solution with an increase in [Ca2+](c). About half of the alpha-cells (47.6%) responded to the addition of alanine (5-10 mmol/l) to 3 mmol/l glucose solution with an increase in [Ca2+](c), In contrast, <13% of beta-cells responded to the addition of alanine (5-10 mmol/l) or arginine (5-10 mmol/l) to 3 mol/l glucose with an increase in [Ca2+](c). More than one-fourth of alpha-cells responded with an increase in [Ca2+](c) when glucose concentration in perifusion solution was reduced from 11 to 0 mmol/l, These results indicate that [Ca2+](c) changes in islet cells stimulated by glucose or amino acid were characteristic of the cell species, at least in the alpha- and beta-cell, This technique provides a useful tool to investigate not only the intracellular signal transduction but also the intercellular signal transmission in the intact islet.