Identification of alpha- and beta-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 (approximately 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.
Asada
N
N
Medical Sciences Laboratory, Hokkaido University of Education at Asahikawa, Japan. asada@asa.hokkyodai.ac.jp
Shibuya
I
I
Iwanaga
T
T
Niwa
K
K
Kanno
T
T
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D018613:Microscopy, Confocal; D013194:Staining and Labeling
D002118:Calcium