Calcium uptake during mitogenic stimulation of human lymphocytes: characterization of intracellular calcium compartments and demonstration of the presence of immunoreactive calrecticulin.
Phytohemagglutinin (PHA) stimulation of human peripheral blood lymphocytes (HPBL) rapidly increases 45Ca2+ uptake into intracellular pools. Detectable increase in 45Ca2+ uptake occurred only on exposure to mitogenic lectins but not with non-mitogenic lectins. However, intracellular free Ca2+ concentration [(Ca2+)i] increased comparably on exposure to either mitogenic or non-mitogenic lectins. Permeabilization of 45Ca2+ loaded cells revealed distinct pools of Ca2+ uptake. The highly digitonin sensitive pool #I (permeabilized by 0.02% digitonin) exchanged slowly and included a part that represented endoplasmic reticulum. Pool II was defined by lower digitonin sensitivity, had a much faster initial uptake. Pool III was digitonin-resistant and predominantly non-vesicular. During the first 120 min of PHA stimulation, significant increase in 45Ca2+ uptake occurred only into pool II. Progressive increase in uptake into pool I then occurred so that by 24 hours, this pool constituted the major fraction of PHA induced increment in total 45Ca2+ uptake. Using specific antibody to the calcium binding protein calreticulin, an analogous immunoreactive protein was detectable in resting HPBL. PHA stimulation led to a striking increase in abundance of immunoreactive calreticulin so that 24 hrs after PHA stimulation, there was a 28 and 3.4 fold increase in the amount of immunoreactive calreticulin present in the non-particulate fraction and the total particulate membrane fraction, respectively. A major part (72%) of the total cellular immunoreactive calreticulin in PHA stimulated cells at 24 hrs was released into the medium after permeabilization of lymphocytes with 0.02% digitonin, corresponding to the location of calcium uptake pool I.
Mookerjee
B K
BK
Department of Medicine, VA Medical Center, Buffalo, New York.
Chakrabarti
R
R
Lee
T P
TP
Michalak
M
M
Ballard
J
J
D001324:Autoantigens / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037282:Calreticulin; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D004072:Digitonin / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D016257:Fura-2; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*
D001324:Autoantigens; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D012261:Ribonucleoproteins; D004072:Digitonin; D002118:Calcium; D016257:Fura-2