Calcium Uptake During Mitogenic Stimulation of Human Lymphocytes: Characterization of Intracellular Calcium Compartments and Demonstration of the Presence of Immunoreactive Calrecticulin

Abstract

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 #1 (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.