Effect of cholinergic stimulation on free intracellular Ca2+ concentration in human lymphocytes

Binding of ligand to receptor, in various types of cells, results in changes in calcium concentration, which is an important factor in cellular signal transduction. Lymphocytes receive signals from the parasympathetic nervous system through the cholinergic receptors. Cholinergic receptors mediate response to stimuli through changes of the IP3, or cAMP level and Ca2+ mobilization in various types of cells. The aim of this work was to measure changes in calcium concentration in cytosol of lymphocytes stimulated with acetylcholine agonists - carbachol (analogue of acetylcholine) or nicotine. Human peripheral blood mononuclear cells (PMBC) and lymphocytes from the leukemic cell lines Jurkat and Raji were used. Immune cells were preactivated with mitogen phytohemagglutinin (PHA) for PBMC and Jurkat cells or lipopolysaccharide for Raji cells. Two methods of measurement of calcium concentration were used. With the first, calcium concentration was measured in the suspension of cells loaded with the fluorescent dye Fura-2AM. With the other method, calcium concentration was assessed in single cells loaded with the fluorescent dye Indo-1AM. Using the method of single cell investigation, we observed an increase in the level of calcium concentration induced by carbachol and nicotine. The method of measuring the Ca2+ concentration in a cell suspension was found to be not sensitive enough for this purpose. The increase in calcium concentration resulted both from the stimulation of Ca2+ influx and from the release of Ca2+ from intracellular stores, most likely due to the increase in the concentration of IP3. In conclusion, we suggest that the lymphocytes activated with PHA respond to cholinergic stimulation with an increase in their free cytoplasmic Ca2+ concentration.