ABSTRACT
It is known that lymphocytes express functional muscarinic cholinergic receptors. In this study, RT-PCR method was applied to study the presence and relative levels of mRNA encoding muscarinic receptor subtypes in human peripheral blood mononuclear cells (PBMCs). Our results, confirmed by DNA sequencing, demonstrate the presence of m2, m3, m4, and m5 receptor subtypes in human PBMCs. The relative levels of muscarinic receptor subtypes fit the following pattern: m3 > m5 > m4 > m2. Our data provide strong evidence confirming previous pharmacological studies that suggested the existence of several subtypes of muscarinic receptors on human PBMCs. We cannot exclude the possibility that expression of receptor subtype depends on the lineage and/or activation status of the cell.
Subject(s)
Lymphocytes/chemistry , Receptors, Muscarinic/genetics , Receptors, Muscarinic/immunology , Cholinergic Agonists/pharmacology , DNA Primers , Gene Expression/immunology , Humans , Lymphocytes/drug effects , Lymphocytes/immunology , Neuroimmunomodulation/genetics , Neuroimmunomodulation/immunology , Parasympathetic Nervous System/chemistry , Parasympathetic Nervous System/immunology , RNA, Messenger/analysis , Receptor, Muscarinic M2 , Receptor, Muscarinic M3 , Receptor, Muscarinic M4 , Receptor, Muscarinic M5 , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
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.
Subject(s)
Calcium/metabolism , Carbachol/pharmacology , Lymphocytes/metabolism , Nicotine/pharmacology , Humans , Lymphocytes/drug effectsABSTRACT
The interrelationships between the immune and neurohormonal system are the subject of this work. In this context, the formation of inositol-1,4,5-trisphosphate (IP3) in response to cholinergic stimulation was studied in activated human peripheral blood mononuclear cells (PBMC) and lymphocytes from the lymphoblastoid cell lines Jurkat and Raji. The cells were stimulated with mitogen and then treated with either the cholinergic agonist carbachol or nicotine. The radioreceptor assay was used to determine IP3. Cholinergic agonists increased the level of IP3 in all studied lymphocytes. Maximal level of IP3 was observed in PBMC after stimulation with nicotine, whereas in Raji cells IP3 level significantly increased after stimulation with carbachol and nicotine. These results indicate that the effect of stimulation of cholinergic receptors present on the activated human lymphocytes appears to be followed by an enhancement in the rate of polyphosphoinositide hydrolysis.
Subject(s)
Carbachol/pharmacology , Inositol 1,4,5-Trisphosphate/blood , Lymphocytes/drug effects , Nicotine/pharmacology , Receptors, Cholinergic/drug effects , Atropine/pharmacology , Cell Line , Cholinergic Fibers/drug effects , Dose-Response Relationship, Drug , Humans , Lymphocytes/immunology , Radioligand Assay , Tubocurarine/pharmacologyABSTRACT
The interrelationships between the immune and neurohormonal system are the subject of this work. The formation of inositol 1,4,5-triphosphate (IP3) in response to cholinergic stimulation was studied in the human peripheral blood mononuclear cells (PBMC) and lymphocytes from the lymphoblastoid cell lines Jurkat and Raji. The cells were stimulated with mitogen and then treated with cholinergic agonist-carbachol or nicotine. The radioreceptor assay was used to determine the amount of IP3 in all studied lymphocytes. The results, combined with the presence of muscarinic and nicotinic receptors on the surface of these lymphocytes, suggest that the parasympathetic nervous system is involved in the immune-neurohormonal crosstalk.