Functional and immunological responses of Jurkat lymphocytes transfected with the substance P receptor.
1. We have transfected the rat substance P receptor (SPR) cDNA into the leukemic T-lymphocyte cell line Jurkat (J-wt) in order to study the effects of substance P (SP) on lymphocyte signaling mechanisms and the resultant neuropeptide-induced immunological changes. 2. The SPR cDNA was transfected into J-wt by the method of electroporation. Clones expressing SPRs were selected using a functional assay that measured SP-induced mobilization of intracellular Ca2+ ([Ca2+]i) in a fluorescence activated cell sorter (FACS) and by their expression of specific 125I-SP binding. 3. One clone, J-SPR, was identified and shown by Northern blot and 125I-SP saturation binding techniques to express the 2.2-kb SPR message and approximately 50,000 SPRs/cell with a Kd of 0.3 nM, respectively. Stimulation of J-SPR by SP resulted in the rapid mobilization of [Ca2+]i. This response was dose dependent in the range 10(-11)-10(-6) M SP and was maximal at 10(-7) M SP, with an EC50 of 0.3-0.5 nM SP. We further demonstrated that the SPR is rapidly desensitized following SP stimulation and by activation of the cell's T-cell receptor (TCR). Whole-cell patch-clamp experiments on J-SPR show that SP stimulation induces a Cl- current by a Ca2+ mediated process dependent on Ca2+/calmodulin-dependent protein kinase (CaMK). 4. Stimulation of J-SPR by SP results in changes in the cell surface expression of a number of molecules that play important roles in cell adhesion and activation: the expression of LFA-1 is decreased, and CD2 and IL-2 receptors are increased by 30 min, 6 hr, and 24 hr, respectively, following stimulation, as assessed by antibody staining in a FACS. 5. The expression of functional SPRs in Jurkat lymphocytes will not permit a detailed examination of how the activation of SPRs result in altered immune responses and further elucidate the role this neuropeptide receptor plays in inflammation.
D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D015815:Cell Adhesion Molecules / Q000096:biosynthesis; D018118:Chloride Channels; D004247:DNA / Q000235:genetics; D006801:Humans; D018799:Intercellular Adhesion Molecule-1; D015640:Ion Channel Gating / Q000187:drug effects; D008213:Lymphocyte Activation / Q000187:drug effects*; D016169:Lymphocyte Function-Associated Antigen-1 / Q000096:biosynthesis; D008565:Membrane Proteins / Q000187:drug effects*; D009363:Neoplasm Proteins / Q000096:biosynthesis; D011494:Protein Kinases / Q000378:metabolism; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000096:biosynthesis / Q000187:drug effects*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects*; D013373:Substance P / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology; D014162:Transfection; D014407:Tumor Cells, Cultured
D015815:Cell Adhesion Molecules; D018118:Chloride Channels; D016169:Lymphocyte Function-Associated Antigen-1; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D011994:Recombinant Proteins; D018799:Intercellular Adhesion Molecule-1; D013373:Substance P; D000001:Calcimycin; D004247:DNA; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium