ABSTRACT
HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations alpha3beta2 or alpha4beta2. [(3)H]-(+/-)Epibatidine ([(3)H]-(+/-)EPI) bound to membranes from A3B2 (alpha3beta2) and A4B2.2 (alpha4beta2) cells with K(d) values of 7.5 and 33.4 pM and B(max) values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca(2+) concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-beta-erythroidine (DHbetaE) in A3B2 cells and MEC=DHbetaE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in alpha3beta2 and alpha4beta2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca(2+), respectively. Our results indicate that stably expressed alpha3beta2 and alpha4beta2 human nAChRs are pharmacologically and functionally distinct.
Subject(s)
Receptors, Nicotinic/metabolism , Blotting, Northern , Blotting, Western , Calcium/metabolism , Cell Line , Electric Stimulation , Electrophysiology , Humans , Kidney/metabolism , Ligands , Membranes/drug effects , Membranes/metabolism , Nicotinic Agonists/pharmacology , Patch-Clamp Techniques , RNA/biosynthesis , RNA/isolation & purification , Radioligand Assay , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/genetics , Recombinant Proteins/chemistryABSTRACT
BACKGROUND: The signal transduction pathways involved in interleukin-2 (IL-2) synthesis have become a focus of interest for pharmacological intervention. Regulation of synthesis of IL-2 requires costimulation of two cell surface receptors, the T cell receptor and an appropriate costimulatory receptor. Antibodies such as anti-CD3 to stimulate the T cell receptor and anti-CD28 can be used to induce this costimulation. Previous methodologies employed antibody coupled to polystyrene microtiter plates or solution phase stimulation. METHODS: Here, a method using magnetic beads to present anti-CD3 and anti-CD28, to the Jurkat T cell line was developed. Conditions were also developed for simultaneous analysis of the effect of test compounds on cell viability. The IL-2 synthesis methods and cell viability methods have been used to evaluate signal transduction inhibitors. RESULTS: This method was then employed to test the effects of various signal transduction inhibitors on IL-2 synthesis. Agents which increase cAMP, cyclosporin and inhibitors of calmodulin, tyrosine kinases/phosphatases, protein kinase C, and PC-PL-C decrease IL-2 synthesis at concentrations which do not affect cell viability. CONCLUSION: These results found with this novel system of stimulation with antibody to CD3 and CD28 compare favorably with activities reported in the literature thus validating this rapid facile system for evaluation of the effect of signal transduction inhibitors in IL-2 synthesis.
