guanosine-5--o-(3-thiotriphosphate) and Lymphoma--Large-B-Cell--Diffuse

Permeabilized human leukemia HL-60 and U-937 cells suspended in an acidic or alkaline medium release various unsaturated fatty acids, most abundantly oleic and arachidonic acids. Concomitant production of lysophospholipids suggests that phospholipases A2 play a major role in this fatty acid release reaction. The fatty acid release at acidic conditions depends on the intracellular Ca2+ concentrations at the 10(-8)-10(-7) M range and is enhanced by membrane-permeant diacylglycerols, although this enhancement seems independent of protein kinase C activation. On the other hand, the fatty acid release at alkaline conditions is potentiated by vanadate, and this potentiation is counteracted by genistein, suggesting a role of tyrosine phosphorylation in this release reaction. GTP[gamma S], an activator of G proteins, greatly enhances the fatty acid release. Aluminum fluoride, another activator of heterotrimeric G proteins, also greatly potentiates this release reaction. Phorbol ester increases the fatty acid release at alkaline conditions, to some extent, whereas it counteracts the vanadate-induced potentiation of fatty acid release. The results imply that several phospholipases A2 are coupled to receptors for their activation, thereby functioning in the transmembrane control of cellular events.

Topics: Calcium; Cell Line; Cell Membrane Permeability; Diglycerides; Electroporation; Fatty Acids, Nonesterified; Genistein; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Homeostasis; Humans; Hydrogen-Ion Concentration; Isoflavones; Kinetics; Leukemia, Promyelocytic, Acute; Lymphoma, Large B-Cell, Diffuse; Phospholipases A; Phospholipases A2; Protein-Tyrosine Kinases; Signal Transduction; Sodium Fluoride; Tetradecanoylphorbol Acetate; Thionucleotides; Tumor Cells, Cultured; Vanadates

Histamine H1 receptors were identified in U937 human histiocytic lymphoma cells using a radioligand binding technique with [3H]mepyramine. Reversible high-affinity binding with this ligand was obtained, and specificity of binding for selected H1 agonists and antagonists was demonstrated. Competition binding experiments with mepyramine and histamine yielded results consistent with single-site binding for mepyramine and two-site binding for histamine. Dissociation constants for the high- and low-affinity histamine binding states were 6.8 X 10(-6) and 2.4 X 10(-4) M respectively. The high-affinity state for histamine binding was abolished when the membranes were coincubated with 100 microM guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S). Saturation binding studies yielded an average of 66 fmol/mg protein binding sites (6000 receptors per cell) with a [3H]mepyramine KD of 9.6 nM. When differentiation of these cells was induced by phorbol-myristate-acetate, receptor density increased by 73% to 114 fmol/mg protein. This increase in receptor density was inhibited by actinomycin D and cycloheximide. Exposure of native and differentiated U937 cells to 10(-5) and 10(-4) M histamine for 24 hr resulted in a dose-dependent down-regulation in receptor density. The data indicate that U937 cells may provide a model cell line for the study of histamine receptor gene expression.

Topics: Cycloheximide; Dactinomycin; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Histamine; Humans; Lymphoma, Large B-Cell, Diffuse; Pyrilamine; Receptors, Histamine H1; Tetradecanoylphorbol Acetate; Thionucleotides; Tumor Cells, Cultured