concanavalin-a and 2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein

A fluorimetric multi-parameter cell sensor at the single cell level is presented which makes it possible to observe the physiological behavior of different cell lines, different physiological parameters, and statistical data at the same time. Different cell types were immobilized at predefined positions with high accuracy using optical tweezers and adhesion promoting surface layers. The process is applicable to both adherent and non-adherent cells. Coating of the immobilization area with mussel adhesive protein was shown to be essential for the process. Intracellular proton and calcium concentrations in different cell classes were simultaneously imaged and the specific activation of T lymphocytes was demonstrated. This method should be especially useful for drug screening due to the small sample volume and high information density.

Topics: Adhesives; Animals; Bivalvia; Calcium; Cell Adhesion; Cell Line; Cell Survival; Cells, Cultured; Concanavalin A; Drug Evaluation, Preclinical; Fluoresceins; Fluorometry; Humans; Hydrogen-Ion Concentration; Lasers; Lymphocyte Activation; Mice; Proteins; T-Lymphocytes; Tumor Cells, Cultured

The effects of arachidonic acid (AA) and the lectin mitogens, concanavalin A (Con A) and phytohemagglutinin (PHA), on [Ca2+]i and pHi in rat thymocytes have been studied by using the intracellular fluorescent probes, Fura-2 and BCECF. It was revealed that exogenous AA (3 microM), in addition to the well-known changes in basal [Ca2+]i and pHi, also caused a complete blockade of [Ca2+]i and pHi signals induced by Con A (10 micrograms/ml) and PHA (10 micrograms/ml). In contrast, exposure of thymocytes to mitogens did not prevent the AA-induced increase in [Ca2+]i and decrease in pHi. In experiments with sodium propionate, the similarity between AA action and EIPA (ethylisopropylamiloride), an inhibitor of Na+/H+ exchangers, was revealed. It is proposed that the inhibitory effect of AA on mitogen-induced lymphocyte proliferation is due primarily to the blockade of transmembrane [Ca2+]i and pHi signals, associated with a sustained cytosolic acidification.

Topics: Amiloride; Animals; Arachidonic Acid; Calcium; Concanavalin A; Cytosol; Fluoresceins; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Mitogens; Phytohemagglutinins; Rats; Rats, Wistar; Thymus Gland

Interaction of some mitogenic lectins and growth factors with the cell surface leads to activation of the Na+/H+ antiport and a resultant cytoplasmic alkalinization. Because amiloride inhibits both Na+/H+ exchange and cell proliferation, it has been hypothesized that activation of the antiport is an obligatory requirement and may, perhaps, be the "trigger" for proliferation. However, concentrations of amiloride which inhibit the antiport also inhibit several other intracellular processes, including protein synthesis and phosphorylation. To determine whether activation of the Na+/H+ antiport is necessary for lectin-induced proliferation, we examined the inhibitory activity of a series of potent amiloride analogs by measuring [3H]thymidine incorporation, cell cycle progression, and induction of the interleukin 2 (IL 2) receptor on human lymphocytes. In medium containing bicarbonate, and at concentrations at least 10 times higher than required to inhibit the antiport, these drugs did not inhibit the proliferative response of human peripheral blood T cells to the mitogen phytohemagglutinin. The amiloride analogs also failed to inhibit induction of the IL 2 receptor. Similarly, with human thymocytes, the amiloride analogs did not inhibit the co-mitogenic effects of the lectins phytohemagglutinin and concanavalin A together with IL 2 or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. This finding suggests that Na+/H+ exchange through the antiport is not an obligatory requirement for activation or proliferation of human lymphocytes or thymocytes.

Topics: Amiloride; Biological Transport, Active; Cell Cycle; Concanavalin A; Fluoresceins; Humans; Interleukin-2; Lymphocyte Activation; Phytohemagglutinins; Protons; Receptors, Immunologic; Receptors, Interleukin-2; Sodium; T-Lymphocytes; Tetradecanoylphorbol Acetate