gramicidin-a and Leukemia--Promyelocytic--Acute

Exposure to hypotonic stress produces a transient increase in cell volume followed by a regulatory volume decrease (RVD) in both THP-1 and HL-60 cells. In contrast, cells exposed to hypotonic stress in a high K/low Na Hanks' solution not only failed to volume regulate, but displayed a secondary swelling. Thus, while an outward K gradient was required for RVD, the secondary swelling indicated that hypotonic stress increased permeability in the absence of a negative membrane potential. The K channel blocker quinine (1-4 mM) blocked RVD in both cell types. Gramicidin's ability to overcome the quinine block of RVD indicated that RVD is mediated by a quinine-sensitive cation transport mechanism that is independent of the swelling-induced anion transport mechanism. Barium (1-4 mM), another K channel blocker, slowed the rate of RVD, while 4-aminopyridine, charybdotoxin, tetraethylammonium chloride, tetrabutylammonium chloride, and gadolinium had no effect on RVD. Furthermore, RVD was not mediated by calcium-activated conductances, since it occurred normally in Ca-free medium, in medium containing cadmium, and in BAPTA-loaded cells. Gramicidin produced little or no volume change in isotonic medium, suggesting that basal C1 permeability of both THP-1 and HL-60 cells is low. However, swelling induced an anion efflux pathway that is permeable to both chloride and bromide, but is impermeable to methanesulfonate and glutamate. The anion channel blocker 3,5-diiodosalicylic acid (DISA) antagonized RVD in both cell types. In conclusion, RVD in THP-1 and HL-60 cells is mediated by independent anion and cation transport mechanisms that involve both a DISA-sensitive anion pathway and a quinine-inhibitable K efflux pathway, neither of which requires increases in intracellular calcium to be activated.

Topics: Anions; Calcium; Cell Line; Cell Membrane Permeability; Cell Survival; Fura-2; Gramicidin; Homeostasis; Humans; Hypotonic Solutions; Kinetics; Leukemia, Promyelocytic, Acute; Potassium Channels; Quinine; Time Factors; Tumor Cells, Cultured

We examined the kinetics of polyamine uptake by human myeloid cells at different stages of maturity. The Km values of putrescine, spermidine and spermine transport by HL-60 cells were 52, 7.9 and 8.1 microM, respectively. These values decreased to 5.1, 1.7 and 0.77 microM, respectively, in HL-60 cells induced to mature past the promyelocyte stage by DMSO. In human PMNs, the respective Km values were 501, 479 and 381 microM. Transport by HL-60 cells was enhanced when intracellular polyamine levels were reduced with difluoromethylornithine. Thus, HL-60 cell maturation is accompanied by an increase in the affinity of their polyamine transport system. This system is much more efficient than that found in end-stage PMNs, suggesting that it plays a more important role in supporting the metabolic requirements of HL-60 cells. Alternatively, the low affinity of the PMN polyamine transport system could represent an adaptation to the high polyamine concentrations found at infection sites.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Biological Transport; Cell Line; Genistein; Gramicidin; Humans; In Vitro Techniques; Ionomycin; Isoflavones; Isoquinolines; Kinetics; Leukemia, Promyelocytic, Acute; Neutrophils; Piperazines; Protein Kinase Inhibitors; Putrescine; Spermidine; Spermine; Sulfonamides; Temperature; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The effects of plasma membrane depolarization on cytosolic free calcium ([Ca2+]i) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) generation were investigated in the human promyelocytic cell line HL-60 differentiated with either dimethyl sulfoxide or retinoic acid into neutrophil-like cells. Increases in [Ca2+]i and accumulation of Ins(1,4,5)P3 were triggered by two chemoattractants fMet-Leu-Phe and leukotriene B4. Plasma membrane potential was depolarized by isoosmotic substitution of NaCl with KCl, by the pore-forming ionophore gramicidin D, or by long term treatment with ouabain. Both Ca2+ mobilization from intracellular stores and Ca2+ influx across the plasma membrane were reduced by prior depolarization of plasma membrane potential regardless of the procedure employed to collapse it. Agonist-induced generation of Ins(1,4,5)P3 was also reduced in parallel in pre-depolarized HL-60 cells. The present findings provide further evidence suggesting that plasma membrane potential can be an important modulator of agonist-activated second messenger generation in myelocytic cells.

Topics: Calcium; Cell Line; Cell Membrane; Cytosol; Dimethyl Sulfoxide; Egtazic Acid; Gramicidin; Humans; Inositol 1,4,5-Trisphosphate; Ionomycin; Kinetics; Leukemia, Promyelocytic, Acute; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Ouabain; Potassium Chloride; Second Messenger Systems; Tretinoin; Tumor Cells, Cultured