monensin and 5-dimethylamiloride

Possible involvement of cell membrane ion transport systems in the uptake and extrusion of Tc-99m-MIBI was investigated by using various buffers with or without Na+ and Ca++, and ion transport inhibitors in a tumor cell line. The ion transport modulators dimethyl amiloride (DMA), verapamil, flunarizine and monensin were used. The uptake of Tc-99m-MIBI was significantly increased in all buffers containing either Na+ or Ca++ alone or none of them. There was significantly increased uptake of Tc-99m-MIBI especially in buffers without Na+. Verapamil, a L-type Ca++ channel blocker, increased Tc-99m-MIBI uptake in all buffers. Flunarizine, which inhibits Na+/ Ca++ channels, caused significantly increased accumulation of Tc-99m-MIBI only in buffer containing both Na+ and Ca++. Monensin, a sodium ionophore, significantly increased uptake of Tc-99m-MIBI. DMA, a potent Na+/H+ antiport inhibitor, significantly inhibited the uptake of Tc-99m-MIBI in all buffers. In conclusion, Tc-99m-MIBI behaves like Na+ during its uptake and extrusion. Extrusion of Tc-99m-MIBI may involve both verapamil- and flunarizine-sensitive pathways.

Topics: Adrenal Cortex Neoplasms; Amiloride; Calcium; Calcium Channel Blockers; Calcium Channels; Carcinoma, Small Cell; Flunarizine; Humans; Monensin; Sodium; Sodium Channels; Technetium Tc 99m Sestamibi; Tumor Cells, Cultured; Verapamil

The mechanisms of uptake and intracellular distribution of 99mTc-tetrofosmin, 99mTc-MIBI and 201TI and the behaviors of 99mTc-tetrofosmin and 99mTc-MIBI in relation to Na+ were studied with primary cultures of myocardial cells.. Both the uptake and the washout of the tracers were sequentially measured. The cells were treated with ouabain, bumetanide, tetrodotoxin, dimethyl amiloride (DMA), nigericin and carbonyl cyanide m-chlorophenylhydrazone (CCCP) to observe the effects of the uptake and intracellular distribution of the traders. Cells equilibrated in buffers with or without Na+ were treated with monensin and DMA to evaluate the effect of Na+ on the accumulation of the tracers.. Despite the similarities in uptake kinetics, there was a higher level of retention of 99mTc-tetrofosmin inside the cells. Ouabain, bumetanide and tetrodotoxin did not show any inhibitory effect on the uptake of 99mTc-tetrofosmin and 99mTc-MIBI, whereas they produced various degrees of inhibition of 201TI uptake. DMA produced approximately 35% inhibition of 99mTc-tetrofosmin uptake and 50% inhibition of 99mTc-MIBI uptake. Nigericin increased the uptake of 99mTc-MIBI by the cells. The addition of CCCP produced the release of 38% of the accumulated 99mTc-tetrofosmin and 52%-70% of the accumulated 99mTc-MIBI, indicating that these percentages of accumulation were related to mitochondrial uptake. Neither Na+-free buffer nor monensin had any significant effect on 99mTc-tetrofosmin accumulation, but they both caused increased accumulation of 99mTc-MIBI.. The uptake of both 99mTc-tetrofosmin and 99mTc-MIBI through the cell membrane is partly related to the Na+/H+ antiporter system. Only part of the accumulated 99mTc-tetrofosmin inside the cells enters into mitochondria; most of the accumulated 99mc-MIBI is related to mitochondrial uptake. This uptake may be related to Na+.

Topics: Amiloride; Animals; Bumetanide; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Survival; Cells, Cultured; Heart; Ion Transport; Membrane Potentials; Mitochondria, Heart; Monensin; Myocardium; Nigericin; Organophosphorus Compounds; Organotechnetium Compounds; Ouabain; Radionuclide Imaging; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Sodium; Technetium Tc 99m Sestamibi; Tetrodotoxin; Thallium Radioisotopes

The roles of Na+/H+ antiport and intracellular pH in apoptosis of HL-60 cells were investigated here. We found that dimethyl amiloride, a specific Na+/H+ antiport inhibitor, induced intracellular acidification but not apoptosis; while sodium ionophore, monensin caused intracellular alkalinization as well as apoptosis in HL-60 cells. Br-A23187 and thapsigargin could induce a various degree of intracellular alkalinization through the stimulation of Na+/H+ antiport. Dimethyl amiloride blocked the intracellular alkalinization and inhibited apoptosis induced by Br-A23187 and thapsigargin. PMA also stimulated Na+/H+ antiport and induced intracellular alkalinization which was completely blocked by dimethyl amiloride and partially attenuated by PKC inhibitors. PMA could inhibit apoptosis in HL-60 cells. PMA-induced suppression of apoptosis was, however, not interfered by dimethyl amiloride, but could be abolished by PKC inhibitors. These results indicate that pHi alkalinization and/or the stimulation of Na+/H+ antiport, instead of intracellular acidification, are contributory to the induction of apoptosis. PMA-induced inhibition of apoptosis is not necessarily associated with intracellular alkalinization, but primarily due to activation of PKC. We suggest that stimulation of Na+/H+ antiport and pHi alkalinization act as facilitating factors in the induction of apoptosis.

Topics: Amiloride; Apoptosis; DNA Damage; Enzyme Inhibitors; HL-60 Cells; Humans; Hydrogen-Ion Concentration; Indoles; Ionophores; Maleimides; Monensin; Protein Kinase C; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate

Na+-H+ exchange may proceed via an endogenous antiporter or by exposure to the Na+ ionophore monensin. We investigated the characteristics of Na+-H+ exchange induced by antiporter stimulation and by monensin in FRTL-5 rat thyroid cells. We also examined the effects of intracellular pH (pHi) changes on iodide uptake and efflux. pHi was determined using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The resting pHi was 7.33 +/- 0.02 units; this level correlated directly with extracellular pH. In acid-loaded cells, Km for external Na+ activation of the antiporter was 7.1 mM and maximum velocity was 0.3801 delta pH units/min. Dimethylamiloride was 42 times more potent than amiloride in inhibiting sodium-dependent recovery in acidified cells. Metabolic inhibition reduced the initial alkalinization rate. Monensin increased pHi, and this response was dependent on extracellular Na+ and HCO3- but not on antiporter function. Low-dose monensin (1 microM) and 1 mM NH4Cl enhanced 125I uptake. High-dose monensin (100 microM), but not NH4Cl, reduced iodide uptake. Neither NH4Cl nor monensin altered 125I efflux. Thus FRTL-5 cells possess an amiloride-sensitive Na+-H+ exchanger, which is not essential for maintaining basal pHi but is affected by ATP depletion. Monensin also alkalinizes these cells but independently of the antiporter. Iodide uptake, but not efflux, is affected by changes in intracellular Na+ and H+ levels.

Topics: Amiloride; Ammonium Chloride; Animals; Carrier Proteins; Cell Line; Cytosol; Hydrogen-Ion Concentration; Iodides; Kinetics; Monensin; Ouabain; Rats; Sodium; Sodium-Hydrogen Exchangers; Thyroid Gland

Ticlopidine is a potent inhibitor of ADP-induced aggregation of rabbit platelets ex vivo. In vivo, however, multiple agonists play a role in platelet activation. In this study, we examined the effect of epinephrine on the antiplatelet action of ticlopidine in rabbit platelets. Epinephrine reversed the inhibitory effect of drug on ADP-induced platelet aggregation. The potentiating effect of epinephrine was mediated through alpha 2-adrenergic receptors, was reversed by pretreatment with the Na+/H+ exchange inhibitor dimethylamiloride, and was mimicked by agents that increased intracellular sodium or pH. Ticlopidine had no effect on resting intracellular pH, an indication that the effect of epinephrine was not compensating for a drug-induced intracellular acidification. While this potentiation was also found to be inhibited by aspirin, it did not involve enhanced release of thromboxane A2. Our results demonstrate that epinephrine can overcome the inhibitory effect of ticlopidine on ADP-induced aggregation through a mechanism involving activation of Na+/H+ exchange and through an as yet unidentified mechanism sensitive to aspirin.

Topics: Adenosine Diphosphate; Amiloride; Animals; Aspirin; Drug Interactions; Epinephrine; Hydrogen-Ion Concentration; Male; Methylamines; Monensin; Platelet Aggregation; Platelet Aggregation Inhibitors; Rabbits; Receptors, Adrenergic, alpha; Sodium; Thromboxane A2; Ticlopidine

The growth of the human leukemia cell line AML-193 in a serum-free medium is strictly dependent on the presence of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), which is one of the major regulators of the myelomonocytic lineage. At present, little is known about the mechanisms by which this growth factor transduces the signal intracellularly. The results of this study demonstrate that GM-CSF needs the operation of a Na+/H+ exchanger, which is located in the plasma membrane of almost every vertebrate cell. In fact, the GM-CSF-dependent proliferation of AML-193 cells is strongly reduced in the presence of the amiloride analog EIPA, a specific inhibitor of the Na+/H+ exchanger. When acidified, AML-193 cells are able to recover the original pHi in a Na(+)-dependent and EIPA-inhibitable way; this demonstrates for the first time the presence of the Na+/H+ exchanger in these cells. Finally, GM-CSF, at doses superimposable to those needed for triggering proliferation, induces in AML-193 cells a sustained alkalinization, which is dependent on a operating Na+/H+ exchange, as it is inhibited by EIPA. These results suggest that GM-CSF, like other growth factors in other cell systems, exerts its mitogenic activity in AML-193 cells by inducing a Na+/H+ exchanger-mediated rise in pHi.

Topics: Amiloride; Carrier Proteins; Cell Division; Cell Survival; Colony-Stimulating Factors; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Hydrogen-Ion Concentration; Leukemia, Monocytic, Acute; Monensin; Sodium-Hydrogen Exchangers; Tumor Cells, Cultured