Characterization of the Na+/H+ exchanger during maturation of HL-60 cells induced by dimethyl sulfoxide

We have studied the activity of the Na+/H+ exchanger during dimethyl sulfoxide (Me2SO)-induced maturation of the human promyelocytic leukemia cell line HL-60. 22Na uptake was measured in cells preloaded with Li+ or NH+4 in order to specifically activate the Na+/H+ exchanger. Measurement of the rate of uptake as a function of sodium concentration revealed a decrease in Km for Na+ from 38 +/- 3 to 13 +/- 1 mM after 20-24-h treatment with Me2SO. Vmax was not changed significantly. Inhibition of the exchanger by dimethylamiloride (DMA) and by acidic external pH was similar in treated and untreated cells. Thus it is unlikely that the Na+ binding site is altered. A change, however, was observed in the regulation of the exchanger by intracellular pH. In control cells maximal stimulation of the Na+ uptake was observed when the intracellular pH decreased from 7.25 to 7.00. In Me2SO-treated cells the 22Na uptake at intracellular pH 7.00 was greater than in the control and continued to increase as the intracellular pH was adjusted below 7.00, down to 6.75. This suggests that the Na+/H+ exchanger in Me2SO-treated cells is altered structurally in its allosteric H+ binding site. The appearance of this modified exchanger preceded by a period of days the appearance of a functional property characteristic of mature granulocytes, that is, the capability to produce superoxide, suggesting that the modified exchanger may be required for the expression of the mature phenotype. A second modification, a decrease in the Vmax of the 22Na uptake, occurred after 2 days treatment with Me2SO. This reduction may reflect a decrease in the number of functioning exchangers per cell.     

Multiple receptors linked to inhibition of adenylate cyclase accelerate Na+/H+ exchange in neuroblastoma x glioma cells via a mechanism other than decreased cAMP accumulation

alpha 2-Adrenergic receptors, a population of receptors linked to inhibition of adenylate cyclase, accelerate Na+/H+ exchange in NG108-15 neuroblastoma x glioma cells (Isom, L. L., Cragoe, E. J., Jr., and Limbird, L. E. (1987) J. Biol. Chem. 262, 6750-6757). We now report that two other receptor populations linked to inhibition of adenylate cyclase, muscarinic cholinergic and delta-opiate receptors, also alkalinize the interior of NG108-15 cells, as measured with the pH-sensitive fluorescent probe, 2,7-biscarboxyethyl-5(6)-carboxy-fluorescein. Manipulations that block Na+/H+ exchange, i.e. removal of extracellular Na+, reduction of extracellular pH to equal that of intracellular pH, and addition of 5-amino-substituted analogs of amiloride, all block alpha 2-adrenergic, delta-opiate, or muscarinic cholinergic receptor-induced alkalinization in a parallel fashion. These data suggest that all three populations of receptors alkalinize NG108-15 cells by acceleration of Na+/H+ exchange and do so via a shared or similar mechanism. Although these three receptor populations are linked to inhibition of adenylate cyclase, decreased production of cAMP does not appear to be the mechanism responsible for receptor-accelerated Na+/H+ exchange. Thus, ADP-ribosylation of intact NG108-15 cells with Bordetella pertussis islet-activating protein prevents attenuation of prostaglandin E1-stimulated cAMP accumulation by alpha 2-adrenergic, muscarinic, and delta-opiate agonists but has no measurable effect on the ability of these agonists to accelerate Na+/H+ exchange. Similarly, manipulations that block receptor-accelerated Na+/H+ exchange influence but do not block receptor-mediated attenuation of cAMP accumulation. Thus, the present data suggest that these two receptor-mediated biochemical events, acceleration of Na+/H+ exchange and attenuation of cAMP accumulation, occur through divergent mechanisms in NG108-15 cells.     

Inhibition of na/h antiport activity in sugar beet tonoplast by analogs of amiloride

The effects of amiloride and a series of amiloride analogs have been tested on the Na⁺/H⁺ antiport activity in intact vacuoles and tonoplast vesicles isolated from sugar beet cell suspension cultures. There is a competitive interaction between amiloride analogs and sodium. Substitution of one or both H-atoms of the 5-amino group of amiloride (apparent K_i about 150 micromolar) resulted in a 3- to 200-fold increase in inhibitory potency of the antiport activity.     

Volume changes in activated human neutrophils: The role of Na+/H+ exchange

The apparent volume of neutrophils, as measured electronically with the Coulter counter, has been reported to increase upon treatment with chemotactic factors. The occurrence of a volume change was confirmed by forward angle light scattering and by isotopic measurements of intracellular water space in cells treated with 12-O-tetradecanoylphorbol 13, acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP). Cell swelling was associated with an increase in the osmotic content of the cells, determined from Boyle-van't Hoff plots, and with an increase in Na⁺ content, measured by flame photometry. The volume change was inhibited by replacement of extracellular Na⁺ with K⁺ or N-methyl-D-glucamine⁺, or by addition of amiloride. Swelling was also inhibited by the 5-N-substituted analogs of amiloride, which are potent specific inhibitors of the Na⁺/H⁺ antiport. This pathway is activated in neutrophils by both TPA and FMLP. Activation of Na⁺/H⁺ exchange, determined as a Na⁺-dependent and amiloride-sensitive cytoplasmic alkalinization, was also found when neutrophils were treated with hypertonic solutions. The hypertonic activation of the antiport was similarly followed by cell swelling, detectable by electronic sizing. The results indicate that activation of Na⁺/H⁺ exchange can lead to significant cell swelling in neutrophils.     

Intracellular pH and Catecholamine Secretion from Bovine Adrenal Chromaffin Cells

To study the role of intracellular pH (pHi) in catecholamine secretion and the regulation of pHi in bovine chromaffin cells, the pH-sensitive fluorescent indicator [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] was used to monitor the on-line changes in pHi. The pHi of chromaffin cells at resting state is approximately 7.2. The pHi was manipulated first by incubation of the cells with NH4+, and then the solution was replaced with a NH4(+)-free solution to induce acidification of the cytoplasm. The pHi returned toward the basal pH value after acidification within 5-10 min in the presence of Na+ or Li+, but the pHi stayed acidic when Na(+)-free buffers were used or in the presence of amiloride and its analogues. These results suggest that the pH recovery process after an acid load is due to the Na+/H+ exchange activity in the plasma membrane of the chromaffin cells. The catecholamine secretion evoked by carbachol and Na+ removal was enhanced after the cytoplasm had been made more acidic. It appears that acidic pH favors the occurrence of exocytosis.     

Regulation of intracellular pH in cultured hippocampal neurons by an amiloride-insensitive Na+/H+ exchanger.

Regulation of intracellular pH (pHi) in single cultured rat hippocampal neurons was investigated using the fluorescent pHi indicator dye bis-carboxyethylcarboxyfluorescein. Resting pHi was dependent on the presence of bicarbonate and external Na+ but was not altered significantly by removal of Cl- or treatment with the anion exchange inhibitor diisothiocyanatostilbene-2,2'-disulfonate. Recovery of pHi from acute acid loading was due, in large part, to a pharmacologically distinct variant of the Na+/H+ antiporter. In nominally HCO3(-)-free solutions, this recovery exhibited a saturable dose dependence on extracellular Na+ (Km = 23-26 mM) or Li+. The antiporter was activated by decreasing pHi and was unaffected by collapse of the membrane potential with valinomycin. Like the Na+/H+ antiporter described in other cell systems, the hippocampal activity was inhibited by harmaline, but in sharp contrast, neither amiloride nor its more potent 5-amino-substituted analogues were able to prevent the recovery from an acid load. These data indicate that Na(+)-dependent mechanisms dominate pHi regulation in hippocampal neurons and suggest a role for a novel variant of the Na+/H+ antiporter.     

Identification of apical membranes from tight epithelia using spin-labeled amiloride and electron paramagnetic resonance spectroscopy

Summary Apical cell membranes from Na⁺-transporting epithelia were identified in centrifugal fractions prepared from homogenates of rainbow trout kidney, gill and frog skin using a spinlabeled, nitroxide derivative of amiloride and electron paramagnetic resonance spectroscopy. Spin-labeled amiloride (ASp) is a potent inhibitor of Na⁺ transport. Frog skin shortcircuit current was inhibited by 50% in the presence of 7×10^–8 m ASp, whereas 4×10^–7 m amiloride was required to obtain the same effect. ASp is a suitable probe for the amiloride binding site based on analytical criteria: Unbound ASp produces an EPR signal linear with concentration and detectable at micromolar concentrations. Estimates of ASp binding can usually be made on less than 100 g of membrane protein. While ASp binds nonspecifically to many materials, amiloride- or benzamil-displaceable binding occurred only in trout gill and kidney, and in frog skin, but not in trout skeletal muscle. ASp binds to membrane fractions produced by differential centrifugation of trout gill, kidney and frog skin. In trout gill and kidney, 81% and 91%, respectively, of the amiloride-displaceable ASp binding is found in the 10,000 xg fraction. All of the ASp binding in frog skin is found in the 10,000 xg fraction. These data indicate that spin-labeled amiloride is a useful probe for the identification of the amiloride binding site, and electron paramagnetic resonance spectroscopy will allow the amiloride binding site to be used as a molecular marker for apical membranes.     

Interleukin-3-stimulated haemopoietic stem cell proliferation. Evidence for activation of protein kinase C and Na+/H+ exchange without inositol lipid hydrolysis.

Interleukin 3 (IL-3) is an important regulator of haemopoietic stem cell proliferation both in vivo and in vitro. Little is known about the possible mechanisms whereby this growth factor acts on stem cells to stimulate cell survival and proliferation. Here we have investigated the role of intracellular pH and the Na+/H+ antiport in stem cell proliferation using the multipotential IL-3-dependent stem cell line, FDCP-Mix 1. Evidence is presented that IL-3 can stimulate the activation of an amiloride-sensitive Na+/H+ exchange via protein kinase C activation. IL-3-mediated activation of the Na+/H+ exchange is not observed in FDCP-Mix 1 cells where protein kinase C levels have been down-modulated by treatment with phorbol esters. Also the protein kinase C inhibitor H7 can inhibit IL-3-mediated increases in intracellular pH. This activation of Na+/H+ exchange via protein kinase C has been shown to occur with no measurable effects of IL-3 on inositol lipid hydrolysis or on cytosolic Ca2+ levels. Evidence is also presented that this IL-3-stimulated alkalinization acts as a signal for cellular proliferation in stem cells.     

Amiloride analogs stimulate protein phosphorylation and serotonin release in human platelets

We examined the effects of newly exploited amiloride analogs on protein phosphorylation and serotonin secretion in human platelets. 5-(N-methyl-N-isobutyl) amiloride (IBA) and, to a lesser extent, 5-(N-methyl-N-isopropyl) amiloride (IPA), highly specific inhibitors of Na+/H+-pump, induced the phosphorylation of 47K-dalton protein and myosin light chain (20K). The phosphorylation was inhibited by apyrase. On the other hand, 3', 4'-dichlorobenzamil (DCB) and 2', 4'-dimethylbenzamil (DMB), highly specific inhibitors of Na+/Ca2+-pump, and to a lesser extent amiloride analogs induced serotonin secretion. Apparently there was dissociation between the phosphorylation and the serotonin release induced by the analogs.     

The role of protein kinase C in T lymphocyte proliferation. Existence of protein kinase C-dependent and -independent pathways

The mouse cytotoxic T cell clone (CTLL-2) was able to grow in the presence of culture medium supplemented only with transferrin, 2-mercaptoethanol, and recombinant interleukin 2 (IL-2). This lymphokine stimulated the synthesis of DNA in these cells. Similarly, phorbol esters, which activate protein kinase C, induced DNA synthesis in this clone. Furthermore, this later proliferation was not blocked by anti-IL-2 receptor antibodies, which inhibited IL-2-induced proliferation, suggesting that it was not indirectly due to the secretion of IL-2 by the cells. CTLL-2 cells pretreated with high doses of phorbol esters for 48 h down regulated protein kinase C and were depleted of this enzyme. This was shown by: 1) purification and in vitro assay of protein kinase C; 2) the lack of effect of phorbol esters in the stimulation of the Na+/H+ anti-porter which has been directly linked to the activation of protein kinase C. As expected, those protein kinase C-depleted cells no longer synthesized DNA and proliferated in response to phorbol esters. However, they proliferated identically to control cells in response to IL-2. Therefore, our results suggest two different pathways for T cell proliferation, one which involves protein kinase C and the other which does not.