valinomycin and ethylisopropylamiloride

The energy transduction by complex I from Rhodothermusmarinus was addressed by studying the influence of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on the activities of this enzyme. EIPA is an inhibitor of both Na(+)/H(+) antiporter and complex I NADH:quinone oxidoreductase activity. We performed studies of NADH:quinone oxidoreductase and H(+) and Na(+) translocation activities of complex I from R. marinus at different concentrations of EIPA, using inside-out membrane vesicles. We observed that the oxidoreductase activity and both H(+) and Na(+) transports are inhibited by EIPA. Most interestingly, the catalytic and the two transport activities showed different inhibition profiles. The transports are inhibited at concentrations of EIPA at which the catalytic activity is not affected. In this way the catalytic and transport activities were decoupled. Moreover, the inhibition of the catalytic activity was not influenced by the presence of Na(+), whereas the transport of H(+) showed different inhibition behaviors in the presence and absence of Na(+). Taken together our observations indicate that complex I from R. marinus performs energy transduction by two different processes: proton pumping and Na(+)/H(+) antiporting. The decoupling of the catalytic and transport activities suggests the involvement of an indirect coupling mechanism, possibly through conformational changes.

Topics: Amiloride; Biological Transport; Catalysis; Electron Transport Complex I; Hydrogen; Hydrogen-Ion Concentration; Ion Transport; Membrane Potentials; Quinone Reductases; Rhodothermus; Sodium; Sodium-Hydrogen Exchangers; Valinomycin

The mechanism of mammalian polyamine transport is poorly understood. We have investigated the role of plasma-membrane potential (DeltaPsipm) in putrescine and spermidine uptake in ZR-75-1 human breast cancer cells. The rate of [3H]putrescine and [3H]spermidine uptake was inversely correlated to extracellular [K+] ([K+]o) and to DeltaPsipm, as determined by the accumulation of [3H]tetraphenylphosphonium bromide (TPP). Inward transport was unaffected by a selective decrease in mitochondrial potential (DeltaPsimit) induced by valinomycin at low [K+]o, but was reduced by approximately 60% by the rheogenic protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which rapidly (<=15 min) collapsed both DeltaPsipm and DeltaPsimit. Plasma-membrane depolarization by high [K+]o or CCCP did not enhance putrescine efflux in cells pre-loaded with [3H]putrescine, suggesting that decreased uptake caused by these agents did not result from a higher excretion rate. On the other hand, the electroneutral K+/H+ exchanger nigericin (10 microM) co-operatively depressed -3H-TPP, [3H]putrescine and [3H]spermidine uptake in the presence of ouabain. Suppression of putrescine uptake by nigericin+ouabain was Na+-dependent, suggesting that plasma-membrane repolarization by the electrogenic Na+ pump was required upon acidification induced by nigericin, due to the activation of the Na+/H+ antiporter. The sole addition of 5-N, N-hexamethylene amiloride, a potent inhibitor of the Na+/H+ antiporter, strongly inhibited putrescine uptake in a competitive fashion -Ki 4.0+/-0.9 (S.D.) microM-, while being a weaker antagonist of spermidine uptake. The potency of a series of amiloride analogues to inhibit putrescine uptake was clearly different from that of the Na+/H+ antiporter, and resembled that noted for Na+ co-transport proteins. These data demonstrate that putrescine and spermidine influx is mainly unidirectional and strictly depends on DeltaPsipm, but not DeltaPsimit. This report also provides first evidence for a high-affinity amiloride-binding site on the putrescine carrier, which provides new insight into the biochemical properties of this transporter.

Topics: Amiloride; Animals; Binding Sites; Biological Transport; Breast Neoplasms; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane; Female; Humans; Indicators and Reagents; Kinetics; Mammals; Membrane Potentials; Onium Compounds; Organophosphorus Compounds; Potassium; Putrescine; Sodium-Hydrogen Exchangers; Spermidine; Tumor Cells, Cultured; Valinomycin

We have characterized a Na+/H+ exchanger in the membrane of isolated zymogen granules (ZG) from rat exocrine pancreas and investigated its role in secretagogue-induced enzyme secretion. ZG Na+/H+ exchanger activity was estimated by measuring Na+ or Li+ influx and consequent osmotic swelling and lysis of ZG incubated in Na- or Li-acetate. Alternatively, intragranule pH was investigated by measuring absorbance changes in ZG which had been preloaded with the weak base acridine orange. Na+- or Li+-dependent ZG lysis was enhanced by increasing inward to outward directed H+ gradients. Na+-dependent ZG lysis was not prevented by an inside-positive K+ diffusion potential generated by valinomycin which argues against parallel operation of separate electrogenic Na+ and H+ permeabilities and for coupled Na+/H+ exchange through an electroneutral carrier. Na+- and Li+-dependent ZG lysis was inhibited by EIPA (EC50 approximately 25 microM) and benzamil (EC50 approximately 100 microM), but only weakly by amiloride. Similarly, absorbance changes due to release of acridine orange from acidic granules into the medium were obtained with Na+ and Li+ salts only, and were inhibited by EIPA, suggesting the presence of a Na+/H+ exchanger in the membrane. Na+ dependent lysis of ZG was inhibited by 0.5 mm MgATP and MgATP-gamma-S by about 60% and 35%, respectively. Inhibition by MgATP was prevented by incubation of ZG with alkaline phosphatase (100 U/ml), or by the calmodulin antagonists calmidazolium (0.75 microM), trifluoperazine (100 microM) and W-7 (500 microM), suggesting that the ZG Na+/H+ exchanger is regulated by a ZG membrane-bound calmodulin-dependent protein kinase. Na+ dependence of secretagogue (CCK-OP)-stimulated amylase secretion was investigated in digitonin permeabilized rat pancreatic acini and was higher in acini incubated in Na+ containing buffer (30 mm NaCl/105 mm KCl buffer; 6.4 +/- 0.4% of total amylase above basal) compared to buffer without Na+ (0 mm NaCl/135 mm KCl buffer; 4.7 +/- 0.4% of total amylase above basal, P < 0.03). EIPA (50 microM) reduced CCK-OP-induced amylase secretion in Na+ containing buffer from 7.5 +/- 0.6% to 4.1 +/- 0.8% (P < 0.02). In the absence of Na+ in the buffer, CCK-OP-stimulated amylase release was not inhibited by 50 microM EIPA. The data suggest that an amiloride insensitive, EIPA inhibitable Na+/H+ exchanger is present in ZG membranes, which is stimulated by calmodulin antagonists and could be involved in secretagogue-induced

Topics: Adenine Nucleotides; Amiloride; Amylases; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cations, Monovalent; Cytoplasmic Granules; Electric Conductivity; Hydrogen-Ion Concentration; Intracellular Membranes; Kinetics; Lithium; Magnesium; Male; Membrane Potentials; Pancreas; Rats; Rats, Wistar; Sodium; Sodium-Hydrogen Exchangers; Valinomycin

Proton-dependent, ethylisopropylamiloride (EIPA)-sensitive Na+ uptake (Na+/H+ antiporter) studies were performed to examine if saliva, and ionophores which alter cellular electrolyte balance, could influence the activity of the cheek cell Na+/H+ antiporter. Using the standard conditions of 1 mmol/l Na+, and a 65:1 (inside:outside) proton gradient in the assay, the uniport ionophores valinomycin (K+) and gramicidin (Na+) increased EIPA-sensitive Na+ uptake by 177% (p < 0.01) and 227% (p < 0.01), respectively. The dual antiporter ionophore nigericin (K(+)-H+) increased EIPA-sensitive Na+ uptake by 654% (p < 0.01), with maximal Na+ uptake achieved by 1 min and at an ionophore concentration of 50 mumol/l, with an EC50 value 6.4 mumol/l. Pre-incubation of cheek cells with saliva or the low molecular weight (MW) components of saliva (saliva activating factors, SAF) for 2 h at 37 degrees C, also significantly stimulated EIPA-sensitive Na+ uptake. This stimulation could be mimicked by pre-incubation with 25 mmol/l KCl or K(+)-phosphate buffer. Pre-incubating cheek cells with SAF and the inclusion of 20 mumol/l nigericin in the assay, produced maximum EIPA-sensitive Na+ uptake. After pre-incubation with water, 25 mmol/l K(+)-phosphate or SAF, with nigericin in all assays, the initial rate of proton-gradient dependent, EIPA-sensitive Na+ uptake was saturable with respect to external Na+, with Km values of 0.9, 1.7, and 1.8 mmol/l, and Vmax values of 13.4, 25.8, and 31.1 nmol/mg protein/30 sec, respectively. With 20 mumol/l nigericin in the assay, Na+ uptake was inhibited by either increasing the [K+]o in the assay, with an ID50 of 3 mmol/l. These results indicate that nigericin can facilitate K+i exchange for H+o and the attending re-acidification of the cheek cell amplifies 22Na+ uptake via the Na+/H+ antiporter. The degree of stimulation of proton-dependent, EIPA-sensitive Na+ uptake is therefore dependent, in part, on the intracellular [K+]i.

Topics: Adult; Amiloride; Cheek; Electrolytes; Gramicidin; Humans; In Vitro Techniques; Ionophores; Kinetics; Mouth Mucosa; Nigericin; Potassium; Saliva; Sodium; Sodium-Hydrogen Exchangers; Valinomycin

The purpose of these studies was to document the existence of electrogenic Na+ uptake by membrane vesicles of rabbit alveolar type II (ATII) cells and the extent to which this process was inhibited by amiloride. ATII cells (greater than 85% pure) were obtained by elastase digestion of lung tissue followed by Percoll centrifugation, and an enriched plasma membrane vesicle fraction was obtained by differential centrifugation. 22Na+ uptake into these vesicles was measured in the presence of a negative inside membrane potential, produced by the addition of the K+ ionophore valinomycin (10 microM) after all external K+ was removed. Electrogenic (valinomycin-sensitive) Na+ uptake (ELNa) was defined as the difference in uptake in the presence and absence of valinomycin. ELNa, normalized per milligram protein, was twice as high across ATII cells than alveolar macrophage membrane vesicles, was inhibited by amiloride (50% inhibitory concentration = 10 microM), and was decreased in the presence of an outwardly directed proton gradient (pHin 6.8; pHout 7.8), suggesting that it was not mediated by Na(+)-H+ antiport. Furthermore, ELNa was equally inhibited by increasing concentrations of amiloride and benzamil but was more sensitive to 5-(N-ethyl-N-isopropyl)-2'-4'-amiloride in concentrations of 10-1,000 microM. These findings indicate that a fraction of Na+ transport across ATII membrane vesicles occurs through a conductive pathway, probably a channel, that has different sensitivity to amiloride and its analogues than the previously described epithelial high amiloride-affinity Na+ channel.

Topics: Alkaline Phosphatase; Amiloride; Animals; Biological Transport, Active; Cell Membrane; Cell Separation; Cells, Cultured; Hydrogen-Ion Concentration; Kinetics; Macrophages; Pulmonary Alveoli; Rabbits; Sodium; Time Factors; Valinomycin

Na-H exchange is present in apical membrane vesicles (AMV) isolated from distal colon of normal rats. Because in intact tissue aldosterone both induces amiloride-sensitive electrogenic sodium transport and inhibits electroneutral sodium absorption, these studies with AMV were designed to establish the effect of aldosterone on sodium transport. An outward-directed proton gradient stimulated 22Na uptake in AMV isolated from distal colon of normal and dietary sodium depleted (with elevated aldosterone levels) experimental rats. Unlike normal AMV, proton gradient-dependent 22Na uptake in experimental AMV was inhibited when uptake was measured under voltage-clamped conditions. 10 microM amiloride inhibited the initial rate of proton gradient-dependent 22Na uptake in AMV of normal and experimental rats by 30 and 75%, respectively. In contrast, 1 mM amiloride produced comparable inhibition (90 and 80%) of 22Na uptake in normal and experimental AMV. Intravesicular-negative potential stimulated 22Na uptake in experimental but not in normal AMV. This increase was inhibited by 90% by 10 microM amiloride. An analogue of amiloride, 5-(N-ethylisopropyl) amiloride (1 microM), a potent inhibitor of electroneutral Na-H exchange in AMV of normal rat distal colon, did not alter potassium diffusion potential-dependent 22Na uptake. Increasing sodium concentration saturated proton gradient-dependent 22Na uptake in normal AMV. However, in experimental AMV, 22Na uptake stimulated by both proton gradient and potassium diffusion potential did not saturate as a function of increasing sodium concentration. We conclude from these results that an electrically sensitive conductive channel, not electroneutral Na-H exchange, mediates 22Na uptake in AMV isolated from the distal colon of aldosterone rats.

Topics: Aldosterone; Amiloride; Animals; Cell Membrane; Colon; Diffusion; Electrochemistry; Fluorescent Antibody Technique; Kinetics; Microscopy, Electron; Potassium; Protons; Rats; Rats, Inbred Strains; Sodium; Sodium Radioisotopes; Valinomycin

Electrical potential driven 22Na+ fluxes were measured in membrane vesicles prepared from a number of cultured and naturally occurring epithelia. In all preparations a rheogenic pathway blocked by 200 microM (but not by 1.5 microM) amiloride was noted. This transporter was characterized in membranes prepared from cultured LLC-PK1 cells. In this preparation more than 50% of the rheogenic 22Na+ uptake was blocked by amiloride (IC50 approximately 30 microM), phenamil (IC50 approximately 66 microM), or ethylisopropylamiloride (IC50 approximately 5 microM). This amiloride-sensitive flux was not seen if the vesicles were partially depolarized by external Na+ or K+. It could not be driven by a pH gradient, did not require the presence of Ca2+, sugars, or amino acids, and showed little dependence on temperature (25 versus 0 degrees C). The data suggest the existence of an epithelial amiloride-blockable Na+ transporter different from the previously characterized Na+ channel, Na+/H+ and Na+/Ca2+ exchangers, and the Na+-hexose co-transporter. In rat kidney cortex membranes prepared by Mn2+ precipitation, this transporter is primarily located in the brush-border fraction.

Topics: Amiloride; Animals; Cell Line; Cell Membrane; Epithelium; Erythrocyte Membrane; Female; Humans; Kidney Cortex; Kinetics; Male; Membrane Potentials; Microvilli; Rabbits; Rana ridibunda; Rats; Rats, Inbred Strains; Sodium; Sodium Channels; Valinomycin