Competitive effects of some cations on active potassium transport in the human red blood cell

The effect of some cations on the active potassium transport system of the human red blood cell has been investigated. At low extracellular potassium concentrations, extracellular sodium competitively inhibits the active potassium influx at all sodium concentrations investigated, and tetraethylammonium behaves in a fashion similar to that of sodium. At low extracellular concentrations of potassium, ammonium at low concentrations at first stimulates the active potassium influx, but at higher concentrations inhibits it. Tetramethylammonium at most slightly stimulates the active potassium influx, and calcium is without effect. The behavior is consistent with a model in which potassium is required at more than one site before transport occurs, and the sites are indistinguishable as far as their behavior toward the ions investigated is concerned. The affinity of the alkali metal cations for the sites appears to be explicable in terms of their physical characteristics.     

Ouabain Binding and Cation Transport in Human Erythrocytes

In the present studies we have explored the relation between ouabain binding and the inhibition of potassium influx in intact human erythrocytes. The rate at which bound ouabain molecules dissociate from the erythrocyte membrane is not altered by complete replacement of choline with sodium or by partial replacement with potassium. These findings indicate that the effects of these cations on ouabain binding reflect alterations in the rate of association of ouabain molecules with the erythrocyte membrane. Variations in the cation composition of the incubation solution did not alter the relation between the fraction of the glycosidebinding sites occupied by ouabain or the fraction of ouabain-sensitive potassium influx which was inhibited. That is, irrespective of the affinity of the erythrocyte membrane for ouabain molecules and irrespective of the magnitude of glycoside-sensitive potassium influx, occupation of a given fraction of the glycoside-binding sites by ouabain results in the inhibition of an equal fraction of the ouabain-sensitive potassium transport sites.     

The Concentration Dependence of Active Potassium Transport in the Human Red Blood Cell

The relation between the active potassium influx in the human red blood cell and the extracellular potassium concentration does not appear to be consistent with the Michaelis-Menten model, but is adequately described by a model in which two potassium ions are required simultaneously at some site or sites in the transport mechanism before transport occurs. The same type of relation appears to exist between that portion of the sodium outflux that requires the presence of extracellular potassium and the extracellular potassium concentration. Rubidium, cesium, and lithium, which are apparently transported by the same system that transports potassium, stimulate the potassium influx when both potassium and the second ion are present at low concentrations, as is predicted by the two-site model.     

Effects of ethacrynic acid and furosemide on respiration of isolated kidney tubules: the role of ion transport and the source of metabolic energy

In order to investigate the mechanism of action of ethacrynic acid and furosemide, experiments were designed to determine whether these drugs directly inhibit active transport or energy metabolism. The effects of these diuretics on the respiration of tubule suspensions isolated from renal cortex (of rats and rabbits) and outer medulla (of rabbits) were measured. The respiration of tubules prepared from renal outer medulla was stimulated by the presence of chloride in the incubation medium, whereas the respiration of cortical tubules was unaffected by chloride. Both ethacrynic acid and furosemide produced the greatest inhibition of respiration on tubules from outer medulla suspended in chloride-containing media; this result suggests that the diuretics directly inhibit chloride transport. The source of metabolic energy for ion transport was varied by using substrates which donate electrons to the respiratory chain at different phosphorylation sites. Both ethacrynic acid and furosemide inhibit respiration supported by beta-hydroxybutyrate, but there was little or no inhibition of respiration with succinate or tetramethylphenylenediamine ascorbate. Similarly, ouabain inhibited respiration with beta-hydroxybutyrate, but not with the other substrates. Therefore, both diuretics inhibited respiration in a fashion similar to ouabain. It is concluded from both types of experiments that ethacrynic acid and furosemide may directly inhibit active chloride transport.     

Cytosine Arabinoside Influx and Nucleoside Transport Sites in Acute Leukemia

Although cytosine arabinoside (araC) can induce a remission in a majority of patients presenting with acute myeloblastic leukemia (AML), a minority fail to respond and moreover the drug has less effect in acute lymphoblastic leukemia (ALL). The carrier-mediated influx of araC into purified blasts from patients with AML, ALL, and acute undifferentiated leukemia (AUL) has been compared to that of normal lymphocytes and polymorphs. Blasts showed a larger mediated influx of araC than mature cells, since mean influxes for myeloblasts and lymphoblasts were 6- and 2.3-fold greater than polymorphs and lymphocytes, respectively. Also, the mean influx for myeloblasts was fourfold greater than the mean for lymphoblasts. The number of nucleoside transport sites was estimated for each cell type by measuring the equilibrium binding of [³H]nitrobenzylthioinosine (NBMPR), which inhibits nucleoside fluxes by binding with high affinity to specific sites on the transport mechanism. The mean binding site numbers for myeloblasts and lymphoblasts were 5- and 2.8-fold greater, respectively, than for the mature cells of the same maturation series. The mean number of NBMPR binding sites for myeloblasts was fourfold greater than for lymphoblasts. Patients with AUL were heterogeneous since blasts from some gave values within the myeloblastic range and others within the lymphoblastic range. The araC influx correlated closely with the number of NBMPR binding sites measured in the same cells on the same day. Transport parameters were measured on blasts from 15 patients with AML or AUL who were then treated with standard induction therapy containing araC. Eight patients entered complete remission, while seven failed therapy, among whom were the three patients with the lowest araC influx (<0.4 pmol/10⁷ cells per min) and NBMPR binding (<3,000 sites/cell) for the treated group. In summary, myeloblasts have both higher araC transport rates and more nucleoside transport sites than lymphoblasts and this factor may contribute to the greater sensitivity of AML to this drug. AraC transport varied >10-fold between leukemic blasts and normal leukocytes, but transport capacity related directly to the number of nucleoside transport sites on the cell. Finally, low araC transport rates or few NBMPR binding sites on blasts were observed in a subset of patients with acute leukemia who failed to achieve remission with drug combinations containing araC.     

The effects of transport inhibitors on sodium outflux and influx in red blood cells: evidence for exchange diffusion

Active sodium transport (outflux or efflux) in red blood cells generally has been measured by assessing the amount of outflux inhibited by digitalis glycosides (outflux-fraction I). The presence of a ouabain-uninhibited sodium outflux (outflux-fraction II) attributable either to a second active transport mechanism or to exchange diffusion has been the subject of recent investigations. In the present study a variety of transport inhibitors, including ouabain, ethacrynic acid, furosemide, oligomycin, and amiloride, were studied for their effects on these components of sodium transport in RBC.In the presence of ouabain both ethacrynic acid and furosemide exerted similar effects on sodium outflux, inhibiting approximately 0.5 mmoles/L of cells per hr. This component of sodium outflux has been called outflux-fraction II. Ethacrynic acid showed no inhibitory potency when ouabain and furosemide were present, thereby suggesting that the same outflux component (fraction II) was affected by ethacrynic acid and by furosemide. In addition, furosemide reduced sodium influx to the same extent that it reduced sodium outflux. Outflux-fraction II, as defined by furosemide, did not contribute a net sodium outflux. These results of sodium outflux and influx experiments confirm the existence of a transport pathway which does not contribute to net flux and which fits the definition of exchange diffusion.The inhibitory effect of furosemide on outflux-fraction II remained despite the use of a sulfhydryl protective reagent, whereas the effect of ethacrynic acid was obliterated. No combination of inhibitors was found which affected the residual or uninhibited sodium outflux (0.4-0.5 mmoles/liter of cells per hr). Oligomycin possessed an inhibitory potency less than that of ouabain, and it exerted no effect on sodium outflux if it was superimposed upon ouabain inhibition. Amiloride proved to be a very weak inhibitor of sodium outflux in human erythrocytes.     

Abnormal membrane sodium transport in Liddle''s syndrome

We have documented the presence of abnormal sodium transport in Liddle's syndrome by measuring sodium concentration, sodium influx, and fractional sodium outflux in vitro in erythrocytes from normal subjects, two patients with Liddle's syndrome, and one patient with primary hyperaldosteronism. Sodium influx and fractional sodium outflux, but not sodium concentration, were significantly increased in patients with Liddle's syndrome. Sodium outflux in a patient with primary hyperaldosteronism did not differ significantly from normal. These alterations of sodium transport in erythrocytes from patients with Liddle's syndrome were not attributable to circulating levels of aldosterone, renin, angiotensin, or serum potassium. Furthermore, changes in aldosterone secretory rate and levels of circulating renin produced by varying dietary sodium intake, did not alter sodium influx or fractional sodium outflux in either patients with Liddle's syndrome or normal subjects. The response of fractional sodium outflux and sodium influx to ouabain, ethacrynic acid, and to changes in the cation composition of the incubation medium suggests that the increased sodium fluxes in Liddle's syndrome do not result solely from a quantitative increase in those components of sodium transport which occur in normal human erythrocytes. Instead, at least a portion of the increased erythrocyte sodium transport in Liddle's syndrome represents a component of sodium transport which does not occur in normal human erythrocytes.     

The effect of bumetanide on cation transport in human red blood cells.

Bumetanide, a sulfamyl-aminobenzoic acid derivative, is a new and highly effective diuretic agent. The present studies were designed to examine its effects on cation transport in human red cells. At a concentration of 10(-3) M, the drug inhibited both active and passive unidirectional sodium fluxes, as well as active potassium influx. It also caused a significant inhibition of glycolysis. The inhibition caused by bumetanide was less than that seen with ouabain alone, but a bumetanide effect was also present in ouabain-treated cells. Bumetanide had no effect on red cell Na-K adenosine triphosphatase activity and did not affect net transport of sodium in sodium-loaded cells. The data are consistent with a model in which the inhibition of monovalent cation movement in red cells by bumetanide is related to an effect of this compound in decreasing the permeability of the red cell membrane to sodium.     

Effect of diuretics on ADP incorporation in kidney mitochondria

The effect of diuretics on incorporation of ADP in mitochondria isolated from rabbit renal cortex and medulla was examined. Inhibition of incorporation of [14 C]ADP into both types of mitochondria was observed following pretreatment with furosemide, ethacrynic acid and meralluride at high drug concentrations (7.5 x 10-4 M furosemide and ethacrynic acid, 6.4 x 10-3 M meralluride). At lower concentrations (7.5 x 10-5 M furosemide and ethacrynic acid, 6.4 x 10-4 M meralluride), only entry of ADP in medullary mitochondria was inhibited. Chlorothiazide, 1.7 x 10-3 M, did not inhibit incorporation of ADP into either mitochondrial preparation. Atractyloside, a classic inhibitor of ADP-ATP exchange, showed inhibition in both preparations. Furosemide, injected in vivo inhibited incorporation of ADP into medullary but not cortical mitochondria. These results are consistent with the possibility that loop diuretics may reduce tubular sodium reabsorption by inhibiting ADP-ATP exchange across the mitochondrial membrane, thereby depriving active transport processes of ATP. The differential action on cortical and medullary mitochondria by loop diuretics is consistent with their predominant site of action in the tubule and with the different morphologic characteristics of both types of mitochondria.     

Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA.

The canalicular (apical) membrane of the hepatocyte contains an ATP-dependent transport system for organic anions, known as the multispecific organic anion transporter (cMOAT). The deduced amino acid sequence of cMOAT is 49% identical to that of the human multidrug resistance- associated protein (MRP) MRP1, and cMOAT and MRP1 are members of the same sub-family of adenine nucleotide binding cassette transporters. In contrast to MRP1, cMOAT was predominantly found intracellularly in nonpolarized cells, suggesting that cMOAT requires a polarized cell for plasma membrane routing. Therefore, we expressed cMOAT cDNA in polarized kidney epithelial MDCK cell lines. When these cells are grown in a monolayer, cMOAT localizes to the apical plasma membrane. We demonstrate that cMOAT causes transport of the organic anions S-(2,4-dinitrophenyl)-glutathione, the glutathione conjugate of ethacrynic acid, and S-(PGA1)-glutathione, a substrate not shown to be transported by organic anion transporters previously. Transport is inhibited only inefficiently by compounds known to block MRP1. We also show that cMOAT causes transport of the anticancer drug vinblastine to the apical side of a cell monolayer. We conclude that cMOAT is a 5'-adenosine triphosphate binding cassette transporter that potentially might be involved in drug resistance in mammalian cells.     

Glucocorticoid-induced alterations in the sodium potassium pump of the human erythrocyte.

To evaluate the effects of glucocorticoids on the Na-K pump in human subjects, were evaluated the intracellular sodium and potassium, 42K influx across and the [3H]ouabain binding to cell membranes of intact human erythrocytes from a group of subjects taking glucocorticoids and a group of normal subjects. Intracellular sodium concentration was lower (7.2 +/- 0.4 vs. 10.9 +/- 0.2 mmol/liter cell water) and intracellular potassium concentration higher (149.8 +/- 1.5 vs. 137.2 +/- 1.2 mmol/liter cell water) in erythrocytes from steroid-treated patients. In spite of a significantly decrease intracellular sodium which normally diminishes ouabain-sensitive 42K influx, the ouabain-sensitive K influx was unchanged in erythrocytes from the steroid-treated group. Maximum [3H]ouabain binding was markedly higher in the steroid-treated group (835 +/- 44 vs. 449 +/- 11 sites/cell). There was close linear correlation between [3H]ouabain binding and inhibition of K pump, suggesting the specificity of ouabain binding to Na-K pump sites on the cell membrane. Association kinetics for ouabain were similar in the two groups despite the marked difference in the amount of [3H]ouabain binding. External potassium concentration required for half-maximum ouabain-sensitive K influx was identical in the two groups. Thus, the additional Na-K pump sites in the steroid-treated group were qualitatively similar to those in normals. These results suggest that administration of glucocorticoids leads to an increase in the number of Na-K pump sites. The increase in the number of Na-K pump sites may explain the low levels of intracellular sodium and higher cell potassium observed in steroid-treated subjects.     

A common mechanism for transport of di- and tri-peptides by hamster jejunum in vitro.

1. This paper describes the results of a survey of the effects of peptides and amino acids on uptake by rings of everted hamster jejunum in vitro of glycylsarcosylsarcosine, a tripeptide which is taken up by an active mechanism but is very resistant to hydrolysis, appearing intact in the rings. The results of a small number of similar experiments with beta-alanylglycylglycine, another tripeptide which is taken up with very little hydrolysis, are also described. 2. Uptake of the two tripeptides was inhibited by other di- and tri-peptides, but not by free amino acids. The results suggest that dipeptides and tripeptides share a common uptake mechanism. The tetrapeptide glycylsarcosylsarcosylsarcosine did not inhibit uptake of glycylsarcosylsarcosine, and appears to be unable to utilize the uptake mechanism. 3. The results add to information about the influence of molecular structure on intestinal uptake of peptides by the system used by glycylsarcosylsarcosine, which is shared by a wide range of other di- and tri-peptides. In conjunction with previous results, they suggest that substitution of the N-terminal amino or C-terminal carboxyl groups reduces affinity for transport, that the presence of a beta-amino acid residue in a peptide is tolerated by the transport system, and that the presence of a D-amino acid residue reduces affinity for transport. Some peptides containing or made up of basic or acidic amino acid residues appear to have a low affinity for the transport system used by glycylsarcosylsarcosine. 4. Of two biologically active peptides, one, cephalexin, a peptide antibiotic, inhibited uptake of glycylsarcosylsarcosine and is probably transported by the same system. The other, prolylleucylglycineamide, which has the action of a hypothalamic regulatory factor, did not, and its structural features may make it unsuitable for carrier-mediated transport by the small intestine.     

Binding of Dipyridamole to Human Platelets and to α1 Acid Glycoprotein and its Significance for the Inhibition of Adenosine Uptake

The interactions of dipyridamole with α₁ acid glycoprotein of plasma and with human platelets are related to inhibition of adenosine uptake by platelets. Binding studies by equilibrium gel filtration suggested that 1 mol of dipyridamole binds per mol of α₁ acid glycoprotein with a dissociation constant of 1.6 μM. Platelets contain two populations of binding sites, one with high and another with lower affinity for the drug. The binding of dipyridamole to the high-affinity sites follows a Michaelis-Menten binding pattern with a dissociation constant of 0.04 μM. Approximately 2 × 10⁴ dipyridamole molecules are bound at the high-affinity sites of each platelet. The lower affinity sites bind the drug with a dissociation constant of 4 μM. In the presence of α₁ acid glycoprotein of plasma, the binding of dipyridamole to human platelets is inhibited. Correspondingly, the dipyridamole inhibition of adenosine uptake by platelets is reduced 1,000-fold by purified α₁ acid glycoprotein. The binding of dipyridamole to human platelets was found to be essential for its inhibition of adenosine uptake by platelets. Dipyridamole decreases the incorporation of [¹⁴C]adenosine radioactivity in platelet nucleotides and reduces the [¹⁴C]-ATP to [¹⁴C]ADP ratio. Purified α₁ acid glycoprotein reverses these effects of dipyridamole on adenosine metabolism of platelets in a concentration-dependent manner. An equilibrium of dipyridamole binding to α₁ acid glycoprotein and to platelets is proposed.     

Effects of theophylline on inotropic and arrhythmogenic actions of cardiotonic steroids in guinea pig cardiac muscle

This study was designed to examine effects of theophylline, a methylxanthine, on both the positive inotropic and toxic actions of cardiotonic steroids in cardiac muscle isolated from guinea pig heart. In electrically paced left atrial muscle, 0.3 mM theophylline reduced both the maximum developed tension observed in the presence of increasing concentrations of strophanthidin and the dose of this steroid that first elicited extrasystoles. Similarly, 0.3 mM theophylline decreased the time to onset of arrhythmias produced by 5 microM digoxin and the fractional occupancy of specific binding sites on Na,K-adenosine triphosphatase by digoxin at the onset of these dysrhythmic events. A higher level of theophylline (6.5 mM) severely diminished or prevented the positive inotropic and arrhythmogenic actions of cardiotonic steroids while promoting the contracture elicited by these digitalis-like compounds. In spite of the severe contracture observed in the presence of 6.5 mM theophylline plus 5 microM digoxin, the digoxin fractional occupancy was significantly less than that observed at the onset of digoxin-induced extrasystoles and contracture in the absence of theophylline. In radiolabeled ligand binding experiments, 6.5 mM theophylline reduced the affinity of specific binding sites for ouabain while having no effect on receptor density. These results, when considered in light of previous reports by other investigators, suggest that moderate concentrations of methylxanthines promote cardiotonic steroid-induced arrhythmias by increasing Ca++ influx and its uptake into sarcoplasmic reticulum. Higher levels seem to antagonize the arrhythmogenic actions by inhibition of sarcoplasmic reticular Ca++ uptake and by antagonism of receptor binding.     

The effects of harmaline on sodium transport in human erythrocytes: evidence in favor of action at interior sodium-sensitive sites.

The effects of the hallucinogen, harmaline (HME), and its congeners on human red blood cell (RBC) transport were studied. HME reduced sodium efflux by 70% at maximum inhibitory concentrations (6-8 mM). It acted upon the ouabain-sensitive component of sodium efflux since it exerted no inhibitory actions in the presence of ouabain. Several lines of evidence suggested that HME exerted its inhibitory effect at intracellular sodium-sensitive sites. The percent inhibition of Na efflux by 0.1 mM HME was unaffected by increasing extracellular potassium from 10 to 100 mM. When HME was incorporated into RBC ghosts by reversible hemolysis, the degree of inhibition of sodium efflux was comparable to that found with ouabain outside the red cells and was always greater than the inhibition produced with HME outside cells. HME increased membrane permeability to sodium, as shown by enhanced sodium influx into RBC and at concentrations of 10 mM caused rapid increments of intracellular sodium and decrements of intracellular potassium. We conclude that the harmala alkaloids inhibit the active Na-K transport system in human RBCs through their effects on sodium-sensitive transport sites on the interior membrane surface.     

Further properties of stereospecific opiate binding sites in rat brain: on the nature of the sodium effect.

Addition of sodium salts has been reported to enhance stereospecific binding of opiate antagonists while reducing binding of agonists to rat brain homogenate. We have tested, in addition to sodium and potassium, a number of organic cations. Our results support the suggestion that the ability to enhace antagonist binding is not a general characteristic of cations or high ionic strength, but a property of sodium ions. We have shown that the increase in antagonist binding results from an enhancement of binding affinity and not from unmasking of new binding sites. The reduction in etorphine binding in the presence of sodium is due to a decrease in binding affinity. This decrease is largely accounted for by an acceleration in the dissociation rate, while the greater affinity of naltrexone binding appears to be due to an increase in rate of association. Our results are consistent with the hypothesis of a conformational change in opiate binding sites in the presence of sodium, transformed sites exhibiting greater affinity for antagonists and reduced affinity for agonists. Preheating of rat brain P2 fraction at 50 degrees C results in gradual inactivation of stereospecific binding, but an increase in naltrexone binding is consistently observed after heating at 50 degrees C for 2 to 3 minutes, even at optimal concentrations of sodium.     

Transport of imipenem, a novel carbapenem antibiotic, in the rat central nervous system

The transport of imipenem, a novel carbapenem antibiotic, in the rat central nervous system (CNS) was studied using in vivo, in situ and in vitro experimental techniques. After i.v. bolus administration, the imipenem concentration in the cerebrospinal fluid (CSF) rose to a peak within 30 min and declined with time. The CSF/serum unbound concentration ratio of imipenem was 0.22 at 2 hr after i.v. administration, substantially higher than that reported for benzylpenicillin. By using an in situ brain perfusion technique, we found that imipenem was transported through the blood-brain barrier principally via passive diffusion with a permeability-surface area product comparable to that of mannitol. In vitro, imipenem was accumulated by the isolated choroid plexus via an active organic anion transport system, although much less rapidly than benzylpenicillin. In vivo, after i.c.v. administration, imipenem was cleared from the CNS in a manner comparable to that of mannitol with only a small probenecid-sensitive process. Imipenem thus has minimal affinity for the organic anion transport system in the choroid plexus, resulting in the slow elimination of this drug from the CNS. These results suggest that the difference between imipenem and benzylpenicillin in the ratio of CSF to unbound serum drug concentration is determined principally by the efflux process in the choroid plexus rather than the influx process through the blood-brain barrier.     

Equilibrium dialysis, ultrafiltration, and ultracentrifugation compared for determining the plasma-protein-binding characteristics of valproic acid

Equilibrium dialysis, ultrafiltration, and ultracentrifugation were compared to determine their reliability and applicability in the study of binding of an anticonvulsant drug, valproic acid, by plasma proteins. We studied drug binding with pooled serum and with solutions of human serum albumin at physiological concentrations. We compared binding characteristics such as number of binding sites, affinity constants, and percent of binding as measured by each method in the therapeutic range for valproic acid. Results by ultracentrifugation differed from those by equilibrium dialysis and ultrafiltration, which agreed reasonably well with each other.     

On the mechanism of the different sensitivity of Purkinje and myocardial fibers to strophanthidin

The mechanism of the different sensitivity of Purkinje and myocardial fibers to strophanthidin was studied in these tissues isolated from the same hearts. Membrane potentials, force and, in some experiments, intracellular sodium activity were recorded under conditions that vary the sodium load in the absence and presence of strophanthidin. Strophanthidin (0.1-0.3 microM) increased force in percent terms more and at a faster rate in Purkinje than in myocardial fibers. Tetrodotoxin (TTX, 2 microM) markedly reduced whereas high [Na]o (176.6 mM) and veratridine (0.2 microM) potentiated strophanthidin inotropy in Purkinje but not in myocardial fibers. The rate of force development was augmented by high [Na]o and veratridine in Purkinje fibers but in myocardial fibers this effect was absent with high [Na]o and smaller with veratridine. Strophanthidin increased the action potential duration at plateau level in Purkinje and decreased it in myocardial fibers. The effects of TTX, high [Na]o and veratridine on the action potential were more pronounced in Purkinje than in myocardial fibers. TTX decreased far more and adding strophanthidin increased intracellular sodium activity (aiNa) less in Purkinje fibers. Strophanthidin increased aiNa to a similar extent in the presence of high [Na]o and veratridine in the two tissues. Thus, changes in Na influx modify the action potential duration, force and strophanthidin inotropy more in Purkinje than in myocardial fibers. This greater sensitivity of Purkinje fibers to strophanthidin does not appear to be related to a larger increase in aiNa, but rather to the changes in action potential (and consequent changes in calcium influx).