3H]Ouabain binding to human erythrocytes in protein-énergy malnutrition

[3H]Ouabain binding to erythrocytes was determined in normal children and in children suffering from kwashiorkor or marasmus. Scatchard plot analysis of [3H]ouabain binding displayed straight lines with linear slopes in all subjects indicating the presence of a single species of ouabain binding sites on erythrocytes. The number of ouabain binding sites per cell was 385 +/- 26 (mean +/- SEM, n = 3) in normal, 891 +/- 102 (n = 8) (p less than 0.001) in kwashiorkor and 316 +/- 45 (n = 3) in marasmic children. The equilibrium dissociation constant (Kd) for ouabain binding in kwashiorkor (16 nmol/1) was similar to that in control (12 nmol/1). The specific activity of Na+, K+ -ATPase of erythrocyte membrane has been shown to be higher in kwashiorkor children as compared to normal children [3]. This increase in enzyme activity may be considered as a consequence of increase in the enzyme content as indicated by the increased number of ouabain binding sites on red cells. Elevation in the level and activity of erythrocyte Na+, K+ -ATPase in kwashiorkor might represent a compensatory mechanism in response to a primary membrane abnormality, to effect prevention of Na+ accumulation and K+ depletion inside the cell.     

Digitalis toxicity: lack of marked effect on brain na+,k+-adenosine triphosphatase in the cat.

Effect of digitalis on central sympathetic neurons have been proposed to alter sympathetic influences on the heart and to contribute to the induction of arrhythmias. Recently, however, we have presented evidence which indicates that the involvement of a direct central action of digitalis is negligible in the alteration of sympathetic nerve activity after i.v. administration of the drug. Thus, a group of experiments were designed to determine if central drug concentrations or biochemical events in the brain would suggest a central action of the drug. Tritiated digoxin (20 microng/kg) was injected i.v. into cats every 15 minutes until ventricular fibrillation occurred. The concentrations of digoxin in cerebrospinal fluid and serum increased linearly with time as the cumulative dose of digoxin was increased. At the mean arrhythmic dose, 140 microng/kg, cerebrospinal fluid contained approximately 10 nM digoxin whereas free digoxin concentration in serum was approximately 30 nM and total digoxin concentration in serum was approximately 120 nM. Since inhibition of Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) is often associated with the pharmacological effects of digitalis, effects of nanomolar concentrations of digoxin on Na+,K+-ATPase activity were determined in vitro. The concentration of digoxin faund in cerebrospinal fluid at arrhythmia inhibited Na+,K+-ATPase only slightly (5-10%). Activity of Na+,K+-ATP-ase was also examined in brains of cats which had died in ventricular arrhythmias due to treatment with lethal dose of digitoxin. After ventricular fibrillation, the cat brains were removed and Na+,K+-ATPase activity and ouabain binding were determined in eight areas. No reduction in Na+,K+-ATPase activity or [3H]ouabain binding was observed in any area. Thus, it appeared that toxic doses of digitalis did not cause sail to provide evidence for central effects of toxic doses of digoxin or digitoxin.     

Species differences in sodium-potassium adenosine triphosphatase activity in the smooth muscle of the guinea-pig and rat vas deferens.

The acitvities of sodium-potassium-activated adenosine triphosphatase (Na+,K+-activated ATPase) and ouabain-inhibited, sodium-potassium-activated adensoine triphosphatase (Na+,K+-ATPase) in subcellular fractions of guinea-pig and rat vasa deferentia were compared to determine whether the ineffectiveness of ouabain and reduced extracellular potassium in the rat vas deferens observed in the preceding paper occurs because of a relatively low level of Na+,K+-ATPase and/or an insensitivity to ouabain. The results indicate that the specific and total activities of Na+,K+-activated ATPase and Na+,K+-ATPase (i.e., the transport enzyme) in the individual subcellular fractions and in the tissue were higher in the vas deferens of the rat than in the guinea pig. The percentage of inhibition of Na+,K+-activated activity by ouabain (8 x 10(-5) M) varied in the subcellular fractions; it was higher in the guinea-pig (range 31--87%) than in the rat (nonsignificant effect to 40%). A greater percentage of total Na+K+- activated ATPase activity was inhibited in the vas deferens of the guinea pig (56%) than the rat (30%). Differences in the effects of lowered extracellular potassium concentration or ouabain on resting membrane potential (preceding paper) are apparently unrelated to the amount of transport enzyme in the vasa deferentia or the two species, or to its relative sensitivity to ouabain.     

Role of Na(+), K(+)-ATPase in morphine-induced antinociception

We evaluated the modulation by Na+,K+-ATPase inhibitors of morphine-induced antinociception in the tail-flick test and [3H]naloxone binding to forebrain membranes. The antinociception induced by morphine (1-32 mg/kg, s.c.) in mice was dose-dependently antagonized by ouabain (1-10 ng/mouse, i.c.v.), which produced a significant shift to the right of the morphine dose-response curve. The i.c.v. administration of three Na+,K+-ATPase inhibitors (ouabain at 0.1-100, digoxin at 1-1000, and digitoxin at 10-10000 ng/mouse) dose-dependently antagonized the antinociceptive effect of morphine (4 mg/kg, s.c.) in mice, with the following order of potency: ouabain > digoxin > digitoxin. This effect cannot be explained by any interaction at opioid receptors, since none of these Na+,K+-ATPase inhibitors displaced [3H]naloxone from its binding sites, whereas naloxone did so in a concentration-dependent manner. The antinociception induced by morphine (5 mg/kg, s.c.) in rats was antagonized by the i.c.v. administration of ouabain at 10 ng/rat, whereas it was not significantly modified by intrathecally administered ouabain (10 and 100 ng/rat). These results suggest that the activation of Na+,K+-ATPase plays a role in the supraspinal, but not spinal, antinociceptive effect of morphine.     

Increased renal tubular sodium pump and Na+,K+-adenosine triphosphatase in streptozotocin-diabetic rats

The effects of chronic glucose osmotic diuresis on renal tubular sodium pump and Na+,K+-adenosine triphosphatase (ATPase) activities were studied in chronic streptozotocin-induced diabetic rats. Four to seven weeks after streptozotocin (60 mg/kg i.p.) injection, specific renal Na+,K+-ATPase activity showed a 34.8% increase as compared to the saline-citrate treated controls, whereas the nonspecific Mg++-ATPase was not altered. The concentration of Na+,K+-ATPase, estimated from the maximum [3H]ouabain binding site concentration, also showed a significant increase in the chronic streptozotocin-diabetic rats. To determine further the specificity of this increase in Na+,K+-ATPase, the activity of the sodium pump, estimated from ouabain-sensitive 86Rb uptake, was measured in nonenzymatically isolated renal tubules. Again, a significant (+106.4%) increase in the renal tubular sodium pump activity was observed in the streptozotocin-diabetic rats, whereas the nonspecific, ouabain-insensitive 86Rb uptake was not altered. Neither was there any difference in 86Rb uptake by the isolated renal glomeruli. Thus, it appears that chronic streptozotocin-induced diabetes in rats is associated with a significant increase in renal tubular sodium pump and Na+,K+-ATPase. The latter effects may represent an important physiologic adaptation of the kidneys to maintain electrolyte homeostasis in diabetes.     

Effects of ouabain on contractions induced by manganese ions in C2+a-free, isotonic solutions with varying concentrations of K+ in guinea-pig taenia coli

The action of ouabain, a cell membrane Na+, K+-ATPase blocker, on contractions induced by manganese ions (Mn2+) in Ca2+-free, isotonic solutions with varying concentrations of K+ in the external medium were investigated in order to evaluate the underlying role of external Na+ in Mn2+-induced contractions in isolated taenia coli of the guinea-pig. Mn2+ at 5 mM induced greater contractions as external isotonic K+ concentrations progressively increased from 10 to 100 mM. Ouabain (2 x 10(-4) M) completely inhibited tension development stimulated by 5 mM Mn2+ in isotonic, 30 mM K+ (96 mM Na+) medium. Whereas, the tension inhibitory effects of ouabain became progressively weaker as isotonic, external K+ concentrations increased to 60 mM, which successively decreased external Na+ concentrations. Eventually, ouabain failed to affect contractions stimulated by Mn2+ in isotonic, 126 mM K+, Na+-deficient medium. Ouabain caused progressively greater increase in cellular Na+ concentrations as the Na+ concentrations increased in the isotonic, K+ medium. While, pyruvate, which penetrates cell independently of external Na+, reversed the inhibition of tension by ouabain in isotonic, 30 mM K+, Na+-sufficient (96 mM) medium containing 5 mM Mn2+. These results suggested that Mn2+ induced the contraction, which was maintained by glucose transport depending on external Na+, in the case of Na+-sufficient medium in K+-depolarized taenia coli. However, it induced the contraction independent of external Na+, in the case of Na+-deficient, K+ medium. Ouabain might exhibit greater inhibition of the contraction induced by Mn2+ as the decrease in the Na+ gradient across the cell membranes continues.     

Dissociation and redistribution of Na+,K(+)-ATPase from its surface membrane actin cytoskeletal complex during cellular ATP depletion.

Establishment and maintenance of a polar distribution of Na+,K(+)-ATPase is essential for efficient Na+ reabsorption by proximal tubule cells and is dependent upon the formation of a metabolically stable, detergent-insoluble complex of Na+,K(+)-ATPase with the actin membrane cytoskeleton. The present studies show that cellular ATP depletion results in a rapid duration-dependent dissociation of Na+,K(+)-ATPase from the actin cytoskeleton and redistribution of Na+,K(+)-ATPase to the apical membrane. During ATP depletion, total cellular Na+,K(+)-ATPase activity was unaltered, but the Triton-X-100-insoluble fraction (cytoskeleton associated) of Na+,K(+)-ATPase activity decreased (P less than 0.01), with a corresponding increase in the detergent-soluble fraction of Na+,K(+)-ATPase (P less than 0.01). Indirect immunofluorescent studies of cells with depleted ATP revealed a redistribution of Na+,K(+)-ATPase from the basolateral membrane into the apical membrane and throughout the cytoplasm. ATP depletion also resulted in the redistribution of F-actin from a primarily cortical concentration to a perinuclear location. There was also a rapid, duration-dependent conversion of monomeric G-actin to F-actin starting during the first 5 min of ATP depletion. Taken together, these data suggest that ATP depletion causes profound alterations in cell polarity by inducing major changes in the actin cytoskeletal architecture.     

Comparison of the effects of aminosugar cardiac glycosides with ouabain and digoxin on Na+, K+ -adenosine triphosphatase and cardiac contractile force.

Two aminosugar cardiac glycosides, 3-beta-O-(4-amino-4,6-dideoxy-beta-D-galactopyranosyl) digitoxigenin (ASI-222) and its 4-aminoglucose analog (ASI-254) have been shown in our laboratory to have a greater therapeutic index than ouabain (O) or digoxin (D). We have now compared the ability of ASI-222, its nonamino galactose analog (ASI-253), ASI-254, ouabain and digoxin to inhibit swine brain Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) and to increase contractile force of isolated, driven rabbit atria. As inhibitors of Na+,K+ -ATPase, both ASI-222 and ASI-254 were found to be about 10 times more potent than ASI-253, O or D (I50:ASI-222, 1.3 X 10(-7) M; ASI-254, 1.4 X 10(-7) M; ASI-253, 1.15 X 10(-6) M; D, 1.6 X 10(-6) M; O, 1.75 X 10(-6) 7). Moreover the potency of these glycosides in inhibiting Na+, K+ -ATPase correlates closely with the ability of these same glycosides to increase contractile force. The concentration needed to obtain 50% of the maximum increase in contractile force was 9.7 X 10(-8) M for ASI-254, 1.5 X 10(-7) M for ASI-222, 8.8 X 10(-7) M for ASI-253 8.4 X 10(-7) M for O and 1.2 X 10(-6) M for D. Since ASI-253, a nonaminogalactose analog of ASI-222, exhibits a potency in both of our test systems which is similar to the other neutral sugar cardenolides, our data also indicate that the presence of an aminosugar group at position 4 of a sugar in a cardiac glycoside confers greater potency.     

Lack of pharmacodynamic interactions between quinidine and digoxin in isolated atrial muscle of guinea pig heart

Quinidine has been reported to have no effect on the positive inotropic action of digoxin observed in isolated cardiac muscle preparations. This is surprising because quinidine has been shown to reduce Na+ influx in cardiac muscle. The conditions which increase Na+ influx stimulate the glycoside binding to Na+- and K+-activated Mg++-dependent ATP phosphohydrolase (Na+,K+-ATPase), and therefore quinidine may be expected to have an opposite effect. Thus, the effects of quinidine on cardiac muscle and its possible interactions with digoxin were re-evaluated using electrically paced left atrial muscle preparations of guinea pig heart. Quinidine caused a frequency- and concentration-dependent decrease in maximal upstroke velocity and amplitude of the action potential without altering resting membrane potential. In addition, quinidine prolonged action potential duration markedly in a frequency-dependent manner. Despite action potential prolongation, the alkaloid reduced net Na+ influx as determined by a decrease in steady-state ouabain-sensitive 86Rb+ uptake. Under these conditions, however, quinidine failed to reduce the rate of onset or the maximal positive inotropic effect of digoxin; or did it reduce digoxin binding to Na+,K+- ATPase in beating atrial muscle preparations. Benzocaine, which reduced net Na+ influx without increasing the action potential duration, also failed to affect the peak inotropic effect of digoxin or the glycoside binding. Quinidine had no direct effects on glycoside binding to isolated cardiac Na+,K+-ATPase. Moreover, [3H]ouabain binding to isolated enzyme was relatively insensitive to changes in Na+ concentrations between 1 and 8 mM although binding was stimulated clearly by Na+ above 8 mM. These results indicate that quinidine, at therapeutic concentrations, does not interact pharmacodynamically with digoxin in isolated cardiac muscle.     

20-Hydroxyeicosa-tetraenoic acid (20 HETE) activates protein kinase C. Role in regulation of rat renal Na+,K+-ATPase.

It is well documented that the activity of Na+,K+-ATPase can be inhibited by the arachidonic acid metabolite, 20-hydroxyeicosa-tetraenoic acid (20 HETE). Evidence is presented here that this effect is mediated by protein kinase C (PKC). PKC inhibitors abolished 20 HETE inhibition of rat Na+,K+-ATPase in renal tubular cells. 20 HETE caused translocation of PKC alpha from cytoplasm to membrane in COS cells. It also inhibited Na+,K+-ATPase activity in COS cells transfected with rat wild-type renal Na+,K+-ATPase alpha1 subunit, but not in cells transfected with Na+,K+-ATPase alpha1, where the PKC phosphorylation site, serine 23, had been mutated to alanine. PKC-induced phosphorylation of rat renal Na+,K+-ATPase, as well as of histone was strongly enhanced by 20 HETE at the physiologic calcium concentration of 1.3 microM, but not at the calcium concentration of 200 microM. The results indicate that phospholipase A2-arachidonic acid-20 HETE pathway can exert important biological effects via activation of PKC and that this effect may occur in the absence of a rise in intracellular calcium.     

Enhancement of cardiac actions of ouabain and its binding to Na+, K+-adenosine triphosphatase by increased sodium influx in isolated guinea-pig heart

The rate of development of the positive inotropic action of ouabain is enhanced when the heart is stimulated at higher frequencies. A hypothesis that this enhancement is due to a stimulation of the glycoside binding to sarcolemmal Na+,K+-adenosine triphosphatase (ATPase) caused by an increase in intracellular Na+ available to the sodium pump was tested in isolated left atrial muscle preparations of guinea-pig heart, incubated at 30 degrees C and electrically stimulated at 0.5, 1 or 2 Hz. The rate of development of the positive inotropic action of ouabain was dependent on the frequency of stimulation. Each preparation was homogenized at a predetermined time and the fractional occupancy of Na+,K+-ATPase by ouabain was estimated from the decrease in the initial velocity of ATP-dependent [3H]ouabain binding reaction. A parallel relationship was observed between effects of stimulation frequency of the positive inotropic action and those on the occupancy of Na+,K+-ATPase by ouabain. In quiescent preparations, a sodium ionophore, monensin, enhanced the development of contracture caused by a toxic concentration of ouabain and also the glycoside binding to Na+,K+-ATPase. Similar effects on the ouabain-induced contracture and on the glycoside binding were observed with either grayanotoxin I or batrachotoxin, agents known to increase sodium influx, when muscle preparations were exposed to these agents under 1.5 Hz stimulation and were subsequently tested for the actions of ouabain in quiescence. When the exposure to ouabain and either grayanotoxin I or batrachotoxin was restricted to quiescent period, the development of ouabain-induced contracture and glycoside binding to Na+,K+-ATPase were not significantly altered. Monensin, grayanotoxin I or batrachotoxin failed to significantly affect [3H]ouabain binding to muscle homogenates when added to the medium for the labeled glycoside binding assay. These results indicate that intracellular sodium ions promote the ouabain binding to Na+,K+-ATPase and thereby enhance the development of glycoside actions in the isolated atrial muscle of guinea-pig heart. The "beat-dependent" onset of the glycoside action is at least partially explained from the effect of membrane depolarization to increase Na+ available to the sodium pump and to enhance the glycoside binding.     

Effects of quinidine on the renal tubular and biliary transport of digoxin: in vivo and in vitro studies in the dog

Quinidine is known to inhibit the renal clearance of digoxin without affecting glomerular filtration rate. The renal interaction between these drugs was investigated by a combination of in vivo and in vitro methods. The uptake of digoxin by brush border membrane vesicles was not affected by quinidine. Similarly, digoxin did not inhibit the uptake of the cation N-methylnicotinamide by these vesicles and did not alter the binding kinetics of digoxin to the Na+, K+-adenosine triphosphatase by the antiluminal membrane vesicles. By using the in vivo multiple indicator dilution technique transtubular transport of digoxin was documented; renal-artery infusion of quinidine did not affect the recovery of digoxin in the renal vein or urine. Clearance studies documented that the decrease in the renal clearance of digoxin is paralleled by a significant fall in renal blood flow evidenced by a decrease in p-aminohippuric acid clearance. It is concluded that quinidine inhibits the renal excretion of digoxin not by competition at the tubular cell membrane level, but rather by decreasing renal blood flow. A parallel decrease in biliary clearance of digoxin is documented and may suggest a similar mechanism.     

Alpha 2 adrenergic agonists stimulate Na+-H+ antiport activity in the rabbit renal proximal tubule

The role of adrenergic agents in augmenting proximal tubular salt and water flux, was studied in a preparation of freshly isolated rabbit renal proximal tubular cells in suspension. Norepinephrine (NE, 10(-5) M) increased sodium influx (JNa) 60 +/- 5% above control value. The alpha adrenergic antagonist, phentolamine (10(-5) M), inhibited the NE-induced enhanced JNa by 90 +/- 2%, while the beta adrenergic antagonist, propranolol, had a minimal inhibitory effect (10 +/- 2%). The alpha adrenergic subtype was further defined. Yohimbine (10(-5) M), an alpha2 adrenergic antagonist but not prazosin (10(-5) M), an alpha1 adrenergic antagonist completely blocked the NE induced increase in JNa. Clonidine, a partial alpha2 adrenergic agonist, increased JNa by 58 +/- 2% comparable to that observed with NE (10(-5) M). Yohimbine, but not prazosin, inhibited the clonidine-induced increase in JNa, confirming that alpha2 adrenergic receptors were involved. Additional alpha2 adrenergic agents, notably p-amino clonidine and alpha-methyl-norepinephrine, imparted a similar increase in JNa. The clonidine-induced increase in JNa could be completely blocked by the amiloride analogue, ethylisopropyl amiloride (EIPA, 10(-5) M). The transport pathway blocked by EIPA was partially inhibited by Li and cis H+, but stimulated by trans H+, consistent with Na+-H+ antiport. Radioligand binding studies using [3H]prazosin (alpha1 adrenergic antagonist) and [3H]rauwolscine (alpha2 adrenergic antagonist) were performed to complement the flux studies. Binding of [3H]prazosin to the cells was negligible. In contrast, [3H]rauwolscine showed saturable binding to a single class of sites, with Bmax 1678 +/- 143 binding sites/cell and KD 5.4 +/- 1.4 nM. In summary, in the isolated rabbit renal proximal tubular cell preparation, alpha2 adrenergic receptors are the predominant expression of alpha adreno-receptors, and in the absence of organic Na+-cotransported solutes, alpha2 adrenergic agonists enhance 22Na influx into the cell by stimulating the brush border membrane Na+-H+ exchange pathway.     

Effect of red laser light on Na+,K(+)-ATPase activity in human erythrocyte membranes sensitized with Zn-phthalocyanine

OBJECTIVE: The influence of laser light (670 nm) on human erythrocyte membrane Na+,K(+)-ATPase activity in the presence and absence of Zn-phthalocyanine (ZnPc) was studied. BACKGROUND DATA: The response of erythrocyte membranes to low-power laser irradiation has not been fully elucidated. In our study, we focused on the studies on photo-induced changes of Na+,K(+)-ATPase activity. The erythrocyte membrane suspensions were incubated with 2 mM of ZnPc and next irradiated with energy doses of 19.1, 38.2, 57.3, 76.4, and 95.5 J x cm(-2). MATERIALS AND METHODS: The activity of Na+,K(+)-ATPase was assayed colorimetrically at the wavelength of 820 nm and expressed in micromol of inorganic phosphate released from ATP per mg of protein. RESULTS: The measurements of Na+,K(+)-ATPase activity in erythrocyte membranes incubated with ZnPc in the dark demonstrated that all concentrations of the dye (0.5, 1, 2, and 3 microM) stimulated enzyme activity. The concentration of 2 microM caused the smallest increase of enzyme activity, so this concentration was accepted for further studies. Irradiation of erythrocyte membranes in the presence of the dye (2 microM) significantly decreased Na+,K(+)-ATPase activity. Only for energy doses of 19.1 and 38.2 J x cm(-2) was the enzyme activity comparable to the activity of the control. CONCLUSION: It was found that irradiation with all energy doses applied caused a rise of enzyme activity. In the presence of ZnPc, significant decrease of Na+,K(+)-ATPase activity was observed.     

Kinetics of ouabain binding and changes in cellular sodium content, 42K+ transport and contractile state during ouabain exposure in cultured chick heart cells

To define further the mechanism of positive inotropic action of cardiac glycosides, the temporal relationships among ouabain binding, sodium pump inhibition and positive inotropy were examined using cultured chick embryo ventricular cells. In K+-free medium, specific [3H]ouabain binding to intact cells followed pseudo first-order kinetics with saturation of binding sites occurring at 1 microM ouabain. The KD values calculated from the association and dissociation rate constants were 1.4 and 4.9 X 10(-7) M, respectively, in K+-free and 4 mM K+ medium. The Scatchard plot of binding in K+-free medium was linear, consistent with the presence of a single class of binding sites (KD = 1.3 X 10(-7) M). In 4 mM K+, 0.1 microM ouabain occupied 10% of the total binding sites and failed to produce an inotropic effect, inhibit 42K+ uptake or alter [Na+]i. Exposure of cells to 1 microM ouabain caused a significant increase in contractile state after 30 sec, reaching a plateau after 7 min with 50 +/- 6% augmentation of the amplitude of cell motion; the 42K+ uptake rate was concurrently inhibited by 36% accompanied by a 35% increase in [Na+]i and occupation of 38% of total ouabain binding sites. The initial rate of 42K+ uptake in cells loaded with Na+ by incubation in K+-free medium was 4 times greater than that observed without Na+ loading. These results indicate that more than 10% of sodium pump sites must be inhibited to produce an appreciable change in the rate of monovalent cation transport, [Na+]i or contractile state, due to the reserve capacity of uninhibited sodium pumps.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the mechanism of inhibition of the red cell metabolism by cardiac glycosides.

The purpose of this work was to test the previously suggested hypothesis that the inhibitory effect of ouabain on lactate production in human red cells is due to an interaction between phosphoglycerate kinase and (Na+ + k+)-activated adenosine triphosphatase (Na+,K+ATPase). An antibody to red cell phosphoglycerate kingase caused complete inhibition of the purified enzyme, whereas a portion of the phophoglycerate kinase activity of the red cell membranes was resistant to the antibody. When increasing amounts of the purified enzyme were added to the membranes, the antibody-resistant portion of the activity increased. The effects of the antibody and ouabain on lactate production from fructose-6,6-diphosphate in red cell hemolysates were studied. Ouabain, at a maximally effective concentration, produced about 30% inhibition of lactate formation. This value was doubled in the presence of the antibody. Red cell membranes, and rat brain Na+,K+-ATPase, did not catalyze the hydrolysis of 1,3-diphosphoglycerate. Ouabain did not affect the reactions of the Rapport-Luebering pathway of the red cells. These findings provide further support for the view that in red cells a membrane pool of phosphoglycerate kinase is oriented in the vicinity of Na+,K+-ATPase in a way that the product of each enzyme may be used as the immediate substrate of the other and that ouabain inhibits glycolysis by removing the regulatory effect of Na+,K+-ATPase on that portion of glycolysis which is channeled through this pool of phosphoglycerate kinase.     

On the Mechanism of Action of Triamterene: Effects on Transport of Na+, K+, and HVHCO-3 -ions.

The rat salivary duct epithelium, which actively transports Na+, K+, and H+/HCO3- in a manner similar to renal distal tubules, was used as a model tissue to study the mechanism of action of triamterene on electrolyte transport. 10(-4) M triamterene completely blocked Na+ resorption and lowered net K+ secretion to half that of controls, whereas HCO3- accumlated in the lumen, probably due to a decrease in H+ secretion. The rates of K+ and H+/HCO3- transport in the presence of triamterene did not differ from those determined after omission of Na+ from the luminal fluid. This was considered to be evidence against a direct action of triamterene on transport of K+ and H+/HCO3-. Triamterene rapidly and reversibly reduced the transepithelial electrical potential difference. This was due to almost complete abolition of Na+ conductance of the luminal membrane at 10(-4) M triamterene, whereas K+ conductance was not altered. Triamterene, administered in vitro from the interstitial side of the isolated duct epithelium was ineffective even at the highest concentrations. The activities of the Na-K-ATPase, the Mg-ATPase and the microsomal HCO3-ATPase were influenced by 10(-4) M triameterene in a similiar fashion. These effects were clearly demonstrated only in the homogenate of the duct tissue and not in intact cells in the isolated duct preparation. Therefore they were considered unspecific. The transport studied demonstrate a primary effect of triamterene on Na+ entry from lumen to cell. Influences on net K+ and H+/HCO3 transport are secondary consequences of functional coupling between movement of Na+ and movement of K+ and H+ across the luminal cell membrane.     

Effect of inhibition of Na+/K(+)-adenosine triphosphatase on vascular action of vasopressin.

The present study was undertaken to examine the cellular interaction between a Na+/K(+)-ATPase inhibitor, ouabain, and arginine vasopressin (AVP) in rat vascular smooth muscle cells (VSMC) in culture. Preincubation with 10(-5) M ouabain for 60 min increased basal cytosolic free Ca2+ [( Ca2+]i) concentration and intracellular 45Ca2+ uptake. Ouabain, however, did not affect basal 45Ca2+ efflux or AVP-stimulated 45Ca2+ efflux. As assessed by cell shape change, preincubation with 10(-5) M ouabain for 60 min also enhanced the sustained cellular contractile effect of a submaximal (10(-8) M AVP, 21.5% vs. 30.5%, P less than 0.01) but not maximal dose of 10(-6) M AVP. Preincubation with 10(-5) M ouabain for 60 min did not change AVP-induced V1-specific surface receptor binding or AVP-induced inositol phosphate production but did however potentiate the mobilization of [Ca2+]i induced by a submaximal (10(-8) M AVP, 301 vs. 385 nM, P less than 0.01) but not a maximal dose of AVP. These effects of ouabain on the mobilization of [Ca2+]i were abolished by incubation in Ca2(+)-free buffer or 5 X 10(-5) M verapamil. Ouabain (10(-5) M) also enhanced the sustained cellular contractile effect of a direct protein kinase C activator, phorbol 12-myristate 13-acetate. The present results therefore indicate that the inhibition of Na+/K(+)-ATPase may enhance the vascular action of AVP, and perhaps other vasoconstrictors, by increasing the AVP-induced mobilization of [Ca2+]i and by potentiating the activity of protein kinase C stimulated by AVP through enhancing basal and AVP-stimulated cellular Ca2+ uptake.     

A rapid assay for the measurement of Na+,K(+)-ATPase inhibitors.

A rapid and automated assay for inhibitors of Na+, K(+)-ATPase was developed by determining the amount of inorganic phosphorus (Pi) released by Na+,K(+)-ATPase in a centrifugal analyzer. This method avoids long incubation, strong acids and centrifugation as in the conventional manual method. The coefficients of variation of intra- and inter-assay at ouabain concentration 0.5 mumol/L were 1.0 and 1.4%, respectively. The method is quick, reproducible and easy compared with current techniques.     

Cardiac Na+, K+-adenosine triphosphatase inhibition by ouabain and myocardial sodium: a computer simulation.

The major evidence against the hypothesis that Na+, K+-adenosine triphosphatase (Na+, K+-ATPase) inhibition is the mechanism of the positive inotropic action of digitalis is that the myocardial sodium content does not increase at the time of the inotropic response. In order to understand the relationship between sodium pump inhibition and myocardial sodium content, a computer simulation of the intracellular sodium concentration ([Na+]i) during a cycle of myocardial function was performed. The model for the computer simulation is a small compartment adjacent to the inner surface of the sarcolemma. The change in [Na+]i in this compartment is determined by the rate of sodium influx (published data utilized) and the rate of active sodium transport was estimated from the activities of partially purified dog heart Na+, K+-ATPase preparations assayed with various concentrations of sodium and ouabain. The initial rapid sodium influx results in maximal sodium pump activation, but the pump activity decreases with time as the [Na+]i decreases. Thus, the sodium pump functions at a rate close to its maximal velocity during the initial phase of each cycle but at reduced rates during the later phase. Inhibition of Na+, K+-ATPase by ouabain decreases the maximal velocity during the intiial phase of each cycle but at reduced rates during the later phase. Inhibition of Na+, K+-ATPase by ouabain decreases the maximal velocity of the sodium pump but increases the time in each cycle at which the sodium pump operates at its highest possible rate under these conditions, i.e., a rate close to the inhibited maximal velocity. A 40% inhibition of Na+, K+-ATPase activity, caused by inotropic concentrations of ouabain, increases the peak [Na+]i but fails to cause intracellular sodium accumulation since [Na+]i approaches control levels before the beginning of the next cardiac cycle. With greater enzyme inhibition, caused by arrhythmic concentrations of ouabain, [Na+]i fails to return to the precycle level and thus each subsequent cycle causes a progressive accumulation of myocardial sodium. Computer simulation predicts that a positive inotropic concentration of ouabain causes a myocardial sodium accumulation at a high heart rate but not at a lower heart rate. This was confirmed by experiments with Langendorff preparations of guinea-pig hearts. It is concluded that a moderate sodium pump inhibition by inotropic concentrations of ouabain enhances the intracellular sodium transient (a transient increase in intracellular sodium concentration associated with each membrane excitation) but does not cause a significant myocardial sodium accumulation at normal heart rates. A progressive myocardial sodium accumulation occurs only when the degree of Na+, K+-ATPase inhibition exceeds a critical magnitude.