Inhibition of Na+-pump enhances carbachol-induced influx of 45Ca2+ and secretion of catecholamines by elevation of cellular accumulation of 22Na+ in cultured bovine adrenal medullary cells

Summary In bovine adrenal medullary cells, we reported that ²²Na⁺ influx via nicotinic receptor-associated Na⁺ channels is involved in ⁴⁵Ca²⁺ influx, a requisite for initiating the secretion of catecholamines (Wada et al. 1984, 1985b).In the present study, we investigated whether the inhibition of Na⁺-pump modulates carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured bovine adrenal medullary cells. We also measured ⁸⁶Rb⁺ uptake by the cells to estimate the activity of Na⁺, K⁺-ATPase. (1) Ouabain and extracellular K⁺ deprivation remarkably potentiated carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion; this potentiation of carbachol-induced ⁴⁵Ca²⁺ influx and catecholamine secretion was not observed in Na⁺ free medium. (2) Carbachol increased the uptake of ⁸⁶Rb⁺; this increase was inhibited by hexamethonium and d-tubocurarine. In Na⁺ free medium, carbachol failed to increase ⁸⁶Rb⁺ uptake. (3) Ouabain inhibited carbachol-induced ⁸⁶Rb⁺ uptake in a concentration-dependent manner, as it increased the accumulation of cellular ²²Na⁺. These results suggest that Na⁺ influx via nicotinic receptor-associated Na⁺ channels increases the activity of Na⁺, K⁺-ATPase and the inhibition of Na⁺, K⁺-ATPase augmented carbachol-induced Ca²⁺ influx and catecholamine secretion by potentiating cellular accumulation of Na⁺. It seems that nicotinic receptor-associated Na⁺ channels and Na⁺, K⁺-ATPase, both modulate the influx of Ca²⁺ and secretion of catecholamines by accomodating cellular concentration of Na⁺.     

Cardiac glycosides: Correlations among Na+, K+-ATPase,sodium pump and contractility in the guinea pig heart

Summary Relationships among positive inotropic response to cardiac glycosides, Na⁺,K⁺-ATPase inhibition and monovalent cation pump activities were studied using paced Langendorff preparations of guinea-pig heart. Na⁺,K⁺-ATPase activity was estimated from the initial velocity of (³H)-ouabain binding in ventricular homogenates, and cation pump activity from ouabain-sensitive ⁸⁶Rb uptake of ventricular slices. These parameters were assayed in control, ouabain- or digitoxintreated hearts either at the time of inotropic response to the cardiac glycosides or during the course of drug washout. Development and loss of the inotropic response during ouabain or digitoxin perfusion and washout was accompanied by reduction and subsequent recovery of the initial ouabain binding velocity, respectively. If homogenates from glycoside-treated hearts were incubated at 37°C for 10 min during ouabain-binding studies, the levels of binding were not different from those of control hearts, indicating a rapid dissociation of the glycosides from cardiac Na⁺,K⁺-ATPase in this species. Despite differences in the time course of the loss of inotropic responses produced by ouabain or digitoxin, the relationship between Na⁺,K⁺-ATPase inhibition and inotropic responses were similar. Inotropic responses to digitoxin during perfusion, and subsequent los during washout, also were accompanied by a reduction and subsequent recovery of ⁸⁶Rb uptake. A correlation between inhibition of cation pump activity and positive inotropy has hitherto not been demonstrated. Thus, it appears that with cardiac glycosides, a relationship exists among contractility, cardiac Na⁺,K⁺-ATPase and monovalent cation pump activities.     

Chronic digoxin treatment on canine myocardial Na+, K+-ATPase

Summary In order to determine whether a prolonged inhibition of cardiac Na⁺, K⁺-ATPase causes a compensatory or adaptive change in this enzyme, the relationships among serum digoxin concentration, binding of digoxin to the enzyme and cardiac Na⁺, K⁺-ATPase and sodium pump activity were studied in dogs chronically treated with digoxin. Digoxin was injected intravenously twice daily up to 4 weeks. Two hours after the injection of a single non-toxic dose of digoxin, Na⁺, K⁺-ATPase and sodium pump activities were inhibited quantitatively in a manner corresponding to the binding of digoxin to the enzyme. The magnitude of sodium pump inhibition was reduced 12 h after the digoxin injection, with simultaneous decreases in serum digoxin concentration and the binding of digoxin to the enzyme. After 1 or 4 weeks of digoxin treatment with non-toxic doses, the relationships among serum digoxin concentration, binding of digoxin to cardiac Na⁺, K⁺-ATPase and the degree of cardiac Na⁺, K⁺-ATPase or sodium pump inhibition remained unchanged. The magnitude of the inhibition was related to serum digoxin concentrations and digoxin binding to cardiac Na⁺, K⁺-ATPase, in a manner similar to that observed after a single digoxin injection. After 4 weeks of digoxin treatment with toxic doses, these relationships were also unaffected. It was concluded that prolonged digoxin treatment fails to alter the inhibition of myocardial Na⁺, K⁺-ATPase by this agent.This work was supported by U.S. Public Health Service Grant HL-16052.     

Platelet 5-HT uptake sites in depression: three concurrent measures using [3H] imipramine and [3H] paroxetine

Platelet 5-HT uptake sites were measured in 40 depressed patients and 40 controls using [³H] imipramine binding, defined with desmethylimipramine (DMI) and Na⁺ dependence, and [³H] paroxetine binding. In control subjects the B_max of DMI defined [³H] imipramine binding was significantly higher than both Na⁺ dependent [³H] imipramine (by 30%) and [³H] paroxetine binding (by 22%). The B_max of Na⁺ dependent [³H] imipramine and [³H] paroxetine binding did not differ significantly. The K_d of Na⁺ dependent [³H] imipramine binding was significantly lower than the K_d of DMI defined [³H] imipramine binding. The binding of DMI defined and Na⁺ dependent [³H] imipramine and [³H] paroxetine did not differ significantly between depressed patients and controls in the total group, in those depressed patients who had never taken antidepressants or in those depressed patients who had been recently with-drawn from antidepressants. This study provides no support for the view that the number of platelet 5-HT uptake sites are reduced in depression.     

Palytoxin binds to and inhibits kidney and erythrocyte Na+, K+-ATPase

Summary Hog kidney Na⁺, K⁺-ATPase, purified to the microsomal stage and activated with detergent, binds palytoxin, as shown by the nearly complete competition of the toxin with ³H-ouabain. The K _i-values of palytoxin, but not of ouabain, depend on the protein concentration; this indicates additional binding sites for the toxin on kidney membranes. — Palytoxin inhibits the enzymatic activity of the detergent-activated preparation nearly completely (IC₅₀ 8·10^–7 mol/l). Inhibition of ATPase activity and of ouabain binding are promoted by borate, a known activator of palytoxin. — Palytoxin also inhibits the Na⁺, K⁺-ATPase of erythrocyte ghosts in the same dose range.The data are discussed in context with the hypothesis (Chhatwal et al. 1983) that palytoxin raises the cellular permeability by altering the state of Na⁺, K⁺-ATPase or its environment.Part of the thesis (Dr. rer. nat.) of H. Böttinger     

Quabain: Temporal relationship between the inotropic effect and the in vitro binding to,and dissociation from, (Na++K+)-activated ATPase

Summary The time course of the inotropic response to ouabain in Langendorff preparations was compared with that of the in vitro ATP-dependent (³H)-ouabain binding to cardiac (Na⁺+K⁺)-activated ATPase preparations, and subsequent dissociation, to determine the temporal relationship between the inotropic response and (Na⁺+K⁺)-activated ATPase inhibition.Species differences were minimal either in the onset of inotropic response or the (³H)-ouabain binding. The rates of both loss of the inotropic response to ouabain during washout and the dissociation of the ouabain-enzyme complex, however, were rapid in guinea pig and rabbit (relatively ouabain-insensitive species) and slow in cat and dog (ouabain-sensitive species). The half-time of the loss of the inotropic response was similar to the half-time of the dissociation of the ouabain-enzyme complex in each species.Since ATP-dependent binding of cardiac glycosides has been related to enzyme inhibition, it was concluded that the time course of the inotropic response to ouabain parallels the time course of (Na⁺+K⁺)-activated ATPase inhibition, and that the dissociation of ouabain from the enzyme may terminate the inotropic response.A part of this study was presented at the Fifth Annual Meeting of the International Study Group for Research in Cardiac Metabolism, Winnipeg, Manitoba, June, 1972.     

AMP-activated protein kinase (AMPK)-dependent and -independent pathways regulate hypoxic inhibition of transepithelial Na(+) transport across human airway epithelial cells

BACKGROUND AND PURPOSE

Pulmonary transepithelial Na⁺ transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na⁺ channels and basolateral Na⁺K⁺ ATPase activity.

EXPERIMENTAL APPROACH

H441 human airway epithelial cells were used to examine the effects of hypoxia on Na⁺ transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS.

KEY RESULTS

AMPK was activated by exposure to 3% or 0.2% O₂ for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm⁻²) was added to the apical surface of cells grown at the air–liquid interface. Only 0.2% O₂ activated AMPK in cells grown at the air–liquid interface. AMPK activation was associated with elevation of cellular AMP : ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive I_sc (I_ouabain) and apical amiloride-sensitive Na⁺ conductance (G_Na+). Modification of AMPK activity prevented the effect of hypoxia on I_ouabain (Na⁺K⁺ ATPase) but not apical G_Na+. Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical G_Na+ (epithelial Na⁺ channels).

CONCLUSIONS AND IMPLICATIONS

Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na⁺ channels and basolateral Na⁺K⁺ ATPase activity to decrease transepithelial Na⁺ transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions.     

Diphenyl ditelluride impairs short-term memory and alters neurochemical parameters in young rats

The aim of this study was to investigate if maternal exposure to 0.03 mg/kg of diphenyl ditelluride (PhTe)₂ during the first 14 days of lactational period in Wistar rats alters recognition memory and neurochemical parameters in young rats. Object recognition memory task, evaluation of synaptosomal [³H]glutamate uptake and release as well as cerebral Na⁺/K⁺ATPase activity were evaluated in 4 week-old rats. There were no significant specific overt signs of maternal intoxication. The body weight gain of rats was similar among groups. (PhTe)₂-exposed group showed a significantly lower time exploring the novel object when compared to the performance of the control group in short-term memory (STM) test. In addition, (PhTe)₂ significantly inhibited synaptosomal [³H]glutamate uptake and cerebral Na⁺/K⁺ATPase activity in animals. The synaptosomal [³H]glutamate release was similar between (PhTe)₂ and control groups. In conclusion, the present study establishes that young rats presented cognitive impairment after exposure to (PhTe)₂ via maternal milk, demonstrated by the performance of animals in object recognition memory task. The possible mechanism involved in (PhTe)₂ action in memory of recognition might involve inhibition of cerebral Na⁺/K⁺ATPase activity and synaptosomal [³H]glutamate uptake.     

Effects of monovalent cations on cardiac Na+, K+-ATPase activity and on contractile force

Summary The relationship between Na⁺, K⁺-ATPase inhibition by monovalent cations and their inotropic effect was studied in guinea pig hearts. The activity of partially purified cardiac enzyme was assayed in the presence of 5.8 mM KCl and either 20 or 150 mM NaCl. Rb⁺ and Tl⁺ inhibited Na⁺, K⁺-ATPase activity, the magnitude of the inhibition by these cations being greater in the assay media containing lower Na⁺ concentrations. Tl⁺ produced a dose-dependent inhibition of Na⁺, K⁺-ATPase activity in the presence of 20 mM Na⁺ and 75 mM K⁺, a cationic condition similar to that of intracellular fluid. Other monovalent cations such as K⁺, Cs⁺, NH₄ ⁺, Na⁺ or Li⁺ produced essentially no effect on the Na⁺, K⁺-ATPase activity or slightly stimulated it. In left atrial strips stimulated with field electrodes and bathed in Krebs-Henseleit solution (5.8 mM K⁺ and 145 mM Na⁺), addition of Cs⁺ failed to alter the isometric contractile force significantly. NH₄ ⁺ and K⁺ caused a transient positive inotropic effect which was partially blocked by propranolol. The positive inotropic response to K⁺ was followed by a negative inotropic response. Rb⁺ produced a sustained, dose-dependent inotropic response reaching a plateau at 1–2 min, whereas Tl⁺ produced a dose-dependent positive inotropic effect which developed slowly over a 30-min period. The positive inotropic effects produced by Rb⁺ and Tl⁺ were insensitive to propranolol pretreatment. Concentrations of Tl⁺ and cardiac glycosides which produce similar inotropic effects appear to cause the same degree of Na⁺-pump inhibition. The onset of the positive inotropic response to Rb⁺ or Tl⁺ was not dependent on the number of contractions which is in contrast to the cardiac glycoside-induced inotropic response. Substitution of 20 mM LiCl for an equimolar amount of NaCl in Krebs-Henseleit solution produced a significantly greater inotropic response than that observed when sucrose was substituted for NaCl. It appears that, among monovalent cations, only sodium pump inhibitors produce a sustained positive inotropic response.     

Inhibition of human colonic (Na++K+)-ATPase by arachidonic and linoleic acid

Summary The sodium pump, (Na⁺+K⁺)-ATPase, which is involved in the transport of cations and water movement by the colonic mucosa, may be decreased in various diarrhoeal states. In this study, we have measured ³H-ouabain binding and (Na⁺+K⁺)-ATPase activity in human colonic biopsy homogenates and the influence of various inflammatory and antiinflammatory compounds on these parameters. ³H-ouabain binds to one site of high affinity (K _D 1.9±0.2×10^–9 mol/l) with a maximal binding capacity of 7.5±0.8×10¹⁴ binding sites/g protein. Both arachidonic and linoleic acid inhibited (Na⁺+K⁺)-ATPase activity (IC₅₀ arachidonic acid: 7.5×10^–5 mol/l, linoleic acid: 6.5×10^–5 mol/l) and Mg²⁺-ATPase activity (IC₅₀ arachidonic acid: 9×10^–5 mol/l, linoleic acid: 4×10^–5 mol/l). Arachidonic acid inhibited ³H-ouabain binding, (IC₅₀ 3.2×10^–5 mol/l). The following antiinflammatory compounds, at concentrations up to 1×10^–3 mol/l, did not influence ATPase activity directly nor reverse the arachidonic acid-induced inhibition: indomethacin (cyclooxygenase inhibitor), nordihydroguaretic acid (lipoxygenase inhibitor), sulphasalazine and its metabolites: 5-aminosalicylic acid, N-acetylaminosalicylic acid and sulphapyridine.These results indicate that human colonic (Na⁺+K⁺)-ATPase is inhibited by the prostanoid precursors, arachidonic and linoleic acid. From a therapeutic point of view (effect on colonic (Na⁺+K⁺)-ATPase and perhaps diarrhoea), the suppression of the production of these prostanoid precursors by drugs may, therefore, be beneficial in the treatment of inflammatory bowel disease.Supported by DFG (Er65/4-4)     

Biochemical basis for the low sensitivity of the rat heart to digitalis

Summary The concentration of cardiac glycosides to produce positive inotropic effects in the rat heart is markedly higher than that in other species. Such a low digitalis sensitivity of the rat heat is attributed to the low affinity of cardiac Na⁺, K⁺-ATPase for digitalis in this species. In the present study the biochemical cause which is responsible for the formation of the unstable complex between the glycosides and Na⁺, K⁺-ATPase or positive inotropic, receptor in the rat heart was examined using Na⁺, K⁺-ATPase preparations obtained from rat hearts, guinea-pig hearts and rat brains as well as isolated, electrically stimulated atrial preparations obtained from these animals. Monensin, which alters transmembrane Na⁺ movements without interacting with the cardiotonic sites on Na⁺, K⁺-ATPase, had equivalent potencies in guinea-pig and rat hearts. Cassaine, which lacks a lactone ring but interacts with cardiotonic sites on Na⁺, K⁺-ATPase, increased the force of contraction in guinea-pig hearts at low, but in rat hearts only at high, concentrations. AY-22,241 (Actodigin) and prednisolone-3,20-bisguanylhydrazone (PBGH) bind to cardiotonic sites on Na⁺, K⁺-ATPase and had a similar spectrum as cassaine in these two species. Actodigin has an altered lactone ring resulting in a marked reduction of the inotropic potency, and PBGH is devoid of this structure. With the latter agent, the rabbit was as insensitive as the rat, although both rabbit and guinea-pig are equally sensitive to digitalis. K⁺ delayed the development of the positive inotropic action of ouabain with a minimal effect on the plateau response in guinea-pig hearts. In rat hearts, however, K⁺ markedly lowered the plateau response without affecting the time course of the response. These results indicate that the low sensitivity of the rat heart to digitalis is due to a difference in the glycoside binding sites on Na⁺, K⁺-ATPase; but the difference cannot be explained by the lack of a lactone ring complementary binding sites. The difference seems to result from the absence of lipid barrier which regulates the rate of release of cardiac glycosides from their binding sites on Na⁺, K⁺-ATPase.This work was supported by U.S. Public Health Service grant, HL-16052 and by the Michigan Heart Association     

Inhibition of Na+, K+-ATPase in rat renal proximal tubules by dopamine involved DA-1 receptor activation

Summary Endogenous kidney dopamine (DA) causes natriuresis and diuresis, at least partly, via inhibition of proximal tubular Na⁺,K⁺-ATPase. The present study was done to identify the dopamine receptor subtype(s) involved in dopamine-induced inhibition of Na⁺,K⁺-ATPase activity. Suspensions of renal proximal tubules from Sprague-Dawley rats were incubated with dopamine, the DA-1 receptor agonist fenoldopam or the DA-2 receptor agonist SK&F 89124 in the presence or absence of either the DA-1 receptor antagonist SCH 23390 or the DA-2 receptor antagonist domperidone. Dopamine and fenoldopam (10^–5 to 10^–8 mol/1) produced a concentration-dependent inhibition of Na⁺,K⁺-ATPase activity. However, SK&F 89124 failed to produce any significant effect over the same concentration range. Incubation with fenoldopam (10^–5 to 10^–8 mol/1) in the presence of SK&F 89124 (10^–6 mol/l) inhibited Na+,K+-ATPase activity to a degree similar to that with fenoldopam alone. Furthermore, DA-induced inhibition of Na⁺,K⁺-ATPase activity was attenuated by SCH 23390, but not by domperidone. Since -adrenoceptor activation is reported to stimulate Na⁺,K⁺-ATPase activity and, at higher concentrations, dopamine also acts as an a-adrenoceptor agonist, the potential opposing effect from -adrenoceptor activation on DA-induced inhibition of Na⁺,K⁺-ATPase activity was investigated by using the -adrenoceptor blocker phentolamine. We found that, in the lower concentration range (10^–5 to 10^–7 mol/1), dopamine-induced inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine was similar in magnitude to that observed with dopamine alone. However, at the highest concentration used (10^–4 mol/1), dopamine produced a significantly larger degree of inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine. These results indicate that the DA-1 dopamine receptor subtype, but not the DA-2 receptor subtype, is involved in dopamine-mediated inhibition of Na⁺,K⁺-ATPase. At higher concentrations of dopamine, the DA-1 receptor-mediated inhibitory effect on Na⁺,K⁺-ATPase activity may be partly opposed by a simultaneous -adrenoceptor-mediated stimulation of the activity of this enzyme.     

Interaction of sanguinarine and its dihydroderivative with the Na/K-ATPase. Complex view on the old problem

The effects of sanguinarine (SG) and its metabolite dihydrosanguinarine (DHSG) on Na⁺/K⁺-ATPase were investigated using fluorescence spectroscopy. The results showed that the enzyme in E1 conformation can bind both charged and neutral (pseudobase) forms of SG with a K_D = 7.2 ± 2.0 μM or 11.7 ± 0.9 μM, while the enzyme in E2 conformation binds only the charged form of SG with a K_D = 4.7 ± 1.1 μM. Fluorescence quenching experiments suggest that the binding site in E1 conformation is located on the surface of the enzyme for both forms but the binding site in E2 conformation is protected from the solvent. We found no evidence for interaction of Na⁺/K⁺-ATPase and DHSG. This implies that any in vivo effect of SG attributable to inhibition of Na⁺/K⁺-ATPase can be considered only prior to SG → DHSG transformation in the gastro-intestinal tract and/or blood. Hence, Na⁺/K⁺-ATPase inhibition will be effective in SG topical application but its duration will be very limited in SG oral or parenteral administration.     

Activation by reduced glutathione of methotrexate transport into isolated rat liver cells

The uptake of methotrexate (MTX) by isolated rat hepatocytes and its changes under the influence of exogenous GSH have been studied under various conditions: GSH concentration, pH of incubation medium, preincubation of cells prior to MTX and GSH addition, ionic composition of the incubation medium (standard saline, Na⁺-free, Na⁺ and K⁺-free, or ion-deficient), after prior treatment of cells by membrane -SH blockers (p-CMBS, 4-CMB and DIP²⁺) and ATP.It was found that GSH strongly accelerated MTX uptake. This effect depended on GSH concentration and on preincubation of cells. The GSH effect was not dependent on medium pH in spite of an observed close relationship between pH of incubate and MTX transport itself. Activation by GSH of MTX transport was connected to an increase in intracellular K⁺. It was also noted that while blockers of membrane -SH groups like p-CMBS and 4-CMB inhibited MTX uptake and increased the intracellular Na⁺/K⁺ ratio, both effects were partially overcome by GSH. After treatment by DIP²⁺, Na⁺/K⁺ ratio was unaffected, but MTX uptake inhibited. Still GSH abolished inhibition. Added ATP also inhibited MTX uptake and caused loss of cellular K⁺ and accumulation of Na⁺ Here neither effect could be reversed by GSH; consequently, high cellular amounts of K⁺ and MTX accumulated by previous action of GSH were depleted on subsequent ATP addition. MTX uptake was low in sucrose medium. But in this ion-deficient medium, GSH had the greatest stimulatory effect on MTX uptake.It is concluded that binding GSH can affect the redox state of the -S-S-/-SH groups of the cellular plasma membrane and that this effect of GSH might demonstrate involvement of the redox state in the control of MTX permeability.     

Arachidonic Acid Activates K+-Cl--cotransport in HepG2 Human Hepatoblastoma Cells

K⁺-Cl^--cotransport (KCC) has been reported to have various cellular functions, including proliferation and apoptosis of human cancer cells. However, the signal transduction pathways that control the activity of KCC are currently not well understood. In this study we investigated the possible role of phospholipase A₂ (PLA₂)-arachidonic acid (AA) signal in the regulatory mechanism of KCC activity. Exogenous application of AA significantly induced K⁺ efflux in a dose-dependent manner, which was completely blocked by R-(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl]oxy]acetic acid (DIOA), a specific KCC inhibitor. N-Ethylmaleimide (NEM), a KCC activator-induced K⁺ efflux was significantly suppressed by bromoenol lactone (BEL), an inhibitor of the calcium-independent PLA₂ (iPLA₂), whereas it was not significantly altered by arachidonyl trifluoromethylketone (AACOCF₃) and p-bromophenacyl bromide (BPB), inhibitors of the calcium-dependent cytosolic PLA₂ (cPLA₂) and the secretory PLA₂ (sPLA₂), respectively. NEM increased AA liberation in a dose- and time-dependent manner, which was markedly prevented only by BEL. In addition, the NEM-induced ROS generation was significantly reduced by DPI and BEL, whereas AACOCF₃ and BPB did not have an influence. The NEM-induced KCC activation and ROS production was not significantly affected by treatment with indomethacin (Indo) and nordihydroguaiaretic acid (NDGA), selective inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Treatment with 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, markedly produced ROS and activated the KCC. Collectively, these results suggest that iPLA₂-AA signal may be essentially involved in the mechanism of ROS-mediated KCC activation in HepG2 cells.     

Inhibition of neuronal noradrenaline uptake (uptake1) and desipramine binding by N-ethylmaleimide (NEM)

Summary The inhibition by N-ethylmaleimide (NEM) of uptake₁ and desipramine binding was studied on clonal rat phaeochromocytoma cells (PC12 cells) in different experimental settings: (1) ³H-noradrenaline uptake into intact PC12 cells; (2) ³H-noradrenaline uptake into isolated PC12 plasma membrane vesicles; (3) ³H-desipramine binding to isolated PC12 plasma membrane vesicles.In plasma membrane vesicles, NEM inhibited ³H-desipramine binding and ³H-noradrenaline uptake with similar potency (the IC₅₀'s were 1.36 mmol/l and 1.04 mmol/l, respectively). However, in intact cells, NEM was about 75 times more potent in inhibiting ³H-noradrenaline uptake (IC₅₀ = 0.014 mmol/l). The increased potency of NEM in intact cells is probably due to an inhibition of the Na^{+/K +}-ATPase and not to a direct interaction with the noradrenaline carrier.The inactivation by NEM of ³H-desipramine binding to PC12 plasma membrane vesicles was irreversible. Both an inhibitor (cocaine, 1 mmol/l) and a substrate of uptake₁ (amezinium, 1 mmol/l) protected desipramine binding from inactivation.These results are compatible with the hypothesis of a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier.This study was supported by the Deutsche Forschungsgemeinschaft (SFB176) Send offprint requests to E. Schömig at the above address     

Functional role of sodium pump in human placental arteries

Summary Ouabain (10^–7 to 10^–4 M) elicited concentration-dependent contractile responses in human placental arteries. The contractions were reduced by 10^–4 M amiloride and Ca²⁺-free medium, but not affected by 10^–6 M nifedipine or 10^–6 M Bay-K-8644, which markedly reduced or potentiated 75 mM K⁺-induced contractions, respectively. After contracting the vessels with 10^–6 M prostaglandin F₂ in a K⁺-free medium, the subsequent addition of 7.5 mM K⁺ induced a marked relaxation, which was blocked by 10^–6 M ouabain. This glycoside (10^–8 to 10^–4 M) also produced a concentration-dependent reduction of ⁸⁶Rb⁺ uptake. Scatchard analysis of the [³H]-ouabain binding to membrane fractions from human placental arteries suggests a single class of binding sites with a KD of 88.3 nM and a B_max of 345 fmol/mg. 5-Hydroxytryptamine (5-HT; 10^–9 to 10^–5 M) caused concentration-dependent contractions. Single concentrations produced transient responses composed of an initial contraction, followed by a slow fall in tension. Ouabain (10^–8 to 10^–6 M), K⁺-free medium or the reduction of bath temperature (28°C) did not modify contractions but inhibited the relaxant phase of the response. 5-HT (10^–8 to 10^–6 M) increased both total and ouabain-insensitive ⁸⁶Rb⁺ uptake, but the difference between them was not modified. These data indicate that: (1) human placental arteries possess an important sodium pump activity, inhibition or stimulation of which markedly alters vascular tone, (2) ouabain-evoked contractions are produced by Ca²⁺ entry mainly through Na⁺-Ca²⁺ exchange, secondary to intracellular Na⁺ accumulation, (3) the relaxant component of 5-HT response is dependent on the activity of the sodium pump, (4) the activation of Na⁺,K⁺-ATPase activity by this amine is not apparently due to direct effect, and (5) the inhibition of the sodium pump can cause long lasting increases of placental vascular resistance in the presence of physiological concentrations of 5-HT. Send offprint requests to J. Marin at the above address     

High- and low-affinity sites for sodium in δ-OR-Gi1α (Cys351-Ile351) fusion protein stably expressed in HEK293 cells; functional significance and correlation with biophysical state of plasma membrane

The effect of sodium, potassium, and lithium on δ-opioid receptor ligand binding parameters and coupling with the cognate G proteins was compared in model HEK293 cell line stably expressing PTX-insensitive δ-OR-G_i1α (Cys³⁵¹-Ile³⁵¹) fusion protein. Agonist [³H]DADLE binding was decreased in the order Na⁺???Li⁺?>?K⁺?>?(+)NMDG. When plotted as a function of increasing NaCl concentrations, the binding was best-fitted with a two-phase exponential decay considering two Na⁺-responsive sites (r ²?=?0.99). High-affinity Na⁺-sites were characterized by K_d?=?7.9 mM and represented 25 % of the basal level determined in the absence of ions. The remaining 75 % represented the low-affinity sites (K_d?=?463 mM). Inhibition of [³H]DADLE binding by lithium, potassium, and (+)-NMDG proceeded in low-affinity manner only. Surprisingly, the affinity/potency of DADLE-stimulated [³⁵S]GTPγS binding was increased in a reverse order: Na⁺?<?K⁺?<?Li⁺. This result was demonstrated in PTX-treated as well as PTX-untreated cells. Therefore, it is not restricted to G_i1α(Cys³⁵¹-Ile³⁵¹) within the δ-OR-G_i1α fusion protein, but is also valid for stimulation of endogenous G proteins of G_i/G_o family in HEK293 cells. Biophysical studies of interaction of ions with polar head-group region of lipids using Laurdan generalized polarization indicated the low-affinity type of interaction only proceeding in the order: Cs⁺?<?K⁺?<?Na⁺?<?Li⁺. The results are discussed in terms of interaction of Na⁺, K⁺ and Li⁺ with the high- and low-affinity sites located in water-accessible part of δ-OR binding pocket. We also consider the role of negatively charged Cl^?, Br^?, and I^? counter anions in inhibition of both [³H]DADLE and [³⁵S]GTPγS binding.     

Correlation between inhibition of (Na+, K+)-membrane-ATPase and positive inotropic activity of cardenolides in isolated papillary muscles of guinea pig

Summary Concentrations of 17 cardenolides, cardenolide glucuronides and sulfates producing halfmaximal inhibition of (Na⁺, K⁺)-membrane-ATPase from different organs and animal species were determined in vitro. In addition the concentrations that increased the contractility of guinea pig isolated papillary muscles to a particular level were investigated. Comparisons between ATPase-inhibiting and positive inotropic cardiac activities showed extensive parallelism: the correlation coefficients after log/log transformation were between 0.92 and 0.97. The same close correlations are found if dissociation constants of cardenolide receptor complexes and concentrations causing ⁸⁶Rb-uptake inhibition in human erythrocytes are examined.The concentrations necessary for inhibition of (Na⁺, K⁺)-membrane-ATPase of the guinea pig heart and the concentrations required to achieve a defined positive inotropic effect in guinea pig papillary muscle showed a log/log correlation coefficient of 0.97 (P<0.001). In both tests the potencies covered more than three orders of magnitude. The results support Repke's hypothesis on the digitalis receptor.     

The interaction of canrenone with the Na+,K+ pump in human red blood cells

Summary Canrenone inhibits 30–40% of ouabain-sensitive Na⁺ efflux in human red cells. Half-maximal inhibition was obtained with a canrenone concentration=86±37 mol/l (mean±SD of 13 experiments). The partial inhibition of the Na⁺,K⁺ pump appears to be mediated at the digitalis receptor site with an apparent dissociation constant (K _C)=200±130 mol/l (mean±SD). Further evidence suggesting that canrenone is a partial agonist at the digitalis receptor site was obtained by the observation that it decreases the apparent affinity of the Na⁺,K⁺ pump for external K⁺. However, in contrast to ouabain, canrenone decreases the apparent pump affinity for internal Na⁺.Our results show that, at physiological cell Na⁺ levels canrenone is able to enhance the inhibition of the Na⁺,K⁺ pump by low doses of ouabain. Conversely, in cells treated with high concentrations of cardiac glycosides (in which cell Na⁺ content increases), canrenone is able to restimulate the blocked pumps.