Vanadyl (VO2+) and vanadate (VO-3) ions inhibit the brain microsomal Na,K-ATPase with similar affinities. Protection by transferrin and noradrenaline

The activity of Na,K-ATPase was measured in brain microsomes as the function of increasing concentrations of vanadyl (VOSO4, V4+) and the vanadate (NaVO3, V5+) ions. Both forms of vanadium inhibited the Na,K-ATPase activity with high affinity -Ki (vanadate) = 3 X 10(-7)M and Ki (vanadyl = 1 X 10(-6)M. The stability of V4+ in ATPase reaction media (Tris buffers) was measured by electron spin resonance spectroscopy. Without any reducing agent, V4+ was quickly oxidised by atmospheric oxygen. When a reducing agent such as dithiothreitol was added, the V4+ was stable for at least 30 min and the inhibition pattern of Na,K-ATPase by V4+ was not changed. The blocking effect of V4+ in the presence of dithiothreitol was counteracted by pre-incubation with equimolar concentrations of transferrin or 100 times excess of noradrenaline. The regulation of brain Na,K-ATPase by vanadate may be represented by competition between low-capacity inhibitory binding sites localized on the enzyme molecule and high-capacity sites of intracellular proteins. Preferential binding of vanadyl to the latter type of sites will decrease the intracellular concentration of the free metal and thus eliminate the enzyme inhibition.     

Catecholamines and the brain microsomal Na, K-adenosinetriphosphatase--I. Protection against lipoperoxidative damage

The brain microsomal Na,K-ATPase is selectively inactivated by endogenous inhibitory system comprised from ascorbic acid and ferrous ions. The mechanism of this inhibition appears to be identical with the radical damage of unsaturated membrane phospholipids (lipoperoxidation) induced by these agents. Both adrenaline and EDTA block the manifestation of the lipoperoxidative capacity of brain subcellular fractions and simultaneously protect the Na,K-ATPase activity against inhibition. A possible biological importance of these data is discussed.     

Differential effect of ethacrynic acid on microsomal Mg2+-ATPase.

The effect of ethacrynic acid (EA) was studied on microsomal ATPase in various tissues of the rat and in kidneys of different species. Inhibition of Na,K-ATPase in microsomes of kidney medulla and cortex was observed only at 5 × 10^?3 M EA. At concentrations of 10^?5?10^?4 M, EA caused some enhancement of Na,K-ATPase activity. Microsomal Mg-ATPase of kidney medulla and cortex was inhibited by EA at concentrations of 10^?5?10^?3 M in various rodents (rat, mouse, guinea-pig and rabbit) as well as in human and cat kidney. EA showed the same differential effect on kidney microsomal Mg-ATPase when microsomes were prepared without treatment with desoxycholate, when chloride was replaced by sulfate in the medium. EA inhibited preferentially Mg-ATPase vs Na, K-ATPase in micro-somes of rat brain and rat submaxillary gland.     

Catecholamines and the brain microsomal Na, K-adenosinetriphosphatase--II. The mechanism of action

In the presence of ATP and an endogenous inhibitory system, presumably ascorbic acid and Fe²⁺ cation, the brain microsomal Na, K-ATPase is slowly inhibited. The inhibition rate is diminished in the presence of catecholamines and EDTA. This effect is not mediated by any specific adrenergic receptor. At present, the slow type of inhibition appears to be best explained by the lipoperoxidative degradation of the unsaturated phospholipid component of Na, K-ATPase. In the absence of ATP, the fast type of inhibition of Na, K-ATPase occurs. The inhibition could also be restrained by catecholamines but it is not directly related to the lipoperoxidation. The underlying mechanism is largely unknown but could be partially explained by divalent metal inhibition of Na, K-ATPase.     

Action of antiestrogens on the (Ca2+ + Mg2+)-ATPase and Na+/Ca2+ exchange of brain cortex membranes

The effect of tamoxifen (TAM) and other antiestrogens on the Ca2+ transport activity of synaptic plasma membranes (SPM) and microsomal membranes isolated from sheep brain cortex was investigated. The maximal (Ca2+ + Mg2+)-ATPase activity of SPM, which is reached at a pCa of about 6.0-6.5, is decreased by about 30% in the presence of 50 microM TAM, whereas the (Ca2+ + Mg2+)-ATPase activity of microsomes, which is maximal at a pCa of about 5.0, is decreased by about 90% by 50 microM TAM. In parallel experiments, we observed that the ATP-dependent Ca2+ uptake is also affected differently by TAM in the two membrane preparations. We found that 50 microM TAM inhibits SPM Ca2+ uptake by about 25-30%, whereas the ATP-dependent Ca2+ uptake by the microsomal fraction is inhibited by about 60%. No significant effect of TAM was observed on the Na+/Ca2+ exchange of either membrane system. The results indicate that TAM is a more potent inhibitor of the active, calmodulin-independent Ca2+ transport system of the intracellular membranes than of that of the plasma membranes, which is calmodulin-dependent. It appears that TAM inhibits calmodulin-mediated reactions, probably through its binding to calmodulin, as we showed previously. However, the Ca2+ transport system of microsomes, which does not depend on calmodulin, is also particularly sensitive to TAM.     

Developmental lead exposure inhibits adult rat retinal, but not kidney, Na+,K(+)-ATPase.

Neonatal lead exposure produces selective rod degeneration and functional deficits in adult hooded rats. Similar alterations occur following retinal exposure to ouabain. This study determined whether there were long-term effects of neonatal lead exposure on rat retinal or renal Na+,K(+)-ATPase (Na,K-ATPase) activity and employed in vitro studies to examine the mechanism of ionic lead (Pb2+)-induced inhibition of retinal Na,K-ATPase. Pups, exposed to lead only via the milk of dams consuming 0, 0.02, or 0.2% lead solutions, had mean blood lead concentrations of 1.2, 18.8, and 59.4 micrograms/dl at weaning, respectively, and 5-7 micrograms/dl as adults. Prior lead exposure produced significant dose-dependent decreases in isolated retinal Na,K-ATPase activity (-11%; -26%) whereas activity in the kidney was unchanged. In contrast, Na,K-ATPase from both isolated control tissues was inhibited by Pb2+. The half-maximal inhibitory dose (I50) of Pb2+ for retinal and renal Na,K-ATPase was 5.21 x 10(-7) and 1.25 x 10(-5) M, respectively. The Hill coefficient of the retina was 0.42 whereas it was 0.88 in the kidney. With MgATP as a substrate, the Pb(2+)-induced inhibition of retinal Na,K-ATPase was competitive and reversible with a Ki of 2.1 x 10(-7) M. Retinal and renal Na,K-ATPase were 20-fold and 1.1-fold more sensitive to inhibition by Pb2+ than by Ca2+, respectively. The Pb(2+)-induced inhibition of retinal Na,K-ATPase was antagonized by Na+, potentiated by Mg2+, not altered by K+ or Ca2+, and prevented by ATP. Kinetic and competition studies with the retinal Na,K-ATPase establish that the Pb(2+)-induced inhibition is complex. The increased sensitivity of retinal, compared to renal, Na,K-ATPase to inhibition following in vivo or in vitro lead exposure may relate to their different alpha subunit composition. This is speculated to play a fundamental role in the target organ toxicity of lead.     

Studies on the stable inhibition of Na+ + K+-ATPase by cassaine.

Exposure of rat brain Na+ + K+-ATPase (ATP phosphohydrolase E.C. 3.6.1.3) to concentrations of cassaine greater than 1 x 10(-4) M resulted in a poorly reversible inhibition of this enzyme. Inhibition did not require the presence of ATP and developed rapidly, but the final amount of inhibition observed was independent of time. The amount of inhibition observed at a given concentration of cassaine was reduced by increasing the concentration of membranes in the system. The inhibition of Na+ + K+-ATPase activity was associated with equivalent inhibition of the phosphorylation and (3H)-ouabain binding reactions of this enzyme, while the uninhibited enzyme was apparently kinetically normal. Concentrations of cassaine which produced this stable inhibition of Na+ + K+-ATPase had no effect on the Mg2+-activated ATPase or the NADH cytochrome-c-reductase activities of crude rat brain microsomal preparations. Cassaine inhibited the cholinesterase activity of rat brain microsomes with a Ki of about 5 x 10(-5) M, but his inhibition was fully reversible. The poorly reversible inhibitory actions of cassaine, thus, appeared specific for Na+ + K+-ATPase. Because this stable pattern of inhibition of the Na+ + K+-ATPase by cassaine required drug concentrations at least one hundred-fold greater than those which produce positive inotropic effects, it appears unlikely that this pattern of Na+ + K+-ATPase inhibition is involved in the cardiotonic actions of this drug.     

Different effects of paraquat on microsomal lipid peroxidation in mouse brain, lung and liver

Paraquat stimulates NADPH-Fe(2+)-dependent microsomal lipid peroxidation in mouse brain and strongly inhibits it in the liver. In lung microsomes, the lipid peroxidation was stimulated by paraquat at 10(-4) M, but not at higher doses. An antioxidant action of paraquat seemed to account, at least in part, for the lack of stimulation in lung microsomes, but it was inappropriate to explain the result in hepatic microsomes. There was no apparent correlation between the effects of paraquat on the lipid peroxidation and on the activity of NADPH-cytochrome P-450 reductase, the enzyme which initiates redox cycling of paraquat, resulting in generation of active oxygen species. In fact, the effect of paraquat on the lipid peroxidation was independent of paraquat radical production, an intermediate in the cycle. However, the inhibitory potency of N-ethylmaleimide on NADPH-cytochrome P-450 reductase activity paralleled that on the lipid peroxidation stimulated by paraquat in brain and lung. These findings indicate that the effect of paraquat on microsomal lipid peroxidation differs among the organs and that other factors, besides NADPH-cytochrome P-450 reductase, might be involved in the stimulation of lipid peroxidation by paraquat.     

Inhibitory characteristics of lead chloride in sodium- and potassium- dependent adenosinetriphosphatase preparations derived from kidney, brain, and heart of several species.

Inhibition of adenosinetriphosphatase (ATPase) by lead chloride (PbCl2) was studied in microsomal fractions or tissue homogenates of kidney, brain, and heart of several species, including humans. The concentration of PbCl2 causing 50% inhibition (I50) of Na+ + K+ ATPase activity varied from 8 X 10(-6) to 8 X 10(-5) M, depending on the species and organ of origin of the enzyme. The enzyme preparations derived from various parts of the kidney showed no differential sensitivity to PbCl2. These differences in sensitivity to lead were not related to specific activity of the enzyme or to the protein content of the preparations studied. Mg2+ ATPase, which contaminated the enzyme preparations to a variable degree, was 10--100 times more resistant to PbCl2 than was Na+ + K+-activated ATPase. The following more detailed studies were performed on the dog brain and/or kidney enzyme. The inhibition of microsomal Na+ + K+ ATPase was characterized by reversible kinetics. The inhibitory effect was antagonized by Na+, increased by Mg2+, and not altered by K+. ATP alone, or together with Mg2+, antagonized the inhibition. Disodium edetate prevented or reversed the inhibition. These inhibitory characteristics suggest that Pb2+ inhibits Na+ + K+ ATPase at the Na+-dependent phosphorylation site, and that ATP chelates Pb2+ in competition with Mg2+. Combining Pb2+ with ATP may not only result in a reduction of ATPase activity but also cause a relative ATP deficiency if lead is present in sufficiently high concentration.     

Noradrenaline release induced by ouabain and vanadate in cat cerebral and peripheral arteries

The effects of 10(-4) M ouabain and 10(-3) M vanadate (Na3VO4) on [3H]noradrenaline release from cat cerebral and femoral arteries was studied. Ouabain induced tritium secretion in cerebral arteries, but not in femoral ones, which was reduced by Ca suppression and potentiated by extracellular Na reduction to 11.9 mM. However, vanadate evoked tritium release from both kinds of vessels was unaffected under these experimental conditions. These data suggest: ouabain elicited secretion from adrenergic nerve endings is likely due to inhibition of the Na, K-ATPase and subsequent Ca influx through Na-Ca exchange, and vanadate action is mediated by another mechanism different to the Na pump blockade.     

Functional proteins involved in regulation of intracellular Ca(2+) for drug development: pharmacology of SEA0400, a specific inhibitor of the Na(+)-Ca(2+) exchanger

The Na(+)-Ca(2+) exchanger (NCX) is involved in regulation of intracellular Ca(2+) concentration. A specific inhibitor of NCX has been required for clarification of the physiological and pathological roles of NCX. We have developed 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400), a highly potent and selective inhibitor of NCX. SEA0400 in the concentration range that inhibits NCX exhibits negligible affinities for the Ca(2+) channels, Na(+) channels, K(+) channels, noradrenaline transporter, and 14 receptors; and it does not affect the activities of the store-operated Ca(2+) channel, Na(+)-H(+) exchanger, and several enzymes including Na(+),K(+)-ATPase and Ca(2+)-ATPase. Furthermore, recent studies show that SEA0400 attenuates ischemia-reperfusion injury in the brain, heart, and kidney and radiofrequency lesion-induced edema in rat brain. These findings suggest that NCX plays a key role in ischemia-reperfusion injury and may be a target molecule for treatment of reperfusion injury-related diseases.     

Effect of dronedarone on Na+, Ca2+ and HCN channels

Previous studies showed that amiodarone causes state-dependent inhibition of Na(+) channels thereby mediating an atrial-selective drug effect. The aim of the present study was to investigate the impact of the new antiarrhythmic compound dronedarone on Na(+), Ca(2+) and hyperpolarization-activated cyclic nucleotide-gated ion channels. Monophasic action potentials (MAP) and effective refractory period (ERP) were studied in arterially perfused left atria and ventricular wedge preparations of the pig. Fast Na(+) and Ca(2+) currents in isolated guinea pig ventricular myocytes as well as human HCN4 channels expressed in Chinese hamster ovary (CHO) cells were investigated with the patch-clamp technique. In left atrial epicardial tissue, dronedarone (3?μM) had no effect on the MAP duration, but the drug caused a significant prolongation of the ERP from 145?±?9 to 184?±?17?ms (n?=?6; p?相似文献   

Intrarenal distribution and ATPase inhibiting activity of ouabain in dogs.

Experiments were undertaken to substantiate the hypothesis that the mechanism of the direct effect of ouabain on the renal excretion of electrolytes is the result of inhibition of the transport enzyme, (Na, K)-ATPase. In dogs hydrated with saline, an injection of 3H-ouabain into the unilateral renal artery produced a continuing marked increase in excretion of water and sodium from the kidney, but not from the counter kidney. At maximal diuresis--90 min after ouabain injection, both kidneys were removed to assay microsomal ATPase activity and determine radioactivity distributed in subcellular structures. It was demonstrated that 3H-ouabain was deposited in the microsome fraction obtained from the injected kidney in concentrations ranging from 10(-7) to 10(-6) M/kg wet weight, and (Na, K)-ATPase activity of this fraction was inhibited as compared with that of the microsomal fraction obtained from control kidneys. Since (Na, K)-ATPase activity of renal microsomes was significantly inhibited in vitro by more than 10(-7) M of ouabain, ouabain concentration in microsomes obtained from the injected kidney was considered to be sufficient to inhibit ATPase activity. These findings indicate that ouabain diuresis under the present condition is closely related to direct inhibitory effect of ouabain on (Na, K)-ATPase activity of microsomes in tubular cells.     

Effect of pyridine and quinoline N-oxides on microsomal Na,K-ATPase activity

Some heteroaromatic N-oxides of pyridine and quinoline derivatives at concentrations ranging from 10⁻⁴ to 10⁻¹⁰ mole/liter inhibit Na,K-ATPase activity in cattle brain microsomes stronger than does strophanthin K (a drug used for the treatment of cardiac insufficiency). A new Na,K-ATPase activator, 2-(2,4-dimethoxystyryl)quinoline-N-oxide), has been found, which is capable of acting at concentrations within 10⁻⁶–10⁻⁹ mole/liter. Since these compounds activate the enzyme in very low concentrations, they can probably be effective for the treatment of some disorders involving violation of the Na,K-ATPase function. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 40, No. 7, pp. 3–4, July, 2006.     

Influence of prenylated and non-prenylated flavonoids on liver microsomal lipid peroxidation and oxidative injury in rat hepatocytes.

Prenylated chalcones from hops and beer were compared with non-prenylated flavonoids [chalconaringenin (CN), naringenin (NG), genistein (GS) and quercetin (QC)] for their ability to inhibit lipid peroxidation in rat liver microsomes. Chalcones with prenyl- or geranyl-groups (5 and 25 microM) were more effective inhibitors of microsomal lipid peroxidation than CN, NG or GS induced by Fe(2+)/ascorbate. Prenylated chalcones were effective inhibitors of microsomal lipid peroxidation induced by Fe(3+)-ADP/NADPH and by tert-butyl hydroperoxide (TBH) but to a lesser extent compared to the Fe(2+)/ascorbate system. An increase of prenyl substituents decreased antioxidant activity in the lipid peroxidation systems. Certain flavonoids behaved as prooxidants in the iron-dependent lipid peroxidation systems. For example, at 5 microM, NG enhanced iron/ascorbate-induced lipid peroxidation whereas CN, diprenylxanthohumol and tetrahydroxanthohumol enhanced Fe(3+)-ADP/NADPH-induced lipid peroxidation. None of the flavonoids (25 microM), except QC, inhibited NADPH cytochrome P450-reductase activity of rat liver microsomes, suggesting that the mechanism of inhibition of lipid peroxidation induced by Fe(3+)-ADP/NADPH is not due to inhibition of the reductase enzyme. Chalcones exhibiting antioxidant activity against TBH-induced lipid peroxidation such as xanthohumol and 5'-prenylxanthohumol, and NG, with no antioxidant property at 5 microM concentration protected cultured rat hepatocytes from TBH toxicity. Other antioxidants (desmethylxanthohumol and CN) in the TBH system were not cytoprotective. These results demonstrate the importance of prenyl groups in the antioxidant activity of hop chalcones in the various in vitro systems of lipid peroxidation. Furthermore, the antioxidant activity of the flavonoids has little or no bearing on their ability to protect rat hepatocytes from the toxic effects of TBH.     

Characterization of palytoxin-induced catecholamine secretion from cultured bovine adrenal chromaffin cells. Effects of Na(+)- and Ca(2+)-channel blockers.

The effect of palytoxin (PTX), a potent marine toxin, on catecholamine (CA) secretion from cultured bovine adrenal chromaffin cells was examined. PTX at concentrations of over 10(-10) M induced CA secretion concentration-dependently. About 40-50% of the total cellular CA was secreted during 20-min incubation with 3 x 10(-8) M PTX. PTX-induced CA secretion was dependent on both extracellular Na+ and Ca2+. PTX caused increases in [22Na](+)- and [45Ca](2+)-influxes into the cells. Increase in [22Na](+)-influx was observed at concentrations of over 10(-11) M PTX and was maximal at 10(-10) M PTX and then gradually decreased at higher concentrations that induced [45Ca](2+)-influx and CA secretion. On the other hand, increase in [45Ca](2+)-influx was observed at concentrations of over 10(-10) M PTX and increased with increase in concentration of PTX. This concentration-response curve for PTX-induced [45Ca](2+)-influx was similar to that for PTX-induced CA secretion. The CA secretion and [22Na](+)- and [45Ca](2+)-influxes induced by PTX were not affected by tetrodotoxin (TTX), but were significantly inhibited by quinidine and aprindine(mexiletine), antiarrythmic drugs known to block Na(+)-channels. Ca(2+)-channel blockers such as nifedipine, verapamil, Co2+, Cd2+, inhibited both CA secretion and [45Ca](2+)-influx induced by PTX. These results indicate that PTX-induced CA secretion is mediated by activation of Na(+)-dependent, TTX-insensitive voltage-dependent Ca(2+)-channels, and is inhibited by quinidine and aprindine through their inhibitory effects on the Na(+)- and Ca(2+)-influxes into the cells induced by PTX.     

Actions of vanadate on muscles

Vanadate, a trace element of biological tissues and fluid, has been known to be a potent inhibitor of Na, K-ATPase in various tissues. In skeletal muscle, it inhibits the Na, K-ATPase of the membrane fraction, whereas it can not inhibit the Na, K-pump in an intact preparation. It inhibits the Ca-ATPase of the sarcoplasmic reticulum and that of contractile proteins. Vanadate potentiates the contraction of some heart muscles, while it depresses the contraction in some other heart muscles. The positive and negative inotropic effects are mediated by changes in the action potential. The inhibition of Na, K-ATPase is not always involved in the inotropic actions. The inhibition of the Ca-ATPase of the sarcoplasmic reticulum and plasma membrane can be the causes of positive inotropic action. Actions on adenylate cyclase can also be the cause of inotropism. In smooth muscle, vanadate induces contractions with and/or without membrane excitation. The contractions are initiated by both the influx of extracellular Ca2+ and the release of intracellular bound Ca2+. The inhibition of Na, K-ATPase is not involved in the contraction but the inhibition of the Ca-pump of membranous systems can be the cause of the contraction. Vanadate is a useful tool for studies on the excitation-contraction coupling in muscles.     

Effects of dihydropyridine calcium antagonists on rabbit renal Na,K-ATPase activity in vitro

Dihydropyridines are reported to have a stimulatory effect on vascular smooth muscle Na,K-ATPase activity in vitro. We studied the effects of the dihydropyridine calcium antagonists nimodipine, nitrendipine, nisoldipine, niludipine, nifedipine and felodipine (10(-5) M) on purified Na,K-ATPase isolated from rabbit kidney outer medulla. We were unable to detect an effect of the drugs on the enzyme activity, either under optimal or suboptimal substrate conditions. Likewise, Na,K-ATPase activity, partly inhibited by the addition of ouabain (10(-6) M), Ca2+ (10(-3) M) or arachidonic acid (4 x 10(-5) M), was not influenced by the dihydropyridines. The absence of a stimulatory effect of dihydropyridines on renal Na,K-ATPase is in agreement with the known diuretic and natriuretic effects of the drugs in normotensive and hypertensive men.     

Cardioprotection by ouabain and digoxin in perfused rat hearts

This work was aimed at determining the cardioprotective effect of digitalis glycosides in rat heart, and to relate it with Na, K-ATPase inhibition and ERK1/2 activation. Isolated working rat hearts were perfused in the presence of ouabain or digoxin, which were used at concentrations ranging from 10 to 10 M. The hearts were then subjected to 30 minutes of global normothermic ischemia followed by 120 minutes of retrograde reperfusion; irreversible tissue injury was determined on the basis of triphenyltetrazolium chloride staining. Significant cardioprotection was observed with 10 M and 10 M ouabain (ischemic injury averaged 7.0 +/- 3.5% and 8.3 +/- 0.6% versus 37.3 +/- 2.0% in controls, P < 0.01 in each case). Hearts treated with digoxin showed decreased ischemic injury at 10 M and 10 M (18.0 +/- 1.5% and 14.2 +/- 1.0%, P < 0.01 versus control in both cases). In parallel experiments, ERK2 phosphorylation was increased by 10 to 10 M ouabain, while ERK1 and ERK2 phosphorylation was increased by 10 to 10 M digoxin. The cardioprotective effect was not related to Na, K-ATPase inhibition, since Rbuptake was not significantly different between control and treated hearts.     

Inhibition of rat liver microsomal cytochrome P-450 steroid hydroxylase reactions by imidazole antimycotic agents

The imidazole antimycotic agents ketoconazole, miconazole and clotrimazole were tested for their abilities to inhibit the reactions involved in the oxidative metabolism of androst-4-ene-3,17-dione by rat liver microsomal cytochromes P-450. All three compounds were found to function as potent inhibitors of steroid hydroxylase reactions, producing 50% inhibition of 6 beta-, 16 beta-, and 16 alpha-hydroxylase activities at concentrations between 10(-7) and 10(-5) M. The antimycotic agents, when added to liver microsomes, bound to cytochrome P-450 with high affinity to produce a "type II" spectral complex. These agents showed differential inhibition of the various steroid hydroxylases and were found not to affect the activities of the liver microsomal steroid 5 alpha-reductase or the androst-4-ene-3,17-dione 17-oxidoreductase. The results presented demonstrate an interaction of these imidazole antimycotic agents with the various cytochromes P-450 of liver microsomes, resulting in selective inhibition of monooxygenase activity.