Ouabain-insensitive ATPase Activity Research Articles

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

We investigated modulation by ATP, Mg²⁺, Na⁺, K⁺ and NH₄⁺ and inhibition by ouabain of (Na⁺,K⁺)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na⁺,K⁺)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K(M) = 0.09 ± 0.01 mmol L⁻¹) of the decapodid III (Na⁺,K⁺)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na⁺,K⁺-ATPase activity by K⁺ also revealed a threefold greater affinity for K⁺ (K₀.₅ = 0.91 ± 0.04 mmol L⁻¹) in decapodid III than in other stages; NH₄⁺ had no modulatory effect. The affinity for Na⁺ (K₀.₅ = 13.2 ± 0.6 mmol L⁻¹) of zoea I (Na⁺,K⁺)-ATPase was fourfold less than other stages. Modulation by Na⁺, Mg²⁺ and NH₄⁺ obeyed cooperative kinetics, while K⁺ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg²⁺ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg²⁺-stimulated ATPases other than (Na⁺,K⁺)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na⁺-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH₄⁺-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.

Na+,K+-ATPase Activity in the Posterior Gills of the Blue Crab, Callinectes ornatus (Decapoda, Brachyura): Modulation of ATP Hydrolysis by the Biogenic Amines Spermidine and Spermine

We investigated the effect of the exogenous polyamines spermine, spermidine and putrescine on modulation by ATP, K⁺, Na⁺, NH₄⁺ and Mg²⁺ and on inhibition by ouabain of posterior gill microsomal Na⁺,K⁺-ATPase activity in the blue crab, Callinectes ornatus, acclimated to a dilute medium (21‰ salinity). This is the first kinetic demonstration of competition between spermine and spermidine for the cation sites of a crustacean Na⁺,K⁺-ATPase. Polyamine inhibition is enhanced at low cation concentrations: spermidine almost completely inhibited total ATPase activity, while spermine inhibition attained 58%; putrescine had a negligible effect on Na⁺,K⁺-ATPase activity. Spermine and spermidine affected both V and K for ATP hydrolysis but did not affect ouabain-insensitive ATPase activity. ATP hydrolysis in the absence of spermine and spermidine obeyed Michaelis-Menten behavior, in contrast to the cooperative kinetics seen for both polyamines. Modulation of V and K by K⁺, Na⁺, NH₄⁺ and Mg²⁺ varied considerably in the presence of spermine and spermidine. These findings suggest that polyamine inhibition of Na⁺,K⁺-ATPase activity may be of physiological relevance to crustaceans that occupy habitats of variable salinity.

Response of Na+-dependent ATPase Activities to the Contaminant Ammonia Nitrogen in Tapes philippinarum: Possible ATPase Involvement in Ammonium Transport

In vivo and in vitro experiments elicited different responses to ammonia nitrogen (ammonia-N) of gill and mantle Na,K-ATPase and ouabain-insensitive Na-ATPase activities in the Philippine clam Tapes philippinarum. Short-term (120 h) exposed clams to sublethal ammonia-N (NH(3)+NH (4) (+) ) concentrations (1.5 and 3.0 mg/L ammonia-N) showed enhanced gill and mantle ouabain-insensitive ATPase activity and decreased mantle Na,K-ATPase activity with respect to unexposed clams, while gill Na,K-ATPase was unaffected. In vitro experiments showed that NH (4) (+) could efficiently replace Na(+) in ouabain-insensitive ATPase activation and K(+), but not Na(+), in Na, K-ATPase activation. Simple saturation kinetics was constantly followed with similar K (0.5) values to that of the substituted cation. The same maximal ouabain-insensitive ATPase activation was obtained at 80 mM Na(+) or NH (4) (+) in the gills and at 50 mM Na(+) or NH (4) (+ ) in the mantle and that of Na,K-ATPase at 10 mM K(+) or NH (4) (+) in the presence of 100 mM Na(+) in both tissues. The two coexistent ATPase activities maintained their typical response to ouabain also when stimulated by NH (4) (+) : when activated by Na(+)+K(+) or by Na(+)+NH (4) (+) the ATPase activity was completely suppressed by 10(-3 )M ouabain, whereas the Na(+)- or NH (4) (+) -stimulated ATPase activity was unaffected by up to 10(-2 )M ouabain. The whole of the data suggests a possible involvement of the two ATPase activities in NH (4) (+) transmembrane transport.

β-Adrenergic Receptor Stimulation and Adenoviral Overexpression of Superoxide Dismutase Prevent the Hypoxia-mediated Decrease in Na,K-ATPase and Alveolar Fluid Reabsorption

Hypoxia has been shown to cause lung edema and impair lung edema clearance. In the present study, we exposed isolated rat lungs to pO(2) = 40 mm Hg for 60 min or rats to 8% O(2) for up to 24 h and then measured changes in alveolar fluid reabsorption (AFR) and Na,K-ATPase function. Low levels of oxygen severely impaired AFR in both ex vivo and in vivo models. The decrease in AFR was associated with a decrease in Na,K-ATPase activity and protein abundance in the basolateral membranes from peripheral lung tissue of hypoxic rats. Beta-adrenergic agonists restored AFR in rats exposed to 8% O(2) (from 0.02 +/- 0.07 ml/h to 0.59 +/- 0.03 ml/h), which was associated with parallel increases in Na,K-ATPase protein abundance in the basolateral membrane. Hypoxia is associated with increased production of reactive oxygen species. Therefore, we examined whether overexpression of SOD2, manganese superoxide dismutase, would prevent the hypoxia-mediated decrease in AFR. Spontaneously breathing rats were infected with a replication-deficient human type 5 adenovirus containing cDNA for SOD2. An otherwise identical virus that contained no cDNA was used as a control (Adnull). Hypoxic Adnull rats had decreased rates of AFR (0.12 +/- 0.1 ml/h) as compared with hypoxic AdSOD2 and normoxic control rats (0.47 +/- 0.04 ml/h and 0.49 +/- 0.02 ml/h, respectively), with parallel changes in Na,K-ATPase.

Mechanisms and physiological significance of the transport of the glutathione conjugate of 4-hydroxynonenal in human lens epithelial cells.

To study the mechanism and physiological significance of the transport of the glutathione (GSH) conjugate of 4-hydroxynonenal (4-HNE) from lens epithelial cells. HLE B-3 cells were treated with [(3)H] 4-HNE and efflux of its GSH conjugate, [(3)H] GS-HNE, into the medium was quantitated and characterized by HPLC and mass spectrometry. Inside-out vesicles (IOVs) were prepared from HLE B-3 cell membranes. The kinetics of adenosine triphosphate (ATP)-dependent uptake of GS-HNE and dinitrophenyl S-glutathione (DNP-SG) by these IOVs and inhibition of GS-HNE uptake by anti-RLIP76 IgG was studied. Localization of RLIP76 was studied by immunogold electron microscopy and kinetics of the adenosine triphosphatase (ATPase) activity of purified RLIP76 was determined. 4-HNE-induced apoptosis was compared in HLE-B3 cells coated with anti-RLIP76 IgG or preimmune IgG, by caspase activation assay. The results showed the presence of RLIP76 in plasma membranes of HLE B-3 cells and that it mediated ATP-dependent transport of GS-HNE as well as of DNP-SG. The transport was saturable with respect to GS-HNE (K(m) = 8.4 micro M), DNP-SG (100 micro M) as well as to ATP (K(m) 0.45 mM) and was sensitive to temperature and osmolarity of the medium. Anti-RLIP76 IgG inhibited approximately 65% of the transport of GS-HNE and DNP-SG, indicating that most of the transport was mediated by RLIP76. Compatible with its transport function, the EGTA- and ouabain-insensitive ATPase activity of purified RLIP76 was stimulated by DNP-SG and GS-HNE. 4-HNE-induced caspase activation in HLE-B3 cells was exacerbated when the transport of GS-HNE from these cells was blocked by anti-RLIP76 IgG. RLIP76 provides a defense against the deleterious effects of 4-HNE by transporting GS-HNE and can modulate apoptotic signaling by regulating the intracellular concentrations of 4-HNE.

Characterization of (Na +, K +)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na +, K +)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na +, K +)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na +, K +)-ATPase activity. Western blotting showed that M. olfersii gill contains a single α-subunit isoform of about 110 kDa. The (Na +, K +)-ATPase hydrolyzed ATP with Michaelis–Menten kinetics with K 0.5=165±5 μM and V max=686.1±24.7 U mg −1. Stimulation by potassium ( K 0.5=2.4±0.1 mM) and magnesium ions ( K 0.5=0.76±0.03 mM) also obeyed Michaelis–Menten kinetics, while that by sodium ions ( K 0.5=6.0±0.2 mM) exhibited site–site interactions ( n=1.6). Ouabain ( K 0.5=61.6±2.8 μM) and vanadate ( K 0.5=3.2±0.1 μM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A 1. These data suggest that the (Na +, K +)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F 0F 1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na +, K +)-ATPase activity may be regulated by the Na + concentration of the external medium.

The effects of a purified toxic extract of Prymnesium patelliferum on transport of ions through the plasma membrane of synaptosomes

Extract of the ichthyotoxic marine alga Prymnesium patelliferumhas been shown to have several different effects on the transport of neurotransmitters across nerve membranes. It inhibits the sodium dependent uptake of l-glutamate and GABA and enhances the calcium-dependent release of acetylcholine. We have therefore investigated the effects of a purified toxic extract of P. patelliferum on some membrane properties using rat brain synaptosomes. We found that under conditions where the algal extract inhibited the uptake of l-glutamate, it increased the intracellular concentrations of Na + and Ca 2+, stimulated efflux of K + determined as 86Rb efflux, and depolarized the synaptosomal membrane. There was no effect on Na +/K + -ATPase or ouabain-insensitive ATPase activities. Further, there was no leakage of the cytosolic marker LDH, indicating that the various effects of the algal extract were not due to nonspecific leakage or lysis of the synaptosomes. The rise in the cytosolic concentration of free Ca 2+ induced by the algal extract was dependent on extracellular Ca 2+, and was inhibited by flunarizine (1–100 μM) but not by the Ca 2+ channel blockers ω-conotoxin GVIA (1 μM), diltiazem (100 μM), nifedipine (100 μM) or verapamil (100–500 μM). The increase in Na + influx induced by the algal extract was insensitive to tetrodotoxin (3 μM) and procaine (100 μM), whereas both the Na + influx and the membrane depolarization were inhibited by flunarizine (1–100 μM). The increase in K + efflux was insensitive to flunarizine (5–100 μM). From these results it appears that the toxic extract of P. patelliferum increases the permeability of synaptosomes to Ca 2+, Na + and K + and that these effects may be responsible for the plasma membrane depolarization and the disturbance of the neurotransmitter transport processes.

Metabolic studies of rat renal tubule cells loaded with cystine: the cystine dimethylester model of cystinosis.

The cause of Fanconi syndrome in cystinosis is enigmatic. It has previously been shown that renal tubules could be loaded with cystine by incubating them with cystine dimethylester (CDE), mimicking the biochemical hallmark of cystinosis. Such tubules have impaired transport, decreased whole-cell O2 consumption, and substrate utilization. In this study, the metabolic disturbances in cystine-loaded renal tubule cells were further characterized. Isolated rat renal tubules were loaded with cystine by incubating them with 2 mM CDE for 10 min. This had no significant effect on total ATPase, Na(+)-K(+)-ATPase, or the ouabain-insensitive ATPase activity of renal tissue homogenates from these cystine-loaded tubules. Intracellular K was significantly lower in the cystine-loaded tubules (37 +/- 2 versus 47 +/- 3 nEq/mg; P < 0.008). Intracellular ATP was reduced by 39% in the cystine-loaded tubules (23.7 +/- 2.4 versus 38.1 +/- 3.3 nmol/mg of protein; P < 0.0025). CDE (2 mM) reduced isolated mitochondrial O2 consumption with glutamate as the substrate by 66% (4.7 +/- 0.7 versus 13.9 +/- 0.8 nm/min per mg of protein, P < 0.001) but had no effect on mitochondrial O2 consumption with succinate as the substrate. It was speculated that the impaired transport from cystine loading with CDE is secondary to a decrease in energy generation.

Two Distinct K +-ATPase Activities in Rabbit Distal Colon

The distribution of K +-ATPase activity in surface and crypt cells from rabbit distal colon was studied. Separation of surface and crypt cells was validated using the multidrug resistance gene ( mdr I) product. P-glycoprotein, as marker for differentiated surface epithelial cells. Western blot analysis revealed a 6-fold higher expression level of P-glycoprotein in colonic surface cells. K +-stimulated ouabain-insensitive ATPase activity was present in surface and in crypt cells. In surface cells, this K +-ATPase activity was only partly inhibitable by 10 μM SCH 28080, while in crypt cells K +-ATPase activity equalled SCH 28080-sensitive ATPase activity. These results strongly suggest the presence of two distinct K +-ATPases in colonic epithetial cells.

Identification and properties of pathways for K+ transport in guinea-pig and rat alveolar epithelial type II cells.

86Rb+ was used to study potassium uptake and efflux in type II pneumocytes freshly isolated from adult guinea-pig and rat lung. Both species exhibited a substantial ouabain-sensitive component of potassium influx. In rats, most of the ouabain-resistant influx was abolished by bumetanide and removal of extracellular chloride elicited no further effect. In contrast, only a proportion of the ouabain-insensitive uptake was inhibitable by bumetanide in guinea-pigs and this species showed an additional component of influx, which was chloride dependent and which was reduced by either the K(+)-H(+)-ATPase inhibitor, omeprazole, or by the stilbene derivative, 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS). The chloride-dependent component was also apparent in efflux experiments in guinea-pigs, but was absent in rats. Ouabain-insensitive ATPase activity was assayed in highly purified apical membranes from guinea-pig type II pneumocytes. This activity was inhibitable by omeprazole (apparent inhibition constant, Ki, was approximately 40 microM), was potassium dependent (apparent activation constant, Ka, was approximately 200 microM) and was doubled by the addition of nigericin. While potassium transport in rat type II cells is adequately accounted for by Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- cotransport, our data suggest the additional presence of K(+)-Cl- cotransport and K(+)-H(+)-ATPase in guinea-pig type II pneumocytes. A model of how alveolar subphase acidification may occur is proposed.

Nephron segment-specific inhibition of Na+/K+-ATPase activity by cyclosporin A

Decreased kaliuresis and hyperkalemia are common complications of cyclosporin A (CsA) therapy. If CsA significantly inhibits renal tubular Na+/K(+)-ATPase activity, the alteration in transepithelial K+ secretion and K+ homeostasis could result in hyperkalemia. To investigate this possibility, we tested the effects of CsA on Na+/K(+)-ATPase activity in microdissected rat tubules. CsA, at a "toxic" concentration of 600 ng/ml, significantly inhibited Na+/K(+)-ATPase in cortical collecting ducts (CCD), medullary thick ascending limbs (mTAL), and outer medullary collecting ducts from the outer stripe (OMCDos) by 35%, 53%, and 39%, respectively. Cremophore, the commercial vehicle for CsA, did not change Na+/K(+)-ATPase activity in any nephron segment tested. To determine whether CsA inhibits Na+/K(+)-ATPase activity in a dose-dependent manner, microdissected CCD's were incubated with 300, 600, and 2500 ng/ml of CsA for 30 minutes. Na+/K(+)-ATPase activity was inhibited at 600 and 2500 mg/ml, but not at 300 ng/ml. No further inhibition of enzyme activity was noted at 2500 ng/ml. CsA did not change Na+/K(+)-ATPase activity in proximal tubule S1, S2, and S3 subsegments; cortical thick ascending limbs (cTAL), connecting tubules (CNT), or outer medullary collecting ducts from the inner stripe (OMCDis). Prolonging the incubation of CsA with S2 subsegments to 60 minutes did not result in inhibition of Na+/K(+)-ATPase activity. Ouabain-insensitive ATPase activity was unaffected by CsA or its vehicle in any nephron segment tested. In summary, CsA specifically inhibits Na+/K(+)-ATPase activity in the CCD, mTAL, and OMCDos.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of the acute neurotoxic effects of phenytoin on rat peripheral nerve by H7, an inhibitor of protein kinase C

The neurotoxic effects of a single dose of phenytoin (150 mg/kg body weight) alone or 30 min after H7 (a protein kinase C inhibitor) injection (20 mg/kg body weight) were investigated in terms of peripheral neuromuscular function and Na +,K +-ATPase activity of the sciatic nerve. This intraperitoneal injection of phenytoin induced complete blockade of muscle action potentials in the dorsal segmental muscles of the rat tail evoked by electric stimulation of the caudal nerve and a 40% decrease in the Na +,K +-ATPase activity of the rat sciatic nerve when compared with control values, measured as the difference between total and ouabain-insensitive ATPase activity. Prior administration of H7 resulted in the complete prevention of both effects. Implication of protein kinase C inhibition in phenytoin neurotoxicity are discussed.

Aldose reductase inhibition with imirestat-effects on impulse conduction and insulin-stimulation of Na+/K(+)-adenosine triphosphatase activity in sciatic nerves of streptozotocin-diabetic rats.

This study describes reduced motor nerve conduction velocity and increased resistance to hypoxia-induced conduction failure in sciatic nerves of rats after four weeks of streptozotocin-induced diabetes (both effects were significant at p less than 0.05). These changes occurred in the absence of any deficit in the steady-state ouabain-sensitive adenosine triphosphatase (ATPase) activity of sciatic nerve endoneurial homogenates. The addition of 10 nmol/l insulin to endoneurial homogenates from control animals resulted in a 34% increase in ouabain-sensitive ATPase activity and a 19% reduction in ouabain-insensitive ATPase activity (both p less than 0.01). This stimulation of ouabain-sensitive ATPase activity by insulin did not occur in homogenates from diabetic rats. Treating diabetic rats daily with the aldose reductase inhibitor, imirestat (1 mg/kg) improved nerve conduction velocity (p less than 0.05) but was without effect upon the resistance to hypoxic conduction blockade or the deficit in insulin-stimulated ouabain-sensitive ATPase activity. These data suggest that in streptozotocin-diabetic rats the functional disorders of reduced motor nerve conduction velocity and increased resistance to hypoxic conduction blockade do not share a common aetiology and that impaired nerve conduction is not related to reduced maximal potential ouabain-sensitive ATPase activity.

Studies on the subcellular distribution of (Na + + K +)-ATPase, K +-stimulated ATPase and HCO 3−-stimulated ATPase activities in malpighian tubules of Locusta migratoria L.

The present study confirms previous reports of the presence of (Na + + K +)-ATPase and anion-stimulated ATPase activity in Malpighian tubules of Locusta. In addition, the presence of a K +-stimulated, ouabain-insensitive ATPase activity has been identified in microsomal fractions. Differential and sucrose density-gradient centrifugation of homogenates has been used to separate membrane fractions which are rich in mitochondria, apical membranes and basolateral membranes; as indicated by the presence of succinate dehydrogenase and the presence or absence of non-specific alkaline phosphatase activity, respectively. Relatively high specific (Na + + K +)-ATPase activity was associated with the basolateral membrane-rich fractions with only low levels of this activity being associated with the apical membrane-rich preparation. K +-stimulated ATPase activity was also associated, predominantly, with the basolateral membrane-rich fractions. However, comparison of the distribution of this activity with that of the (Na + + K +)-ATPase suggests that the two enzymes did not co-separate. The possibility that the K +-stimulated ATPase was not associated with the basolateral plasma membrane is discussed. Anion-stimulated ATPase activity was found in the apical and basolateral membrane-rich fractions and in the fraction contaning mainly mitochondria. Nevertheless, the fact that this bicarbonate-stimulated activity did not co-separate with succinate dehydrogenase activity suggests that it was not exclusively mitochondrial in origin. These results are consistent with physiological studies indicating a basolateral (Na + + K +)-ATPase but do not support the K +-stimulated ATPase as a candidate for the apical electrogenic pump. The possible role of the bicarbonate-stimulated ATPase activity in ion transport across both the basolateral and apical cell membranes is discussed.

Ultrastructural Localization of ATPases in the Gastrodermis of Megalodiscus temperatus with Additional Observations on Schistosoma japonicum (Trematoda: Digenea)

Ultrastructural Localization of ATPases in the Gastrodermis of Megalodiscus temperatus with Additional Observations on Schistosoma japonicum (Trematoda: Digenea)

Reduced brain Na+, K+-ATPase activity in rats with galactosamine-induced hepatic failure: relationship to encephalopathy and cerebral oedema.

Previously we have shown that sera from patients with fulminant hepatic failure (FHF) will inhibit partially purified rat brain Na+, K+-ATPase and sodium efflux from human leucocytes in vitro. Similar inhibition may be involved in the pathogenesis of encephalopathy and cerebral oedema in these patients. In the present study we have attempted to establish whether the activity of brain Na+, K+-ATPase is decreased in vivo in rats with D-galactosamine induced hepatic failure using homogenates of snap-frozen brains. Na+, K+-ATPase activity was significantly reduced in the forebrain region at the stage of mild encephalopathy (43 h after injection), while at the deeper stage of coma (43-53 h after injection) enzyme activity was further reduced in the forebrain region and was also significantly reduced in the hindbrain region. Ouabain insensitive ATPase activity was not significantly altered at any time. While a significant increase in the water content (0.5%) of the hindbrain region was found 43 h after galactosamine, there was no clear correlation between the development of cerebral oedema and the reduction of Na+, K+-ATPase activity. The activity of partially purified normal rat brain Na+, K+-ATPase was 15% lower when incubated with sera from rats in the deep stage of coma compared with control sera. These data support other evidence that the reduction in brain Na+, K+-ATPase is likely to be due to toxic substance circulating in serum which have been shown to inhibit this enzyme in vitro and to cause coma when administered to normal animals.

Adaption to underfeeding in adult rats

The mode of adaption in adult rats subjected to 50% energy restriction was studied. Basal O 2 consumption decreased by 17%, the magnitude of increase in O 2 consumption over basal level, in response to norepinephrine (NE) stimulation decreased by 45%. interscapular brown adipose tissue (IBAT) mass decreased by 30%. The specific activity of oubain sensitive ATPase increased in liver by 35% but not in kidney. The ouabain insensitive ATPase activity decreased in muscle was unaltered in the restricted rats. The mitochondrial respiration rates were unaltered. The total body fat content in the restricted rats decreased by 35% and the total protein content decreased by 8% compared to the initial value. The control rats had put on 30% more weight whereas the restricted rats lost 23% of the initial weight. Although alterations in the above parameters do indicate the mode of adaptation to underfeeding in adult rats, most of the observed changes in biochemical parameters however do not help in understanding the underlying mechanisms.

Evidence for a Na-K-ATPase-inhibitor in erythrocytes of patients with essential hypertension.

ATPase activities were determined in haemolysed and dialysed erythrocytes and in haemoglobin-free membranes of twenty patients with essential hypertension and twenty normotensive controls. Ouabain-sensitive ATPase (Na-K-ATPase) activity of haemolysate but not that of membranes was decreased in hypertensives whereas ouabain-insensitive ATPase (Mg-ATPase + some residual Ca-ATPase) activity was increased in both enzyme preparations when measurements were preformed in the absence of Ca2+-chelating substances. In haemolysed erythrocytes ouabain-sensitivity as a percentage of total ATPase activity was a good discriminator between both groups and may be a possible marker for essential hypertension. The decreased activity of Na-K-ATPase in haemolysate is apparently due to a non dialysable inhibitor of Na-K-ATPase which is either tightly bound to the erythrocyte membrane or dissolved in the cytoplasm. Following haemolysis with subsequent centrifugation the Na-K-ATPase inhibitor is removed, at least in part, and thus differences in Na-K-ATPase activity demonstrable in haemolysed and dialysed erythrocytes are no longer apparent in haemoglobin-free membranes.

ATP-dependent proton transport by isolated brain clathrin-coated vesicles. Role of clathrin and other determinants of acidification

We have systematically investigated certain characteristics of the ATP-dependent proton transport mechanism of bovine brain clathrin-coated vesicles. H + transport specific activity was shown by column chromatography to co-purify with coated vesicles, however, the clathrin coat is not required for vesicle acidification as H + transport was not altered by prior removal of the clathrin coat. Acidification of the vesicle interior, measured by fluorescence quenching of acridine orange, displayed considerable anion selectively (CI − > Br −⪢NO 3 −⪢ gluconate, SO 4 2-, HPO 4 2-, mannitol; K m for Cl - ≈- 15 mM), but was relatively insensitive to cation replacement as long as Cl - was present. Acidification was unaffected by ouabain or vanadate but was inhibited by N-ethylmaleimide (IC 50 < 10 μM), dicyclohexylcarbodiimide (DCCD) (lC 50 ≈- 10 μM), chlorpromazine (lC 50 ≈- 15 μM), and oligomycin (lC 50 ≈- 3 μM). In contrast to N-ethylmaleimide, chlorpromazine rapidly dissipated preformed pH gradients. Valinomycin stimulated H + transport in the presence of potassium salts (gluconate ⪢ NO 3 − > Cl −), and the membrane-potential-sensitive dye Oxonol V demonstrated an ATP-dependent interior-positive vesicle membrane potential which was greater in the absence of permeant anions (mannitol > potassium gluconate > KCI) and was abolished by N-ethylmaleimide, protonophores or detergent. Total vesicle-associated ouabain-insensitive ATPase activity was inhibited 64% by 1 mM N-ethylmaleimide, and correlated poorly with H + transport, however N-ethylmaleimide-sensitive ATPase activity correlated well with proton transport ( r = 0.95) in the presence of various Cl - salts and KNO 3- Finally, vesicles prepared from bovine brain synaptic membranes exhibited H + transport activity similar to that of the coated vesicles. Collectively these findings indicate that: (1) the H + transport mechanism of bovine brain clathrin-coated vesicles is not dependent upon the clathrin coat, and closely resembles that of rat liver clathrin-coated vesicles, (2) the H + transport mechanism is intrinsically electrogenic and pH and electrical gradients established by the proton transport mechanism vary inversely in the presence of permeable or impermeable anions, (3) at concentrations above 3 μM, oligomycin inhibits vesicle acidification, (4) chlorpromazine inhibits coated vesicle acidification, at least in part, by increasing proton conductance, and (5) other smooth membranes from bovine brain, including synaptic membranes, exhibit a similar H + transport mechanism.

Clathrin-coated vesicles from rat liver: enzymatic profile and characterization of ATP-dependent proton transport.

Clathrin-coated vesicles isolated from rat liver exhibited an enzymatic profile distinct from that of rat liver plasma membranes, lysosomes, microsomes, and mitochondria. The coated vesicles catalyzed ATP-dependent proton transport that acidified the vesicle interior, as measured by the fluorescence quenching of acridine orange. H+ transport by coated vesicles was not inhibited by vanadate (0.1 mM) or ouabain (2 mM) and differed from H+ transport by rat liver submitochondrial particles in its greater resistance to inhibition by oligomycin (10 pM to 10 microM) and N,N'-dicyclohexylcarbodiimide (DCCD) (0.1-100 microM) and its sensitivity to N-ethylmaleimide (0.1-2 mM). H+ transport was stimulated by valinomycin in the presence of K+, exhibited no specific cation requirement, but was dependent upon the presence of a permeant anion, with Cl- and Br- being the most effective of the anions studied. Finally, H+ transport was poorly supported by GTP, UTP, or ADP and exhibited no consistent relationship to the coated vesicle-associated ouabain-insensitive ATPase activity.