Ouabain-insensitive ATPase Research Articles

Increased susceptibility of brain acetylcholinesterase activity to methylmalonate in young rats with renal failure

Tissue methylmalonic acid (MMA) accumulation is the biochemical hallmark of methylmalonic acidemia. Clinically, the disease is characterized by progressive neurological deterioration and renal failure, whose pathophysiology is still undefined. In the present study we investigated the effect of acute MMA administration on some important parameters of brain neurotransmission in cerebral cortex of rats, namely Na(+), K(+)-ATPase, ouabain-insensitive ATPases and acetylcholinesterase activities, in the presence or absence of kidney injury induced by gentamicin administration. Initially, thirty-day old Wistar rats received one intraperitoneal injection of saline or gentamicin (70 mg/kg). One hour after, the animals received three consecutive subcutaneous injections of MMA (1.67 μmol/g) or saline, with an 11 h interval between each injection. One hour after the last injection the animals were killed and the cerebral cortex isolated. MMA administration by itself was not able to modify Na(+), K(+)-ATPase, ATPases ouabain-insensitive or acetylcholinesterase activities in cerebral cortex of young rats. In rats receiving gentamicin simultaneously with MMA, it was observed an increase in the activity of acetylcholinesterase activity in cerebral cortex, without any alteration in the activity of the other studied enzymes. Therefore, it may be speculated that cholinergic imbalance may play a role in the pathogenesis of the brain damage. Furthermore, the pathophysiology of tissue damage cannot be exclusively attributed to MMA toxicity, and control of kidney function should be considered as a priority in the management of these patients, specifically during episodes of metabolic decompensation when MMA levels are higher.

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.

Effect of alcohol and kolanut interaction on brain sodium pump activity in wistar albino rats.

Effect of alcohol-kolanut interaction on sodium pump activity in wistar albino rats was studied. Thirty wistar albino rats were divided into six groups of five (5) rats per group and used for the study. The control group (1) received via oral route a placebo (4 ml of distilled water). Groups 2 to 6 were treated for a period of 21 days, with (10% v/v) of alcohol (group 2), 50mg/kg body weight of kolanut (group 3), 50 mg/kg body weight of caffeine (group 4), 4 ml of 10% v/v of alcohol and 50 mg/kg body weight kolanut (group 5), 4 ml of 10% v/v of alcohol and 50 mg/kg body weight of caffeine in 4.0 ml of the vehicle via gastric intubation respectively. A day after the final exposure, the brain of each rat was harvested and processed to examine several biochemical parameters, i.e., total ATpase, ouabain-insensitive ATpase, ouabain sensitive ATpase (Na(+)-K(+)ATPase), non-enzymatic breakdown of ATP and inorganic phosphate (Pi) released. The results showed that the essential enzyme of the brain responsible for neuronal function, Na(+)-K(+)ATPase, was inhibited by alcohol-kolanut co-administration relative to control, resulting in a decrease in Na(+)-K(+)ATPase activity, ATP production, ion transport and action potential, leading to loss of neuronal activities.

Interaction of alcohol and kola nut on brain sodium pump activity in Wistar rats

The effect of interaction of alcohol and kola nut on sodium pump activity was studied in Wistar rats. Thirty Wistar rats were divided into six groups of five rats per group. Control received a placebo (4 mL of distilled water). Groups 2–6 were treated for a period of 21 days with 10% (v/v) alcohol, 50 mg caffeine/kg, 50 mg kola nut/kg, or a combination of 10% (v/v) alcohol + 50 mg kola nut/kg, or 10% (v/v) alcohol + 50 mg caffeine/kg, respectively. One day after the final exposure, brains were harvested and several biochemical parameters examined including activities of total ATPase, ouabain-insensitive ATPase, ouabain sensitive ATPase (Na+–K+-ATPase) and levels of nonenzymatic breakdown of ATP and inorganic phosphate (Pi) released. Results showed that the essential enzyme of the brain responsible for neuronal function, Na+–K+-ATPase, was inhibited by alcohol–kola nut co-administration relative to control, resulting in a decreased ATP production, ion transport and action potential, leading to loss of neuronal activities.

Inhibition of mammal Na(+)K(+)-ATPase by diterpenes extracted from the Brazilian brown alga Dictyota cervicornis.

The dolastane diterpenes 4-acetoxy-9,14-dihydroxydolast-1(15),7-diene (1) and 4,7-diacetoxy-14-hydroxydolast-1(15),8-diene (2) were isolated from specimens of the alga Dictyota cervicornis collected from the Rio de Janeiro coast, Brazil. Chemical structures of the diterpenes were assigned by 1D and 2D NMR spectral data for the first time. Both substances inhibited Na(+)K(+)-ATPase preparations from guinea-pig brain or kidney, with the same inhibitory potency towards enzyme isoforms. The maximal inhibition obtained for 1 was 40% at a concentration of 0.5 mm in the incubation mixture, while it reached 80% for compound 2 at this concentration. Ouabain insensitive ATPases were inhibited by 1, but not by 2. Data comparing the inhibitory potency of these compounds with that of ouabain and oleic acid suggest a higher degree of selectivity of 2 towards the Na(+)K(+)-pump. Cardiac glycosides such as ouabain are used classically in the treatment of heart failure, but alterations of Na(+)K(+)-pump activity are also involved in several other diseases. Therefore, the study of compounds interfering with this pump activity is gaining further importance.

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.

Decreased gill ATPase activities in the freshwater fish Channa punctata (Bloch) exposed to a diluted paper mill effluent

Aquatic habitat is affected by paper mill effluent discharge in many ways. The effect of paper mill effluent on the gill ATPases was studied in freshwater fish Channa punctata (Bloch) exposed to 1%(v/v) of effluent for 15, 30, and 60 days. There was a time-dependent significant ( P<0.05–0.001) decrease in all the ATPase activities measured, viz., total, Na +, K +- and ouabain-insensitive ATPase in gill. ATPases play an important role in maintenance of functional integrity of plasma membrane and in several intracellular functions and are considered to be a sensitive indicator of toxicity. In addition to this, branchial ATPases are intimately involved in osmoregulation, acid–base regulation, and respiration of fish. The inhibition of ATPases in gills by, e.g., paper mill effluent could cause disruption of these processes. It is suggested that measurement of ATPases could also be used as a surrogate biomarker of exposure to chemical pollutants.

Regional intestinal adaptations in Na+,K+‐ATPase in experimental colitis and the contrasting effects of interferon‐γ

This study evaluated Na+,K+-ATPase activity and the abundance of alpha1 subunit Na+,K+-ATPase in experimental colitis and gathered evidence on the effects of interferon-gamma (IFN-gamma) on intestinal Na+,K+-ATPase. Colitis was induced by the intrarectal administration of 2,4,6-trinitrobenzene sulphonic acid (TNBS, 30 mg/250 microL). Na+,K+-ATPase activity was determined as the difference between total and ouabain-insensitive ATPase. The abundance of Na+,K+-ATPase was analysed by immunoblotting. Na+,K+-ATPase activity was markedly reduced in the proximal colonic mucosa of TNBS-treated rats, whereas upstream in the terminal ileal mucosa a marked increase in sodium pump activity was observed. At the jejunal level no significant changes in Na+,K+-ATPase activity were observed between TNBS-treated rats and corresponding controls (ethanol-treated rats). No changes were observed in the abundance of alpha1 subunit Na+,K+-ATPase in the proximal colon, terminal ileum and jejunum. The administration of IFN-gamma (50,000 U) 48 h before sacrifice reduced both Na+,K+-ATPase activity and the abundance of alpha1 subunit Na+,K+-ATPase in the proximal colon. Dexamethasone prevented colonic inflammation and decreases in proximal colonic Na+,K+-ATPase activity in TNBS-treated rats, but did not affect the INF-gamma-induced decrease in colonic Na+,K+-ATPase activity. The increase in ileal Na+,K+-ATPase activity upstream to the lesioned colonic mucosa, where Na+,K+-ATPase activity was markedly reduced, might indicate a compensatory process to counteract the decrease in water and electrolyte absorption at the colonic level. This decrease in colonic Na+,K+-ATPase activity is likely not related to INF-gamma-induced downregulation of 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.

Apical ouabain-sensitive and ouabain-insensitive ATPases in rat colonic epithelium

Active resorption of potassium ions in the colon is mediated by ouabain-sensitive and ouabain-insensitive H,K-ATPases localized in the apical membrane of colonic enterocytes (colonocytes). The present study was performed to investigate distribution patterns of apical ATPases using catalytic histochemistry and immunohistochemistry. Activity of the ATPases was localized both at the apical and basolateral regions of these cells. The basolateral activity was almost completely inhibited by ouabain, but apical ATPase activity was only partially inhibited by ouabain. Ultracytochemically, activity was localized at the cytoplasmic side of the apical and basolateral membrane and thus we assume that the activity represents isoforms of apical H,K-ATPases and basolateral Na,K-ATPase. With the use of a polyclonal antibody raised against H,K-ATPase alpha-subunit, we demonstrated immunostaining only in the apical region of colonic enterocytes whereas positive staining was not observed at the basolateral membrane. On the other hand, when antibodies against alpha1-subunit Na,K-ATPase were applied, immunostaining was localized only in the basolateral membrane domain. Therefore, we conclude that ATPases as demonstrated histochemically in the present study were identified immunohistochemically as colonic alpha-subunit H,K-ATPase (in the apical cell membrane of colonocytes along the entire length of crypts) and as alpha1-subunit Na,K-ATPase (in the basolateral membrane).

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.

Inhibition of Na + /K + -ATPase and of active ion-transport functions in the gills of the shore crab Carcinus maenas induced by cadmium

Inhibition of Na+/K+-ATPase from gill plasma membranes of the shore crab Carcinus maenas by cadmium was investigated and compared with inhibitory effects by known antagonists (ouabain and Ca2+). For comparative considerations the Cd2+-inhibition of the enzyme from dog kidney was also tested. Na+/K+-ATPase from dog kidney and from crab gill differed greatly in sensitivity against ouabain. The inhibition constant Ki of the dog enzyme amounted to 9.1 × 10−7 mol l−1, i.e. more than 300-fold smaller than the Ki of 2.9 × 10−4 mol l−1 determined for the crab enzyme. Ca2+ inhibited the activity of Na+/K+-ATPase from crab gill plasma membranes with a Ki of 4.3 × 10−4 mol l−1. The Na+/K+-ATPase from crab gill was inhibited by Cd2+ with a Ki of 9.1 × 10−5 mol l−1. Cd2+ inhibited the Na+/K+-ATPase from dog kidney with a Ki (6.4 × 10−5 mol l−1) comparable to that observed in the crab gill enzyme. Under experimental conditions Cd2+-inhibition of Na+/K+-ATPase was irreversible. Repeated washing, centrifugation and homogenization of the plasma membranes (four times) with Cd2+-free buffer did not restore any activity lost in the presence of 1 × 10−3 mol l−1 Cd2+. Since ouabain-insensitive (nonspecific) ATPases in the plasma membrane fraction of crab gills were inhibited by Cd2+ in the same way as Na+/K+-ATPase, the heavy metal is considered as an unspecific ATPase inhibitor. Comparing these results with literature data on Cd2+-binding to electrophoretically separated proteins suggests that Na+/K+-ATPase is a Cd2+-binding enzyme. The results obtained on Na+/K+-ATPase were reflected by Cd2+-inhibition of the branchial ion-transport functions depending on this enzyme. The transepithelial short-circuit current of isolated gill half lamellae, a direct measure of area-specific active ion uptake, and the transepithelial potential difference of isolated, perfused whole gills, also indicative of active ion uptake, were inhibited by the heavy metal in a time- and dose-dependent mode. Remarkably these inhibitions were also irreversible. These findings are ecologically and biomedically significant: even when the actual environmental or tissue concentrations measured are low, biological microstructures such as Na+/K+-ATPase may accumulate the heavy metal by tight binding over prolonged periods until the first inhibitory effects occur.

Direct modulation of G-proteins by polyunsaturated fatty acids: a novel eicosanoid-independent regulatory mechanism in the developing lung

Basal and fatty-acid-modulated G-protein function was studied in 1-3-day-pre-term, fetal guinea-pig, type II (fATII) pneumocyte apical membrane. Unstimulated (tonic) high-affinity GTPase activity (measured as [gamma-32P]GTP hydrolysis rate) was high and 77% pertussis toxin-insensitive. Alteration of this activity was used as a marker of G-protein regulation. Arachidonic acid (AA) showed a dose-dependent (IC50 = 48+/-8 microM) inhibition of activity at concentrations significantly below critical micellar concentrations; this effect was mimicked by other polyunsaturated fatty acids (IC50 for linoleic acid = 47 +/- 2 microM; IC50 for oleic acid = 106 +/- 11 microM). Saturated fatty acids showed no effect. The effect of AA on ouabain-insensitive ATPases in the same preparation was significantly lower, suggesting a specificity of the GTPase modulation effect. AA modulation of GTPase activity was not attenuated by blocking eicosanoid metabolism with inhibitors of 5'-lipoxygenase, cyclo-oxygenase and P-450. In order to explore further the mechanism of AA-G-protein interaction, the effect of AA on the time course and equilibrium binding of [35S]GTP[S] to apical membrane was studied. Consistent with our GTPase assay data, AA inhibited binding with an IC50 value of 71+/-1 microM; stearic acid did not mimic this effect. This is the first report of unsaturated-fatty-acid-specific modulation of lung G-protein function: since AA also up-regulates perinatal lung alveolar Na+ transport, we suggest this lipid/G-protein switch helps maintain pulmonary fluid homoeostasis around birth.

Selective Effect of Salt Stress on the Activity of Two ATPases in the Cell Membrane of &lt;italic&gt;Nostoc muscorum&lt;/italic&gt;

ATPase activity in the cell membrane of a salt-stressed cyanobacterium, Nostoc muscorum M-14, was examined cytochemically by three different staining protocols. Application of Hulstaert's method resulted in distinct precipitation of the reaction products of ATPase inside the cell membrane exclusively. No reaction products were formed when ATP was replaced by GTP or when dicyclohexylcarbodiimide or N-ethylmaleimide was present in the reaction mixture. By contrast, low levels were detectable after the reaction in the presence of ouabain. Bafilomycin did not affect the formation of products. Mayahara's method, which is considered to demonstrate the reaction of Na+,K+-ATPase activity, revealed the presence of a ouabain-sensitive Na+,K+-ATPase in the cell membrane, while Wachstein-Meisel's method revealed the presence of an ATPase activity that was resistant to ouabain. It appears, therefore, that cell membranes of Nostoc muscorum contain both ouabain-sensitive ATPase and ouabain-insensitive ATPase. Comparison of the staining profiles of salt-stressed cells with those of control cells suggested that a high-salt environment activates the ouabain-sensitive Na+,K+-ATPase, which seems likely to be involved in the efflux of Na+ ions.

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.

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.

Mechanisms of cell volume regulation in the proximal segment of the Malpighian tubule of Rhodnius neglectus.

The cell volume regulation of the lower segment cells of the Malpighian tubule of Rhodnius neglectus in anisosmotic media was evaluated by using video-optic techniques. When the medium osmolality was increased with addition of 100 mM mannitol the cells shrank to a minimum of 16.84 +/- 2.62% and subsequently swelled towards their initial volume undergoing a typical regulatory volume increase (RVI). Replacement of either K+ or Cl- or HCO(3-) by Na+, gluconate and phosphate, respectively, abolished the RVI response. Furthermore, the substitution of Na+ by tetramethylammonium (TMA+) in isosmotic conditions led to cellular swelling and death. Addition of either amiloride 10(-4) M, anthracene-9-COOH 5 x 10(-4) M, furosemide 5 x 10(-4) M or ethacrynic acid 5 x 10(-5) M, also abolished RVI. On the other hand, addition of either Ba2+ 10(-3) M, SITS 5 x 10(-4) M, ouabain 10(-3) M or vanadate 10(-3) M, did not change the RVI response. When the tubules were incubated in hyperosmotic media with EGTA 2 mM or verapamil 10(-6) M, the RVI response was abolished. In contrast, a decrease of NaCl concentration from 129 to 79 mM induced a cell swelling to a maximum of 33.11 +/- 1.73%, but the cells maintained swollen, only partially regulating their volume. These results show that the proximal cells of Malpighian tubule of R. neglectus are able to regulate their volume in hyperosmotic but only partially regulating in hyposmotic solutions. The mechanisms in RVI involve Na+, K+, Cl-, Ca2+ and HCO(3-) transport pathways and a ouabain-insensitive ATPase stimulated by Na+.

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.