Relative humidity affects the intercellular spaces and cell contacts of the kidney epithelium of the terrestrial snail Helix aspersa müller

The effects of relative humidity on hemolymph osmolarity and on kidney ultrastructure are explored in Helix aspersa. The snails are active at 95% relative humidity and less active at 50% relative humidity. The hemolymph osmotic pressure increases with the decrease of relative humidity. Pericardial fluid and hemolymph collected from the heart contain similar amounts of total proteins, and both fluids display hemocyanin molecules in negatively stained preparations. When the snails are kept in an atmosphere of 95% relative humidity, numerous wide intercellular spaces are observed in the single-layered-kidney epithelium. The spaces are almost absent when the snails are kept at 50% relative humidity. It is suggested that prourine is formed through a paracellular junctional pathway across the single-layered kidney epithelium, and that the pericardial cavity is not the site of prourine formation. The septate junctions joining the kidney epithelial cells form a continuous belt of intimate contact in the paracellular pathway of prourine. Long septate junctions with many septa are present in the kidneys of snails from the atmosphere of 50% relative humidity, whereas short septate junctions with fewer septa are found in the kidneys of snails from the atmosphere of 95% relative humidity. It is possible that the longer septate junctions with many septa reduce prourine formation across the kidney sac epithelium.     

The effect of Na+-K+ ATPase inhibition by ouabain on histamine release from human cutaneous mast cells

Background: There are controversial reports on the effect of sodium-potassium adenosine triphosphatase (Na⁺-K⁺ ATPase) inhibition on mast cell mediator release. Some of them have indicated that ouabain (strophanthin G), a specific Na⁺-K⁺ ATPase inhibitor, inhibited the release, whereas the others have shown that ouabain had no effect or even had a stimulatory effect on the mediator secretion. Most of these studies have utilized animal-derived mast cells. The aim of this study was to determine the effect of Na⁺-K⁺ ATPase inhibition on human skin mast cells. Methods: Unpurified and purified mast cells were obtained from newborn foreskins and stimulated by calcium ionophore A23187 (1 μM) for 30 min following a 1 hr incubation with various concentrations (10⁻⁴ to 10⁻⁸ M) of ouabain. Histamine release was assayed by enzyme-linked immunosorbent assay (ELISA). Results: The results indicated that ouabain had no significant effect on the non-immunologic histamine release from human skin mast cells, in vitro. Conclusions: Na⁺-K⁺ ATPase inhibition by ouabain had no significant effect on the non-immunologic histamine release from human cutaneous mast cells and suggested differences between human and animal mast cells.     

Responses of Na+-K+ ATPase activities from dog olfactory tissue to selected odorants.

Na⁺-K⁺ ATPase activity from nerve ending particle (NEP) fractions of dog olfactory tissue homogenates showed different patterns of response to odorants. Similar turbinal groupings were removed from the right and left sides of the septum in the nasal cavity and NEP preparations were tested with eight different odor compounds, including 2-keto alkane homologs and the optical isomers d- and l-carvone. Odorant stimulation of Na⁺-K⁺ ATPase activity from paired turbinal groupings did not show bilateral symmetry. Different patterns of stimulation were observed for each turbinal grouping and for each odorant. A stimulation of over 200% was observed in one preparation in response to 2-nonanone.A study of the response of Na⁺-K⁺ ATPase activity from individual turbinals showed that the enzyme in each turbinal had a different response pattern to six different odorants. Inhibitory and stimulatory responses were observed for the individual turbinal NEP preparations. These results support the proposal that odor sensing initiation may occur through odorant perturbation of the Na⁺-K⁺ ATPase activity.     

Studies on the glycoprotein component of (Na+-K+)ATPase from guinea pig brain

In this study an attempt was made to elucidate the possible mechanism of the brain microsomal (Na⁺-K⁺)ATPase inhibition based on the assumption that glycoprotein part of the enzyme is exposed on the outer membrane surface. In our experiments the modification with concanavalin A of sugar end groups exposed by neuraminidase treatment resulted in a significant decrease of the brain (Na⁺-K⁺)ATPase activity. The percentage of the enzyme inhibition by concanavalin A binding to the neuraminidase-treated preparation corresponds to the amount of liberated sialic acids. The modification of the glycoprotein part of the brain (Na⁺-K⁺)ATPase complex by neuraminidase and concanavalin A treatments did not affect K⁺-nitrophenylphosphatase activity.     

EFFECT OF UNILATERAL VISUAL DEPRIVATION AND VISUAL STIMULATION ON THE ACTIVITIES OF ALKALINE PHOSPHATASE, ACID PHOSPHATASE, Na+−K+ ACTIVATED Mg2+ CATALYSED ADENOSINE TRIPHOSPHATASE AND ON THE CONTENT OF SODIUM AND POTASSIUM IONS OF THE OPTIC LOBE OF ADULT PIGEON

—A study was made of the effects of unilateral visual deprivation and stimulation upon the activities of alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2), Na⁺-K⁺ activated Mg²⁺ catalysed ATPase (EC 3.6.1.4) and upon the Na⁺ and K⁺ contents of the optic lobe of adult pigeon (Columba livia). Visual deprivation was achieved by eyelid suturing or by enucleation and maintained for 1–9 weeks. Unilateral visual stimulation was maintained for 75 min following 72 h of darkness. A statistically significant increase in the activity of alkaline phosphatase activity was observed in the optic lobe after unilateral visual deprivation whereas unilateral visual stimulation resulted in the opposite effect. Acid phosphatase activity was found to be unchanged under all experimental conditions. Na⁺-K⁺ ATPase activity was found to increase significantly following unilateral visual stimulation and following eyelid suturing in the corresponding optic lobes; unilateral enucleation resulted in a decrease in the Na⁺-K⁺ ATPase activity. An increase in the enzyme activity was found to be associated with an increase in the level of Na⁺-ion and a decrease in the level of K⁺-ion, and vice versa.     

Variations in myocardial CPK and Na+-K+ ATPase following long-term freezing.

The activity of the soluble enzyme CPK and the membrane bound enzyme Na⁺-K⁺ ATPase as a function of storage temperature, time of storage and cryoprotectant type and concentration in canine myocardial tissue was invesigated. Activity of CPK is well preserved at ?196 °C and ?79 °C and falls off during one month storage at ?40 °, ?20 °, and 0 °C. Na⁺-K⁺ ATPase demonstrates a greater liability. After an initial cryoprotectant “activation,” activity drops. In all cases, however, addition of the cryoprotectant preserved activity better than in samples stored only in buffer.     

Na+-K+ ATPase and carbonic anhydrase activities in larvae,postlarvae and adults of the shrimp Penaeus japonicus (Decapoda,Penaeidea)

• 1.1. Activities of Na⁺-K⁺ ATPase and carbonic anhydrase were measured through the early post-embryonic development of Penaeusjaponicus. In adults, only the Na⁺-K⁺ ATPase activity was measured.
• 2.2. ATPase activity was variable in the successive development stages. From zero in nauplii, the activity slightly increased in zoeae, and rose sharply in mysis stages 2 and 3.
• 3.3. A further significant increase in activity was noted at the transition from late mysis to early postlarvae, concomitant with a change from the larval osmoconforming pattern of osmoregulation to the postlarval and adult hyper-hyporegulating pattern.
• 4.4. The activity of Na⁺-K⁺ ATPase, measured in isolated cephalothorax, increased from PL3 to PL4 to its maximum value in PL5; at this stage, osmoregulatory capacity was fully efficient.
• 5.5. In young stages of P. japonicus, the variations in Na⁺-K⁺ ATPase activity appear correlated with the development of osmoregulatory ultrastructures, and with osmoregulation and salinity tolerance.
• 6.6. These results are discussed with regard to their ecological and physiological implications.
• 7.7. In adults, the activity of Na⁺-K⁺ ATPase was high in gills and epipodites and no activity was detected in branchiostegites. These results are related to the ultrastructure of these organs.
• 8.8. The activity of carbonic anhydrase did not change significantly in larval and postlarval stages.
• 9.9. From these results, it is proposed that the effector sites of osmoregulation are located in branchiostegites, pleurae and epipodites in postlarvae, and in epipodites and mainly in gills in adults.

     

Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: Implications for angiotensin II-induced vascular dysfunction

Glutathionylation of the Na⁺-K⁺ pump’s β₁-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na⁺-K⁺ pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na⁺-K⁺ pump activity is an important determinant of vascular tone through effects on [Ca²⁺]_i, we have examined the role of oxidative regulation of the Na⁺-K⁺ pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β₁-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na⁺-K⁺ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K⁺-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na⁺-K⁺ ATPase and decrease in K⁺-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β₁-subunit. Knock-out of FXYD1 dramatically decreased K⁺-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8 mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β₁-subunit glutathionylation, and enhanced K⁺-induced vasorelaxation. Ang II inhibits the Na⁺-K⁺ pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump’s β₁-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na⁺-K⁺ pump and may have an important protective role in the vasculature under conditions of oxidative stress.     

The Extrusion of Sodium Ions from Presynaptic Nerve Endings of Rat Cerebral Cortex

(Na⁺-K⁺) ATPase is present in synaptosomal preparations and it is assumed to represent the sodium-potassium pump. 10 μm -noradrenaline activates (Na⁺-K⁺) ATPase approximately 100%, but 50 μm -noradrenaline does not stimulate the rate of ²²Na extrusion from synaptosomes. The results suggest that it is unlikely that the noradrenaline stimulation of (Na⁺-K⁺) ATPase is part of a feedback mechanism whereby released noradrenaline can influence the activity of the presynaptic sodium pump.     

The expression of gastric H+-K+-ATPase mRNA and protein in developing rat fundic gland

The proton pump H⁺-K⁺-ATPase is the final common pathway mediating the production and secretion of hydrochloric acid by gastric parietal cells. The present studies were undertaken to examine whether the expression of gastric H⁺-K⁺-ATPase mRNA and protein changes are associated with the development of H⁺-K⁺-ATPase activity in the rat fundic gland. H⁺-K⁺-ATPase activity was examined in rat fundic gland at different stages from gestational day 18.5 to postnatal 8 weeks. The expression of H⁺-K⁺-ATPase mRNA was detected by in situ hybridization using a digoxigenin-labelled RNA probe with a tyramide signal amplification system. The expression of H⁺-K⁺-ATPase protein was evaluated by immunoblotting and immunohistochemistry using antibodies against H⁺-K⁺-ATPase - and -subunits. We found that H⁺-K⁺-ATPase enzyme activity was detectable from the onset of gland formation (day 19.5 of gestation) and increased with age in the developing rat fundic gland. Expression of mRNA and protein was also discernible at the same time, and a progressive increase in expressions was observed as rats developed. Our results suggested that in developing rat fundic gland, the expression of both mRNA and protein of H⁺-K⁺-ATPase increased with age in a manner that parallels the development of H⁺-K⁺-ATPase enzymebreak activity.     

Response to environmental salinity of Na-KATPase activity in individual gills of the euryhaline crab Cyrtograpsus angulatus

The occurrence, localization and response to environmental salinity changes of Na⁺-K⁺ATPase activity were studied in each of the individual gills 4-8 of the euryhaline crab Cyrtograpsus angulatus from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). Na⁺-K⁺ATPase activity appeared to be differentially sensitive to environmental salinity among gills. Upon an abrupt change to low salinity, a differential response of Na⁺-K⁺ATPase activity occurred in each individual gill which could suggest a differential role of this enzyme in ion transport process in the different gills of C. angulatus. With the exception of gill 8, a short-term increase of Na⁺-K⁺ATPase specific activity was observed in posterior gills, which is similar to adaptative variations of this activity described in other euryhaline crabs. However, and conversely to that described in other hyperregulating crabs, the highest increase of activity occurred in anterior gills 4 by 1 day after the change to dilute media which could suggest also a role for these gills in ion transport processes in C. angulatus. The fact that variations of Na⁺-K⁺ATPase activity in anterior and posterior gills were concomitant with the transition to hyperregulation indicate that this enzyme could be a component of the branchial ionoregulatory mechanisms at the biochemical level in this crab. The results suggest a differential participation of branchial Na⁺-K⁺ATPase activity in ionoregulatory mechanisms of C. angulatus. The possible existence of functional differences as well as distinct regulation mechanisms operating in individual gills is discussed.     

Early effects of decapitation on the Mg2+-K+ ATPase and cation contents in lateral buds of the aquatic fern,Marsilea drummondii A. Br.

Summary A detailed comparison of Mg²⁺-K⁺ ATPase activity and cation fluxes in terminal buds, inhibited buds or buds released from apical dominance was carried out. Light microscope observations indicate intense reaction at the plasmalemma of stelar cells, pericyclic, phloem and xylem transfer cells at nodes along the main rhizome. In intact plants, with the exception of pericyclic cells, bud branches show no ATPase activity. Excision of the terminal bud results in rapid (within 5–15 minutes) stimulation of ATPase activity at nodes and all bud axes. As the subapical bud gains precedence, ATPase stimulation ceases and returns to its initial level in the older median and basal buds. Enzyme activation is kinetically correlated with K⁺ flux. X-ray microanalysis confirms that K⁺ accumulates in the stele at the node and the bud branch with the same lag period. This data increases the evidence for close association between ATP (Sossountzov et al. 1982), ATPase activity and K⁺ flux. The kinetics strengthen the impression that these factors may be involved very early in bud outgrowth regulation.     

Effects of batrachotoxin on electroplax Na + -K + -ATPase and levels of ATP in rat muscle

—Batrachotoxin (BTX) in low concentrations (20 nm ) depolarizes electrically excitable membranes (Albuquerque , Daly and Witkop , 1971). At these levels, BTX does not inhibit Na⁺-K⁺-ATPase. At much higher concentrations (60 μm ) BTX partially inhibits Na⁺-K⁺-ATPase from electroplax of Electrophorus electricus. In contrast to inhibition by cardiac glycosides, the inhibition of Na⁺-K⁺-ATPase by batrachotoxin is not antagonized by KCl. BTX had no effect on ATP levels in stimulated nerve muscle preparations at the time when sustained contracture was initiated by the drug. Phosphocreatine levels were decreased and levels of glucose-6-phosphate and 6-phosphogluconate were increased, while levels of fructose-1,6-diphosphate and α-ketoglutarate were unchanged. It is concluded that the inhibition of Na⁺-K⁺-ATPase or lowering of ATP levels by BTX is not significantly involved in the membrane depolarization produced by the toxin.     

Evidence implicating a membrane ATPase in the control of passive permeability of excitable cells

The temperature sensitivity of the ATPase enzyme systems in a muscle microsomal preparation from the crayfish, Astacus pallipes, was studied. Preincubation of the enzyme preparation in the range 33–36°C produced a marked inactivation of the ATPases; the Mg⁺⁺-dependent ATPase was very much more sensitive to this treatment than the Na⁺-K⁺-Mg⁺⁺-dependent ATPase. Thus, the Arrhenius μ for the inactivation of the Mg⁺⁺-dependent ATPase produced by eight minute preincubation is > 100 Kcals. These results are compared with the changes that are observed during the heat death of the whole animal, where exposure to 35°C produces a dramatic change in Na⁺ permeability within five minutes. Arrhenius μ for heat death is also > 100 Kcals and operates over the identical critical temperature range. It is suggested that the Mg⁺⁺-dependent ATPase controls passive permeability in these excitable cells and the results also confirm the view that Mg⁺⁺ and Na⁺-K⁺-Mg⁺⁺ ATPases are separate enzymes.     

Irreversible zinc ion inhibition of (Na+ -K+)-adenosinetriphosphatase, Na+ -phosphorylation, and K+-p-nitrophenylphosphatase of Electrophorus electricus electroplax.

Zinc ion in micromolar concentrations is an irreversible inhibitor of Electrophorus electricus electroplax microsomal (Na⁺-K⁺)-ATPase. The rate of inhibition is dependent on [ZnCl₂] and the extent of inhibition varies with the ratio of ZnCl₂ to microsomal protein. The same kinetics are observed for inhibition of K⁺ -p-nitrophenylphosphatase and steady-state levels of Na⁺ -dependent enzyme phosphorylation. The observations suggest that a Zn²⁺ -sensitive conformational restraint is important to both kinase and phosphatase activities. The fact that inhibition is irreversible has implications for models seeking to relate zinc effects in tissue to inhibition of (Na⁺-K⁺)-ATPase.     

Carotenoid oxidative degradation products inhibit Na+-K+-ATPase

This study investigates the biological significance of carotenoid oxidation products using inhibition of Na⁺-K⁺-ATPase activity as an index. β-Carotene was completely oxidized by hypochlorous acid and the oxidation products were analyzed by capillary gasliquid chromatography and high performance liquid chromatography. The Na⁺-K⁺-ATPase activity was assayed in the presence of these oxidized carotenoids and was rapidly and potently inhibited. This was demonstrated for a mixture of β-carotene oxidative breakdown products, β-Apo-10′-carotenal and retinal. Most of the β-carotene oxidation products were identified as aldehydic. The concentration of the oxidized carotenoid mixture that inhibited Na⁺-K⁺-ATPase activity by 50% (IC₅₀) was equivalent to 10μM non-degraded β-carotene, whereas the IC₅₀ for 4-hydroxy-2-nonenal, a major lipid peroxidation product, was 120 μM. Carotenoid oxidation products are more potent inhibitors of Na⁺-K⁺-ATPase than 4-hydroxy-2-nonenal. Enzyme activity was only partially restored with hydroxylamine and/or β-mercaptoethanol. Thus, in vitro binding of carotenoid oxidation products results in strong enzyme inhibition. These data indicate the potential toxicity of oxidative carotenoid metabolites and their activity on key enzyme regulators and signal modulators.     

Activation of Na+-K+-adenosine triphosphatase by spermine.

Spermine activated Na⁺-K⁺-ATPase when the concentrations of K⁺ and ATP were low, whereas it inhibited K⁺-dependent and ouabain-inhibitable monophosphatase. The activating effect of sperimine was not due to the substitution for K⁺ or Na⁺. Excess K⁺ inhibited Na⁺-K⁺-ATPase partially, and reduced the spermine activation. When 1 mM ATP was used, spermine at higher concentrations inhibited Na⁺-K⁺-ATPase, and did not activate at all. It is suggested that the K⁺-sites essential to Na⁺-K⁺-ATPase and the K⁺-phosphatase co-exist at different places of the enzyme.     

Exogeneous diacylglycerols downregulate the activity of Na-K pump in Xenopus laevis oocytes

In this study, cell permeable diacyglycerols, sn-1,2-dioctanoglycerol (DiC8), and sn-1-oleoyl-2-acetylglycerol (OAG) were found to downregulate the activity of Na⁺-K⁺ pump in Xenopus laevis oocytes. Both DiC8 and OAG decreased the binding of [³H]ouabain to intact oocytes while phorbol esters did not appreciably influence the same. These diacylglycerols inhibited the amiloride-sensitive ²²Na⁺ influx and ouabain-sensitive ⁸⁸Rb⁺ uptake in the oocytes. Furthermore, DiC8 prevented the ²²Na⁺ efflux from the oocytes preloaded with ²²Na⁺. Addition of H-7 to DiC8- and OAG-treated oocytes stimulated the pump activity curtailed by the two latters. The impairment of Na⁺-K⁺ pump activity by diacylglycerols suggests that protein kinase C activators may stimulate endocytosis of membrane-coupled Na⁺-K⁺ ATPase.     

SGK1 activates Na+-K+-ATPase in amphibian renal epithelial cells

Serum- and glucocorticoid-induced kinase 1 (SGK1) is thought to be an important regulator of Na⁺ reabsorption in the kidney. It has been proposed that SGK1 mediates the effects of aldosterone on transepithelial Na⁺ transport. Previous studies have shown that SGK1 increases Na⁺ transport and epithelial Na⁺ channel (ENaC) activity in the apical membrane of renal epithelial cells. SGK1 has also been implicated in the modulation of Na⁺-K⁺-ATPase activity, the transporter responsible for basolateral Na⁺ efflux, although this observation has not been confirmed in renal epithelial cells. We examined Na⁺-K⁺-ATPase function in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible promoter. The results showed that expression of a constitutively active mutant of SGK1 (SGK1^T_S425D) increased the transport activity of Na⁺-K⁺-ATPase 2.5-fold. The increase in activity was a direct consequence of activation of the pump itself. The onset of Na⁺-K⁺-ATPase activation was observed between 6 and 24 h after induction of SGK1 expression, a delay that is significantly longer than that required for activation of ENaC in the same cell line (1 h). SGK1 and aldosterone stimulated the Na⁺ pump synergistically, indicating that the pathways mediated by these molecules operate independently. This observation was confirmed by demonstrating that aldosterone, but not SGK1^T_S425D, induced an 2.5-fold increase in total protein and plasma membrane Na⁺-K⁺-ATPase ₁-subunit abundance. We conclude that aldosterone increases the abundance of Na⁺-K⁺-ATPase, whereas SGK1 may activate existing pumps in the membrane in response to chronic or slowly acting stimuli. sodium transport; serum- and glucocorticoid-induced kinase; A6 cells; sodium pump     

In vitro binding of inorganic mercury to the plasma membrane of rat platelet affects Na+-K+-ATPase activity and platelet aggregation

Hg²⁺ binding to ouabain-sensitive Na⁺-K⁺-ATPase of rat platelet membrane was specific with a K_a of 1.3×10⁹ moles and B_max of 3.8 nmoles/mg protein. The binding of mercury to Na⁺-K⁺-ATPase also inhibits the enzyme significantly (P<0.001), which is greater than its ouabain sensitivity. Further in the cytosol of washed platelets conjugation of reduced glutathione (GSH) to Hg²⁺ is correlated dose dependently (25, 50 and 100 pmoles) to enhanced GSH-S-transferase (GST) activity. It may be concluded from the present in vitro experiments that mercury binds specifically to thiol groups present in the platelet membrane Na⁺-K⁺-ATPase, inhibits the enzyme and induces changes in platelet function, namely, platelet aggregation by interfering with the sodium pump.