Dependence of the CO<Subscript>2</Subscript> Uptake in a Plant Cell on the Plasma Membrane H<Superscript>+</Superscript>-ATPase Activity: Theoretical Analysis

A decrease of the plasma membrane H⁺-ATPase activity in plant cells is associated with the formation of response to adverse factors and reception of signals. A theoretical analysis of the influence of the H⁺-ATPase activity on the flow of CO₂ into a plant cell has been conducted. With this purpose the model of transport processes and electrogenesis in the plant cell developed previously was used, which takes into account transport systems, including H⁺-ATPase, in the plasma membrane and tonoplast. The CO₂ fraction (\({P_{c{o_2}}}\)) in the total amount of inorganic carbon (C_i) in the external medium was used as an indicator of the CO₂ amount entering the plant cell; this parameter depends on the extracellular pH, which, in particular, is influenced by the H⁺-ATPase activity. Excitable and non-excitable cells were simulated. It was shown that a decrease of the H+-ATPase activity causes a \({P_{c{o_2}}}\)reduction in both variants of the model, and this reduction has an extremum: after passing through minimal values, \({P_{c{o_2}}}\)reaches a stationary level. The dynamics of \({P_{c{o_2}}}\)decrease may be related to the Ca²⁺ influx into the cytoplasm of the plant cell. The reduction of \({P_{c{o_2}}}\)depended on the extent of the H⁺-ATPase inactivation and on its initial activity. As a whole, it was shown that the inactivation of the H⁺-ATPase can affect the CO₂ uptake in a plant cell and thereby regulate photosynthetic processes.     

Purification and characterization of ferredoxin-NADP<Superscript>+</Superscript> reductase encoded by <Emphasis Type="Italic">Bacillus subtilis yumC</Emphasis>

From Bacillus subtilis cell extracts, ferredoxin-NADP⁺ reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing. YumC is a 94-kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit. In a diaphorase assay with 2,6-dichlorophenol-indophenol as electron acceptor, the affinity for NADPH was much higher than that for NADH, with K_m values of 0.57 M vs >200 M. K_cat values of YumC with NADPH were 22.7 s^–1 and 35.4 s^–1 in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively. The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low. The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR. A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and which have been annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc. These genes are found notably in gram-positive bacteria, except Clostridia, and less frequently in archaea and proteobacteria. We propose that YumC and C. tepidum FNR constitute a new group of FNR that should be added to the already established plant-type, bacteria-type, and mitochondria-type FNR groups.     

Vasotocin has the potential to inhibit basolateral Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-pump current across isolated skin of tree frog in vitro<Emphasis Type="Italic">,</Emphasis> via its V<Subscript>2</Subscript>-type receptor/cAMP pathway

Adult frog skin transports Na⁺ from the apical to the basolateral side across the skin. Antidiuretic hormone (ADH) is involved in the regulation of Na⁺ transport in both mammals and amphibians. We investigated the effect of arginine vasotocin (AVT), the ADH of amphibians, on the short-circuit current (SCC) across intact skin and on the basolateral Na⁺/K⁺-pump current across apically nystatin-permeabilized skin of the tree frog, Hyla japonica, in which the V₂-type ADH receptor is expressed in vitro. In intact skin, 1 pM AVT had no effect on the SCC, but 10 nM AVT was sufficient to stimulate the SCC since 10 nM and 1 μM of AVT increased the SCC 3.2- and 3.4-fold, respectively (P > 0.9). However, in permeabilized skin, AVT (1 μM) decreased the Na⁺/K⁺-pump current to 0.79 times vehicle control. Similarly, 500 μM of 8Br-cAMP increased the SCC 3.2-fold, yet 1 mM of 8Br-cAMP decreased the Na⁺/K⁺-pump current to 0.76 times vehicle control. Arachidonic acid (10⁻⁵ M) tended to decrease the Na⁺/K⁺-pump current. To judge from these in vitro experiments, AVT has the potential to inhibit the basolateral Na⁺/K⁺-pump current via the V₂-type receptor/cAMP pathway in the skin of the tree frog.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange activity in the alkaliphile halotolerant cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

The activity of Na⁺/H⁺ exchanger to remove toxic Na⁺ is important for growth of organisms under high salinity. In this study, the halotolerant cyanobacterium Aphanothece halophytica was shown to possess Na⁺/H⁺ exchange activity since exogenously added Na⁺ could dissipate a pre-formed pH gradient, and decrease extracellular pH. Kinetic analysis yielded apparent K _m (Na⁺) and V _max of 20.7 ± 3.1 mM and 3,333 ± 370 nmol H⁺ min⁻¹ mg⁻¹, respectively. For cells grown under salt-stress condition, the apparent K _m (Na⁺) and V _max was 18.3 ± 3.5 mM and 3,703 ± 350 nmol H⁺ min⁻¹ mg⁻¹, respectively. Three cations with decreasing efficiency namely Li⁺, Ca²⁺, and K⁺ were also able to dissipate pH gradient. Only marginal exchange activity was observed for Mg²⁺. The exchange activity was strongly inhibited by Na⁺-gradient dissipators, monensin, and sodium ionophore as well as by CCCP, a protonophore. A. halophytica showed high Na⁺/H⁺ exchange activity at neutral and alkaline pH up to pH 10. Cells grown at pH 7.6 under high salinity exhibited higher Na⁺/H⁺ exchange activity than those grown under low salinity during 15 days of growth suggesting a role of Na⁺/H⁺ exchanger for salt tolerance in A. halophytica. Cells grown at alkaline pH of 9.0 also exhibited a progressive increase of Na⁺/H⁺ exchange activity during 15 days of growth.     

The Expression Pattern of NAD(P)H Oxidases and the Cyclic Electron Transport Pathway around Photosystem I of Synechocystis sp. PCC6803 Depend on Growth Conditions

Cyclic electron transport and NADH and/or NADPH (NAD(P)H)-oxidizing activities were investigated in Synechocystis sp. PCC6803 grown under various stressed conditions and in ndhB-less (M55) and ycf33-deletion mutants. Activity staining and inhibitor data suggested that the ferredoxin-quinone reductase (FQR) route is the main pathway in ycf33-deletion and high-light (300 μE m^?2 s^?1)-grown cells as well as in M55 cells. The FQR route was highly sensitive to HgCl₂, but not to diphenyleneiodonium (DPI). On the other hand, cells grown under low CO₂ (0.03%) or normal (100 μE m^?2 s^?1, 3% CO₂) conditions were found perhaps to use the complex I-type NAD(P)H dehydrogenase route, which was found to be highly sensitive to DPI but not to HgCl₂. In high-salt (0.55 M NaCl)-grown cells, the amount of ferredoxin-NADP⁺ oxidoreductase (FNR) increased, and the main cyclic electron flow was perhaps the FNR route. Both DPI and HgCl₂ were strong inhibitors of the FNR route.     

Relationship between expression of the PM H<Superscript>+</Superscript>-ATPase,growth and ion partitioning in the leaves of salt-treated <Emphasis Type="Italic">Medicago</Emphasis> species

The role of the plasma membrane (PM) H⁺-ATPase (E.C. 3.6.1.3) in the plants response to salt stress was studied in the perennial leguminosae forage Medicago arborea L. and its close relative Medicago citrina (Font-Quer) Greuter, a species exposed to saline conditions in its original habitat. Plants were solution cultured for 8 days in 1 or 100 mM NaCl. Leaf growth and CO₂ assimilation were more inhibited by salt in M. arborea than in M. citrina. Both species were able to osmoregulate, and salt-treated plants maintained turgor potentials, with no differences between species. Contrasting ion distribution patterns showed that M. citrina was able to exclude Na⁺ from the leaves more selectively, while M. arborea had a greater buildup of leaf blade Na⁺. Isolation of purified PM and quantification of H⁺-ATPase protein by Western blot analysis against the 46E5B11D5 or AHA3 antibodies showed an increase in response to salt stress in the expanding (92%) and expanded leaves (87%) of M. citrina, while no differences were found in the corresponding leaves of M. arborea. The assay of H⁺-ATPase specific activity of the two leaf types in salinized M. citrina confirmed this increase, as activities increased with 55% and 104% for the expanded and expanding leaves, respectively, while no significant differences were found for either leaf type of salinized M. arborea. A possible role of the increased expression of the PM H⁺-ATPase for leaf expansion and ion exclusion in salt-stressed plants is discussed.     

Theoretical assessment of oscillations in the concentrations of P<Stack><Subscript>700</Subscript><Superscript>+</Superscript></Stack> and intermediates of the Calvin-Benson cycle

A theoretically obtained oscillatory mode of photosynthesis caused by long-wave illumination is discussed. Periodical variations in the operation of photosystem (PS) I, the electron transport chain, and the Calvin-Benson cycle take place in this mode. The changes in PS II operation are not repetitive. Damped oscillations of P₇₀₀⁺ concentration and CO₂ assimilation rate are observed in the theoretical curves. The results of modeling are quite consistent with the P₇₀₀⁺ kinetics obtained earlier by measuring the induction of the PS I EPR signal in leaves at different temperatures.     

Metagenome Cloning and Functional Analysis of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Genes from Keke Salt Lake in China

Na⁺/H⁺ antiporters are ubiquitous membrane proteins and play a central role in cell homeostasis including pH regulation, osmoregulation, and Na⁺/Li⁺ tolerance in bacteria. The microbial communities in extremely hypersaline soil are an important resource for isolating Na⁺/H⁺ antiporter genes. A metagenomic library containing 35,700 clones was constructed by using genomic DNA obtained from the hypersaline soil samples of Keke Salt Lake in Northwest of China. Two Na⁺/H⁺ antiporters, K1-NhaD, and K2-NhaD belonging to NhaD family, were screened and cloned from this metagenome by complementing the triple mutant Escherichia coli strain KNabc (nhaA ⁻ , nhaB ⁻ , chaA ⁻) in medium containing 0.2 M NaCl. K1-NhaD and K2-NhaD have 75.5% identity at the predicted amino acid sequence. K1-NhaD has 78% identity with Na⁺/H⁺ antiporter NhaD from Halomonas elongate at the predicted amino acid sequence. The predicted K1-NhaD is a 53.5 kDa protein (487 amino acids) with 13 transmembrane helices. K2-NhaD has 73% identity with Alkalimonas amylolytica NhaD. The predicted K2-NhaD is a 55 kDa protein (495 amino acids) with 12 transmembrane helices. Both K1-NhaD and K2-NhaD could make the triple mutant E. coli KNabc (nhaA ⁻ , nhaB ⁻ , chaA⁻) grow in the LBK medium containing 0.2–0.6 M Na⁺ or with 0.05–0.4 M Li⁺. Everted membrane vesicles prepared from E. coli KNabc cells carrying K1-NhaD or K2-NhaD exhibited Na⁺/H⁺ and Li⁺/H⁺ antiporter activities which were pH-dependent with the highest activity at pH 9.5. Little K⁺/H⁺ antiporter activity was also detected in vesicles form E. coli KNabc carrying K1-NhaD or K2-NhaD.     

Isovaleric Acid Reduces Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity in Synaptic Membranes from Cerebral Cortex of Young Rats

1. Patients affected by isovaleric acidemia (IVAcidemia) suffer from acute episodes of encephalopathy. However, the mechanisms underlying the neuropathology of this disease are poorly known. The objective of the present study was to investigate the in vitro effects of the metabolites that predominantly accumulate in IVAcidemia, namely isovaleric acid (IVA), 3-hydroxyisovaleric acid (3-OHIVA) and isovalerylglycine (IVG), on important parameters of energy metabolism, such as ¹⁴CO₂ production from acetate and the activities of the respiratory chain complexes I–IV, creatine kinase and Na⁺, K⁺-ATPase in synaptic plasma membranes from cerebral cortex homogenates of 30-day-old rats. 2. We observed that 3-OHIVA acid and IVG did not affect all the parameters analyzed. Similarly, ¹⁴CO₂ production from acetate (Krebs cycle activity), the activities of creatine kinase, and of the respiratory chain complexes was not modified by IVA. In contrast, IVA exposition to cortical homogenates provoked a marked inhibition of Na⁺, K⁺-ATPase activity. However, this activity was not changed when IVA was directly exposed to purified synaptic plasma membranes, suggesting an indirect effect of this organic acid on the enzyme. Furthermore, pretreatment of cortical homogenates with α-tocopherol and creatine totally prevented IVA-induced inhibition on Na⁺, K⁺-ATPase activity from synaptic plasma membranes, whereas glutathione (GSH) and the NO synthase inhibitor N^ω-nitro-l-arginine methyl ester (L-NAME) did not alter this inhibition. 3. These data indicate that peroxide radicals were probably involved in this inhibitory effect. Since Na⁺, K⁺-ATPase is a critical enzyme for normal brain development and functioning and necessary to maintain neuronal excitability, it is presumed that the inhibitory effect of IVA on this activity may be involved in the pathophysiology of the neurological dysfunction of isovaleric acidemic patients.     

Effects of elevated CO<Subscript>2</Subscript> and/or O<Subscript>3</Subscript> on hormone IAA in needles of Chinese pine

This study examined the effects of season-long exposure of Chinese pine (Pinus tabulaeformis) to elevated carbon dioxide (CO₂) and/or ozone (O₃) on indole-3-acetic acid (IAA) content, activities of IAA oxidase (IAAO) and peroxidase (POD) in needles. Trees grown in open-top chambers (OTC) were exposed to control (ambient O₃, 55 nmol mol⁻¹ + ambient CO₂, 350 μmol mol⁻¹, CK), elevated CO₂ (ambient O₃ + high CO₂, 700 μmol mol⁻¹, EC) and elevated O₃ (high O₃, 80 ± 8 nmol mol⁻¹ + ambient CO₂, EO) OTCs from 1 June to 30 September. Plants grown in elevated CO₂ OTC had a growth increase of axial shoot and needle length, compared to control, by 20% and 10% respectively, while the growth in elevated O₃ OTC was 43% and 7% less respectively, than control. An increase in IAA content and POD activity and decrease in IAAO activity were observed in trees exposed to elevated CO₂ concentration compared with control. Elevated O₃ decreased IAA content and had no significant effect on IAAO activity, but significantly increased POD activity. When trees pre-exposed to elevated CO₂ were transferred to elevated O₃ (EC–EO) or trees pre-exposed to elevated O₃ were transferred to elevated CO₂ (EO–EC), IAA content was lower while IAAO activity was higher than that transferred to CK (EC–CK or EO–CK), the change in IAA content was also related to IAAO activity. The results indicated that IAAO and POD activities in Chinese pine needles may be affected by the changes in the atmospheric environment, resulting in the change of IAA metabolism which in turn may cause changes in Chinese pine’s growth. An erratum to this article can be found at     

Enhanced rates of P700<Superscript>+</Superscript> dark-reduction in leaves of<Emphasis Type="Italic"> Cucumis sativus</Emphasis> L. photoinhibited at chilling temperature

The changes in electron transport within photosystem I (PSI) were studied in detached leaves of Cucumis sativus L. during the course of irradiation with moderate white light (300 mol photons m^–2 s^–1) at 4°C. When intact leaves were exposed to the combination of moderate light and low temperature, the amplitude of far-red light-induced P700 absorbance changes at 820 nm (A₈₂₀), a relative measure of PSI, progressively decreased as the light treatment time increased. Almost no oxidation of P700 was noticeable after 5 h. Methyl viologen accelerated the oxidation of P700 to a steady-state level and also increased the magnitudes of A₈₂₀ changes in photoinhibited leaves, reflecting the rapid removal of electrons from native carriers. Photoinhibition under moderate light and chilling temperature also accelerated the rate of P700⁺ reduction after far-red light excitation as the half-times of the two exponential components of P700⁺ decay curves decreased relative to the control ones. A detailed analysis of the kinetics of P700⁺ reduction using diuron alone or the combination of diuron and methyl viologen strongly favours an increased rate of electron donation from stromal reductants to PSI through the plastoquinone pool following photoinhibitory treatment. Importantly, the marked acceleration of P700⁺ re-reduction is the consequence of the irradiation of leaf segments at low temperature and not caused by chilling stress alone.Abbreviations A ₀ and A ₁ Primary acceptor chlorophyll and secondary electron acceptor phylloquinone - FR Far-red light - F _X , F _A , and F _B Iron–sulfur centers - MT Multiple-turnover flash - MV Methyl viologen - Ndh NAD(P)H-dehydrogenase - PQ Plastoquinone - PS Photosystem - P700 Reaction-center chlorophyll of PSI - ST Single-turnover flash     

Co-regulation of dark and light reactions in three biochemical subtypes of C<Subscript>4</Subscript> species

Regulation of light harvesting in response to changes in light intensity, CO₂ and O₂ concentration was studied in C₄ species representing three different metabolic subtypes: Sorghum bicolor (NADP-malic enzyme), Amaranthus edulis (NAD-malic enzyme), and Panicum texanum (PEP-carboxykinase). Several photosynthetic parameters were measured on the intact leaf level including CO₂ assimilation rates, O₂ evolution, photosystem II activities, thylakoid proton circuit and dissipation of excitation energy. Gross rates of O₂ evolution ( J_\textO2 J_{{{\text{O}}_{2} }} , measured by analysis of chlorophyll fluorescence), net rates of O₂ evolution and CO₂ assimilation responded in parallel to changes in light and CO₂ levels. The C₄ subtypes had similar energy requirements for photosynthesis since there were no significant differences in maximal quantum efficiencies for gross rates of O₂ evolution (average value = 0.072 O₂/quanta absorbed, ~14 quanta per O₂ evolved). At saturating actinic light intensities, when photosynthesis was suppressed by decreasing CO₂, ATP synthase proton conductivity (g _H ⁺) responded strongly to changes in electron flow, decreasing linearly with J_\textO2 J_{{{\text{O}}_{2} }} , which was previously observed in C₃ plants. It is proposed that g _H ⁺ is controlled at the substrate level by inorganic phosphate availability. The results suggest development of nonphotochemical quenching in C₄ plants is controlled by a decrease in g _H ⁺, which causes an increase in proton motive force by restricting proton efflux from the lumen, rather than by cyclic or pseudocyclic electron flow.     

Fumonisin B<Subscript>1</Subscript>, a sphingoid toxin,is a potent inhibitor of the plasma membrane H<Superscript>+</Superscript>-ATPase

Fumonisin B₁ (FB₁) is an amphipathic toxin produced by the pathogenic fungus Fusarium verticillioides which causes stem, root and ear rot in maize (Zea mays L.). In this work, we studied the action of FB₁ on the plasma membrane H⁺-ATPase (EC 3.6.1.34) from germinating maize embryos, and on the fluidity and lipid peroxidation of these membranes. In maize embryos the toxin at 40 M inhibited root elongation by 50% and at 30 M decreased medium acidification by about 80%. Irrespective of the presence and absence of FB₁, the H⁺-ATPase in plasma membrane vesicles exhibited non-hyperbolic saturation kinetics by ATPH-Mg, with Hill number of 0.67. Initial velocity studies revealed that FB₁ is a total uncompetitive inhibitor of this enzyme with an inhibition constant value of 17.5±1 M. Thus FB₁ decreased V_max and increased the apparent affinity of the enzyme for ATP-Mg to the same extent. Although FB₁ increased the fluidity at the hydrophobic region of the membrane, no correlation was found with its effect on enzyme activity, since both effects showed different FB₁-concentration dependence. Peroxidation of membrane lipids was not affected by the toxin. Our results suggest that, under in vivo conditions, the plasma membrane H⁺-ATPase is a potentially important target of the toxin, as it is inhibited not only by FB₁ but also by its structural analogs, the sphingoid intermediates, which accumulate upon the inhibition of sphinganine N-acyltransferase by this toxin.     

The peroxidase activity of cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from spinach chloroplasts

The cytochrome b ₆ f (Cyt b ₆ f) complex, which functions as a plastoquinol-plastocyanin oxidoreductase and mediates the linear electron flow between photosystem II (PSII) and photosystem I (PSI) and the cyclic electron flow around PSI, was isolated from spinach (Spinacia oleracea L.) chloroplasts using n-octyl-β-D-glucopyranoside (β-OG). The preparation was also able to catalyze the peroxidase-like reaction in the presence of hydrogen peroxide (H₂O₂) and guaiacol. The optimal conditions for peroxidase activity of the preparation included: pH 3.6, ionic strength 0.1, and temperature 35°C. The apparent Michaelis constant (K _m) values for H₂O₂ and guaiacol were 50 mM and 2 mM, respectively. The bimolecular rate constant (k _obs) was about 26 M⁻¹ s⁻¹ and the turnover number (K _cat) was about 60 min⁻¹ (20 mM guaiacol, 100 mM sodium phosphate, pH 3.6, 25°C, [H₂O₂]<100mM). These parameters were similar to those of several other heme-containing proteins, such as myoglobin and Cyt c.     

Functional Characterization of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchangers in Primary Cultures of Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. We previously reported Na⁺/H⁺ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I_sc, (11.6 ± 0.5 µA · cm^–2), potential differences, V_t (2.1 ± 0.2 mV), and resistance, R_t (169 ± 12 · cm²). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable ²²Na⁺ uptake under a H⁺ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of ²²Na⁺ uptake was 21.4 ± 1.3 nmol · mg prot^–1 · min^–1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (V_max 9.2 ± 0.3 nmol · mg prot^–1 · min^–1; K_m 11.4 ± 1.4 mM Na⁺). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na⁺ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.     

Stimulation of Transepithelial Na<Superscript>+</Superscript> Current by Extracellular Gd<Superscript>3+</Superscript> in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Alveolar Epithelium

In the present study we investigated the effect of extracellular gadolinium on amiloride-sensitive Na⁺ current across Xenopus alveolar epithelium by Ussing chamber experiments and studied its direct effect on epithelial Na⁺ channels with the patch-clamp method. As observed in various epithelia, the short-circuit current (I _sc) and the amiloride-sensitive Na⁺ current (I _ami) across Xenopus alveolar epithelium was downregulated by high apical Na⁺ concentrations. Apical application of gadolinium (Gd³⁺) increased I _sc in a dose-dependent manner (EC ₅₀ = 23.5 µM). The effect of Gd³⁺ was sensitive to amiloride, which indicated the amiloride-sensitive transcellular Na⁺ transport to be upregulated. Benz-imidazolyl-guanidin (BIG) and p-hydroxy-mercuribenzonic-acid (PHMB) probably release apical Na⁺ channels from Na⁺-dependent autoregulating mechanisms. BIG did not stimulate transepithelial Na⁺ currents across Xenopus lung epithelium but, interestingly, it prevented the stimulating effect of Gd³⁺ on transepithelial Na⁺ transport. PHMB increased I _sc and this stimulation was similar to the effect of Gd³⁺. Co-application of PHMB and Gd³⁺ had no additive effects on I _sc. In cell-attached patches on Xenopus oocytes extracellular Gd³⁺ increased the open probability (NP _o) of Xenopus epithelial sodium channels (ENaC) from 0.72 to 1.79 and decreased the single-channel conductance from 5.5 to 4.6 pS. Our data indicate that Xenopus alveolar epithelium exhibits Na⁺-dependent non-hormonal control of transepithelial Na⁺ transport and that the earth metal gadolinium interferes with these mechanisms. The patch-clamp experiments indicate that Gd³⁺ directly modulates the activity of ENaCs.     

Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Activity in the Posterior Gills of the Blue Crab, <Emphasis Type="Italic">Callinectes ornatus</Emphasis> (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.     

Neurochemical Evidence that Pristanic Acid Impairs Energy Production and Inhibits Synaptic Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity in Brain of Young Rats

Pristanic acid (Prist) accumulates in some peroxisomal disorders characterized by neurologic dysfunction and brain abnormalities. The present work investigated the in vitro effects of Prist on important parameters of energy metabolism in brain cortex of young rats. CO₂ production from labeled acetate and the activities of the respiratory chain complexes I–IV, creatine kinase and synaptic Na⁺, K⁺-ATPase were measured. Prist decreased CO₂ production and the activities of complexes I, II and II–III. Prist also reduced Na⁺, K⁺-ATPase activity, but did not affect the activity of creatine kinase. Considering the importance of the citric acid cycle and the electron flow through the respiratory chain for brain energy production and of Na⁺, K⁺-ATPase for the maintenance of membrane potential, the present data indicate that Prist compromises brain bioenergetics and neurotransmission. It is presumed that these pathomechanisms may be involved in the neurological damage found in patients affected by disorders in which Prist accumulates.