Effect of Hypoxia on Na+-K+-Cl− Cotransport in Cultured Brain Capillary Endothelial Cells of the Rat

Abstract: The effect of hypoxia on Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity in cultured rat brain capillary endothelial cells (RBECs) was investigated by measuring ⁸⁶Rb⁺ uptake as a tracer for K⁺. RBECs expressed both Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity (4.6 and 5.5 nmol/mg of protein/min, respectively). Hypoxia (24 h) decreased cellular ATP content by 43.5% and reduced Na⁺,K⁺-ATPase activity by 38.9%, whereas it significantly increased Na⁺-K⁺-Cl^? cotransport activity by 49.1% in RBECs. To clarify further the mechanism responsible for these observations, the effect of oligomycin-induced ATP depletion on these ion transport systems was examined. Exposure of RBECs to oligomycin led to a time-dependent decrease of cellular ATP content (by ～65%) along with a complete inhibition of Na⁺,K⁺-ATPase and a coordinated increase of Na⁺-K⁺-Cl^? cotransport activity (up to 100% above control values). Oligomycin augmentation of Na⁺-K⁺-Cl^? cotransport activity was not observed in the presence of 2-deoxy-d -glucose (a competitive inhibitor of glucose transport and glycolysis) or in the absence of glucose. These results strongly suggest that under hypoxic conditions when Na⁺,K⁺-ATPase activity is reduced, RBECs have the ability to increase K⁺ uptake through Na⁺-K⁺-Cl^? cotransport.     

Modification of isolated subunit c of the F1F0-ATPase from Propionigenium modestum by dicyclohexylcarbodiimide

Subunit c of the F₁F₀-ATPase from Propionigenium modestum was extracted from the particulate cell fraction with chloroform/methanol. The protein was further purified by carboxymethyl cellulose chromatography and anion exchange HPLC in the organic solvent. SDS-PAGE of the purified protein indicated a single stained protein band migrating as expected for the c-subunit. Incubation of isolated subunit c in chlorform/methanol or aqueous buffer containing dodecyl-β- -maltoside with [¹⁴C]dicyclohexylcarbodiimide (DCCD) resulted in the incorporation of radioactivity into the protein. The rate of this reaction depended on the external pH; it was significantly faster in the more acidic than in the alkaline pH range. In the presence of Na⁺ subunit c was partially protected from labeling with [¹⁴C]DCCD at pH 6.1 and at pH 7.5, whereas no protection was evident at pH 5.5. At pH 7.5, the rate of subunit c labeling by [¹⁴C]DCCD in the presence of 20 mM NaCl was about 50% lower than in the absence of Na⁺ ions. The isolated c-subunit therefore apparently retains in part the Na⁺ binding site which, when occupied, diminishes the reactivity of the protein towards DCCD.     

Brain ATP Depletion Induced by Acute Ammonia Intoxication in Rats Is Mediated by Activation of the NMDA Receptor and Na+, K+-ATPase

Abstract: Injection of large doses of ammonia into rats leads to depletion of brain ATP. However, the molecular mechanism leading to ATP depletion is not clear. The aim of the present work was to assess whether ammonium-induced depletion of ATP is mediated by activation of the NMDA receptor. It is shown that injection of MK-801, an antagonist of the NMDA receptor, prevented ammonia-induced ATP depletion but did not prevent changes in glutamine, glutamate, glycogen, glucose, and ketone bodies. Ammonia injection increased Na⁺,K⁺-ATPase activity by 76%. This increase was also prevented by previous injection of MK-801. The molecular mechanism leading to activation of the ATPase was further studied. Na⁺,K⁺-ATPase activity in samples from ammonia-injected rats was normalized by "in vitro" incubation with phorbol 12-myristate 13-acetate, an activator of protein kinase C. The results obtained suggest that ammonia-induced ATP depletion is mediated by activation of the NMDA receptor, which results in decreased protein kinase C-mediated phosphorylation of Na⁺,K⁺-ATPase and, therefore, increased activity of the ATPase and increased consumption of ATP.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

The involvement of a vanadate-sensitive ATPase in plasma membranes of a salt tolerant cyanobacterium

Plasma membranes of the marine cyanobacterium Spirulina subsalsa were tested for ATPase activity, and for involvement in salt stress. Transition of cells from saline to hypersaline medium enhances the respiratory activity associated with extrusion of Na⁺ and Cl⁻, and persisting salt stress induces synthesis of respiratory enzymes in the plasma membranes. The membranes possess an ATPase, specific for ATP and Mg²⁺ and sensitive to orthovanadate and dicyclohexylcarbodiimide. Immunoblot analysis of plasma membrane polypeptides from Spirulina subsalsa with anti- Arabidopsis H⁺-ATPase serum identified a single polypeptide of 100 kDa, which cross-reacted with the antibodies. An unusual feature of this ATPase is a specific stimulation by Na⁺ ions. Prolonged adaptation of S. subsals cells to hypersaline conditions induced an increase in ATPase activity in subsequent plasma membrane preparations, as well as a higher content of the 100 kDa polypeptide. It is suggested that the ATPase investigated is an H⁺-pump, which is involved in extrusion of Na⁺ and in conferring resistance to salt stress.     

Interaction of phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, with the NADPH-binding site of mammalian catalases.

Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity.     

Phosphorylation of the Na,K-ATPase by Ca,phospholipid-dependent andcAMP-dependent protein kinases. Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase

Ca,phospholipid-dependent (PKC) andcAMP-dependent (PKA) protein kinases phosphorylate the -subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol³²P/mol of -subunit, respectively. PKA (in contrast to PKC) phosphorylates the -subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that³²P is incorporated into the N-terminal 41-kDa fragment of the -subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the -subunit molecule. These findings suggest that PKC phosphorylates the -subunit of the Na,K-ATPase within the region restricted by C₃ and T₁ cleavage sites.     

Heterogeneous Na+ Sensitivity of Na+,K+-ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na⁺ sensitivity of whole brain membrane Na⁺,K⁺-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na⁺, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10⁻⁵ and 10⁻⁷ M ), we were able to discriminate Na⁺ sensitivity of Na⁺, K⁺-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na⁺ sensitivity [ K _a of 3.88 ± 0.25 m M Na⁺ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na⁺ sensitivities, a high ( K _a of 4.98 ± 0.2 m M Na⁺ and n of 1.34 ± 0.10) and a low ( K _a of 28 ± 0.5 m M Na⁺ and an n of 1.92 ± 0.18) Na⁺ sensitivity activated above a thresh old (22 ± 0.3 m M Na⁺); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na⁺ sensitivities (apparent K _a values of 3.5 and 20 m M Na⁺). We show that Na⁺ dependence in the absence of ouabain is the result of at least of five Na⁺ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.     

Productivity of outdoor algal cultures in unstable weather conditions

In the outdoor cyclic fed batch cultures of Chlorella pyrenoidosa, some typical growth kinetics patterns in unstable weather conditions were observed. On cloudy days, the biomass output rate (R) was low, but the bioenergetic growth yield (Y) was generally high. In the cloudy morning-sunny afternoon condition, the values of Y were low, especially in the afternoon. In the sunny morning-cloudy afternoon condition, both R and Y were high. A few very high short-term Y values were measured during the cloudy the cloudy afternoon. (c) 1993 Wiley & Sons, Inc.