Insulin effect in vitro on human erythrocyte plasma membrane

Summary The effect of porcine insulin has been tested in vitro on human erythrocyte plasma membrane (Na⁺–K⁺) and Mg²⁺-ATPase activities as well as on membrane fluidity. The results indicate that the hormonal treatment significantly inhibits (Na⁺–K⁺)-ATPase activity, and at the same time decreases membrane fluidity.This investigation has been supported by Consiglio Nazionale delle Richerche, Rome, Italy.     

C-peptide stimulates Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase via activation of ERK1/2 MAP kinases in human renal tubular cells

Proinsulin-connecting peptide (C-peptide) exerts physiological effects partially via stimulation of Na⁺, K⁺-ATPase. We determined the molecular mechanism by which C-peptide stimulates Na⁺, K⁺-ATPase in primary human renal tubular cells (HRTCs). Incubation of the cells with 5 nM human C-peptide at 37°C for 10 min stimulated ⁸⁶Rb⁺ uptake by 40% (p<0.01). The carboxy-terminal pentapeptide was found to elicit 57% of the activity of the intact molecule. In parallel with ouabain-sensitive ⁸⁶Rb⁺ uptake, C-peptide increased subunit phosphorylation and basolateral membrane (BLM) abundance of the Na⁺, K⁺-ATPase ₁ and ₁ subunits. The increase in BLM abundance of the Na⁺, K⁺-ATPase ₁ and ₁ subunits was accompanied by depletion of ₁ and ₁ subunits from the endosomal compartments. C-peptide action on Na⁺, K⁺-ATPase was ERK1/2-dependent in HRTCs. C-peptide-stimulated Na⁺, K⁺-ATPase activation, phosphorylation of ₁-subunit and translocation of ₁ and ₁ subunits to the BLM were abolished by a MEK1/2 inhibitor (20 M PD98059). C-peptide stimulation of ⁸⁶Rb⁺ uptake was also abolished by preincubation of HRTCs with an inhibitor of PKC (1 M GF109203X). C-peptide stimulated phosphorylation of human Na⁺, K⁺-ATPase subunit on Thr-Pro amino acid motifs, which form specific ERK substrates. In conclusion, C-peptide stimulates sodium pump activity via ERK1/2-induced phosphorylation of Thr residues on the subunit of Na⁺, K⁺-ATPase.Received 15 June 2004; received after revision 14 September 2004; accepted 14 September 2004     

Inhibition of hepatic Na+, K+-adenosinetriphosphatase in taurolithocholate-induced cholestasis in the rat

Summary Na⁺, K⁺-adenosinetriphosphatase (Na⁺, K⁺-ATPase) activity was decreased in liver plasma membranes from rats in which cholestasis had been induced by i.v. administration of sodium taurolithocholate (5 moles/100 g b. wt). Incubation of liver plasma membranes with taurolithocholate (10–1300 M) caused significant and dose dependent reductions of Na⁺, K⁺-ATPase activity at taurolithocholate concentrations above 100 M. These findings lend support to the hypothesis that cholestasis induced by monohydroxy bile acids is at least partially the result of an inhibition of hepatic Na⁺, K⁺-ATPase activity.This work was supported by the Swiss National Science Foundation.The authors thank Mr H. Sägesser and Miss B. Schütz for technical assistance.     

Evidence against a role of (Na++K+-ATPase in the alpha-adrenoceptor mediated positive inotropic effect of phenylephrine

Summary Phenylephrine (0.1–100 M) in the presence of 1 M propranolol increased the force of contraction in electrically driven papillary muscles from cats. This presumably alpha-adrenoceptor mediated positive inotropic effect of phenylephrine occurred without any influence on (Na⁺+K⁺-ATPase activity.This work was supported by the Deutsche Forschungsgemeinschaft.     

Imbalance in the activities of alkaline phosphatase and Na+-K+-ATPase in the brain of experimentally induced phenylketonuric squirrels (Funambulus palmarum)

Summary Phenylketonuric squirrels have shown marked inhibition of alkaline phosphatatase in the olfactory lobes and cerebral hemispheres, whereas the Na⁺-K⁺-ATPase remained less altered. In the pathogenesis of phenylketonuria inhibition of alkaline phosphatase at the level of Blood-Brain Barrier (BBB), leads transport system to impaired functioning.This work was done in the Department of Zoology, University of Udaipur, Udaipur-313001, India.     

Na+,K+-ATPase as a docking station: protein–protein complexes of the Na+,K+-ATPase

The Na⁺,K⁺-ATPase, or sodium pump, is well known for its role in ion transport across the plasma membrane of animal cells. It carries out the transport of Na⁺ ions out of the cell and of K⁺ ions into the cell and thus maintains electrolyte and fluid balance. In addition to the fundamental ion-pumping function of the Na⁺,K⁺-ATPase, recent work has suggested additional roles for Na⁺,K⁺-ATPase in signal transduction and biomembrane structure. Several signaling pathways have been found to involve Na⁺,K⁺-ATPase, which serves as a docking station for a fast-growing number of protein interaction partners. In this review, we focus on Na⁺,K⁺-ATPase as a signal transducer, but also briefly discuss other Na⁺,K⁺-ATPase protein–protein interactions, providing a comprehensive overview of the diverse signaling functions ascribed to this well-known enzyme.     

Involvement of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase and its inhibitors in HuR-mediated cytokine mRNA stabilization in lung epithelial cells

Increasing evidence demonstrates that Na⁺, K⁺-ATPase plays an important role in pulmonary inflammation, but the mechanism remains largely unknown. In this study, we used cardiotonic steroids as Na⁺, K⁺-ATPase inhibitors to explore the possible involvement of Na⁺, K⁺-ATPase in pulmonary epithelial inflammation. The results demonstrated that mice after ouabain inhalation developed cyclooxygenase-2-dependent acute lung inflammation. The in vitro experiments further confirmed that Na⁺, K⁺-ATPase inhibitors significantly stimulated cyclooxygenase-2 expression in lung epithelial cells of human or murine origin, the process of which was participated by multiple cis-elements and trans-acting factors. Most importantly, we first described here that Na⁺, K⁺-ATPase inhibitors could evoke a significant Hu antigen R nuclear export in lung epithelial cells, which stabilized cyclooxygenase-2 mRNA by binding with a proximal AU-rich element within its 3′-untranslated region. In conclusion, HuR-mediated mRNA stabilization opens new avenues in understanding the importance of Na⁺, K⁺-ATPase, as well as its inhibitors in inflammation.     

2-Carboxyethylgermanium sesquioxide,a synthetic organogermanium compound,as an inducer of contrasuppressor T cells

2-Carboxyethylgermanium sesquioxide (Ge-132), a synthesized organogermanium compound with immunomodulaing activities, was shown to be an inducer of anti-suppressor T cells in normal mice. The suppressor cell activity of T6S cells, a clone of burn-induced CD8⁺ IL-4-producing suppressor T cells, was clearly inhibited when a mixed lymphocyte-tumor cell reaction of the clone was conducted with splenic mononuclear cells from mice treated orally with a 100 mg/kg dose of Ge-132. The activity of anti-suppressor cells was demonstrated in spleens of mice 2 days after treatment with Ge-132 and reached its peak on day 3. The anti-suppressor cells induced by the compound were of a contrasuppressor T cell-linage, because they were characterized as CD4⁺ CD28⁺ TCR/⁺ Vicia villosa lectin-adherent T cells. These cells produced IFN- but did not produce IL-2, IL-4, IL-6 or IL-10 in their culture fluids. CD4⁺ anti-suppressor T cells induced by Ge-132 may be different from other subsets of CD4⁺ T cells because Th1 and Th2 cells generated in our laboratory did not adhere toVicia villosa lectin-coated petri dishes, and each produced specific cytokines. Th1 cells produced IFN- and IL-2 while Th2 cells produce IL-4 and IL-10 in vitro. These results suggest that Ge-132 may be useful as an inducer of contrasuppressor T cells in immunocompromised individuals bearing suppressor T cells. To eliminate suppressor T cells from immunocompromised hosts may result in improved resistance from various opportunistic infections.     

Dissociation between compensatory renal growth and induction of (Na++K+-ATPase in rat kidney after uninefrectomy

Zusammenfassung Nach Uninephrektomie und bilateraler Adrenalektomie entsteht eine Dissoziation zwischen der Nierenvergrösserung und der (Na⁺+K⁺)-ATPase Aktivität. Es besteht wahrscheinlich kein Zusammenhang zwischen der Nierenvergrösserung und der Induktion von (Na⁺+K⁺)-ATPase. Auch in der vergrösserten Niere sind die Nebennierenhormone notwendig, um die (Na⁺+K⁺)-ATPase zu erhalten.

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The work was supported by a grant from P. Carl Petersen's Fund. Mrs.Birthe Bagge Hansen provided excellent technical assistance.     

Effect of free fatty acids and cholesterol in vitro on liver plasma membrane-bound enzymes

The effect of cholesterol and fatty acid treatment in vitro was tested on rat liver plasma membrane-bound enzymes and lipid fluidity. The observed alterations of membrane fluidity affect both (Na+-K+)-ATPase and Mg2+-ATPase activities but not 5'-nucleotidase; basal adenylate cyclase as well as its hormonal sensitivity were differentially affected by changes of membrane microenvironment.     

The (Na++K+)-ATPase activity in brain of quaking mice

Summary The (Na⁺+K⁺)- and Mg²⁺-dependent ATPase distribution in several brain areas has been investigated in Quaking mutant mice characterized by myelin deficiency. A marked decrease of (Na⁺+K⁺)-ATPase activity has been found in limbic structures, hypothalamus and cerebellum. The Mg²⁺-dependent activity did not change. A possible involvement of the impairment of the (Na⁺+K⁺)-ATPase activity in the seizure susceptibility of this mice is discussed.Chargée de Recherche au CNRS.     

Induction of lymphokine-activated killer cells from nude mouse spleen cells by interleukin-4

The induction of lymphokine-activated killer (LAK) cells from natural killer (NK) lineage cells by interleukin-4 (IL-4) was studied in vitro. Activation of nude mouse spleen cells by IL-4 generated cytotoxic cells, capable of killing NK-sensitive as well as NK-resistant tumor cells. The induction of peak lytic activity was demonstrated after 3 days of culture with IL-4. Surface marker analysis indicated that the majority of precursor cells were aGM1⁺, Thy1^–, and the majority of effector cells were aGM1⁺, Thy1⁺, suggesting that IL-4 induced LAK cells from nude mouse spleen cells were similar to those from normal mouse spleen cells. The induction of nude mouse LAK cells by IL-4 was partially inhibited by anti-IL-4 or anti-interferon (IFN)-, antibody, and it was further inhibited by the combination of two antibodies, suggesting that IFN-, production was associated with LAK induction of NK lineage cells by IL-4.     

Differential chemotactic potential of mouse platelet basic protein for thymocyte subsets

Mouse platelet basic protein (CXCL7/mPBP) was cloned from thymic stromal cells and further identification indicated that it was expressed in thymic monocytes/macrophages (Mo/Ms). Recombinant mPBP was chemoattractive for target cells of polymorphonuclear leucocytes, peritoneal Mo/Ms and splenic lymphocytes with distinct potencies. CXCR2 was identified to be a cognate receptor for mPBP. Mouse thymocyte subsets of CD4^-CD8^- double-negative (DN), CD4⁺CD8⁺ double-positive (DP), CD4⁺CD8^- single-positive (CD4SP) and CD4^-CD8⁺ single-positive (CD8SP) expressed cell surface CXCR2 with different positive percentages and expression levels. mPBP was chemoattractive for thymocyte subsets with the potency order DN>DP> CD8SP>CD4SP, consistent with the levels of CXCR2 expressed on the respective cells. Thus, mPBP in thymus is functionally redundant with chemokine CXCL12/ SDF-1. Moreover, our finding that thymic Mo/Ms can produce mPBP implies that they may have other functions apart from acting as scavengers in thymus.Received 25 March 2004; received after revision 10 May 2004; accepted 8 June 2004     

Effect of mycotoxin (T-2 toxin) on catecholamine and Na+, K+-ATPase levels in rat epididymis

Summary The effect of mycotoxin (T-2 toxin) on catecholamines and Na⁺, K⁺-ATPase activities in rat epididymis has been evaluated. Dopamine and norepinephrine levels were significantly elevated in the caput and corpus regions whereas their levels remained unchanged in the caudal part of the epididymis. Na⁺, K⁺-ATPase activity was significantly increased in all the three regions of rat epididymis as a result of the toxin treatment. These changes may suggest an adverse effect on epididymal functions in rats.     

Aspartate 338 contributes to the cationic specificity and to driver-amino acid coupling in the insect cotransporter KAAT1

To investigate the peculiar ionic specificity of KAAT1, an Na⁺- and K⁺-coupled amino acid cotransporter from Lepidoptera, a detailed analysis of membrane topology predictions was performed, together with sequence comparison with strictly Na⁺-dependent mammalian cotransporters from the same family. The analysis identified aspartate 338, a residue present also in the other cotransporter accepting K⁺ (CAATCH1), but absent in most mammalian transporters that have, instead, an asparagine in the corresponding position. Mutation of D338 in KAAT1 led either to non-functional transporters (D338G, D338C), or to an altered ionic selectivity (D338E, D338N), observable in uptake experiments and in electrophysiological properties. In particular, in D338E, the transport activity, while persisting in the presence of Na⁺, appeared to be completely abolished in the presence of K⁺. D338E also showed uncoupling between transport-associated current and uptake. The opposite mutation in the -aminobutyric acid transporter rGAT-1 (N327D) resulted in complete loss of function. In conclusion, aspartate 338 in KAAT1 appears to be important in allowing K⁺, in addition to Na⁺, to drive the transport mechanism, although other residues in different parts of the protein may also play a role in the complete determination of ionic selectivity.Received 23 September 2003; received after revision 11 November 2003; accepted 25 November 2003     

Na+/NH 4 + co-transport in isolated perfused gills of the Chinese crabEriocheir sinensis acclimated to fresh water

Summary In isolated perfused posterior gills ofE. sinensis acclimated to fresh water, NH ₄ ⁺ may be used as a counter-ion for Na⁺ active transport. This Na⁺/NH ₄ ⁺ coupled transport can, however, only account for a small part of the Na⁺ total active influx.Chargé de Recherches du FNRS-Acknowledgments. This work has been aided by a grant crédit aux chercheurs from the FNRS and by a grant No. 2.4511.76 from the FRFC.     

Studies on the metal-complex of acetyl salicylic acid (aspirin)

Summary The present communication deals with the isolation of acetyl salicylic acid (aspirin) complexes with Bi⁺³, Zn⁺² and UO ₂ ⁺² . The characterization of 12 complexes have been carried out with the help of conductometric, pH metric, elemental analysis and IR spectral studies. Spectrophotometric studies in case of UO ₂ ⁺² (the only colored complex) in range of 4.2 to 5.5 pH show absorption at 490 nm and complex obey Beers Law at the concentration range of 0.01 M to 0.1 M.     

Lack of Na+,K+-ATPase expression in intercalated cells may be compensated by Na+-ATPase: A study on MDCK – C11 cells

The lack of Na⁺,K⁺-ATPase expression in intercalated cells (IC) is an intriguing condition due to its fundamental role in cellular homeostasis. In order to better understand this question we compared the activities of Na⁺,K⁺-ATPase and Na⁺-ATPase in two MDCK cell clones: the C11, with IC characteristics, and the C7, with principal cells (PC) characteristics. The Na⁺,K⁺-ATPase activity found in C11 cells is far lower than in C7 cells and the expression of its β-subunit is similar in both cells. On the other hand, a subset of C11 without α-subunit expression has been found. In C11 cells the Na⁺-ATPase activity is higher than that of the Na⁺,K⁺-ATPase, and it is increased by medium alkalinization, suggesting that it could account for the cellular Na⁺-homeostasis. Although further studies are necessary for a better understanding of these findings, the presence of Na⁺-ATPase may explain the adequate survival of cells that lack Na⁺,K⁺-ATPase. Received 09 July 2008; received after revision 03 August 2008; accepted 12 August 2008     

The partitioning ofΔ 1-tetrahydrocannabinol into erythrocyte membranes in vivo and its effect on membrane fluidity1into erythrocyte membranes in vivo and its effect on membrane fluidity1

Summary ¹-Tetrahydrocannabinol ( ¹-THC) has been quantified directly in erythrocyte membranes from drug-treated mice using gas chromatography/mass spectrometry. Concentrations of approximately 6 ng ¹-THC/mg membrane protein (10^–5 M) were found when effects of the drug on behavior were prevalent. At these concentrations the drug produced a decrease in membrane order as measured by ESR.This work was supported by grants from the Medical Research Council, the Wellcome Trust and the E. P. Abraham Cephalosporin Trust     

Vorgänge bei pH-Änderung und bei Belichtung eines Anthocyanidins

Summary The anthocyanidin investigated here exists below pH 4 as a cationAH ₂ ⁺ (wave length of absorption maximum _max=459 mµ), between pH 5 and 7 in the neutral formAH ( _max=492 mµ) and above pH 8 as an anionA ^– ( _max=537 mµ). At pH 5 the freshly dissolved substance is partially converted into a colourless formBH ( _max=372 mµ) and a chemical equilibrium betweenAH andBH is reached within 1 h. A kinetic study of the process of formation ofBH shows that an intermediate productZ is formed. This process can be reversed by light exposure. It can be concluded from a kinetic investigation by using flash light thatBH is transformed by the absorbing light into a new substanceL ( _max 275 mµ and 225 mµ), andL changes partially intoBH, partially intoZ, which itself is transformed partially intoAH ₂ ⁺ ,AH,A ^–, partially intoBH. The reactionZ AH,AH ₂ ⁺ ,A ^– is proportional to the concentration of protons [H ⁺], the reactionZ BH independent of [H ⁺]. Thus a photochemical production ofAH ₂ ⁺ ,AH,A ^– fromBH is readily obtained in the presence ofH ⁺ and not obtained in the absence ofH ⁺.