Antibiotic activity of isoxazolylnaphthoquinone imines on mice infected with Staphylococcus aureus

I. ALBESA, P. BOGDANOV, A. ERASO, N.R. SPERANDEO AND M.M. DE BERTORELLO. 1995. The antibiotic activity of new synthetic isoxazolylnaphthoquinone imines was studied. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were resistant to the four compounds studied (MIC > 128 µg ml⁻¹), but Staphylococcus aureus ATCC 25923, ATCC 29213 and 30 clinical isolates of Staph. aureus were inhibited by 2-hydroxy- N -(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (I). This compound diminished bloodstream infection of mice injected i.m. with Staph. aureus; septicaemia decayed significantly when I was applied at the beginning of the infection while when I was given 3 d after bacterial challenge, a significant protection was afforded. Bactericidal activity in serum increased during the 5 h after I was administered i.p.
The acetyl derivative of I had a high MIC but when inoculated orally in mice decreased the Staph. aureus counts in circulation. This protection occurred only when the schedule of administration started close to the bacterial challenge. Antibiotic activity in vivo may be associated with in vitro effects.     

Intracellular sodium sensing: SIK1 network,hormone action and high blood pressure

Sodium is the main determinant of body fluid distribution. Sodium accumulation causes water retention and, often, high blood pressure. At the cellular level, the concentration and active transport of sodium is handled by the enzyme Na⁺,K⁺-ATPase, whose appearance enabled evolving primitive cells to cope with osmotic stress and contributed to the complexity of mammalian organisms. Na⁺,K⁺-ATPase is a platform at the hub of many cellular signaling pathways related to sensing intracellular sodium and dealing with its detrimental excess. One of these pathways relies on an intracellular sodium-sensor network with the salt-inducible kinase 1 (SIK1) at its core. When intracellular sodium levels rise, and after the activation of calcium-related signals, this network activates the Na⁺,K⁺-ATPase and expel the excess of sodium from the cytosol. The SIK1 network also mediates sodium-independent signals that modulate the activity of the Na⁺,K⁺-ATPase, like dopamine and angiotensin, which are relevant per se in the development of high blood pressure. Animal models of high blood pressure, with identified mutations in components of multiple pathways, also have alterations in the SIK1 network. The introduction of some of these mutants into normal cells causes changes in SIK1 activity as well. Some cellular processes related to the metabolic syndrome, such as insulin effects on the kidney and other tissues, also appear to involve the SIK1. Therefore, it is likely that this protein, by modulating active sodium transport and numerous hormonal responses, represents a “crossroad” in the development and adaptation to high blood pressure and associated diseases.     

The 14-3-3 protein translates the NA+,K+-ATPase {alpha}1-subunit phosphorylation signal into binding and activation of phosphoinositide 3-kinase during endocytosis

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the alpha-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na(+),K(+)-ATPase alpha-subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na(+),K(+)-ATPase alpha-subunit. The functional relevance of this interaction was established in opossum kidney cells expressing a 14-3-3 dominant negative mutant, where dopamine failed to decrease Na(+),K(+)-ATPase activity and to promote its endocytosis. The phosphorylated Ser-18 residue within the alpha-subunit N terminus is critical for 14-3-3 binding. Activation of phosphoinositide 3-kinase by dopamine during Na(+),K(+)-ATPase endocytosis requires the binding of the kinase to a proline-rich domain within the alpha-subunit, and this effect was blocked by the presence of a 14-3-3 dominant negative mutant. Thus, the 14-3-3 protein represents a critical linking mechanism for recruiting phosphoinositide 3-kinase to the site of Na(+),K(+)-ATPase endocytosis.     

G-protein-coupled receptor-mediated traffic of Na,K-ATPase to the plasma membrane requires the binding of adaptor protein 1 to a Tyr-255-based sequence in the alpha-subunit

Motion of integral membrane proteins to the plasma membrane in response to G-protein-coupled receptor signals requires selective cargo recognition motifs that bind adaptor protein 1 and clathrin. Angiotensin II, through the activation of AT1 receptors, promotes the recruitment to the plasma membrane of Na,K-ATPase molecules from intracellular compartments. We present evidence to demonstrate that a tyrosine-based sequence (IVVY-255) present within the Na,K-ATPase alpha1-subunit is involved in the binding of adaptor protein 1. Mutation of Tyr-255 to a phenylalanine residue in the Na,K-ATPase alpha1-subunit greatly reduces the angiotensin II-dependent activation of Na,K-ATPase, recruitment of Na,K-ATPase molecules to the plasma membrane, and association of adaptor protein 1 with Na,K-ATPase alpha1-subunit molecules. To determine protein-protein interaction, we used fluorescence resonance energy transfer between fluorophores attached to the Na,K-ATPase alpha1-subunit and adaptor protein 1. Although angiotensin II activation of AT1 receptors induces a significant increase in the level of fluorescence resonance energy transfer between the two molecules, this effect was blunted in cells expressing the Tyr-255 mutant. Thus, results from different methods and techniques suggest that the Tyr-255-based sequence within the NKA alpha1-subunit is the site of adaptor protein 1 binding in response to the G-protein-coupled receptor signals produced by angiotensin II binding to AT1 receptors.     

Agonist-dependent regulation of renal Na+,K+-ATPase activity is modulated by intracellular sodium concentration.

We tested the hypothesis that the level of intracellular sodium modulates the hormonal regulation of the Na(+),K(+)-ATPase activity in proximal tubule cells. By using digital imaging fluorescence microscopy of a sodium-sensitive dye, we determined that the sodium ionophore monensin induced a dose-specific increase of intracellular sodium. A correspondence between the elevation of intracellular sodium and the level of dopamine-induced inhibition of Na(+),K(+)-ATPase activity was determined. At basal intracellular sodium concentration, stimulation of cellular protein kinase C by phorbol 12-myristate 13-acetate (PMA) promoted a significant increase in Na(+),K(+)-ATPase activity; however, this activation was gradually reduced as the concentration of intracellular sodium was increased to become a significant inhibition at concentrations of intracellular sodium higher than 16 mm. Under these conditions, PMA and dopamine share the same signaling pathway to inhibit the Na(+),K(+)-ATPase. The effects of PMA and dopamine on the Na(+),K(+)-ATPase activity and the modulation of these effects by different intracellular sodium concentrations were not modified when extracellular and intracellular calcium were almost eliminated. These results suggest that the level of intracellular sodium modulates whether hormones stimulate, inhibit, or have no effect on the Na(+),K(+)-ATPase activity leading to a tight control of sodium reabsorption.     

Growth impairment after TBI of leukemia survivors children: a model- based investigation

Background

Children receiving Total Body Irradiation (TBI) in preparation for Hematopoietic Stem Cell Transplantation (HSCT) are at risk for Growth Hormone Deficiency (GHD), which sometimes severely compromises their Final Height (FH). To better represent the impact of such therapies on growth we apply a mathematical model, which accounts both for the gompertzian-like growth trend and the hormone-related ‘spurts’, and evaluate how the parameter values estimated on the children undergoing TBI differ from those of the matched normal population.

Methods

25 patients long-term childhood lymphoblastic and myeloid acute leukaemia survivors followed at Pediatric Onco-Hematology, Stem Cell Transplantation and Cellular Therapy Division, Regina Margherita Children’s Hospital (Turin, Italy) were retrospectively analysed for assessing the influence of TBI on their longitudinal growth and for validating a new method to estimate the GH therapy effects. Six were treated with GH therapy after a GHD diagnosis.

Results

We show that when TBI was performed before puberty overall growth and pubertal duration were significantly impaired, but such growth limitations were completely reverted in the small sample (6 over 25) of children who underwent GH replacement therapies.

Conclusion

Since in principle the model could account for any additional growth ‘spurt’ induced by therapy, it may become a useful ‘simulation’ tool for paediatricians for comparing the predicted therapy effectiveness depending on its timing and dosage.

     

Tyrosine 537 within the Na+,K+-ATPase alpha-subunit is essential for AP-2 binding and clathrin-dependent endocytosis

In renal epithelial cells endocytosis of Na(+),K(+)-ATPase molecules is initiated by phosphorylation of its alpha(1)-subunit, leading to activation of phosphoinositide 3-kinase and adaptor protein-2 (AP-2)/clathrin recruitment. The present study was performed to establish the identity of the AP-2 recognition domain(s) within the Na(+),K(+)-ATPase alpha(1)-subunit. We identified a conserved sequence (Y(537)LEL) within the alpha(1)-subunit that represents an AP-2 binding site. Binding of AP-2 to the Na(+),K(+)-ATPase alpha(1)-subunit in response to dopamine (DA) was increased in OK cells stably expressing the wild type rodent alpha-subunit (OK-WT), but not in cells expressing the Y537A mutant (OK-Y537A). DA treatment was associated with increased alpha(1)-subunit abundance in clathrin vesicles from OK-WT but not from OK-Y537A cells. In addition, this mutation also impaired the ability of DA to inhibit Na(+),K(+)-ATPase activity. Because phorbol esters increase Na(+),K(+)-ATPase activity in OK cells, and this effect was not affected by the Y537A mutation, the present results suggest that the identified motif is specifically required for DA-induced AP-2 binding and Na(+),K(+)-ATPase endocytosis.     

Dopamine-induced exocytosis of Na,K-ATPase is dependent on activation of protein kinase C-epsilon and -delta

The purpose of this study was to define mechanisms by which dopamine (DA) regulates the Na,K-ATPase in alveolar epithelial type 2 (AT2) cells. The Na,K-ATPase activity increased by twofold in cells incubated with either 1 μM DA or a dopaminergic D₁ agonist, fenoldopam, but not with the dopaminergic D₂ agonist quinpirole. The increase in activity paralleled an increase in Na,K-ATPase α1 and β1 protein abundance in the basolateral membrane (BLM) of AT2 cells. This increase in protein abundance was mediated by the exocytosis of Na,K-pumps from late endosomal compartments into the BLM. Down-regulation of diacylglycerol-sensitive types of protein kinase C (PKC) by pretreatment with phorbol 12-myristate 13-acetate or inhibition with bisindolylmaleimide prevented the DA-mediated increase in Na,K-ATPase activity and exocytosis of Na,K-pumps to the BLM. Preincubation of AT2 cells with either 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983), a selective inhibitor of PKC-δ, or isozyme-specific inhibitor peptides for PKC-δ or PKC-ε inhibited the DA-mediated increase in Na,K-ATPase. PKC-δ and PKC-ε, but not PKC-α or -β, translocated from the cytosol to the membrane fraction after exposure to DA. PKC-δ– and PKC-ε–specific peptide agonists increased Na,K-ATPase protein abundance in the BLM. Accordingly, dopamine increased Na,K-ATPase activity in alveolar epithelial cells through the exocytosis of Na,K-pumps from late endosomes into the basolateral membrane in a mechanism-dependent activation of the novel protein kinase C isozymes PKC-δ and PKC-ε.     

Salt-inducible kinase 1 is present in lung alveolar epithelial cells and regulates active sodium transport

Salt-inducible kinase 1 (SIK1) in epithelial cells mediates the increases in active sodium transport (Na⁺, K⁺-ATPase-mediated) in response to elevations in the intracellular concentration of sodium. In lung alveolar epithelial cells increases in active sodium transport in response to β-adrenergic stimulation increases pulmonary edema clearance. Therefore, we sought to determine whether SIK1 is present in lung epithelial cells and to examine whether isoproterenol-dependent stimulation of Na⁺, K⁺-ATPase is mediated via SIK1 activity. All three SIK isoforms were present in airway epithelial cells, and in alveolar epithelial cells type 1 and type 2 from rat and mouse lungs, as well as from human and mouse cell lines representative of lung alveolar epithelium. In mouse lung epithelial cells, SIK1 associated with the Na⁺, K⁺-ATPase α-subunit, and isoproterenol increased SIK1 activity. Isoproterenol increased Na⁺, K⁺-ATPase activity and the incorporation of Na⁺, K⁺-ATPase molecules at the plasma membrane. Furthermore, those effects were abolished in cells depleted of SIK1 using shRNA, or in cells overexpressing a SIK1 kinase-deficient mutant. These results provide evidence that SIK1 is present in lung epithelial cells and that its function is relevant for the action of isoproterenol during regulation of active sodium transport. As such, SIK1 may constitute an important target for drug discovery aimed at improving the clearance of pulmonary edema.