Insulin-induced Stimulation of Na+,K+-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Phosphorylation of the α-subunit of Na+,K+-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K+-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K+-ATPase α-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the α-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat α-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive 86Rb uptake in opossum kidney cells expressing mutant rat α1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive 86Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K+-ATPase α-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+,K+-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

Luminal Heterodimeric Amino Acid Transporter Defective in Cystinuria

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b0,+ type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b0,+AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b0,+ amino acid transporter complex. We demonstrate its b0,+-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b0,+AT protein is the product of the gene defective in non-type I cystinuria.

Role of transforming growth factor-α in von Hippel– Lindau (VHL)−/− clear cell renal carcinoma cell proliferation: A possible mechanism coupling VHL tumor suppressor inactivation and tumorigenesis

Mutations of the VHL tumor suppressor gene occur in patients with VHL disease and in the majority of sporadic clear cell renal carcinomas (VHL−/− RCC). Loss of VHL protein function is associated with constitutive expression of mRNAs encoding hypoxia-inducible proteins, such as vascular endothelial growth factor. Overproduction of angiogenic factors might explain why VHL−/− RCC tumors are so highly vascularized, but whether this overproduction is sufficient for oncogenesis still remains unknown. In this report, we examined the activity of transforming growth factor-α (TGF-α), another VHL-regulated growth factor. We show that TGF-α mRNA and protein are hypoxia-inducible in VHL−/− RCC cells expressing reintroduced VHL. In addition to its overexpression by VHL−/− RCC cells, TGF-α can also act as a specific growth-stimulatory factor for VHL−/− RCC cells expressing reintroduced wild-type VHL, as well as primary renal proximal tubule epithelial cells, the likely site of origin of RCC. This role is in contrast to those of other growth factors overexpressed by VHL−/− RCC cells, such as vascular endothelial growth factor and TGF-β1, which do not stimulate RCC cell proliferation. A TGF-α-specific antisense oligodeoxynucleotide blocked TGF-α production in VHL−/− RCC cells, which led to the dependence of those cells on exogenous growth factors to sustain growth in culture. Growth of VHL−/− RCC cells was also significantly reduced by a drug that specifically inhibits the epidermal growth factor receptor, the receptor through which TGF-α stimulates proliferation. These results suggest that the generation of a TGF-α autocrine loop as a consequence of VHL inactivation in renal proximal tubule epithelial cells may provide the uncontrolled growth stimulus necessary for the initiation of tumorigenesis.

Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis.

Kidney cortex is a main target for circulating vitamin B12 (cobalamin) in complex with transcobalamin (TC). Ligand blotting of rabbit kidney cortex with rabbit 125I-TC-B12 and human TC-57Co-B12 revealed an exclusive binding to megalin, a 600-kDa endocytic receptor present in renal proximal tubule epithelium and other absorptive epithelia. The binding was Ca2+ dependent and inhibited by receptor-associated protein (RAP). Surface plasmon resonance analysis demonstrated a high-affinity interaction between purified rabbit megalin and rabbit TC-B12 but no measurable affinity of the vitamin complex for the homologous alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor related protein (LRP). 125I-TC-B12 was efficiently endocytosed in a RAP-inhibitable manner in megalin-expressing rat yolk sac carcinoma cells and in vivo microperfused rat proximal tubules. The radioactivity in the tubules localized to the endocytic compartments and a similar apical distribution in the proximal tubules was demonstrated after intravenous injection of 125I-TC-B12. The TC-B12 binding sites in the proximal tubule epithelium colocalized with megalin as shown by ligand binding to cryosections of rat kidney cortex, and the binding was inhibited by anti-megalin polyclonal antibody, EDTA, and RAP. These data show a novel nutritional dimension of megalin as a receptor involved in the cellular uptake of vitamin B12. The expression of megalin in absorptive epithelia in the kidney and other tissues including yolk sac and placenta suggests a role of the receptor in vitamin B12 homeostasis and fetal vitamin B12 supply.

In vivo suppression of the renal Na+/Pi cotransporter by antisense oligonucleotides.

A 20-mer phosphorothioate oligonucleotide (AS1) was designed to hybridize to the message for the rat kidney sodium phosphate cotransporter NaPi-2 close to the translation initiation site. Single intravenous doses of this oligonucleotide were given to rats maintained on a low phosphorus diet to increase NaPi-2 expression. At 3 days after oligonucleotide infusion, rats receiving 2.5 micromol of AS1 exhibited a reduction in renal NaPi-2 to cyclophilin mRNA ratio by 40% +/- 17%, and rats receiving 7.5 micromol of AS1 exhibited a reduction in NaPi-2 to cyclophilin mRNA ratio by 46% +/- 21%. Reversed-sequence AS1 was without effect. The higher dose of 7.5 micromol of AS1 also reduced the rate of phosphate uptake into renal brush border membrane vesicles and the expression of NaPi-2 protein detected by Western blotting in these vesicles. Reversed sequence AS1 was again without effect on these parameters. These results suggest that systemically infused oligonucleotides can exert antisense effects in the renal proximal tubule.

Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury.

Disruption of the renal proximal tubule (PT) brush border is a prominent early event during ischemic injury to the kidney. The molecular basis for this event is unknown. Within the brush border, ezrin may normally link the cytoskeleton to the cell plasma membrane. Anoxia causes ezrin to dissociate from the cytoskeleton and also causes many cell proteins to become dephosphorylated in renal PTs. This study examines the hypothesis that ezrin dephosphorylation accompanies and may mediate the anoxic disruption of the rabbit renal PT. During normoxia, 73 +/- 3% of the cytoskeleton-associated (Triton-insoluble) ezrin was phosphorylated, but 88 +/- 6% of dissociated (Triton-soluble) ezrin was dephosphorylated. Phosphorylation was on serine/threonine resides, since ezrin was not detectable by an antibody against phosphotyrosine. After 60 min of anoxia, phosphorylation of total intracellular ezrin significantly decreased from 72 +/- 2% to 21 +/- 9%, and ezrin associated with the cytoskeleton decreased from 91 +/- 2% to 58 +/- 2%. Calyculin A (1 microM), the serine/threonine phosphatase inhibitor, inhibited the dephosphorylation of ezrin during anoxia by 57% and also blocked the dissociation of ezrin from the cytoskeleton by 53%. Our results demonstrate that (i) the association of ezrin with the renal microvillar cytoskeleton is correlated with phosphorylation of ezrin serine/threonine residues and (ii) anoxia may cause disruption of the renal brush border by dephosphorylating ezrin and thereby dissociating the brush border membrane from the cytoskeleton.

Overexpression of csk inhibits acid-induced activation of NHE-3.

Opossum kidney OKP cells express an apical membrane Na+/H+ antiporter that is encoded by NHE-3 (for Na+/H+ exchanger 3) and is similar in many respects to the renal proximal tubule apical membrane Na+/H+ antiporter. Chronic incubation of OKP cells in acid medium for 24 hr increases Na+/H(+)-antiporter activity and NHE-3 mRNA abundance. The increase in Na+/H(+)-antiporter activity was not prevented by H7, a protein kinase C/protein kinase A inhibitor, but was prevented by herbimycin A, a tyrosine kinase inhibitor. Incubation of cells in acid medium increased c-src activity, and this was inhibited by herbimycin A. To determine the role of the src family of nonreceptor protein-tyrosine kinases, Csk (for carboxyl-terminal src kinase), a physiologic inhibitor of these kinases, was overexpressed in OKP cells. In three clones overexpressing csk, acid-induced increases in Na+/H(+)-antiporter activity and NHE-3 mRNA abundance were inhibited. In these clones, inhibition of acid activation of Na+/H(+)-antiporter activity paralleled inhibition of acid activation of c-src. Neither herbimycin A nor overexpression of csk inhibited dexamethasone-induced increases in Na+/H(+)-antiporter activity. These studies show that decreases in pH activate c-src and that the src family nonreceptor protein-tyrosine kinases play a key role in acid activation of NHE-3.

Arachidonic acid mediates angiotensin II effects on p21ras in renal proximal tubular cells via the tyrosine kinase-Shc-Grb2-Sos pathway

In kidney epithelial cells, an angiotensin II (Ang II) type 2 receptor subtype (AT2) is linked to a membrane-associated phospholipase A2 (PLA2) and the mitogen-activated protein kinase (MAPK) superfamily. However, the intervening steps in this linkage have not been determined. The aim of this study was to determine whether arachidonic acid mediates Ang II’s effect on p21ras and if so, to ascertain the signaling mechanism(s). We observed that Ang II activated p21ras and that mepacrine, a phospholipase A2 inhibitor, blocked this effect. This activation was also inhibited by PD123319, an AT2 receptor antagonist but not by losartan, an AT1 receptor antagonist. Furthermore, Ang II caused rapid tyrosine phosphorylation of Shc and its association with Grb2. Arachidonic acid and linoleic acid mimicked Ang II-induced tyrosine phosphorylation of Shc and activation of p21ras. Moreover, Ang II and arachidonic acid induced an association between p21ras and Shc. We demonstrate that arachidonic acid mediates linkage of a G protein-coupled receptor to p21ras via Shc tyrosine phosphorylation and association with Grb2/Sos. These observations have important implications for other G protein-coupled receptors linked to a variety of phospholipases.

Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

Chloroperoxidase is a versatile heme enzyme which can cross over the catalytic boundaries of other oxidative hemoproteins and perform multiple functions. Chloroperoxidase, in addition to catalyzing classical peroxidative reactions, also acts as a P450 cytochrome and a potent catalase. The multiple functions of chloroperoxidase must be derived from its unique active site structure. Chloroperoxidase possesses a proximal cysteine thiolate heme iron ligand analogous to the P450 cytochromes; however, unlike the P450 enzymes, chloroperoxidase possesses a very polar environment distal to its heme prosthetic group and contains a glutamic acid residue in close proximity to the heme iron. The presence of a thiolate ligand in chloroperoxidase has long been thought to play an essential role in its chlorination and epoxidation activities; however, the research reported in this paper proves that hypothesis to be invalid. To explore the role of Cys-29, the amino acid residue supplying the thiolate ligand in chloroperoxidase, Cys-29 has been replaced with a histidine residue. Mutant clones of the chloroperoxidase genome have been expressed in a Caldariomyces fumago expression system by using gene replacement rather than gene insertion technology. C. fumago produces wild-type chloroperoxidase, thus requiring gene replacement of the wild type by the mutant gene. To the best of our knowledge, this is the first time that gene replacement has been reported for this type of fungus. The recombinant histidine mutants retain most of their chlorination, peroxidation, epoxidation, and catalase activities. These results downplay the importance of a thiolate ligand in chloroperoxidase and suggest that the distal environment of the heme active site plays the major role in maintaining the diverse activities of this enzyme.

Membrane Tubule-mediated Reassembly and Maintenance of the Golgi Complex Is Disrupted by Phospholipase A2 Antagonists

Although membrane tubules can be found extending from, and associated with, the Golgi complex of eukaryotic cells, their physiological function has remained unclear. To gain insight into the biological significance of membrane tubules, we have developed methods for selectively preventing their formation. We show here that a broad range of phospholipase A2 (PLA2) antagonists not only arrest membrane tubule–mediated events that occur late in the assembly of the Golgi complex but also perturb its normal steady-state tubulovesicular architecture by inducing a reversible fragmentation into separate “mini-stacks.” In addition, we show that these same compounds prevent the formation of membrane tubules from Golgi stacks in an in vitro reconstitution system. This in vitro assay was further used to demonstrate that the relevant PLA2 activity originates from the cytoplasm. Taken together, these results demonstrate that Golgi membrane tubules, sensitive to potent and selective PLA2 antagonists, mediate both late events in the reassembly of the Golgi complex and the dynamic maintenance of its steady-state architecture. In addition, they implicate a role for cytoplasmic PLA2 enzymes in mediating these membrane trafficking events.

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.