The role of reversible phosphorylation in the hormonal control of phenylalanine hydroxylase in isolated rat proximal kidney tubules.
Ontology highlight
ABSTRACT:
SUBMITTER: Richardson SC
PROVIDER: S-EPMC1134225 | biostudies-other | 1993 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Hormonal control of the phosphorylation of phenylalanine hydroxylase was studied by using rat liver cells incubated with [32P]Pi. After immunoprecipitation from cell extracts, the hydroxylase was subjected to proteinase digestion and subsequent sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. V8-proteinase digestion yielded one major 32P-labelled fragment, of approx. 9 kDa. Chymotrypsin digestion gave five 32P-labelled fragments ranging from approx. 39 kDa to approx. 10 kDa. Noradrenaline (10 microM) and glucagon (0.1 microM) enhanced the 32P content of all peptide fragments uniformly. Phorbol ester, in contrast with ionophore A23187, did not stimulate enzyme phosphorylation or enhance phenylalanine metabolism in liver cells. These results are discussed in relation to the nature of the protein kinase(s) that mediate phosphorylation of phenylalanine hydroxylase in liver cells.
| S-EPMC1153054 | biostudies-other
Project description:Intrarenal changes in cytoplasmic calcium levels have a key role in determining pathologic and pharmacologic responses in major kidney diseases. However, cell-specific delivery of calcium-sensitive probes in vivo remains problematic. We generated a transgenic rat stably expressing the green fluorescent protein-calmodulin-based genetically encoded calcium indicator (GCaMP2) predominantly in the kidney proximal tubules. The transposon-based method used allowed the generation of homozygous transgenic rats containing one copy of the transgene per allele with a defined insertion pattern, without genetic or phenotypic alterations. We applied in vitro confocal and in vivo two-photon microscopy to examine basal calcium levels and ligand- and drug-induced alterations in these levels in proximal tubular epithelial cells. Notably, renal ischemia induced a transient increase in cellular calcium, and reperfusion resulted in a secondary calcium load, which was significantly decreased by systemic administration of specific blockers of the angiotensin receptor and the Na-Ca exchanger. The parallel examination of in vivo cellular calcium dynamics and renal circulation by fluorescent probes opens new possibilities for physiologic and pharmacologic investigations.
| S-EPMC4625667 | biostudies-literature
Project description:Autophagy is induced in renal tubular cells during acute kidney injury; however, whether this is protective or injurious remains controversial. We address this question by pharmacologic and genetic blockade of autophagy using mouse models of cisplatin- and ischemia-reperfusion-induced acute kidney injury. Chloroquine, a pharmacological inhibitor of autophagy, blocked autophagic flux and enhanced acute kidney injury in both models. Rapamycin, however, activated autophagy and protected against cisplatin-induced acute kidney injury. We also established a renal proximal tubule-specific autophagy-related gene 7-knockout mouse model shown to be defective in both basal and cisplatin-induced autophagy in kidneys. Compared with wild-type littermates, these knockout mice were markedly more sensitive to cisplatin-induced acute kidney injury as indicated by renal functional loss, tissue damage, and apoptosis. Mechanistically, these knockout mice had heightened activation of p53 and c-Jun N terminal kinase, the signaling pathways contributing to cisplatin acute kidney injury. Proximal tubular cells isolated from the knockout mice were more sensitive to cisplatin-induced apoptosis than cells from wild-type mice. In addition, the knockout mice were more sensitive to renal ischemia-reperfusion injury than their wild-type littermates. Thus, our results establish a renoprotective role of tubular cell autophagy in acute kidney injury where it may interfere with cell killing mechanisms.
| S-EPMC3491167 | biostudies-literature
Project description:Nature exploits cage-like proteins for a variety of biological purposes, from molecular packaging and cargo delivery to catalysis. These cage-like proteins are of immense importance in nanomedicine due to their propensity to self-assemble from simple identical building blocks to highly ordered architecture and the design flexibility afforded by protein engineering. However, delivery of protein nanocages to the renal tubules remains a major challenge because of the glomerular filtration barrier, which effectively excludes conventional size nanocages. Here, we show that DNA-binding protein from starved cells (Dps) - the extremely small archaeal antioxidant nanocage - is able to cross the glomerular filtration barrier and is endocytosed by the renal proximal tubules. Using a model of endotoxemia, we present an example of the way in which proximal tubule-selective Dps nanocages can limit the degree of endotoxin-induced kidney injury. This was accomplished by amplifying the endogenous antioxidant property of Dps with addition of a dinuclear manganese cluster. Dps is the first-in-class protein cage nanoparticle that can be targeted to renal proximal tubules through glomerular filtration. In addition to its therapeutic potential, chemical and genetic engineering of Dps will offer a nanoplatform to advance our understanding of the physiology and pathophysiology of glomerular filtration and tubular endocytosis.
| S-EPMC6715384 | biostudies-literature
Project description:Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge-charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13-Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsin-like enzymes and on the increase in catalytic efficiency.
| S-EPMC2286772 | biostudies-literature
Project description:Diadenosine polyphosphates released into the extracellular environment influence a variety of metabolic and other cellular activities in a wide range of target tissues. Here we have studied the impact of these novel nucleotides on gluconeogenesis in isolated rat proximal tubules. Gluconeogenesis was stimulated following exposure of isolated proximal tubules to a range of adenine-containing nucleotides including ADP, ATP, Ap3A, Ap4A, Ap5A and Ap6A. The concentration-dependence of ATP-, Ap3A- and Ap4A-mediated stimulation of gluconeogenesis was similar and was consistent with a role for these agents in the physiological control of renal metabolism. Nucleotide-stimulated gluconeogenesis was diminished in the presence of agents that interfere with phospholipase C activation or intracellular Ca2+ metabolism, indicative of a role for polyphosphoinositide-mediated Ca2+ mobilization in the mechanism of action of ATP, Ap3A and Ap4A. The characteristics of binding of [2-3H]Ap4A to renal plasma-membrane preparations suggest that Ap4A mediates its effects on proximal tubule gluconeogenesis via interaction with P2y-like purinoceptor(s) also recognized by extracellular ATP.
| S-EPMC1218340 | biostudies-other
Project description:Epigenetic mechanisms may underlie the progression of diabetic kidney disease. Because the kidney is a heterogeneous organ with different cell types, we investigated DNA methylation status of the kidney in a cell type-specific manner. We first identified genes specifically demethylated in the normal proximal tubules obtained from control db/m mice, and next delineated the candidate disease-modifying genes bearing aberrant DNA methylation induced by diabetes using db/db mice. Genes involved in glucose metabolism, including Sglt2, Pck1, and G6pc, were selectively hypomethylated in the proximal tubules in control mice. Hnf4a, a transcription factor regulating transporters for reabsorption, was also selectively demethylated. In diabetic mice, aberrant hypomethylation of Agt, Abcc4, Cyp4a10, Glut5, and Met and hypermethylation of Kif20b, Cldn18, and Slco1a1 were observed. Time-dependent demethylation of Agt, a marker of diabetic kidney disease, was accompanied by histone modification changes. Furthermore, inhibition of DNA methyltransferase or histone deacetylase increased Agt mRNA in cultured human proximal tubular cells. Aberrant DNA methylation and concomitant changes in histone modifications and mRNA expression in the diabetic kidney were resistant to antidiabetic treatment with pioglitazone. These results suggest that an epigenetic switch involving aberrant DNA methylation causes persistent mRNA expression of select genes that may lead to phenotype changes of the proximal tubules in diabetic kidney disease.
| S-EPMC4587689 | biostudies-literature
Project description:The role of polyamines in the control of phenylalanine hydroxylase phosphorylation state and enzymic activity was investigated. Pre-treatment of liver cells with spermine (1 mM) abolishes the glucagon (1 nM)-stimulated increase in hydroxylase phosphorylation. Concurrently there is a decrease in phenylalanine hydroxylation flux, reflecting decreased enzyme activity; 50% inhibition occurs at approx. 10 microM-spermine. These results are discussed in the context of reports concerning the properties of protein phosphatase 2A.
| S-EPMC1146977 | biostudies-other
Project description:Organ shortage leads to an increased utilization of marginal organs which are particularly sensitive to storage-associated damage. Cold incubation and rewarming-induced injury is iron-dependent in many cell types. In addition, a chloride-dependent component of injury has been described. This work examines the injury induced by cold incubation and rewarming in isolated rat renal proximal tubules. The tissue storage solution TiProtec® and a chloride-poor modification, each with and without iron chelators, were used for cold incubation. Incubation was performed 4°C for up to 168 h, followed by rewarming in an extracellular buffer (3 h at 37°C). After 48, 120 and 168 h of cold incubation LDH release was lower in solutions containing iron chelators. After rewarming, injury increased especially after cold incubation in chelator-free solutions. Without addition of iron chelators LDH release showed a tendency to be higher in chloride-poor solutions. Following rewarming after 48 h of cold incubation lipid peroxidation was significantly decreased and metabolic activity was tendentially better in tubules incubated with iron chelators. Morphological alterations included mitochondrial swelling and fragmentation being partially reversible during rewarming. ATP content was better preserved in chloride-rich solutions. During rewarming, there was a further decline of ATP content in the so far best conditions and minor alterations under the other conditions, while oxygen consumption was not significantly different compared to non-stored control tubules. Results show an iron-dependent component of preservation injury during cold incubation and rewarming in rat proximal renal tubules and reveal a benefit of chloride for the maintenance of tubular energy state during cold incubation.
| S-EPMC5495391 | biostudies-literature
Project description:A monoclonal antibody (PH 7), which recognizes the phosphorylated form of phenylalanine hydroxylase from human liver, has been used for the analysis of the enzyme in crude cell extracts from rat. In immunoblot analyses of rat liver cell extracts, the extent of binding of PH 7 closely correlates with the phosphorylation state of phenylalanine hydroxylase, as judged by [32P]Pi incorporation. These observations have made possible the rapid non-radioactive quantification of hormonal effects on phenylalanine hydroxylase phosphorylation state. In particular, the glucagon-dependent phosphorylation of phenylalanine hydroxylase in liver cells was investigated. Epidermal growth factor was shown to modulate this process. In addition, this technique was used to demonstrate, for the first time, that dibutyryl cyclic AMP, unlike the Ca2+ ionophore A23187, stimulates the phosphorylation of phenylalanine hydroxylase in isolated kidney tubules from rat.
| S-EPMC1136920 | biostudies-other