Downregulation of bradykinin B2 receptor in human  fibroblasts during prolonged agonist exposure

Sustained activation of G protein-coupled receptors results in an attenuation of cellular responses, a phenomenon termed desensitization. Whereas mechanisms for rapid desensitization of ligand-receptor-G protein-effector systems are relatively well characterized, much less is known about long-term adaptation processes that occur in the continuous presence of an agonist. Here we have studied the fate of endogenously expressed bradykinin B2 receptors on human fibroblasts during prolonged agonist treatment. Stimulation with bradykinin for up to 24 h resulted in a 50% reduction of surface binding sites that was paralleled by a similar decrease of total B2 receptor protein followed by Western blotting using monoclonal antibodies to the B2 receptor. Whereas B2 receptor mRNA levels did not change during 24 h of agonist treatment, B2receptor de novo synthesis was attenuated by 35–50%, indicating translational control of B2 receptor levels. Furthermore, the half-life of B2 receptor protein was shortened by 20–40% as shown by 35S-labeled pulse-chase and immunoprecipitation experiments. This study demonstrates that bradykinin B2 receptor expression during long-term agonist treatment is primarily regulated on the (post)translational level, i.e., by attenuation of de novo synthesis and by reduction of receptor stability.

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Abstract 112: Myeloid Mineralocorticoid Receptor Controls Inflammatory and Fibrotic Responses After Renal Ischemic Injury via Macrophage Interleukin-4 Receptor

Hypertension ◽

10.1161/hyp.70.suppl_1.112 ◽

2017 ◽

Vol 70 (suppl_1) ◽

Author(s):

Jonatan Barrera-Chimal ◽

Sebastian M Lechner ◽

Soumaya E Moghrabi ◽

Peter Kolkhof ◽

Frédéric Jaisser

Keyword(s):

Mineralocorticoid Receptor ◽

De Novo ◽

Interstitial Fibrosis ◽

Macrophage Polarization ◽

Peritoneal Macrophages ◽

Renal Ischemia ◽

Receptor Expression ◽

Interleukin 4 ◽

Mrna Levels ◽

Kidney Fibrosis

Introduction: Patients who survive an episode of acute kidney injury (AKI) are at high risk of de novo chronic kidney disease (CKD) development. Pharmacological mineralocorticoid receptor (MR) antagonism is useful to prevent CKD after a single episode of ischemic AKI in the rat. Objective: Test the involvement of myeloid MR in the development of kidney fibrosis after an ischemic AKI episode. Methods: We included 18 male C57/B6 mice that were divided in: sham, renal ischemia for 22.5 min and IR plus treatment with the non-steroidal MR antagonist finerenone (10 mg/kg) at -48, -24 and -1 h before IR. MR inactivation in myeloid cells (MR MyKO ) was achieved by crossing mice with the MR alleles flanked by loxP sites (MR f/f ) with mice expressing the Cre recombinase under the LysM promoter activity. In MR f/f and MR MyKO mice we induced renal IR of 22.5 min or sham surgery. The mice were followed-up during 4 weeks to test for AKI to CKD transition. In another set of mice, the macrophages were sorted from kidneys after 24 h of reperfusion and flow cytometry characterization or mRNA extraction was performed. Thyoglycolate elicited peritoneal macrophages were used for in vitro studies. Results: The progression of AKI to CKD after 4 weeks of renal ischemia in the untreated C57/B6 and MR f/f mice was characterized by a 50% increase in plasma creatinine, a 2-fold increase in the mRNA levels of TGF-β and fibronectin as well as by severe tubule-interstitial fibrosis. The mice that received finerenone or MR MyKO mice were protected against these alterations. Increased expression of M2-anti-inflamatory markers in kidney-isolated macrophages from finerenone-treated or MR MyKO mice was observed. The inflammatory population of Ly6C high macrophages was reduced by 50%. In peritoneal macrophages in culture, MR inhibition promoted increased IL-4 receptor expression and activation, facilitating macrophage polarization to an M2 phenotype. Conclusion: MR antagonism or myeloid MR deficiency facilitates macrophage polarization to a M2, anti-inflammatory phenotype after kidney IR, preventing maladaptive repair and chronic kidney fibrosis and dysfunction. MR inhibition acts through the modulation of IL-4 receptor signaling to facilitate macrophage phenotype switching.

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Requirement of de novo synthesis of pyruvate carboxylase in long-term succinic acid production in Corynebacterium glutamicum

Applied Microbiology and Biotechnology ◽

10.1007/s00253-020-10556-5 ◽

2020 ◽

Vol 104 (10) ◽

pp. 4313-4320 ◽

Cited By ~ 2

Author(s):

Hiroto Uchikura ◽

Kazuaki Ninomiya ◽

Kenji Takahashi ◽

Yota Tsuge

Keyword(s):

Succinic Acid ◽

Corynebacterium Glutamicum ◽

Acid Production ◽

Pyruvate Carboxylase ◽

De Novo ◽

De Novo Synthesis ◽

Succinic Acid Production

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Arabidopsis REI-LIKE proteins activate ribosome biogenesis during cold acclimation

Scientific Reports ◽

10.1038/s41598-021-81610-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Bo Eng Cheong ◽

Olga Beine-Golovchuk ◽

Michal Gorka ◽

William Wing Ho Ho ◽

Federico Martinez-Seidel ◽

...

Keyword(s):

Steady State ◽

Feedback Control ◽

Cold Acclimation ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Transcriptional Control ◽

De Novo ◽

De Novo Synthesis ◽

Steady State Temperature

AbstractArabidopsis REIL proteins are cytosolic ribosomal 60S-biogenesis factors. After shift to 10 °C, reil mutants deplete and slowly replenish non-translating eukaryotic ribosome complexes of root tissue, while controlling the balance of non-translating 40S- and 60S-subunits. Reil mutations respond by hyper-accumulation of non-translating subunits at steady-state temperature; after cold-shift, a KCl-sensitive 80S sub-fraction remains depleted. We infer that Arabidopsis may buffer fluctuating translation by pre-existing non-translating ribosomes before de novo synthesis meets temperature-induced demands. Reil1 reil2 double mutants accumulate 43S-preinitiation and pre-60S-maturation complexes and alter paralog composition of ribosomal proteins in non-translating complexes. With few exceptions, e.g. RPL3B and RPL24C, these changes are not under transcriptional control. Our study suggests requirement of de novo synthesis of eukaryotic ribosomes for long-term cold acclimation, feedback control of NUC2 and eIF3C2 transcription and links new proteins, AT1G03250, AT5G60530, to plant ribosome biogenesis. We propose that Arabidopsis requires biosynthesis of specialized ribosomes for cold acclimation.

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Removal of endogenous neuromodulators in a small motor network enhances responsiveness to neuromodulation

Journal of Neurophysiology ◽

10.1152/jn.00383.2017 ◽

2017 ◽

Vol 118 (3) ◽

pp. 1749-1761 ◽

Cited By ~ 5

Author(s):

Kawasi M. Lett ◽

Veronica J. Garcia ◽

Simone Temporal ◽

Dirk Bucher ◽

David J. Schulz

Keyword(s):

Nervous System ◽

De Novo ◽

Normal Activity ◽

Stomatogastric Ganglion ◽

Receptor Expression ◽

Mrna Levels ◽

Stomatogastric Nervous System ◽

Cancer Borealis ◽

The Face ◽

Hormonal Modulation

We studied the changes in sensitivity to a peptide modulator, crustacean cardioactive peptide (CCAP), as a response to loss of endogenous modulation in the stomatogastric ganglion (STG) of the crab Cancer borealis. Our data demonstrate that removal of endogenous modulation for 24 h increases the response of the lateral pyloric (LP) neuron of the STG to exogenously applied CCAP. Increased responsiveness is accompanied by increases in CCAP receptor (CCAPr) mRNA levels in LP neurons, requires de novo protein synthesis, and can be prevented by coincubation for the 24-h period with exogenous CCAP. These results suggest that there is a direct feedback from loss of CCAP signaling to the production of CCAPr that increases subsequent response to the ligand. However, we also demonstrate that the modulator-evoked membrane current ( IMI) activated by CCAP is greater in magnitude after combined loss of endogenous modulation and activity compared with removal of just hormonal modulation. These results suggest that both receptor expression and an increase in the target conductance of the CCAP G protein-coupled receptor are involved in the increased response to exogenous hormone exposure following experimental loss of modulation in the STG. NEW & NOTEWORTHY The nervous system shows a tremendous amount of plasticity. More recently there has been an appreciation for compensatory actions that stabilize output in the face of perturbations to normal activity. In this study we demonstrate that neurons of the crustacean stomatogastric ganglion generate apparent compensatory responses to loss of peptide neuromodulation, adding to the repertoire of mechanisms by which the stomatogastric nervous system can regulate and stabilize its own output.

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Insulin treatment enhances AT1 receptor function in OK cells

AJP Renal Physiology ◽

10.1152/ajprenal.00361.2003 ◽

2005 ◽

Vol 288 (6) ◽

pp. F1213-F1219 ◽

Cited By ~ 22

Author(s):

Anees Ahmad Banday ◽

Athar H. Siddiqui ◽

Michelle M. Menezes ◽

Tahir Hussain

Keyword(s):

Insulin Treatment ◽

Receptor Expression ◽

Receptor Protein ◽

Protein Synthesis Inhibitor ◽

Proximal Tubule Cell ◽

Mrna Levels ◽

Ang Ii ◽

Receptor Function ◽

Ok Cells ◽

Stimulation Of

Increased renal sodium retention is considered a major risk factor contributing to hypertension associated with chronic hyperinsulinemia and obesity. However, the molecular mechanism involved is not understood. The present study investigates the effect of insulin treatment on AT1 receptor expression and ANG II-induced stimulation of Na/H exchanger (NHE) and Na-K-ATPase (NKA) in opossum kidney (OK) cells, a proximal tubule cell line. The presence of the AT1 receptors in OK cells was confirmed by the specific binding of 125I-sar-ANG II and by detecting ∼43-kDa protein on Western blot analysis with AT1 receptor antibody and blocking peptide as well as by expression of AT1 receptor mRNA as determined by RT-PCR. Insulin treatment (100 nM for 24 h) caused an increase in 125I-sar-ANG II binding, AT1 receptor protein content, and mRNA levels. The whole cell lysate and membrane showed similar insulin-induced increase in the AT1 receptor protein expression, which was blocked by genistein (100 nM), a tyrosine kinase inhibitor, and cycloheximide (1.5 μg/ml), a protein synthesis inhibitor. Determination of ethyl isopropyl amiloride-sensitive 22Na+ uptake, a measure of the NHE activity, revealed that ANG II (1–100 pM)-induced stimulation of NHE in insulin-treated cells was significantly greater than in the control cells. Similarly, ANG II (1–100 pM)-induced stimulation of ouabain-sensitive 86Rb+ uptake, a measure of NKA activity in insulin-treated cells, was significantly greater than in the control cells. ANG II stimulation of both the transporters was blocked by AT1 receptor antagonist losartan, suggesting the involvement of AT1 receptors. Thus chronic insulin treatment causes upregulation of AT1 receptors, which evoked ANG II-induced stimulation of NHE and NKA. We propose that insulin-induced increase in the renal AT1 receptor function serves as a mechanism responsible for the increased renal sodium reabsorption and thus may contribute to development of hypertension in conditions associated with hyperinsulinemia.

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Leptin biosynthetic pathway in white adipocytes

Biochemistry and Cell Biology ◽

10.1139/o06-032 ◽

2006 ◽

Vol 84 (2) ◽

pp. 207-214 ◽

Cited By ~ 26

Author(s):

Philippe G Cammisotto ◽

Ludwik J Bukowiecki ◽

Yves Deshaies ◽

Moise Bendayan

Keyword(s):

Endoplasmic Reticulum ◽

Rough Endoplasmic Reticulum ◽

Secretory Pathway ◽

De Novo ◽

Brefeldin A ◽

Mrna Levels ◽

De Novo Synthesis ◽

Cellular Content ◽

Constitutive Secretion

The aim of this study was to determine through morphological and biochemical means the biosynthetic and secretory pathway followed by leptin in adipocytes. Immunocytochemistry revealed the presence of leptin in the rough endoplasmic reticulum, the Golgi apparatus, and in numerous small vesicles along the plasma membrane of white adipo cytes. In vitro, isolated adipocytes under nonstimulated conditions (basal) continuously secreted leptin while their intra cellular content remained unchanged. When adipocytes were stimulated with insulin, leptin cellular content and secretion increased in parallel and were significantly different from basal secretion only after 45 min. L-leucine and L-glutamate also strongly stimulated leptin synthesis and secretion. These stimulating effects were abolished by cycloheximide and brefeldin A. The transcriptional inhibitor actinomycin D did not have any effects in either basal or stimulated conditions. Leptin mRNA levels were not affected by any stimulating or inhibiting agents. Finally, norepinephrine, isoproterenol, CL316243, and palmitate inhibited the effects of insulin, L-leucine, and L-glutamate on leptin synthesis. We thus conclude that (i) adipocytes continuously synthesize and secrete leptin along a rough endoplasmic reticulum–Golgi secretory vesicles pathway, (ii) an increase in leptin secretion requires increased de novo synthesis, and (iii) short-term leptin secretion does not involve changes in mRNA levels.Key words: leptin, vesicles, constitutive secretion, de novo synthesis, transcription.

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Biological Predictors of De Novo Tumors in Solid Organ Transplanted Patients During Oncological Surveillance: Potential Role of Circulating TERT mRNA

Frontiers in Oncology ◽

10.3389/fonc.2021.772348 ◽

2021 ◽

Vol 11 ◽

Author(s):

Michela Cangemi ◽

Stefania Zanussi ◽

Enrica Rampazzo ◽

Ettore Bidoli ◽

Silvia Giunco ◽

...

Keyword(s):

Peripheral Blood ◽

De Novo ◽

Solid Organ Transplantation ◽

Solid Organ ◽

Mrna Levels ◽

Blood Samples ◽

Tumor Onset ◽

Diagnosis Of Cancer

BackgroundDe novo tumors are a major cause of morbidity and mortality after long-term solid organ transplantation. Chronic immunosuppression strongly affects solid organ transplanted (SOT) patients’ immune system by promoting immune evasion strategies and reactivations of viruses with oncogenic potential, ultimately leading to cancer onset. In this scenario, an oncological Surveillance Protocol integrated with biobanking of peripheral blood samples and evaluation of immunovirological and molecular parameters was activated for SOT patients at CRO-IRCCS Aviano, with the aim of identifying suitable biomarkers of cancer development.MethodsAn exploratory longitudinal study was designed based on two serial peripheral blood samples collected at least three months apart. Forty nine SOT patients were selected and stratified by tumor onset during follow-up. Spontaneous T-cell responses to EBV, CMV and tumor associated antigens, EBV-DNA and CMV-DNA loads, and circulating TERT mRNA levels were investigated.ResultsSignificantly higher levels of circulating TERT mRNA were observed 3.5-23.5 months before and close to the diagnosis of cancer as compared to tumor-free patients. Plasmatic TERT mRNA levels >97.73 copies/mL at baseline were significantly associated with the risk of developing de novo tumors (HR=4.0, 95%C.I. = 1.4-11.5, p=0.01). In particular, the risk significantly increased by 4% with every ten-unit increment in TERT mRNA (HR=1.04, 95%C.I. = 1.01-1.07, p=0.01).ConclusionsAlthough obtained in an exploratory study, our data support the importance of identifying early biomarkers of tumor onset in SOT patients useful to modulate the pace of surveillance visits.

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Evidence for a hyporesponsive limbic-hypothalamic-pituitary-adrenal axis following early-life repetitive hypoglycemia in adult male rats

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00678.2010 ◽

2011 ◽

Vol 301 (2) ◽

pp. R484-R490 ◽

Cited By ~ 6

Author(s):

Ashton E. Lehmann ◽

Kathleen Ennis ◽

Michael K. Georgieff ◽

Raghavendra Rao ◽

Phu V. Tran

Keyword(s):

Rat Model ◽

Early Life ◽

Regulatory Protein ◽

Receptor Expression ◽

Male Rats ◽

Mrna Levels ◽

Long Term Effects ◽

Hypothalamic Pituitary Adrenal ◽

Lhpa Axis

The developing limbic-hypothalamic-pituitary-adrenal (LHPA) axis is highly vulnerable to programming by early-life environmental factors, including exposure to synthetic glucocorticoids and nutrient deficiencies. Early-life repetitive hypoglycemia (RHG) is a common complication of insulin therapy for type-1 diabetes that may have long-term consequences in adulthood. Recent observations in a rat model of early RHG suggest persistent changes in LHPA axis function, including changes in relevant hormones and affective behaviors, which support a hyperresponsive LHPA axis. Thus, we hypothesized that early RHG would alter the expression of key genes regulating LHPA axis function in adulthood. The present study employed a rat model of insulin-induced RHG spanning postnatal days (P)24–28, a neurodevelopmental equivalent of early childhood in humans, to assess the long-term effects on mRNA levels for proteins relevant to the LHPA function and the corticosterone responses to ACTH stimulation of dispersed adrenocortical cells in vitro and restraint stress in vivo at adulthood. This early RHG model resulted in a hyporesponsive LHPA axis characterized by impaired corticosterone response, increased hippocampal glucocorticoid and mineralocorticoid receptor (GR and MR), decreased hypothalamic corticotropin-releasing hormone, increased adrenal steroidogenic-acute-regulatory protein and GR, and decreased adrenal MR, melanocortin-type-2 receptor and low-density lipoprotein receptor expression. Our findings highlight a complex environmental-gene interaction between RHG and LHPA axis during development that influences regulation of this axis in adulthood. The findings are consistent with the developmental origins of disease and underscore the influences of early-life events on the programming of a major regulatory system.

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Involvement of a G Protein Regulatory Circuit in Alternative Oxidase Production in Neurospora crassa

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.400522 ◽

2019 ◽

Vol 9 (10) ◽

pp. 3453-3465 ◽

Cited By ~ 1

Author(s):

Natasa Bosnjak ◽

Kristina M. Smith ◽

Iman Asaria ◽

Adrian Lahola-Chomiak ◽

Nishka Kishore ◽

...

Keyword(s):

Electron Transport ◽

Neurospora Crassa ◽

G Protein ◽

Translational Control ◽

Electron Transport Chain ◽

Alternative Oxidase ◽

G Protein Signaling ◽

Mrna Levels ◽

Protein Signaling ◽

Transport Chain

The Neurospora crassa nuclear aod-1 gene encodes an alternative oxidase that functions in mitochondria. The enzyme provides a branch from the standard electron transport chain by transferring electrons directly from ubiquinol to oxygen. In standard laboratory strains, aod-1 is transcribed at very low levels under normal growth conditions. However, if the standard electron transport chain is disrupted, aod-1 mRNA expression is induced and the AOD1 protein is produced. We previously identified a strain of N. crassa, that produces high levels of aod-1 transcript under non-inducing conditions. Here we have crossed this strain to a standard lab strain and determined the genomic sequences of the parents and several progeny. Analysis of the sequence data and the levels of aod-1 mRNA in uninduced cultures revealed that a frameshift mutation in the flbA gene results in the high uninduced expression of aod-1. The flbA gene encodes a regulator of G protein signaling that decreases the activity of the Gα subunit of heterotrimeric G proteins. Our data suggest that strains with a functional flbA gene prevent uninduced expression of aod-1 by inactivating a G protein signaling pathway, and that this pathway is activated in cells grown under conditions that induce aod-1. Induced cells with a deletion of the gene encoding the Gα protein still have a partial increase in aod-1 mRNA levels, suggesting a second pathway for inducing transcription of the gene in N. crassa. We also present evidence that a translational control mechanism prevents production of AOD1 protein in uninduced cultures.

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