N-methyl-D-aspartate (NMDA) receptor expression and function is required for early chondrogenesis

Abstract Background In vitro chondrogenesis depends on the concerted action of numerous signalling pathways, many of which are sensitive to the changes of intracellular Ca2+ concentration. N-methyl-D-aspartate (NMDA) glutamate receptor is a cation channel with high permeability for Ca2+. Whilst there is now accumulating evidence for the expression and function of NMDA receptors in non-neural tissues including mature cartilage and bone, the contribution of glutamate signalling to the regulation of chondrogenesis is yet to be elucidated. Methods We studied the role of glutamatergic signalling during the course of in vitro chondrogenesis in high density chondrifying cell cultures using single cell fluorescent calcium imaging, patch clamp, transient gene silencing, and western blotting. Results Here we show that key components of the glutamatergic signalling pathways are functional during in vitro chondrogenesis in a primary chicken chondrogenic model system. We also present the full glutamate receptor subunit mRNA and protein expression profile of these cultures. This is the first study to report that NMDA-mediated signalling may act as a key factor in embryonic limb bud-derived chondrogenic cultures as it evokes intracellular Ca2+ transients, which are abolished by the GluN2B subunit-specific inhibitor ifenprodil. The function of NMDARs is essential for chondrogenesis as their functional knock-down using either ifenprodil or GRIN1 siRNA temporarily blocks the differentiation of chondroprogenitor cells. Cartilage formation was fully restored with the re-expression of the GluN1 protein. Conclusions We propose a key role for NMDARs during the transition of chondroprogenitor cells to cartilage matrix-producing chondroblasts.

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Role of the 807 C/T Polymorphism of the α2 Gene in Platelet GP Ia Collagen Receptor Expression and Function

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614605 ◽

1999 ◽

Vol 81 (06) ◽

pp. 951-956 ◽

Cited By ~ 59

Author(s):

J. Corral ◽

R. González-Conejero ◽

J. Rivera ◽

F. Ortuño ◽

P. Aparicio ◽

...

Keyword(s):

Venous Thrombosis ◽

Cell Types ◽

Receptor Expression ◽

Case Control Studies ◽

Platelet Surface ◽

Vitro Analysis ◽

And Function ◽

In Vitro Analysis

SummaryThe variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by “in vitro” analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.

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Histamine H2 receptor radioligands: triumphs and challenges

Future Medicinal Chemistry ◽

10.4155/fmc-2021-0058 ◽

2021 ◽

Author(s):

Steffen Pockes ◽

Katharina Tropmann

Keyword(s):

Receptor Expression ◽

In Vitro Assays ◽

Histamine H2 Receptor ◽

Drug Candidates ◽

Carbon 14 ◽

The Central Nervous System ◽

And Function ◽

Role And Function

Since the discovery of the histamine H2 receptor (H2R), radioligands were among the most powerful tools to investigate its role and function. Initially, radiolabeling was used to investigate human and rodent tissues regarding their receptor expression. Later, radioligands gained increasing significance as pharmacological tools in in vitro assays. Although tritium-labeling was mainly used for this purpose, labeling with carbon-14 is preferred for metabolic studies of drug candidates. After the more-or-less successful application of numerous labeled H2R antagonists, the recent development of the G protein-biased radioligand [3H]UR-KAT479 represents another step forward to elucidate the widely unknown role of the H2R in the central nervous system through future studies.

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Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum

Journal of Cell Science ◽

10.1242/jcs.109.6.1479 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1479-1495 ◽

Cited By ~ 4

Author(s):

L.A. Temesvari ◽

J.M. Rodriguez-Paris ◽

J.M. Bush ◽

L. Zhang ◽

J.A. Cardelli

Keyword(s):

Morphological Changes ◽

Specific Inhibitor ◽

Fluid Phase ◽

Contractile Vacuole ◽

Dependent Manner ◽

Concanamycin A ◽

Latex Beads ◽

And Function

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454–3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase.

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Purines as Transmitter Molecules: Electrophysiological Studies on Purinergic Signalling in Different Cell Systems

10.36253/978-88-8453-905-2 ◽

2009 ◽

Author(s):

Elisabetta Coppi

Keyword(s):

Cell Signalling ◽

Human Mesenchymal Stem Cells ◽

Membrane Receptors ◽

Receptor Expression ◽

Purinergic Signalling ◽

Cell Systems ◽

Cellular Processes ◽

Electrophysiological Studies ◽

And Function

Purinergic nucleotides and nucleosides (ATP, ADP, AMP and adenosine) are essential intracellular metabolites involved in a number of cellular processes, from energy supply to protein phosphorylation. However, in the last years, several studies demonstrated their involvement in cell signalling by the activation of specific membrane receptors (P1 and P2) and their role as neurotransmitters began to be investigated. The present work was aimed to clarify the effects of purinergic neurotransmission in different cell systems by using electrophysiological techniques. Relevant results of this research include the observation that P1 and P2 receptors play a deleterious role during "in vitro" ischemia in the rat brain, and the first demonstration of P2 receptor expression and function in a line of adult human mesenchymal stem cells.

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Abstract 281: Long-term Exposure of PM2.5 Causes Hypertension by Impaired Renal D1 Receptor Mediated Sodium Excretion via Up-regulation of GRK4 Expression in SD rats

Hypertension ◽

10.1161/hyp.64.suppl_1.281 ◽

2014 ◽

Vol 64 (suppl_1) ◽

Author(s):

Xi Lu ◽

Ken Chen ◽

Jing Zeng ◽

Hongmei Ren ◽

Chunyu Zeng

Keyword(s):

Sodium Excretion ◽

D1 Receptor ◽

Receptor Expression ◽

Sd Rats ◽

Induced Hypertension ◽

Pm2.5 Exposure ◽

And Function

Introduction: Epidemiological evidence supports an important association between PM2.5 exposure and hypertension. There are reports that PM2.5 induced hypertension with impaired sodium excretion, however, the mechanisms are not clear. Hypothesis: We hypothesize that PM2.5, via increased ROS levels, increased GRK4 expression, consequently impaired renal D1 receptor function, and lead to hypertension. Methods: We used Sprague-Dawley (SD) rats with in-vivo PM2.5 exposure, and immortalized renal proximal tubule (RPT) cells from Wistar-Kyoto (WKY) rats in-vitro, which behave similarly to freshly obtained RPT cells. Results: Our present study found that long-term exposure of PM2.5 caused hypertension and impaired renal sodium excretion, which might be ascribed to lower D1 receptor expression and higher D1 receptor phosphorylation, accompanied with higher GRK4 expression. The in-vivo results were confirmed in in-vitro study, i.e. PM2.5 increased basal Na+-K+ ATPase activity, decreased D1 receptor mediated inhibitory effect on Na+-K+ ATPase activity, decreased D1 receptor expression and increased D1 receptor phosphorylation in RPT cells. The downregulation of D1 receptor expression and function might be due to higher GRK4 expression, because down-regulation of GRK4 by siRNA reversed the D1 receptor expression and function. Due to the role of ROS on D1 receptor dysfunction, we checked ROS levels, and found plasma ROS levels were higher in PM2.5 treated SD rats. Inhibition of ROS by tempol reduced blood pressure and increased sodium excretion in PM2.5 treated SD rats, accompanied by increased the lower D1 receptor expression, and decreased the hyperphosphorylated D1 receptor and GRK4 expression. Conclusions: Long-term exposure of PM2.5 increases blood pressure by decreased D1 receptor expression and function; ROS, via regulation of GRK4 expression, is taken part in the pathogenesis of PM2.5-induced hypertension.

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Dopamine Receptor Expression and Function in Clinically Nonfunctioning Pituitary Tumors: Comparison with the Effectiveness of Cabergoline Treatment

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2003-030859 ◽

2004 ◽

Vol 89 (4) ◽

pp. 1674-1683 ◽

Cited By ~ 83

Author(s):

Rosario Pivonello ◽

Carmela Matrone ◽

Mariagiovanna Filippella ◽

Luigi M. Cavallo ◽

Carolina Di Somma ◽

...

Keyword(s):

Dopamine Receptor ◽

D2 Receptor ◽

Pituitary Tumors ◽

Receptor Expression ◽

Treatment Schedule ◽

Α Subunit ◽

Tumor Mass ◽

Isoform Expression ◽

And Function

Abstract The aim of this study was to correlate dopamine receptors and D2 isoform expression with the cabergoline effect on α-subunit secretion in vitro and tumor mass in vivo in clinically nonfunctioning pituitary tumors. Eighteen patients were subjected to neurosurgery, and a tumor sample was used for dopamine receptor and D2 isoform expression evaluation by RT-PCR and the in vitro functional studies. After neurosurgery, nine of 18 patients with persistent tumor were treated with cabergoline and tumor mass was evaluated before and after 1 yr treatment. D2 receptor was expressed in 67% of cases. D2long was found in 50%, D2short in 17%, and both D2 isoforms in 33% of cases. D4 receptor was also expressed in 17% of cases. The in vitro inhibition of α-subunit concentration was found in 56% of cases and was associated with D2 expression (χ2 = 5.6; P < 0.05). After 1 yr of cabergoline treatment, tumor shrinkage was evident in 56% of patients and was associated with D2 expression (χ2 = 5.6; P < 0.05). The expression of D2short rather than D2long isoform is associated with the most favorable response of the tumor to cabergoline treatment. In conclusion, this study demonstrates D2 receptor expression and function in nearly 70% of cases, suggesting a role of this drug in the treatment schedule of clinically nonfunctioning pituitary tumors.

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Vitamin E-Mediated Modulation of Glutamate Receptor Expression in an Oxidative Stress Model of Neural Cells Derived from Embryonic Stem Cell Cultures

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/6048936 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 6

Author(s):

Afifah Abd Jalil ◽

Huzwah Khaza’ai ◽

Norshariza Nordin ◽

Nur’izzati Mansor ◽

Amirah Salwani Zaulkffali

Keyword(s):

Oxidative Stress ◽

Vitamin E ◽

Glutamate Receptor ◽

Cell Cultures ◽

Embryonic Stem ◽

Receptor Expression ◽

Neural Cells ◽

Es Cell ◽

Neural Lineage

Glutamate is the primary excitatory neurotransmitter in the central nervous system. Excessive concentrations of glutamate in the brain can be excitotoxic and cause oxidative stress, which is associated with Alzheimer’s disease. In the present study, the effects of vitamin E in the form of tocotrienol-rich fraction (TRF) and alpha-tocopherol (α-TCP) in modulating the glutamate receptor and neuron injury markers in an in vitro model of oxidative stress in neural-derived embryonic stem (ES) cell cultures were elucidated. A transgenic mouse ES cell line (46C) was differentiated into a neural lineage in vitro via induction with retinoic acid. These cells were then subjected to oxidative stress with a significantly high concentration of glutamate. Measurement of reactive oxygen species (ROS) was performed after inducing glutamate excitotoxicity, and recovery from this toxicity in response to vitamin E was determined. The gene expression levels of glutamate receptors and neuron-specific enolase were elucidated using real-time PCR. The results reveal that neural cells derived from 46C cells and subjected to oxidative stress exhibit downregulation of NMDA, kainate receptor, and NSE after posttreatment with different concentrations of TRF and α-TCP, a sign of neurorecovery. Treatment of either TRF or α-TCP reduced the levels of ROS in neural cells subjected to glutamate-induced oxidative stress; these results indicated that vitamin E is a potent antioxidant.

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Mesenchymal stem cells protect CNS neurons against glutamate excitotoxicity by inhibiting glutamate receptor expression and function

Experimental Neurology ◽

10.1016/j.expneurol.2012.04.011 ◽

2012 ◽

Vol 236 (1) ◽

pp. 161-170 ◽

Cited By ~ 58

Author(s):

A. Voulgari-Kokota ◽

R. Fairless ◽

M. Karamita ◽

V. Kyrargyri ◽

V. Tseveleki ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Glutamate Receptor ◽

Receptor Expression ◽

Glutamate Excitotoxicity ◽

Cns Neurons ◽

And Function

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Prognostic Value and Function of KLF5 in Papillary Thyroid Cancer

Cancers ◽

10.3390/cancers13020185 ◽

2021 ◽

Vol 13 (2) ◽

pp. 185

Author(s):

Poyil Pratheeshkumar ◽

Abdul K. Siraj ◽

Sasidharan Padmaja Divya ◽

Sandeep Kumar Parvathareddy ◽

Sarah Siraj ◽

...

Keyword(s):

Binding Sites ◽

Specific Inhibitor ◽

Papillary Thyroid ◽

Self Renewal ◽

Functional Correlation ◽

Xenograft Growth ◽

Molecular Therapeutic ◽

And Function

The Krüppel-like factor 5 (KLF5), a zinc-finger transcriptional factor, is highly expressed in several solid tumors, but its role in PTC remains unclear. We investigated the expression of KLF5 protein in a large cohort of PTC patient samples and explored its functional role and mechanism in PTC cell lines in vitro and in vivo. KLF5 overexpression was observed in 65.1% of all PTC cases and it was significantly associated with aggressive clinico-pathological parameters and poor outcome. Given the significant association between KLF5 and HIF-1α overexpression in PTC patients, we investigated the functional correlation between KLF5 and HIF-1α in PTC cells. Indeed, the analysis revealed the co-immunoprecipitation of KLF5 with HIF-1α in PTC cells. We also identified KLF5-binding sites in the HIF-1α promoter that specifically bound to KLF5 protein. Mechanistically, KLF5 promoted PTC cell growth, invasion, migration, and angiogenesis, while KLF5 downregulation via specific inhibitor or siRNA reverses its action in vitro. Importantly, the silencing of KLF5 decreases the self-renewal ability of spheroids generated from PTC cells. In addition, the depletion of KLF5 reduces PTC xenograft growth in vivo. These findings suggest KLF5 can be a possible new molecular therapeutic target for a subset of PTC.

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Malaria Pigment Hemozoin Impairs GM-CSF Receptor Expression and Function by 4-Hydroxynonenal

Antioxidants ◽

10.3390/antiox10081259 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1259

Author(s):

Oleksii Skorokhod ◽

Valentina Barrera ◽

Giorgia Mandili ◽

Federica Costanza ◽

Elena Valente ◽

...

Keyword(s):

Direct Interaction ◽

Cell Types ◽

Receptor Expression ◽

Amino Acid Residues ◽

Gm Csf ◽

Malaria Pigment ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

And Function

Malarial pigment hemozoin (HZ) generates the lipoperoxidation product 4-hydroxynonenal (4-HNE), which is known to cause dysregulation of the immune response in malaria. The inhibition of granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent differentiation of dendritic cells (DC) by HZ and 4-HNE was previously described in vitro, and the GM-CSF receptor (GM-CSF R) was hypothesised to be a primary target of 4-HNE in monocytes. In this study, we show the functional impact of HZ on GM-CSF R in monocytes and monocyte-derived DC by (i) impairing GM-CSF binding by 50 ± 9% and 65 ± 14%, respectively (n = 3 for both cell types); (ii) decreasing the expression of GM-CSF R functional subunit (CD116) on monocyte’s surface by 36 ± 11% (n = 6) and in cell lysate by 58 ± 16% (n = 3); and (iii) binding of 4-HNE to distinct amino acid residues on CD116. The data suggest that defective DC differentiation in malaria is caused by GM-CSF R dysregulation and GM-CSF R modification by lipoperoxidation product 4-HNE via direct interaction with its CD116 subunit.

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