scholarly journals Protein Kinases Mediate Anti-Inflammatory Effects of Cannabidiol and Estradiol Against High Glucose in Cardiac Sodium Channels

2021 ◽  
Vol 12 ◽  
Author(s):  
Mohamed A. Fouda ◽  
Peter C. Ruben
Keyword(s):  
Action Potential ◽  
Protein Kinases ◽  
Inflammatory Mediators ◽  
High Glucose ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Strongly Correlated ◽  
Α Subunit ◽  
Anti Inflammatory ◽  
Cardiac Sodium Channels

Background: Cardiovascular anomalies are predisposing factors for diabetes-induced morbidity and mortality. Recently, we showed that high glucose induces changes in the biophysical properties of the cardiac voltage-gated sodium channel (Nav1.5) that could be strongly correlated to diabetes-induced arrhythmia. However, the mechanisms underlying hyperglycemia-induced inflammation, and how inflammation provokes cardiac arrhythmia, are not well understood. We hypothesized that inflammation could mediate the high glucose-induced biophyscial changes on Nav1.5 through protein phosphorylation by protein kinases A and C. We also hypothesized that this signaling pathway is, at least partly, involved in the cardiprotective effects of cannabidiol (CBD) and 17β-estradiol (E2).Methods and Results: To test these ideas, we used Chinese hamster ovarian (CHO) cells transiently co-transfected with cDNA encoding human Nav1.5 α-subunit under control, a cocktail of inflammatory mediators or 100 mM glucose conditions (for 24 h). We used electrophysiological experiments and action potential modeling. Inflammatory mediators, similar to 100 mM glucose, right shifted the voltage dependence of conductance and steady-state fast inactivation and increased persistent current leading to computational prolongation of action potential (hyperexcitability) which could result in long QT3 arrhythmia. We also used human iCell cardiomyocytes derived from inducible pluripotent stem cells (iPSC-CMs) as a physiologically relevant system, and they replicated the effects produced by inflammatory mediators observed in CHO cells. In addition, activators of PK-A or PK-C replicated the inflammation-induced gating changes of Nav1.5. Inhibitors of PK-A or PK-C, CBD or E2 mitigated all the potentially deleterious effects provoked by high glucose/inflammation.Conclusion: These findings suggest that PK-A and PK-C may mediate the anti-inflammatory effects of CBD and E2 against high glucose-induced arrhythmia. CBD, via Nav1.5, may be a cardioprotective therapeutic approach in diabetic postmenopausal population.

scholarly journals Protein kinases mediate anti-inflammatory effects of cannabidiol and estradiol against high glucose in cardiac sodium channels

2020 ◽  
Author(s):  
Mohamed Fouda ◽  
Peter Ruben
Keyword(s):  
Action Potential ◽  
Protein Kinases ◽  
Inflammatory Mediators ◽  
High Glucose ◽  
Chinese Hamster ◽  
Strongly Correlated ◽  
Α Subunit ◽  
Prolongation Of Action Potential ◽  
Anti Inflammatory ◽  
Cardiac Sodium Channels

Background and purpose. Cardiovascular anomalies are predisposing factors for diabetes-induced morbidity and mortality. Recently, we showed that high glucose induces changes in the biophysical properties of Nav1.5 that could be strongly correlated to diabetes-induced arrhythmia. However, the mechanisms underlying hyperglycemia-induced inflammation, and how inflammation provokes cardiac arrhythmia, are not well understood. We hypothesized that inflammation could mediate the high glucose-induced biophyscial changes on Nav1.5 through protein phosphorylation by protein kinases A and C. We also hypothesized that this signaling pathway is, at least partly, involved in the cardiprotective effects of CBD and E2. Experimental approach. To test these ideas, we used Chinese hamster ovarian (CHO) cells transiently co-transfected with cDNA encoding human Nav1.5 α-subunit under control, a cocktail of inflammatory mediators or 100 mM glucose conditions (for 24 hours). We used electrophysiological experiments and action potential modelling. Key Results. Inflammatory mediators, similar to 100 mM glucose, right shifted the voltage dependence of conductance and steady state fast inactivation and increased persistent current leading to computational prolongation of action potential (hyperexcitability) which could result in long QT3 arrhythmia. In addition, activators of PK-A or PK-C replicated the inflammation-induced gating changes of Nav1.5. Inhibitors of PK-A or PK-C, CBD or E2 mitigated all the potentially deleterious effects provoked by high glucose/inflammation. Conclusions and implications. These findings suggest that PK-A and PK-C may mediate the anti-inflammatory effects of CBD and E2 against high glucose-induced arrhythmia. CBD, via Nav1.5, may be a cardioprotective therapeutic approach in diabetic postmenopausal population.

scholarly journals Protein kinases mediate anti-inflammatory effects of cannabidiol and estradiol against high glucose in cardiac sodium channels

2020 ◽  
Author(s):  
Mohamed A. Fouda ◽  
Peter C. Ruben
Keyword(s):  
Action Potential ◽  
Signaling Pathway ◽  
Protein Kinases ◽  
Inflammatory Mediators ◽  
High Glucose ◽  
Therapeutic Approach ◽  
Morbidity And Mortality ◽  
List Type ◽  
Long Qt ◽  
Anti Inflammatory

AbstractBackground and purposeCardiovascular anomalies are predisposing factors for diabetes-induced morbidity and mortality. Recently, we showed that high glucose induces changes in the biophysical properties of Nav1.5 that could be strongly correlated to diabetes-induced arrhythmia. However, the mechanisms underlying hyperglycemia-induced inflammation, and how inflammation provokes cardiac arrhythmia, are not well understood. We hypothesized that inflammation could mediate the high glucose-induced biophyscial changes on Nav1.5 through protein phosphorylation by protein kinases A and C. We also hypothesized that this signaling pathway is, at least partly, involved in the cardiprotective effects of CBD and E2.Experimental approachTo test these ideas, we used Chinese hamster ovarian (CHO) cells transiently co-transfected with cDNA encoding human Nav1.5 α-subunit under control, a cocktail of inflammatory mediators or 100 mM glucose conditions (for 24 hours). We used electrophysiological experiments and action potential modelling.Key ResultsInflammatory mediators, similar to 100 mM glucose, right shifted the voltage dependence of conductance and steady state fast inactivation and increased persistent current leading to computational prolongation of action potential (hyperexcitability) which could result in long QT3 arrhythmia. In addition, activators of PK-A or PK-C replicated the inflammation-induced gating changes of Nav1.5. Inhibitors of PK-A or PK-C, CBD or E2 mitigated all the potentially deleterious effects provoked by high glucose/inflammation.Conclusions and implicationsThese findings suggest that PK-A and PK-C may mediate the anti-inflammatory effects of CBD and E2 against high glucose-induced arrhythmia. CBD, via Nav1.5, may be a cardioprotective therapeutic approach in diabetic postmenopausal population.Bullet pointsWhat is already known:Arrhythmias are among the common cardiac causes of morbidity and mortality in diabetes-related hyperglycemia.One of the diabetes-induced arrhythmias is long-QT syndrome, caused by gating defects in the cardiac voltage-gated sodium channel (Nav1.5).What this study adds:Inflammation and subsequent activation of PK-A and PK-C mediate the high glucose-induced electrophysiological changes of Nav1.5 in a manner consistent with the gating defects that underlie long-QT arrhythmia.Cannabidiol and estradiol rescue the high glucose induced Nav1.5 gating defects through, at least partly, this signaling pathway.Clinical significance:Inflammation/PK-A and PK-C signaling pathway could be a potential therapeutic target to prevent arrhythmias associated with diabetes.Cannabidiol may be a therapeutic approach to prevent cardiac complications in diabetes, especially in postmenopausal populations due to the decreased levels of the cardioprotective estrogen.

scholarly journals Impediment of Gpibα-Mediated Platelet Clearance By an Anti-Gpibβ Antibody Derivative

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1003-1003
Author(s):  
Wenchun Chen ◽  
Moriah Simone Wilson ◽  
Yingchun Wang ◽  
Francois Lanza ◽  
Renhao Li
Keyword(s):  
Life Span ◽  
Cho Cells ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Chinese Hamster ◽  
Coiled Coil ◽  
Wild Type Mouse ◽  
Interleukin 4 ◽  
Wild Type ◽  
Α Subunit

Abstract Background: Glycoprotein (GP)Ib-IX complex plays a critical role in mediating platelet activation and platelet clearance. Recently, we identified the mechanosensory domain (MSD) in the GPIbα subunit, and demonstrated that unfolding of the MSD and subsequent exposure of the Trigger sequence (residues 473-483) therein activates GPIb-IX and induces rapid platelet clearance. This mechanism could explain acute thrombocytopenia induced by activated VWF, anti-GPIbα antibodies, neuraminidase, and ectodomain shedding of GPIbα. Consistently, platelets in IL4R-IbαTg mice, a transgenic strain in which the entire extracellular domain of human GPIbα except the Trigger sequence was replaced with that of the α-subunit of interleukin-4 receptor, exhibit constitutively more filopodia and are cleared much faster than the wild type. Previously, an anti-GPIbβ antibody RAM.1 was developed. RAM.1 significantly inhibits GPIb-IX-mediated filopodia formation and Ca 2+ signaling in platelets. In addition, it could inhibit GPIb-dependent thrombin generation. These results suggest that targeting GPIbβ could inhibit activation of GPIb-IX induced by MSD unfolding. Objectives: To investigate whether targeting GPIbβ with RAM.1 can impede rapid platelet clearance induced by exposed Trigger sequence and ameliorate related thrombocytopenia. Methods: Spontaneous filopodia in platelets and transfected Chinese hamster ovary (CHO) cells were visualized by fluorescence staining of actin and confocal microscopy. Images were quantified by ImageJ. Platelet signaling events, like P-selectin exposure, β-galactose exposure, and Ca 2+ influx, were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488-labeled anti-mouse GPIX antibody. Results: CHO cells stably expressing the same mutant GPIb-IX complex in IL4R-IbαTg mouse platelets have been successfully obtained. Like IL4R-IbαTg platelets, these IL4R-IbαTg CHO cells exhibited spontaneous filopodia in the absence of any GPIbα ligands. RAM.1 could inhibit spontaneous filopodia formation in these CHO cells and IL4R-IbαTg platelets (Fig. 1, 2). Compared to wild-type mouse platelets, IL4R-IbαTg platelets constitutively exhibited increased P-selectin exposure, increased β-galactose exposure, and elevated intracellular Ca 2+, all of which could be inhibited by treatment of RAM.1 (Fig. 3). Recombinant RAM.1-GCN4 protein (rRAM.1-GCN4), in which the Fc region of RAM.1 heavy chain was replaced with the GCN4 coiled coil dimerizing sequence, has been generated and used as an alternative of the divalent RAM.1-Fab2. It retained the ability of RAM.1 antibody to inhibit GPIb-IX signaling. Injecting rRAM.1-GCN4 into IL4R-IbαTg mice dramatically improved the life span of endogenous IL4R-IbαTg platelets (Fig. 4). Conclusion: These results demonstrate that the exposed Trigger sequence is sufficient to activate GPIb-IX in transfected CHO cells, and that RAM.1 derivatives can impede GPIbα-mediated rapid platelet clearance. Targeting GPIbβ may be a novel approach to treat GPIb-related thrombocytopenia. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Polarized Expression of Na+,K+-ATPase in Epithelia Depends on the Association between β-Subunits Located in Neighboring Cells

2005 ◽  
Vol 16 (3) ◽  
pp. 1071-1081 ◽  
Author(s):  
Liora Shoshani ◽  
Rubén G. Contreras ◽  
María L. Roldán ◽  
Jacqueline Moreno ◽  
Amparo Lázaro ◽  
...  
Keyword(s):  
Cho Cells ◽  
Mdck Cells ◽  
Chinese Hamster ◽  
Physiological Role ◽  
Cell Interaction ◽  
Lateral Side ◽  
Β Cells ◽  
Aggregation Assay ◽  
Α Subunit ◽  
Dog Kidney

The polarized distribution of Na+,K+-ATPase plays a paramount physiological role, because either directly or through coupling with co- and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca2+, K+, Cl-, and CO3H- across the whole epithelium. We report here that the β-subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na+,K+-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na+,K+-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with β1-subunit from the dog kidney (CHO-β). 4) This may be attributed to the adhesive property of the β1-subunit, because an aggregation assay using CHO (mock-transfected) and CHO-β cells shows that the expression of dog β1-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of β1-subunit forces CHO-β cells to coexpress endogenous α-subunit. Together, our results indicate that MDCK cells express Na+,K+-ATPase at a given border provided the contacting cell expresses the dog β1-subunit. The cell–cell interaction thus established would suffice to account for the polarized expression and positioning of Na+,K+-ATPase in epithelial cells.

scholarly journals Human mesenchymal stromal cells broadly modulate high glucose-induced inflammatory responses of renal proximal tubular cell monolayers

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Md Nahidul Islam ◽  
Tomás P. Griffin ◽  
Elizabeth Sander ◽  
Stephanie Rocks ◽  
Junaid Qazi ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Mesenchymal Stromal Cells ◽  
Inflammatory Mediators ◽  
Stromal Cells ◽  
High Glucose ◽  
Cell Monolayers ◽  
Proximal Tubular ◽  
Anti Inflammatory ◽  
Renal Proximal Tubular ◽  
Inflammatory Effect

Abstract Background Renal proximal tubular epithelial cells (RPTEC) are dysfunctional in diabetic kidney disease (DKD). Mesenchymal stromal cells (MSC) may modulate DKD pathogenesis through anti-inflammatory mediators. This study aimed to investigate the pro-inflammatory effect of extended exposure to high glucose (HG) concentration on stable RPTEC monolayers and the influence of MSC on this response. Methods Morphologically stable human RPTEC/TERT1 cell monolayers were exposed to 5 mM and 30 mM (HG) D-glucose or to 5 mM D-glucose + 25 mM D-mannitol (MAN) for 5 days with sequential immunoassays of supernatants and end-point transcriptomic analysis by RNA sequencing. Under the same conditions, MSC-conditioned media (MSC-CM) or MSC-containing transwells were added for days 4–5. Effects of CM from HG- and MAN-exposed RPTEC/MSC co-cultures on cytokine secretion by monocyte-derived macrophages were determined. Results After 72–80 h, HG resulted in increased RPTEC/TERT1 release of interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1 and neutrophil gelatinase-associated lipocalin (NGAL). The HG pro-inflammatory effect was attenuated by concentrated (10×) MSC-CM and, to a greater extent, by MSC transwell co-culture. Bioinformatics analysis of RNA sequencing data confirmed a predominant effect of HG on inflammation-related mediators and biological processes/KEGG pathways in RPTEC/TERT1 stable monolayers as well as the non-contact-dependent anti-inflammatory effect of MSC. Finally, CM from HG-exposed RPTEC/MSC transwell co-cultures was associated with attenuated secretion of inflammatory mediators by macrophages compared to CM from HG-stimulated RPTEC alone. Conclusions Stable RPTEC monolayers demonstrate delayed pro-inflammatory response to HG that is attenuated by close proximity to human MSC. In DKD, this MSC effect has potential to modulate hyperglycemia-associated RPTEC/macrophage cross-talk.

scholarly journals Dissociation of Early Folding Events from Assembly of the Human Lutropin β-Subunit

1998 ◽  
Vol 12 (10) ◽  
pp. 1640-1649
Author(s):  
Mesut Muyan ◽  
Raymond W. Ruddon ◽  
Sheila E. Norton ◽  
Irving Boime ◽  
Elliott Bedows
Keyword(s):  
Cho Cells ◽  
Protein Disulfide Isomerase ◽  
Chinese Hamster ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Disulfide Isomerase ◽  
The Common ◽  
Protein Disulfide ◽  
Gene Expressing

Abstract The human LH of the anterior pituitary is a member of the glycoprotein hormone family that includes FSH, TSH, and placental CG. All are noncovalently bound heterodimers that share a common α-subunit and β-subunits that confer biological specificity. LHβ and CGβ share more than 80% amino acid sequence identity; however, in transfected Chinese hamster ovary (CHO) cells, LHβ assembles with the α-subunit more slowly than does hCGβ, and only a fraction of the LHβ synthesized is secreted, whereas CGβ is secreted efficiently. To understand why the assembly and secretion of these related β-subunits differ, we studied the folding of LHβ in CHO cells transfected with either the LHβ gene alone, or in cells cotransfected with the gene expressing the common α-subunit, and compared our findings to those previously seen for CG. We found that the rate of conversion of the earliest detectable folding intermediate of LH, pβ1, to the second major folding form, pβ2, did not differ significantly from the pβ1-to-pβ2 conversion of CGβ, suggesting that variations between the intracellular fates of the two β-subunits cannot be explained by differences in the rates of their early folding steps. Rather, we discovered that unlike CGβ, where the folding to pβ2 results in an assembly-competent product, apparently greater than 90% of the LH pβ2 recovered from LHβ-transfected CHO cells was assembly incompetent, accounting for inefficient LHβ assembly with the α-subunit. Using the formation of disulfide (S-S) bonds as an index, we observed that, in contrast to CGβ, all 12 LHβ cysteine residues formed S-S linkages as soon as pβ2 was detected. Attempts to facilitate LH assembly with protein disulfide isomerase in vitro using LH pβ2 and excess urinary α-subunit as substrate were unsuccessful, although protein disulfide isomerase did facilitate CG assembly in this assay. Moreover, unlike CGβ, LHβ homodimers were recovered from transfected CHO cells. Taken together, these data suggest that differences seen in the rate and extent of LH assembly and secretion, as compared to those of CG, reflect conformational differences between the folding intermediates of the respectiveβ -subunits.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

Relative Expression of Mouse Udp-glucuronosyl Transferase 2b1 Gene in the Livers, Kidneys, and Hearts: The Influence of Nonsteroidal Anti-inflammatory Drug Treatment

2019 ◽  
Vol 20 (11) ◽  
pp. 918-923 ◽  
Author(s):  
Yazun Jarrar ◽  
Qais Jarrar ◽  
Mohammad Abu-Shalhoob ◽  
Abdulqader abed ◽  
Esra'a Sha'ban
Keyword(s):  
Gene Expression ◽  
Strongly Correlated ◽  
Chain Reaction ◽  
Inflammatory Drug ◽  
Relative Expression ◽  
Elimination Half ◽  
Endogenous Compounds ◽  
Polymerase Chain ◽  
Anti Inflammatory ◽  
Glucuronosyl Transferase

Background: Mouse Udp-glucuronosyl Transferase (UGT) 2b1 is equivalent to the human UGT2B7 enzyme, which is a phase II drug-metabolising enzyme and plays a major role in the metabolism of xenobiotic and endogenous compounds. This study aimed to find the relative expression of the mouse ugt2b1 gene in the liver, kidney, and heart organs and the influence of Nonsteroidal Anti-inflammatory Drug (NSAID) administration. Methods: Thirty-five Blab/c mice were divided into 5 groups and treated with different commonly-used NSAIDs; diclofenac, ibuprofen, meloxicam, and mefenamic acid for 14 days. The livers, kidneys, and hearts were isolated, while the expression of ugt2b1 gene was analysed with a quantitative real-time polymerase chain reaction technique. Results: It was found that the ugt2b1 gene is highly expressed in the liver, and then in the heart and the kidneys. NSAIDs significantly upregulated (ANOVA, p < 0.05) the expression of ugt2b1 in the heart, while they downregulated its expression (ANOVA, p < 0.05) in the liver and kidneys. The level of NSAIDs’ effect on ugt2b1 gene expression was strongly correlated (Spearman’s Rho correlation, p < 0.05) with NSAID’s lipophilicity in the liver and its elimination half-life in the heart. Conclusion: This study concluded that the mouse ugt2b1 gene was mainly expressed in the liver, as 14-day administration of different NSAIDs caused alterations in the expression of this gene, which may influence the metabolism of xenobiotic and endogenous compounds.

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