Abstract:Recently, using an animal model of Charcot-MarieTooth human disorder, we showed that ascorbic acid (AA) represses PMP22 gene expression by acting on intracellular cAMP concentrations. In this work, we present kinetics data on the inhibitory effect of AA upon adenylate cyclase activity. The data show that this molecule acts as a competitive inhibitor of the enzyme, a finding that opens new pharmacological avenues.
“…Indeed, ascorbate was found to partially stabilize cAMP levels in the presence of thrombin in an NO-and cGMP-dependent manner. In previous studies using different cells, ascorbate either decreased basal cAMP (45)(46)(47) or increased cAMP under stimulatory conditions (48,49). Our results clarify previous findings by showing that ascorbate preserves endothelial cAMP in the face of thrombin stimulation.…”
Background: Vitamin C (ascorbate) promotes endothelial barrier stability during inflammation. Results: Ascorbate prevents inflammatory decreases in cAMP. Conclusion: cAMP preserves the cortical actin cytoskeleton and prevents stress fiber formation. Significance: Dietary vitamin C may promote vascular integrity in patients with inflammatory diseases.
“…Indeed, ascorbate was found to partially stabilize cAMP levels in the presence of thrombin in an NO-and cGMP-dependent manner. In previous studies using different cells, ascorbate either decreased basal cAMP (45)(46)(47) or increased cAMP under stimulatory conditions (48,49). Our results clarify previous findings by showing that ascorbate preserves endothelial cAMP in the face of thrombin stimulation.…”
Background: Vitamin C (ascorbate) promotes endothelial barrier stability during inflammation. Results: Ascorbate prevents inflammatory decreases in cAMP. Conclusion: cAMP preserves the cortical actin cytoskeleton and prevents stress fiber formation. Significance: Dietary vitamin C may promote vascular integrity in patients with inflammatory diseases.
“…This suggests that ascorbate action not only involves hydroxylation and stabilization of the collagen triple helix [42], it also involves direct or indirect effects on gene expression and protein secretion. ‘Regulation of cAMP metabolic process’ was detected as an enriched class from 48 h onwards, which pointed towards the modulatory effects of vit C on cAMP, consistent with vit C being proposed as a ‘global regulator’ of intracellular cAMP [43, 44]. This advances the possibility that the modulatory effects of vit C on cAMP might be one of the mechanisms by which vit C regulates gene expression.Fig.…”
BackgroundVitamin C (vit C) is an essential dietary nutrient, which is a potent antioxidant, a free radical scavenger and functions as a cofactor in many enzymatic reactions. Vit C is also considered to enhance the immune effector function of macrophages, which are regarded to be the first line of defence in response to any pathogen. The THP-1 cell line is widely used for studying macrophage functions and for analyzing host cell-pathogen interactions.ResultsWe performed a genome-wide temporal gene expression and functional enrichment analysis of THP-1 cells treated with 100 μM of vit C, a physiologically relevant concentration of the vitamin. Modulatory effects of vitamin C on THP-1 cells were revealed by differential expression of genes starting from 8 h onwards. The number of differentially expressed genes peaked at the earliest time-point i.e. 8 h followed by temporal decline till 96 h. Further, functional enrichment analysis based on statistically stringent criteria revealed a gamut of functional responses, namely, ‘Regulation of gene expression’, ‘Signal transduction’, ‘Cell cycle’, ‘Immune system process’, ‘cAMP metabolic process’, ‘Cholesterol transport’ and ‘Ion homeostasis’. A comparative analysis of vit C-mediated modulation of gene expression data in THP-1cells and human skin fibroblasts disclosed an overlap in certain functional processes such as ‘Regulation of transcription’, ‘Cell cycle’ and ‘Extracellular matrix organization’, and THP-1 specific responses, namely, ‘Regulation of gene expression’ and ‘Ion homeostasis’. It was noteworthy that vit C modulated the ‘Immune system’ process throughout the time-course.ConclusionsThis study reveals the genome-wide effects of physiological levels of vit C on THP-1 gene expression. The multitude of effects impacted by vit C in macrophages highlights its role in maintaining homeostasis of several cellular functions. This study provides a rational basis for the use of the Vitamin C- THP-1 cell model, to study biochemical and cellular responses to stresses, including infection with M. tuberculosis and other intracellular pathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3635-4) contains supplementary material, which is available to authorized users.
“…Moreover, the monolayer leak can be blocked with drugs that elevate intracellular cAMP [43]. This mechanism is unlikely to account for the protective effect of intracellular ascorbate that we observed, however, because ascorbate is a competitive inhibitor of adenylate cyclase and decreases intracellular cAMP levels [44]. A more probable explanation of how intracellular ascorbate stabilizes the endothelial barrier during septic insult involves NADPH oxidase, superoxide, peroxynitrite and PP2A (Fig.…”
Endothelial barrier dysfunction contributes to morbidity in sepsis. We tested the hypothesis that raising the intracellular ascorbate concentration protects the endothelial barrier from septic insult by inhibiting protein phosphatase type 2A. Monolayer cultures of microvascular endothelial cells were incubated with ascorbate, dehydroascorbic acid (DHAA), NADPH oxidase inhibitors apocynin and diphenyliodonium, or PP2A inhibitor okadaic acid, and then were exposed to septic insult (lipopolysaccharide and interferon-γ). Under standard culture conditions that depleted intracellular ascorbate, septic insult stimulated oxidant production and PP2A activity, dephosphorylated phosphoserine and phosphothreonine residues in the tight junction-associated protein occludin, decreased the abundance of occludin at cell borders, and increased monolayer permeability to albumin. NADPH oxidase inhibitors prevented PP2A activation and monolayer leak, showing that these changes required reactive oxygen species. Okadaic acid, at a concentration that inhibited PP2A activity and monolayer leak, prevented occludin dephosphorylation and redistribution, implicating PP2A in the responses of occludin to septic insult. Incubation with ascorbate or DHAA raised intracellular ascorbate concentrations and mitigated the effects of septic insult. In conclusion, ascorbate acts within microvascular endothelial cells to inhibit septic stimulation of oxidant production by NADPH oxidase and thereby prevents PP2A activation, PP2A-dependent dephosphorylation and redistribution of occludin, and disruption of the endothelial barrier.
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