Pitfalls and limitations in using 4,5-diaminofluorescein for evaluating the influence of polyphenols on nitric oxide release from endothelial cells

Uhlenhut, Klaus; Högger, Petra

doi:10.1016/j.freeradbiomed.2012.03.006

Cited by 17 publications

(9 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We cannot rule out a role of prostacyclins, EETs (epoxyeicosatrienoic acid), or other EDHFs (endothelial-derived hyperpolarizing factor); however, our prior studies argue against a role of EETs because the enzyme responsible for their production, CYP450 epoxygenase, is directly inhibited by H 2 O 2 . 60 Although it is not technically feasible to directly assess NO bioavailability in the small tissue samples used, 61 we used DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) as a surrogate marker for NO production in cultured human endothelial cells. We observed an elevation in eNOS message and p-eNOS (phospho-endothelial nitric oxide synthase; ser 1117) after activation of TERT with AGS 499.…”

Section: Discussionmentioning

confidence: 99%

Critical Role for Telomerase in the Mechanism of Flow-Mediated Dilation in the Human Microcirculation

Beyer

Freed

Durand

et al. 2016

Circulation Research

117

View full text Add to dashboard Cite

Not all of the effects of telomerase can be easily explained by its nuclear actions. Growing evidence suggests that TERT, the catalytic subunit of telomerase, can reversibly translocate from the nucleus to organelles (including the mitochondria), in a dose-and time-dependent manner, in response to stressors, Objective: We examined the hypothesis that telomerase activity modulates microvascular flow-mediated dilation, and loss of telomerase activity contributes to the change of mediator from nitric oxide to mitochondrial hydrogen peroxide in patients with coronary artery disease (CAD). Clinical Track Methods and Results:Human coronary and adipose arterioles were isolated for videomicroscopy. Flow-mediated dilation was measured in vessels pretreated with the telomerase inhibitor BIBR-1532 or vehicle. Statistical differences between groups were determined using a 2-way analysis of variance repeated measure (n≥4; P<0.05). L-NAME (N ω -nitro-L-arginine methyl ester; nitric oxide synthase inhibitor) abolished flow-mediated dilation in arterioles from subjects without CAD, whereas polyethylene glycol-catalase (PEG-catalase; hydrogen peroxide scavenger) had no effect. After exposure to BIBR-1532, arterioles from non-CAD subjects maintained the magnitude of dilation but changed the mediator from nitric oxide to mitochondrial hydrogen peroxide (% max diameter at 100 cm H 2 O: vehicle 74.6±4.1, L-NAME 37.0±2.0*, PEG-catalase 82.1±2.8; BIBR-1532 69.9±4.0, L-NAME 84.7±2.2, PEG-catalase 36.5±6.9*). Conversely, treatment of microvessels from CAD patients with the telomerase activator AGS 499 converted the PEG-catalase-inhibitable dilation to one mediated by nitric oxide (% max diameter at 100 cm H 2 O: adipose, AGS 499 78.5±3.9; L-NAME 10.9±17.5*; PEG-catalase 79.2±4.9). Endothelial-independent dilation was not altered with either treatment. Conclusions:

show abstract

Section: Discussionmentioning

confidence: 99%

Critical Role for Telomerase in the Mechanism of Flow-Mediated Dilation in the Human Microcirculation

Beyer

Freed

Durand

et al. 2016

Circulation Research

117

View full text Add to dashboard Cite

show abstract

“…The current observation conrms previously reported observations that 4,5-diaminouorescein-based probes are prone to non-specic interactions with reactive species present in cells and cell culture media, exhibiting low selectivity towards cellular NO at physiologically relevant conditions. 101,102 Finally, we did not observe signs of cytotoxicity when using our family of probes, even aer 18 hour of incubation. Since NO 530 was the best performer in terms of uorescence ratio in NO-dosing studies, we evaluated its cytotoxicity in RAW 264.7 cells at various concentrations with a Calcein Blue/propidium iodide viability assay.…”

Section: Probe Performance In Cellsmentioning

confidence: 56%

2-Amino-3′-dialkylaminobiphenyl-based fluorescent intracellular probes for nitric oxide surrogate N₂O₃

et al. 2020

View full text Add to dashboard Cite

show abstract

“…To obtain direct evidence for the interaction between the NO‐generating system and the H 2 S‐generating system in the muscle layer of the mouse colon muscle, we evaluated NO production in CSE‐KO mouse preparations and WT control mouse preparations using fluorescent DAF‐2 probes. DAF‐2 is nitrosated with an oxidation product of NO to form the highly fluorescent triazolofluorescein DAF‐2T (Uhlenhut & Hogger, ). DAF‐2 probes are widely used to evaluate NO production in living tissue (Sutherland et al .…”

Section: Resultsmentioning

confidence: 99%

Effect of endogenous hydrogen sulfide on the transwall gradient of the mouse colon circular smooth muscle

Sha¹,

Linden²,

Farrugia

et al. 2014

The Journal of Physiology

View full text Add to dashboard Cite

Key pointsr A CO-dependent transwall gradient of resting membrane potential exists across the circular muscle layer in the gastrointestinal tract; this gradient regulates the strength of phasic muscle contraction during electrical slow wave activity.r H 2 S, CO and NO are endogenously generated in the muscle layers of the gut wall. r Inhibiting the H 2 S producing enzyme CSE with the CSE inhibitor PAG had no significant effect on transwall resting membrane potential gradient in control preparations but significantly shifted the entire gradient in the depolarizing direction in preparations pretreated with the NO synthase (NOS) inhibitor L-NNA.r PAG significantly shifted the gradient in the depolarizing direction in mouse preparations lacking neuronal NOS (nNOS-KO) and the gradient was significantly shifted in the depolarizing direction in mouse preparations lacking both nNOS and CSE (CSE-KO-nNOS-KO).r NO production was significantly higher in CSE-KO mouse preparations compared to wild-type mouse preparations and the amplitude of NO-mediated slow inhibitory junction potentials (S-IJPs) was significantly higher in CSE-KO mouse preparations compared to the amplitude of S-IJPs in wild-type mouse preparations.r Nearly all submucosal neurons and myenteric neurons were CSE positive and 11% of submucosal neurons and 50% of myenteric neurons were nNOS positive.r Our novel findings suggested that endogenous H 2 S is a stealth hyperpolarizing factor on smooth muscle cells and that endogenous H 2 S inhibits NO production from nNOS.Abstract A transwall gradient in resting membrane potential (RMP) exists across the circular muscle layer in the mouse colon. This gradient is dependent on endogenous generation of CO. H 2 S is also generated in muscle layers of the mouse colon. The effect of endogenously generated H 2 S on the transwall gradient is not known. The aim was to investigate the role of endogenous H 2 S. Our results showed that the CSE inhibitor DL-propargylglycine (PAG, 500 μM) had no effect on the transwall gradient. However, in preparations pretreated with the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA, 200 μM) and in nNOS-knockout (KO) mouse preparations, PAG shifted the transwall gradient in the depolarizing direction. In CSE-KO-nNOS-KO mice, the gradient was shifted in the depolarizing direction. Endogenous generation of NO was significantly higher in muscle preparations of CSE-KO mice compared to wild-type (WT) mice. The amplitude of NO-mediated slow inhibitory junction potentials (S-IJPs) evoked by electric field stimulation was significantly higher in CSE-KO mouse preparations compared to the amplitude of S-IJPs in wild-type mouse preparations. CSE was present in all submucosal ganglion neurons and in almost all myenteric ganglion neurons. Eleven per cent of CSE positive neurons in the submucosal plexus

show abstract

Pitfalls and limitations in using 4,5-diaminofluorescein for evaluating the influence of polyphenols on nitric oxide release from endothelial cells

Cited by 17 publications

References 53 publications

Critical Role for Telomerase in the Mechanism of Flow-Mediated Dilation in the Human Microcirculation

Critical Role for Telomerase in the Mechanism of Flow-Mediated Dilation in the Human Microcirculation

2-Amino-3′-dialkylaminobiphenyl-based fluorescent intracellular probes for nitric oxide surrogate N₂O₃

Effect of endogenous hydrogen sulfide on the transwall gradient of the mouse colon circular smooth muscle

Contact Info

Product

Resources

About

Pitfalls and limitations in using 4,5-diaminofluorescein for evaluating the influence of polyphenols on nitric oxide release from endothelial cells

Cited by 17 publications

References 53 publications

Critical Role for Telomerase in the Mechanism of Flow-Mediated Dilation in the Human Microcirculation

Critical Role for Telomerase in the Mechanism of Flow-Mediated Dilation in the Human Microcirculation

2-Amino-3′-dialkylaminobiphenyl-based fluorescent intracellular probes for nitric oxide surrogate N2O3

Effect of endogenous hydrogen sulfide on the transwall gradient of the mouse colon circular smooth muscle

Contact Info

Product

Resources

About

2-Amino-3′-dialkylaminobiphenyl-based fluorescent intracellular probes for nitric oxide surrogate N₂O₃