A novel assay for 5′-nucleotidase using 1,<i>N</i>6-etheno-AMP as substrate, and comments on the properties of the reaction product, ethenoadenosine

Jamal, Zahirali; Afkham-Ebrahimi, A; Saggerson, E D

doi:10.1042/bj2500369

Cited by 26 publications

(26 citation statements)

References 36 publications

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“…As shown in Fig. 1A, HMVEC pretreatment with the adenosine A 2 receptor agonist NECA at concentrations that approximate measured adenosine plasma levels (1 M) (17) induced a time-dependent increase in functional CD73 (ANOVA, p Ͻ 0.01) as determined by HPLC analysis of E-AMP conversion to E-adenosine (15), with a 5.4-Ϯ 1.1-fold increase at 48 h following NECA exposure ( p Ͻ 0.001). Shorter periods of incubation with NECA (i.e., Ͻ8 h) revealed no significant change in CD73 activity (data not shown).…”

Section: Adenosine Receptor Activation Induces Endothelial Cd73mentioning

confidence: 99%

“…Endothelial CD73 surface activity was assessed as before (4) by measuring the conversion of etheno-AMP (E-AMP) to ethenoadenosine (E-ADO) (15). Briefly, HBSS with or without ␣,␤-methylene ADP (APCP) was added to HMVEC monolayers on six-well plates.…”

Section: Measurement Of 5-ectonucleotidase Activitymentioning

confidence: 99%

“…Briefly, 1 g of DNase I (GenHunter, Nashville, TN)-treated total RNA-derived HMVEC exposed to experimental conditions was reverse transcribed into cDNA using a reaction mixture consisting of 4 l of 25 mM MgCl 2 , 2 l of 10ϫ reverse transcriptase buffer, 2 l of 10 mM concentrations of each dNTP, 0.5 l of rRNase in RNase inhibitor (20 U total), 15 U of avian myeloblastosis virus reverse transcriptase, and 0.5 g of oligo(dT) 15 primer in a total volume of 20 l. Thermostable Tfl DNA polymerase (Promega, Madison, WI) from Thermus flavus was used for second-strand cDNA synthesis and DNA amplification. The CD73 PCR reaction contained 1 M concentrations each of the sense primer (5Ј-CAC CAA GGT TCA GCA GAT CCG C-3Ј) and the antisense primer (5Ј-GTT CAT CAA TGG GCG ACC GG-3Ј), 10 l of 5ϫ PCR buffer, 1 mM MgSO 4 , 0.2 mM dNTP, and 5 U of Tfl enzyme mix in a total volume of 50 l. Each primer set was amplified using 25 cycles of 94°C for 1 min, 60°C for 2 min, and 72°C for 4 min, followed by a final extension of 72°C for 7 min.…”

Section: Analysis Of Mrna Levels By Pcrmentioning

confidence: 99%

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Regulation of Endothelial CD73 by Adenosine: Paracrine Pathway for Enhanced Endothelial Barrier Function

Narravula

Lennon

Mueller

et al. 2000

The Journal of Immunology

109

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During episodes of inflammation, multiple cell types release adenine nucleotides in the form of ATP, ADP, 5′-AMP, and adenosine. In particular, following activation, polymorphonuclear leukocytes release larger quantities of 5′-AMP. Extracellular 5′-AMP is metabolized to adenosine by surface-expressed 5′-ectonucleotidase (CD73). Adenosine liberated by this process activates surface adenosine A2B receptors, results in endothelial junctional reorganization, and promotes barrier function. We hypothesized that adenosine signaling to endothelia provides a paracrine loop for regulated expression of CD73 and enhanced endothelial barrier function. Using an in vitro microvascular endothelial model, we investigated the influence of 5′-AMP; adenosine; and adenosine analogues on CD73 transcription, surface expression, and function. Initial experiments revealed that adenosine and adenosine analogues induce CD73 mRNA (RT-PCR), surface expression (immunoprecipitation of surface biotinylated CD73), and function (HPLC analysis of etheno-AMP conversion to ethenoadenosine) in a time- and concentration-dependent fashion. Subsequent studies revealed that similar exposure conditions increase surface protein through transcriptional induction of CD73. Analysis of DNA-binding activity by EMSA identified a functional role for CD73 cAMP response element and, moreover, indicated that multiple cAMP agonists induce transcriptional activation of functional CD73. Induced CD73 functioned to enhance 5′-AMP-mediated promotion of endothelial barrier (measured as a paracellular flux of 70-kDa FITC-labeled tracer). These results provide an example of transcriptional induction of enzyme (CD73) by enzymatic product (adenosine) and define a paracrine pathway for the regulated expression of vascular endothelial CD73 and barrier function.

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Section: Adenosine Receptor Activation Induces Endothelial Cd73mentioning

confidence: 99%

Section: Measurement Of 5-ectonucleotidase Activitymentioning

confidence: 99%

Section: Analysis Of Mrna Levels By Pcrmentioning

confidence: 99%

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Regulation of Endothelial CD73 by Adenosine: Paracrine Pathway for Enhanced Endothelial Barrier Function

Narravula

Lennon

Mueller

et al. 2000

The Journal of Immunology

109

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“…It is noteworthy that e-Ado derived nucleotides can be fluorimetrically detected at concentrations as low as !0-8 ~. Another interesting prOperw or ~-(Ap~A) is that ~-Ado produced from it is not further catabolized via adenosine deaminase because of the inability of this enzyme to r¢colnize ~-Ado as a subs/rate [25]; this allows, if desired, to follow specifically tile degradation of adenosine nucleoddes.…”

Section: T-(ap4a) As a Substrate Of Ap4a Cleaving Enzymesmentioning

confidence: 99%

Di(1,N⁶‐ethenoadenosine)5′, 5‴‐P¹,P⁴‐tetraphosphate, a fluorescent enzymatically active derivative of Ap₄A

Rotilán¹,

Ramos²,

Pintor³

et al. 1991

FEBS Letters

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Di(1,N6‐ethenoadenosine) 5′, 5‴‐P1, P4‐tetraphosphate, ϵ‐(Ap4A), a fluorescent analog of Ap4A has been synthesized by reaction of 2‐chloroacetaldehyde with Ap4A. At neutral pH this Ap4A analog presents characteristic maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (λexc 307 nm, λem 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split ϵ(Ap4A) and catabolize the resulting ϵ‐nucleotide moieties up to ϵ‐Ado.

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“…We determined the activity of CD73 exposed on the surface of intact mononuclear cells by quantifying the conversion of 1,N 6 -ethenoadenosine 5Ј-monophosphate to 1,N 6 -ethenoadenosine 21 in the presence and absence of the CD73-specific inhibitor ␣,ß-methyleneadenosine 5Ј-diphosphate. The difference in these 2 activities reflects CD73 activity.…”

Section: Ecto-5-nucleotidase Activitymentioning

confidence: 99%

Upregulation of Ecto-5′-Nucleotidase by Rosuvastatin Increases the Vasodilator Response to Ischemia

et al. 2010

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Abstract-3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins) are effective in the primary and secondary prevention of cardiovascular events. Although originally developed to improve lipid profile, statins have demonstrated a surplus of beneficial pleiotropic effects, including improved endothelial function, reduced inflammation, and increased tolerance to ischemia-reperfusion injury. In preclinical studies, increased ecto-5Ј-nucleotidase activity, the key enzyme in extracellular adenosine formation, plays an important role in these effects. Because human data are absent, we explored the effects of rosuvastatin on ecto-5Ј-nucleotidase activity and the clinical relevance of increased extracellular adenosine during ischemia in humans in vivo. The forearm vasodilator responses to 3 increasing periods of forearm ischemia (2, 5, and 13 minutes) were determined during placebo and caffeine (an adenosine receptor antagonist) infusion into the brachial artery. At the end of an 8-day treatment period with rosuvastatin (20 mg per day), this whole procedure was repeated. During both experiments, ecto-5Ј-nucleotidase activity was determined. Vasodilator responses are expressed as the percentage increase in forearm blood flow ratio from baseline. Rosuvastatin increased ecto-5Ј-nucleotidase activity by 49Ϯ17% and enhanced the vasodilator response after 2, 5, and 13 minutes of ischemia in the absence (146Ϯ19, 330Ϯ26, and 987Ϯ133 to 312Ϯ77, 566Ϯ107, and 1533Ϯ267) but not in the presence of caffeine ( Key Words: adenosine Ⅲ ecto-5Ј-nucleotidase Ⅲ caffeine Ⅲ ischemia Ⅲ reactive hyperemia 3 -Hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins) are effective in the primary and secondary prevention of cardiovascular events. 1,2 Although originally developed to improve lipid profile, statins have demonstrated a surplus of beneficial pleiotropic effects, including improved endothelial function, reduced inflammation, and increased tolerance to ischemia-reperfusion injury. [3][4][5][6][7] These effects are independent of plasma cholesterol lowering and improve outcome after cardiovascular events. 8,9 Increased extracellular adenosine formation has been implicated as one of the underlying mechanisms. This is supported by several preclinical studies demonstrating the activation of ecto-5Ј-nucleotidase (CD73) activity, increased stimulation of adenosine receptors, and adenosine-mediated protection against ischemia-reperfusion injury by statins. 10 -12 The enzyme CD73 dephosphorylates extracellular AMP, which forms the rate-limiting step in the formation of extracellular adenosine. 13 We recently demonstrated increased extracellular adenosine formation in healthy human volunteers after a 1-week treatment with rosuvastatin using a pharmacological approach. In addition, we demonstrated rosuvastatin-induced augmentation of postocclusive reactive hyperemia (PORH). 5 However, we did not assess CD73 activity as a potential source of extracellular adenosine, nor did we investigate the role of adenosine in rosuvasta...

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A novel assay for 5′-nucleotidase using 1,N6-etheno-AMP as substrate, and comments on the properties of the reaction product, ethenoadenosine

Cited by 26 publications

References 36 publications

Regulation of Endothelial CD73 by Adenosine: Paracrine Pathway for Enhanced Endothelial Barrier Function

Regulation of Endothelial CD73 by Adenosine: Paracrine Pathway for Enhanced Endothelial Barrier Function

Di(1,N⁶‐ethenoadenosine)5′, 5‴‐P¹,P⁴‐tetraphosphate, a fluorescent enzymatically active derivative of Ap₄A

Upregulation of Ecto-5′-Nucleotidase by Rosuvastatin Increases the Vasodilator Response to Ischemia

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A novel assay for 5′-nucleotidase using 1,N6-etheno-AMP as substrate, and comments on the properties of the reaction product, ethenoadenosine

Cited by 26 publications

References 36 publications

Regulation of Endothelial CD73 by Adenosine: Paracrine Pathway for Enhanced Endothelial Barrier Function

Regulation of Endothelial CD73 by Adenosine: Paracrine Pathway for Enhanced Endothelial Barrier Function

Di(1,N6‐ethenoadenosine)5′, 5‴‐P1,P4‐tetraphosphate, a fluorescent enzymatically active derivative of Ap4A

Upregulation of Ecto-5′-Nucleotidase by Rosuvastatin Increases the Vasodilator Response to Ischemia

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Di(1,N⁶‐ethenoadenosine)5′, 5‴‐P¹,P⁴‐tetraphosphate, a fluorescent enzymatically active derivative of Ap₄A