Document Detail

A novel method of measuring nitric-oxide-dependent fluorescence using 4,5-diaminofluorescein (DAF-2) in the isolated Langendorff-perfused rabbit heart.
MedLine Citation:
PMID:  18180949     Owner:  NLM     Status:  MEDLINE    
4,5-Diaminofluorescein (DAF-2) has been used to measure nitric oxide (NO) activity from a variety of preparations. The aim of this study was to develop a method to assess changes in NO fluorescence using DAF-2 in isolated rabbit hearts (2.0-2.5 kg, n = 8). Hearts were perfused in constant flow Langendorff mode and instrumented to record aortic perfusion pressure, left ventricular pressure and left ventricular epicardial fluorescence using a bifurcated light guide at excitation wavelengths of 470 +/- 10, 480 +/- 10, 490 +/- 10 and 500 +/- 10 nm collected at 535 nm. DAF-2 DA was loaded using a single bolus 150-microl (1 micromol) injection. Changes in NO-dependent fluorescence were determined using the NO donor sodium nitroprusside (SNP: 100 microM), NO-dependent vasodilator bradykinin (BK: 100 microM) and non-specific NO synthase inhibitor NG-nitro-L-arginine (LNA: 200 microM) before and after loading hearts with DAF-2 DA. Before loading, these agents did not alter epicardial fluorescence. After loading, SNP, BK and LNA produced a consistent change in each excitation wavelength. Together, this suggests that change in fluorescence represents changes in the level of NO. SNP and BK increased whilst LNA significantly decreased left ventricular epicardial NO-dependent fluorescence. At the excitation wavelength of 490 nm, SNP and BK increased fluorescence by 104.7 +/- 18.7 mV (1.1 +/- 0.2%) and 150.7 +/- 26.1 mV (1.5 +/- 0.3%) respectively, whilst LNA significantly decreased fluorescence by 90.3 +/- 17.0 mV (-0.9 +/- 0.2%). Changing the rate of aortic perfusion did not alter fluorescence suggesting that changes in aortic perfusion pressure per se do not contribute to the changes in DAF-2 fluorescence seen with SNP, BK or LNA. Our data suggest that DAF-2 DA is a useful fluorescence indicator for measuring NO activity in isolated hearts.
Vanlata H Patel; Kieran E Brack; John H Coote; G André Ng
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Publication Detail:
Type:  In Vitro; Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-01-05
Journal Detail:
Title:  Pflügers Archiv : European journal of physiology     Volume:  456     ISSN:  0031-6768     ISO Abbreviation:  Pflugers Arch.     Publication Date:  2008 Jun 
Date Detail:
Created Date:  2008-05-01     Completed Date:  2008-09-25     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0154720     Medline TA:  Pflugers Arch     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  635-45     Citation Subset:  IM    
Cardiology Group, Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield Hospital, Leicester, UK.
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MeSH Terms
Bradykinin / pharmacology
Diacetyl / analogs & derivatives,  pharmacology
Enzyme Inhibitors / pharmacology
Fluorescein / administration & dosage*
Fluorescent Dyes / administration & dosage*
Heart Ventricles / drug effects,  metabolism
Myocardium / enzymology,  metabolism*
Nitric Oxide / metabolism*
Nitric Oxide Donors / pharmacology
Nitric Oxide Synthase / antagonists & inhibitors,  metabolism
Nitroarginine / pharmacology
Nitroprusside / pharmacology
Reproducibility of Results
Spectrometry, Fluorescence / methods*
Vasodilator Agents / pharmacology
Grant Support
pg/02/088//British Heart Foundation
Reg. No./Substance:
0/4,5-diaminofluorescein; 0/Enzyme Inhibitors; 0/Fluorescent Dyes; 0/Nitric Oxide Donors; 0/Vasodilator Agents; 10102-43-9/Nitric Oxide; 15078-28-1/Nitroprusside; 2149-70-4/Nitroarginine; 2321-07-5/Fluorescein; 431-03-8/Diacetyl; 57-71-6/diacetylmonoxime; 58-82-2/Bradykinin; EC Oxide Synthase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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