title: A novel method of measuring nitric-oxide-dependent fluorescence using 4,5-diaminofluorescein (DAF-2) in the isolated Langendorff-perfused rabbit heart.
abstract: 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.
authors.affiliation: Cardiology Group, Department of Cardiovascular Sciences, University of Leicester, Clinical Sciences Wing, Glenfield Hospital, Leicester, UK.
authors.forename: Vanlata H
authors.initials: VH
authors.lastname: Patel
authors.forename: Kieran E
authors.initials: KE
authors.lastname: Brack
authors.forename: John H
authors.initials: JH
authors.lastname: Coote
authors.forename: G André
authors.initials: GA
authors.lastname: Ng
mesh_terms: D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D003931:Diacetyl / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019793:Fluorescein / Q000008:administration & dosage*; D005456:Fluorescent Dyes / Q000008:administration & dosage*; D006352:Heart Ventricles / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D007267:Injections; D008297:Male; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D019335:Nitroarginine / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D010477:Perfusion; D011817:Rabbits; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014665:Vasodilator Agents / Q000494:pharmacology
chemical_list: C111205:4,5-diaminofluorescein; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D020030:Nitric Oxide Donors; D014665:Vasodilator Agents; D009599:Nitroprusside; C004717:diacetylmonoxime; D019335:Nitroarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D003931:Diacetyl; D001920:Bradykinin; D019793:Fluorescein