Technical Reference #1852

1852.

Losartan increases NO release in afferent arterioles during regression of L-NAME-induced renal damage Frank Helle; Bjarne M Iversen; and Christos Chatziantoniou, Institut National de la Sante et de la Recherche Medicale - Hopital Tenon, American Journal of Physiology - Renal Physiology, 298(1852), (2010)
Link To Paper

Keywords:
endothelin; nitric oxide; endothelial dysfunction; angiotensin II; vascular remodeling

Materials & Methods:
NO fluorescence and mean lumen diameter measurements. Isolated vessels were attached by self-adhesion to the clean glass surface of a No 1.5 coverslip mounted in a petridish (MatTek P35G-1.5–14-C) containing 3 ml medium without Ca2 . Before the experiments Ca2 concentration in the medium was gradually increased to 2 mmol/l in three steps (20 mol/l 200 mol/l and 2 mmol/l) with 3- to 5-min incubation in between. Vessels were loaded with 5.0 g/ml diaminofluorescein-FM (DAF-FM) diacetate at room temperature for 1 h. After wash-out of the dye vessels were incubated for 15 min at 37°C to ensure complete deesterification since DAF-FM was added to the perfusion bath as an ester to facilitate passage across the cell membrane. If a stable baseline was obtained 300 l medium was aspirated from the dish and the same volume containing 10 6 mol/l ACh 10 8 mol/l endothelin (ET) or 10 6 mol/l ET was gently superfused ( 100 l/s) over the vessels using a 1-ml pipette (Gilson) producing final concentrations of 10 7 mol/l ACh 10 9 mol/l ET or 10 7 mol/l ET respectively in the vessel baths. When obtaining a dose-response curve a stable baseline was first obtained for 2 min and then 300 l were aspirated and the same amount containing 10 9 10 8 10 7 and 10 6 M ET was successively added producing final concentrations of 10 10 10 9 10 8 and 10 7 M ET respectively in the vessel bath. Inhibition of NOS was performed by adding L-NAME (10 4 M) to the vessel bath 15 min before starting the recordings. Experiments with exogenous NO were performed by adding 10 2 mM S-nitroso-Nacetyl- penicillamine (SNAP) to the bath solution 1 min before the start of the recordings.

Microscopic Technique
Confocal and Fluorescence Microscopy

Cell Type(s)
Afferent arteriole cells