Nitric Oxide Stress uncovers pM b2‐adrenergic mediated dilation to isoproterenol mimicked by preventing clathrin endosome formation.

Abstract

Nitric oxide exposure induces an oxidative/nitrosative stress (NOX) causing endothelial dysfunction. Our goal was to determine how this stress impacted vasoactive responses to isoproterenol. Intravital microscopy of the hamster cheek pouch was performed (N=20; pentobarbital 70mg/kg i.p.). NOX was induced by applying nitroprusside (10–4M) for 2 minutes via tissue bath. Before NOX, dilation to micropipette isoproterenol was dose dependent (EC50 10–7M). After NOX, dilation was attenuated in the uM range, and a second dilation appeared as a narrow peak from 10–12 to 10–9M, which was inhibited by the b2‐adrenergic receptor (bAR‐2) inverse agonist, carazolol. Preventing clathrin endosome formation with dynasore attenuated dilation to isoproterenol (uM range), and uncovered dilation (pM range), thus mimicking NOX. Dynasore with NOX further increased the low dose potency and efficacy of dilation to isoproterenol (10–14 to 10–9M). To understand the mechanism, we tested whether the fluorescent adrenergic agonist, curcumin, and the bAR‐2 were co‐internalized using FRET in HeLa cells. Before NOX, curcumin is rapidly internalized and co‐localized with the bAR‐2. Moderate NOX delayed co‐localization, while extensive NOX prevented bAR‐2 internalization altogether. Thus, NOX prevented bAR‐2 internalization, and uncovered a low dose dilation to bAR‐2 linked activity. AHA 0655908T