Abstract
Purpose::
Elevated levels of endothelin-1 (ET-1), a potent vasoconstrictor and stimulator of oxidative stress, are associated with retinal ischemic disease. However, it is unclear whether vasodilator function of retinal resistance arterioles is susceptible to ET-1. Herein, we examined whether ET-1 can affect endothelium-dependent nitric oxide (NO)-mediated dilation of retinal arterioles and whether ischemic-related signaling factors such as Rho kinase (ROCK) and reactive oxygen species superoxide are involved in the ET-1-mediated effect.
Methods::
Porcine retinal arterioles were isolated and pressurized without flow for in vitro study. Videomicroscopic techniques were employed to record diameter change in response to agonists before and after exposure to a sub-vasoactive, pathophysiological concentration of ET-1 (10 pM). Vascular levels of superoxide anions were detected by dihydroethidium (DHE) fluorescence staining. Cellular localization of ROCK isoforms was identified by immunohistochemical analysis.
Results::
All vessels developed basal tone (62 ± 3 µm, resting diameter). Extraluminal treatment with ET-1 did not alter resting diameter (60 ± 4 µm) at 30 min but significantly decreased diameter (54 ± 3 µm) within 90 min. In addition, following the 90-min exposure to ET-1, vasodilation to endothelium-dependent NO-mediated agonist bradykinin, but not to endothelium-independent NO donor sodium nitroprusside, was significantly attenuated. DHE staining revealed that superoxide production in arteriolar endothelial cells was enhanced by ET-1 within 90 min but not 30 min. Co-administration of ET-1 with superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), ROCK inhibitor Y-27632 or NAD(P)H oxidase inhibitor apocynin, reduced the ET-1 induced superoxide production, as well as preserved normal resting basal tone and vasodilation to bradykinin. ROCK-2 but not ROCK-1 expression was localized to retinal endothelial cells.
Conclusions::
A clinically relevant level of ET-1 elicits a temporal increase in basal tone and inhibition of endothelium-dependent NO-mediated dilation of retinal arterioles. Both ET-1-mediated actions appear to be dependent upon ROCK-2 activation and subsequent production of superoxide via NAD(P)H oxidase leading to a reduction in endothelial NO bioavailability. These data may partly explain the impaired retinal flow regulation in ocular diseases associated with an upregulation of the ET system.
Keywords: vascular cells • oxidation/oxidative or free radical damage • blood supply