Sympathetic neurotransmission modulates expression of inflammatory markers in the rat retina

Recent evidence suggests that diabetic retinopathy may involve some components of chronic inflammation. Since surgical sympathectomy produces most of the retinal changes noted in the retina of an STZ-treated rat in a non-diabetic rat, we wanted to determine whether sympathetic neurotransmission regulates gene and protein expression of inducible nitric oxide synthase (iNOS) and the prostaglandin (PGE2) receptor, as well as the levels of PGE2. Real-time PCR was conducted on retinal samples from rats that were surgically sympathectomized to investigate steady-state mRNA expression of iNOS in the sympathectomized and contralateral retina. Western blot analysis was done on protein samples from the sympathectomized and contralateral retina for iNOS and PGE2-EP2 receptor. An ELISA assay was done on retinal supernatant fractions to measure PGE2 levels. Additionally, human retinal endothelial cells were grown in either low (5 mM) or high (25 mM) glucose medium and stimulated with isoproterenol (beta-adrenergic receptor agonist), xamoterol (beta1-adrenergic receptor subtype agonist), or BRL37344 (beta3-adrenergic receptor subtype agonist) and the effects of agonist stimulation on iNOS and PGE2 levels in low and high glucose was investigated. Sympathectomy significantly increases gene and protein expression of iNOS, as well as levels of PGE2 and protein expression of PGE2-EP2 receptor subtype. Isoproterenol treatment for 6 h to human retinal endothelial cells grown in high glucose medium reduced iNOS protein expression, but had no effect on PGE2 levels or PGE2 receptor protein expression. iNOS expression was attenutated by stimulation with xamoterol, while BRL37344 had no effect, suggesting that the iNOS effects are mediated by beta1-adrenergic receptors. These results suggest that loss of sympathetic activity, as occurs in diabetes, results in an upregulation of iNOS and PGE2-EP2 receptor protein expression, as well as PGE2 levels. Isoproterenol stimulation of human retinal endothelial cells cultured in a hyperglycemic environment decreased iNOS expression with no change in PGE2 levels, suggesting that only iNOS expression is modulated by sympathetic neurotransmission in endothelial cells. Overall, these results further the idea that alterations in sympathetic neurotransmission may result in many of the changes noted in the retina of the STZ-treated rat.