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Jan J. Blom, Vanessa Hill, William D. Eldred; Inhibition of the Adrenomedullin/Nitric Oxide Signaling Pathway in Early Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3584.
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Diabetic retinopathy is a leading cause of vision impairment and blindness in adults. Most research has focused on the vascular pathology and its prevention. However, electrophysiological deficits and neurodegeneration occur before these vascular changes and they have a significant impact on vision. The nitric oxide (NO) signaling pathway is integrally involved in visual processing, and changes in the NO pathway are measurable in eyes of diabetic patients. The small peptide adrenomedullin (ADM), can activate calcineurin, a Ca2+ activated phosphatase, that dephosphorylates neuronal nitric oxide synthase (nNOS) at Ser 847 to increase its enzyme activity. ADM levels are elevated in eyes of diabetic patients, and therefore may play a role in the pathology of diabetes and diabetic retinopathy. The goal of this research is to examine the role of the ADM signaling pathway in an in vitro retinal organ culture system and in mice with diabetic retinopathy.
PCR, immunocytochemistry, NO imaging, and a nitrite assay were used to determine the modulation and localization of the components of the ADM signaling pathway in two diabetic models. Retinal cultures incubated for 24 to 96 hours in glucose levels ranging from 5mM to 30mM glucose were used to identify early changes in an in vitro system and the five week streptozotocin induced diabetic mouse model was used for longer time points.
In retinal cultures incubated in high glucose, there was a decrease in nNOS-like immunoreactivity (LI) and an increase in cGMP-LI. Nitrite assays showed an increase in NO in isolated retinas incubated in high glucose, as well as in response to stimulation with ADM. Stimulation with ADM also increased cAMP-LI and cGMP-LI levels in retinal cultures. In retinal cultures or five week diabetic mice, there were increases in ADM-LI, a decrease in phospho-nNOS(p-nNOS)-LI, and an increase in ADM message shown using real time PCR. The inhibition of PKC in culture decreased ADM-LI and increased p-nNOS-LI. When calcineurin was inhibited with FK506, there was an increase in p-nNOS-LI in retinas incubated in high glucose. In five week diabetic mice NO imaging showed dramatic increases in NO. ADM mRNA levels were lowered in diabetic mice treated with the PKC inhibitor LY379176 (Eli Lilly).
These results indicate that the ADM/NO signaling pathway can be modulated in short term retinal organ cultures by high glucose in ways that closely resemble in vivo diabetic retinopathy. Continued exploration of this pathway will provide new information how this signaling pathway can be selectively targeted to reduce the pathological changes in diabetic retinopathy.
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