Increased iNOS activity in vascular smooth muscle cells from diabetic rats: Potential role of Ca2+/calmodulin-dependent protein kinase II delta 2 (CaMKIIδ2)

Objective: Inducible nitric oxide synthase (iNOS) expression may be increased by cytokine plasma levels contributing to vascular damage in diabetes. Besides transcriptional regulation, Ca2+/CaMKII may play a role in post-translationally controlled iNOS activity. We accordingly investigated the involvement of the Ca2+/CaMKII delta(2) signaling pathway in regulating lipopolysaccharide (LPS)-induced iNOS activity in cultured aortic vascular smooth muscle cells (VSMCs) from diabetic rats. Methods and results: VSMCs obtained from 10 diabetic rats (DR) and 10 control rats (CR) were stimulated with 20 mu g/ml LPS. After 24 h, iNOS protein levels were 1.37 fold increased in DR- vs CR-VSMCs (p < 0.05; Western Blot), while iNOS activity (conversion L-(H-3)-arginine into L-(H-3)-citrulline) and intracellular nitrotyrosine levels (immunofluorescence) were about 2.7 fold greater in DR-than in CR-VSMCs. Interestingly, LPS increased intracellular Ca2+ levels (Fluorescence video imaging) more markedly in DR-than in CR-VSMCs. This was associated with CaMKII activation by phosphorylation, a decreased amount of co-immunoprecipitating iNOS/CaMKII delta(2) (Western Blot) and increased iNOS activity. The CaMKII inhibitor KN-93 abolished all the LPS-effects. Conclusion: These results indicate that the Ca2+/CaMKII delta(2) signaling pathway may be an important regulator of iNOS activity in diabetes, and hence contribute to the potential development of innovative therapeutic strategies for vascular complications in diabetes. (C) 2012 Elsevier Ireland Ltd. All rights reserved.