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, Ca(2+)/CaMKII may play a role in post-translationally controlled iNOS activity. We accordingly investigated the involvement of the Ca(2+)/CaMKIIδ(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 μg/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-((3)H)-arginine into l-((3)H)-citrulline) and intracellular nitrotyrosine levels (immunofluorescence) were about 2.7 fold greater in DR- than in CR-VSMCs. Interestingly, LPS increased intracellular Ca(2+) 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/CaMKIIδ(2) (Western Blot) and increased iNOS activity. The CaMKII inhibitor KN-93 abolished all the LPS-effects. CONCLUSION: These results indicate that the Ca(2+)/CaMKIIδ(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.