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iNOS regulation by calcium/calmodulin-dependent protein kinase II in vascular smooth muscle

¹Center for Cardiovascular Sciences, Albany Medical Center, Albany, New York; ²Department of Biology and Physics, Kennesaw State University, Kennesaw, Georgia; and ³Department of Pathology, Emory School of Medicine, Atlanta, Georgia

Submitted 15 November 2006 ; accepted in final form 8 February 2007

Nitric oxide synthase (NOS) expression is regulated transcriptionally in response to cytokine induction and posttranslationally by palmitoylation and trafficking into perinuclear aggresome-like structures. We investigated the effects of multifunctional calcium/calmodulin-dependent protein kinase II protein kinase (CaMKII) on inducible NOS (iNOS) trafficking in cultured rat aortic vascular smooth muscle cells (VSMCs). Immunofluorescence and confocal microscopy demonstrated colocalization of iNOS and CaMKII₂ with a perinuclear distribution and concentration in aggresome-like structures identified by colocalization with -tubulin. Furthermore, CaMKII₂ coimmunoprecipitated with iNOS in a CaMKII activity-dependent manner. Addition of Ca²⁺-mobilizing stimuli expected to activate CaMKII; a purinergic agonist (UTP) or calcium ionophore (ionomycin) caused a general redistribution of iNOS from cytosolic to membrane and nuclear fractions. Similarly, adenoviral expression of a constitutively active CaMKII₂ mutant altered iNOS localization, shifting iNOS from the cytosolic fraction. Suppression of CaMKII₂ using an adenovirus expressing a short hairpin, small interfering RNA increased nuclear iNOS localization in resting cells but inhibited ionomycin-induced translocation of iNOS to the nucleus. Following addition of these chronic and acute CaMKII modulators, there were fewer aggresome-like structures containing iNOS. All of the treatments that chronically affected CaMKII activity or expression significantly inhibited iNOS-specific activity following cytokine induction. The results suggest that CaMKII₂ may be an important regulator of iNOS trafficking and activity in VSMCs.

protein trafficking; inflammatory response; inducible nitric oxide synthase

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