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

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 CaMKIIdelta(2) with a perinuclear distribution and concentration in aggresome-like structures identified by colocalization with gamma-tubulin. Furthermore, CaMKIIdelta(2) coimmunoprecipitated with iNOS in a CaMKII activity-dependent manner. Addition of Ca(2+)-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 CaMKIIdelta(2) mutant altered iNOS localization, shifting iNOS from the cytosolic fraction. Suppression of CaMKIIdelta(2) 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 CaMKIIdelta(2) may be an important regulator of iNOS trafficking and activity in VSMCs.