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VASCULAR BIOLOGY
1Vascular Biology Center and Department of Pharmacology, Medical College of Georgia, Augusta, Georgia; and 2Department of Pharmacology and Molecular Cardiobiology Division, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut
Submitted 7 April 2005 ; accepted in final form 26 May 2005
Mislocalization of endothelial nitric oxide (NO) synthase (eNOS) in response to oxidized low-density lipoprotein, cholesterol depletion, elevated blood pressure, and bound eNOS interacting protein/NOS traffic inducer is associated with reduced NO release via unknown mechanisms. The proper targeting of eNOS to the plasma membrane or intracellular organelles is an important regulatory step controlling enzyme activity. Previous studies have shown that plasma membrane eNOS is constitutively phosphorylated on serine 1179 and highly active. In contrast, the activity of eNOS targeted to intracellular organelles is more complex. The cis-Golgi eNOS is fully activated by Akt-dependent phosphorylation. However, eNOS targeted to the trans-Golgi is decidedly less active in response to all modes of activation, including mutation to the phosphomimetic aspartic acid. In this study, we establish that when expressed within other intracellular organelles, such as the mitochondria and nucleus, the activity of eNOS is also greatly reduced. To address the mechanisms underlying the impaired catalytic activity of eNOS within these locations, we generated subcellular-targeted constructs that express a calcium-independent NOS isoform, iNOS. With the use of organelle specific (plasma membrane, cis- vs. trans-Golgi, plasma membrane, and Golgi, nucleus, and mitochondria) targeting motifs fused to the wild-type iNOS, we measured NO release from intact cells. With the exception of the Golgi lumen, our results showed no impairment in the ability of targeted iNOS to synthesize NO. Confirmation of correct targeting was obtained through confocal microscopy using identical constructs fused to the green fluorescent protein. We conclude that the reduced activation of eNOS within discrete cytoplasmic regions of the Golgi, the mitochondria and the nucleus is primarily due to insufficient access to calcium-calmodulin.
nitric oxide; Akt; Golgi
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