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1 Vascular Biology and Pharmacology, Medical College of Georgia, Augusta, Georgia, USA
* To whom correspondence should be addressed. E-mail: dfulton{at}mcg.edu.
Mislocalization of eNOS in response to oxidized LDL, cholesterol depletion, elevated blood pressure and bound NOSIP/NOSTRIN 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 S1179 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 phospho mimetic aspartic acid. In the current 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. Using 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 (EGFP). 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.
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