The Partitioning and Domain-Specific Multi-Site Phosphorylation "Zip Codes" of Endothelial Nitric Oxide Synthase in Uterine Artery Endothelial Cells.

Abstract

Pregnancy-associated rises in uterine blood flow occurs via changes in endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production at the level of uterine arteries. In arterial endothelial cells, the eNOS protein has been associated with lipid-ordered microdomains called caveolae and is tonically inhibited by the major caveolar scaffolding protein caveolin-1 (cav-1). However, the spatial and temporal dynamics of eNOS, including domain-specific post-translational modification via multi-site phosphorylation that are critical for enzyme activation and NO production, in response to a physiologic calcium mobilizing agonist-like ATP, especially during pregnancy are unknown. The aim of this study was to determine the spatial and temporal dynamics of eNOS in response to the calcium mobilizing physiologic agonist ATP in endothelial cells from the uterine arteries of pregnant ewes when the rises in UBF and NO are greatest. Primary uterine arteries of late gestation ewes were dissected and the endothelial cells were isolated and validated. Temporal and spatial dynamics of eNOS, and domain-specific multi-site phosphorylation were studied by density centrifugation, confocal microscopy, immunoisolation, native gel electrophoresis and Western blotting. Under unstimulated conditions, eNOS was predominantly located in specialized caveolar microdomains. Treatment with ATP resulted in time-dependent re-partitioning of eNOS between the caveolar and non-caveolar domains. The abundance of cav-1 bound eNOS significantly decreased in response to ATP (one way ANOVA, P = 0.039); compared to the unstimulated state, the magnitude of cav-1 bound eNOS was significantly lower at 2.5 (P = 0.002), 5 (P = 0.02) and 10 (P = 0.02) minutes after ATP treatment. eNOS exhibited a domain-specific multi-site phosphorylation "zip code." In unstimulated cells, P-Thr497eNOS but not P-Ser635eNOS or P-Ser1179eNOS was detected in the caveolar domain. In contrast, P-Ser114eNOS was detected only in the non-caveolar pool. Upon ATP stimulation, P-Ser1179eNOS was mainly detectable only in the caveolar domain whereas P-Ser635eNOS was detectable in both the caveolar and the non-caveolar pools. Moreover with ATP stimulation, P-Thr497eNOS was barely detectable in the caveolar domain and P-Ser114eNOS was not detectable in any of the fractions. These observations were confirmed by non-denaturing gel electrophoresis where ATP receptor activation increased cav-1 free-dimerized eNOS by 54% (F1,5 = 15.8; P = 0.011). Further, ATP resulted in a significant increase in the magnitude of P14cav-1. Confocal microscopy utilizing double immunofluorescence staining showed the temporal and spatial partition of post-translationally modified eNOS and total eNOS between the caveolar and cytoplasmic domains to be consistent with immunoblots. Conclusion: This is the first study to systematically investigate the temporal and spatial partition of eNOS in uterine artery endothelial cells. We show the novel observation that multi-site phosphorylation state of eNOS acts as a "zip code" for the intracellular location of the enzyme, and gives direct clues to the state of the enzyme activity. These data suggest that the regulatory mechanisms pertaining temporal and spatial dynamics of eNOS may delineate a greater understanding of the etiologies of gestational diseases. NIH HL49210, HD38843, HL87144, HL86939, HL70562 and HL74947. (poster)