Pregnancy Increases Total S-nitrosylation, but Decreases S-Nitrosylation of Endothelial Nitric Oxide Synthesizing System in Sheep Uterine Artery In Vivo.

Abstract

Pregnancy-associated uterine vasodilatation occurs via enhanced endothelial nitric oxide (NO) synthase (eNOS) expression and activation thereby increasing NO production locally at the level of the uterine artery (UA) endothelium. Indeed local inhibition of uterine vascular NO production with L-NAME in vivo dose dependently decreases ovine uteroplacental blood flow. However, it is unknown if increased NO production directly affects UA protein post-translational modifications and thus their functions. Formation of S-nitrothiols (S-nitrosylation) via addition of NO-derived nitrosyl groups to cysteines regulates the function of a plethora of proteins including eNOS. Hypotheses: total protein S-nitrosylation is upregulated, however S-nitrosylation of the NO synthesizing system is downregulated by pregnancy. Methods: UA segments were obtained from day 120-130 pregnant (P) and day 10-12 luteal phase nonpregnant (NP) ewes (n=3/group). UA endothelium (UAendo) and smooth muscle (UAsm) were mechanically separated. Total levels of nitrosylated proteins were labeled by a 3-step biotin switch technique and determined by immunoblotting with anti-biotin antibody. Biotinylated proteins were then pulled down by NeutrAvidinTM Protein coated beads for measuring levels of nitrosylated proteins of the NO synthesizing system (eNOS and HSP90) by immunoblotting with specific antibodies. Results: Levels of total UAsm nitrosylated proteins were significantly greater in P UAsm that of NP UAsm (p<0.01). By contrast levels of total UAendo nitrosylated proteins did not differ in P vs. NP. Total eNOS protein was upregulated whereas total HSP90 was downregulated in UAendo by P. As expected, eNOS protein was barely detected in UAsm and total HSP90 was downregulated in UAsm by P. Levels of nitrosylated eNOS were downregulated in UAendo by P, and levels of nitrosylated HSP90 were also downregulated in UAendo, but unchanged in UASM. Conclusion: Upregulation of UAsm protein nitrosylation implicates a possible pathway of NO-dependent vasorelaxation, whereas de-nitrosylation of UAendo eNOS and HSP90 implicates a mechanism for increasing UAendo NO production via eNOS activation during pregnancy associated with increased NO production. NIH HL98746, HL74947, HL70562, HL49210, HD38843, HL87144. (poster)