Uterine artery adaptations during gestation facilitate increases in uterine blood flow and fetal growth. Hypothesis: Local expression and distribution of uterine artery connexins play roles in mediating in vivo gestational eNOS activation and Nitric Oxide production. We established an ovine model restricting pregnancy to a single uterine horn and measured uterine blood flow, uterine artery shear stress, connexins 37/43 and P635eNOS protein levels in uterine artery and systemic artery [Omental and Renal] endothelium and connexins in vascular smooth muscle. Uterine blood flow and shear stress were locally (unilaterally) and substantially elevated by gestation. During pregnancy uterine artery endothelial gap junction proteins connexins 37/43 were locally regulated in the gravid horn and elevated 10.3- and 25.6-fold; uterine artery endothelial P635eNOS and total eNOS were elevated 3.3- and 2.9-fold; whereas uterine artery vascular smooth muscle connexins 37/43 were locally elevated 12.5- and 5.9-fold, respectively. Less pronounced changes were observed in systemic vasculature except for significant pregnancy-associated increases in omental artery vascular smooth muscle connexin 43 and omental artery endothelial P635eNOS and total eNOS. Gap junction blockade using connexin 43, but not connexin 37 specific GAP peptides abrogated uterine artery endothelial ATP-induced Ca2+-mediated nitric oxide production. Thus uterine artery endothelial connexin43, but not connexin 37 regulates Ca2+-mediated nitric oxide production required for the vasodilation to accommodate increases in uterine blood flow and shear stress during healthy pregnancies.

ATP leads to endothelial NO synthase (eNOS)/NO-mediated vasodilation, a process hypothesized to depend on the endothelial caveolar eNOS partitioning and subcellular domain-specific multisite phosphorylation state. We demonstrate herein that, in both the absence and presence of ATP, the uterine artery endothelial caveolae contain specific protein machinery related to subcellular partitioning and act as specific focal “hubs” for NO- and ATP-related proteins. ATP-induced eNOS regulation showed a complex set of multisite posttranslational phosphorylation events that were closely associated with the enzyme’s partitioning between caveolar and noncaveolar endothelial subcellular domains. The comprehensive model that we present demonstrates that ATP repartitioned eNOS between the caveolar and noncaveolar subcellular domains; specifically, the stimulatory PSer635eNOS was substantially higher in the caveolar pool with subcellular domain-independent increased levels on ATP treatment. The stimulatory PSer1179eNOS was not altered by ATP treatment. However, the inhibitory PThr495eNOS was regulated predominantly in the caveolar domain with decreased levels on ATP action. In contrast, the agonist-specific PSer114eNOS was localized in the noncaveolar pool with increased levels on ATP stimulation. Thus, the endothelial caveolar membrane system plays a pivotal role(s) in ATP-associated subcellular partitioning and possesses the relevant protein machinery for ATP-induced NO regulation. Furthermore, these subcellular domain-specific phosphorylation/dephosphorylation events provide evidence relating to eNOS spatio-temporal dynamics.

In the vasculature, gap junctions (GJ) play a multifaceted role by serving as direct conduits for cell–cell intercellular communication via the facilitated diffusion of signaling molecules. GJs are essential for the control of gene expression and coordinated vascular development in addition to vascular function. The coupling of endothelial cells to each other, as well as with vascular smooth muscle cells via GJs, plays a relevant role in the control of vasomotor tone, tissue perfusion and arterial blood pressure. The regulation of cell-signaling is paramount to cardiovascular adaptations of pregnancy. Pregnancy requires highly developed cell-to-cell coupling, which is affected partly through the formation of intercellular GJs by Cx43, a gap junction protein, within adjacent cell membranes to help facilitate the increase of uterine blood flow (UBF) in order to ensure adequate perfusion for nutrient and oxygen delivery to the placenta and thus the fetus. One mode of communication that plays a critical role in regulating Cx43 is the release of endothelial-derived vasodilators such as prostacyclin (PGI2) and nitric oxide (NO) and their respective signaling mechanisms involving second messengers (cAMP and cGMP, respectively) that are likely to be important in maintaining UBF. Therefore, the assertion we present in this review is that GJs play an integral if not a central role in maintaining UBF by controlling rises in vasodilators (PGI2 and NO) via cyclic nucleotides. In this review, we discuss: (1) GJ structure and regulation; (2) second messenger regulation of GJ phosphorylation and formation; (3) pregnancy-induced changes in cell-signaling; and (4) the role of uterine arterial endothelial GJs during gestation. These topics integrate the current knowledge of this scientific field with interpretations and hypotheses regarding the vascular effects that are mediated by GJs and their relationship with vasodilatory vascular adaptations required for modulating the dramatic physiological rises in uteroplacental perfusion and blood flow observed during normal pregnancy.

Uterine vascular adaptations facilitate rises in uterine blood flow (UBF) during pregnancy, which are associated with gap junction connexin (Cx) proteins and eNOS. In uterine artery endothelial cells (UAECs) ATP activates eNOS in a pregnancy (P) specific manner that is dependent on Cx43 function. Caveolar subcellular domain partitioning plays key roles in ATP-induced eNOS activation and NO production. Little is known regarding the partitioning of Cx proteins to caveolar domains or their dynamics upon ATP treatment. We observed that Cx43-mediated gap junction function upon ATP stimulation is associated with Cx43 re-partitioning between the non-caveolar and caveolar domains. Compared to UAECs from nonpregnant (NP) ewes, levels of ATP, PGI2, cAMP, NOx, and cGMP were 2-fold higher (P<0.05) in P-UAECs. In P-UAECs ATP increased lucifer yellow dye transfer, a response abrogated by Gap27, but not Gap 26 indicating involvement of Cx43 but not Cx37. Confocal microscopy revealed domain partitioning of Cx43 and Cav-1. In P-UAECs LC/MS/MS analysis revealed only Cx43 in the caveolar domain. In contrast, Cx37 was located only in the non-caveolar pool. Western analysis revealed that ATP increased Cx43 distribution (1.7-fold;P=0.013) to the caveolar domain, but had no effect on Cx37. These data demonstrate rapid ATP-stimulated repartitioning of Cx43 to the caveolae, where eNOS resides and plays an important role in NO-mediated increasing UBF during pregnancy.

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