Domain-Specific Partitioning of Uterine Artery Endothelial Connexin43 and Caveolin-1

Abstract

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.

Cyclic Nucleotides Differentially Regulate Cx43 Gap Junction Function in Uterine Artery Endothelial Cells From Pregnant Ewes

“…3 Although repeated doses of ATP itself show modest reductions in subsequent Ca 2+ bursting by ≈10%, the pretreatment with cAMP completely abrogated this (Figure 6). Indeed, compared with control burst numbers, P-UAECs exposed to 1 μmol/L and 1 mmol/L 8-Br-cAMP increased the number of ATP-mediated sustained Ca 2+ bursts by 5.4% and 27.9%, respectively ( P <0.01).…”

“…UAECs were scraped with a sterile razor blade and incubated for 10 minutes with 0.05% Lucifer yellow and tetramethylrhodamine dextran (3 kDa), 1,3,25 washed with Ca 2+ -free Krebs buffer containing an additional 50 μmol/L ethylene glycol tetraacetic acid, 1,3 and imaged in triplicate using fluorescence microscope. Areas of dye spread (fluorescent areas) were quantified using ImageJ software.…”

“…Cell to cell communication is vital for the development of cardiovascular adaptations during gestation and are reliant on GJ (gap junction) intercellular communication (GJIC). 1 – 3 GJs are specialized membrane hemichannels composed of Cx (connexin) proteins that direct cell–cell diffusion of ions, small molecules, and second messengers (eg, IP3, Ca 2+ , cAMP, cGMP, ATP) 4 – 6 that are important for synchronizing endothelial cell vasodilatory responses. Pregnancy is a physiological state of vasodilation that requires heightened cell coupling to maintain substantial increases in blood flow necessary for fetal growth and development.…”

“… 1 , 2 , 7 , 8 Endothelial-derived vasodilators, such as prostacyclin (PGI 2 ) and nitric oxide (NO), are enhanced in the uterine circulation during pregnancy and serve as a pregnancy-adapted vasodilatory phenotype that sets the stage for the pronounced gestational elevations in uterine blood flow. 3 , 8 We have shown that Cx43 is the major GJ protein present in uterine artery endothelial cells (UAECs) and controls pregnancy-specific Ca 2+ –mediated activation of endothelial NO synthase and NO production in vitro 1 and ex vivo. 2 In hypertensive states of pregnancy, reduced vasodilation is related to endothelial cell dysfunction characterized by reduced bioavailability of endothelium-derived vasodilators, in particular NO.…”

“… 10 Because endothelial cell dysfunction is linked to impaired endothelium-dependent vasodilation mechanisms, hypertensive diseases of pregnancy, such as preeclampsia, 9 , 11 , 12 may very well occur at least in part because of a failure to enhance the vasodilatory phenotype through Cx43 closure or internalization. 3 …”