Ampey Bryan C, Ampey Amanda C, Lopez Gladys E, Bird Ian M, Magness Ronald R
From the Department of Obstetrics and Gynecology, Perinatal Research Labs University of Wisconsin, Madison (B.C.A., A.C.A., G.E.L., I.M.B., R.R.M.); and Department of Obstetrics and Gynecology, Perinatal Research Center Tampa, University of South Florida, (R.R.M.).
Hypertension. 2017 Aug;70(2):401-411. doi: 10.1161/HYPERTENSIONAHA.117.09113. Epub 2017 May 30.
Cell-cell communication is dependent on GJ (gap junction) proteins such as Cx43 (connexin 43). We previously demonstrated the importance of Cx43 function in establishing the enhanced pregnancy vasodilatory phenotype during pregnancy in uterine artery endothelial cells from pregnant (P-UAEC) ewes. Cx43 is regulated by elevating cAMP and PKA (protein kinase A)-dependent Cx43 S365 phosphorylation-associated trafficking and GJ open gating, which is opposed by PKC (protein kinase C)-dependent S368 phosphorylation-mediated GJ turnover and closed gating. However, the role of cyclic nucleotide-mediated signaling mechanisms that control Cx43 and GJ function in P-UAECs is unknown. We hypothesize that cAMP will mediate increases in S365 phosphorylation, thereby, enhancing GJ trafficking and open gating, while cGMP will stimulate S368, but not S365, phosphorylation to enhance GJ turnover and closed gating in P-UAECs. Treatment with 8-Bromo (8-Br)-cAMP signal significantly (<0.05) increased nonphosphorylated S365 signal and total Cx43 phosphorylation, but not S368 phosphorylation, while 8-Br-cGMP significantly (<0.05) increased Cx43 C-terminus-S365 signal, S368, and total Cx43 phosphorylation. Inhibition of PKA, but not PKG (protein kinase G), abrogated the 8-Br-cAMP-stimulated increase in nonphosphorylated S365 and total Cx43 phosphorylation and inhibited S368 below basal levels, whereas inhibition of PKG blocked (<0.05) the 8-bromo-cGMP-stimulated rises in nonphosphorylated S365, total Cx43, and S368 phosphorylation levels in P-UAECs. Functional studies showed that 8-Br-cAMP increased dye transfer and sustained calcium bursts, while 8-Br-cGMP decreased both. Thus, in P-UAECs, only 8-Br-cAMP and not 8-Br-cGMP effectively enhances nonphosphorylated S365 and total Cx43 expression that correspondingly reduces S368 phosphorylation, allowing increased GJ communication. This provides new insights into the regulatory mechanisms behind Cx43 function and GJ communication.
细胞间通讯依赖于缝隙连接（GJ）蛋白，如连接蛋白43（Cx43）。我们之前证明了Cx43功能在妊娠母羊子宫动脉内皮细胞（P-UAEC）建立增强的妊娠血管舒张表型中的重要性。Cx43受cAMP升高和蛋白激酶A（PKA）依赖性的Cx43 S365磷酸化相关的转运及缝隙连接开放门控的调节，而蛋白激酶C（PKC）依赖性的S368磷酸化介导的缝隙连接周转和关闭门控则与之相反。然而，在P-UAEC中，控制Cx43和缝隙连接功能的环核苷酸介导的信号传导机制的作用尚不清楚。我们假设，cAMP将介导S365磷酸化增加，从而增强缝隙连接的转运和开放门控，而cGMP将刺激S368而非S365的磷酸化，以增强P-UAEC中缝隙连接的周转和关闭门控。用8-溴（8-Br）-cAMP信号处理显著（<0.05）增加了非磷酸化S365信号和总Cx43磷酸化，但未增加S368磷酸化，而8-Br-cGMP显著（<0.05）增加了Cx43 C末端-S365信号、S368和总Cx43磷酸化。抑制PKA而非蛋白激酶G（PKG）消除了8-Br-cAMP刺激的非磷酸化S365和总Cx43磷酸化增加，并将S368抑制至基础水平以下，而抑制PKG则阻断了（<0.05）8-溴-cGMP刺激的P-UAEC中非磷酸化S365、总Cx43和S368磷酸化水平的升高。功能研究表明，8-Br-cAMP增加了染料转移并维持了钙爆发，而8-Br-cGMP则降低了两者。因此，在P-UAEC中，只有8-Br-cAMP而非8-Br-cGMP有效地增强了非磷酸化S365和总Cx43的表达，相应地降低了S368磷酸化，从而增加了缝隙连接通讯。这为Cx43功能和缝隙连接通讯背后的调节机制提供了新的见解。
