The prejunctional norepinephrine transporter (NET) is responsible for the clearance of released norepinephrine (NE) back into the sympathetic nerve terminal. NET regulation must be tightly controlled as variations could have important implications for neurotransmission. Thus far, the effects of sympathetic neuronal activity on NET function have been unclear. Here, we optically monitor single-terminal cardiac NET activity ex vivo in response to a broad range of sympathetic postganglionic action potential (AP) firing frequencies.

Isolated murine left atrial appendages were loaded with a fluorescent NET substrate [Neurotransmitter Transporter Uptake Assay (NTUA)] and imaged with confocal microscopy. Sympathetic APs were induced with electrical field stimulation at 0.2–10 Hz (0.1–0.2 ms pulse width). Exogenous NE was applied during the NTUA uptake- and washout phases to investigate substrate competition and displacement, respectively, on transport.

Single-terminal NET reuptake rate was rapidly suppressed in a frequency-dependent manner with an inhibitory EF₅₀ of 0.9 Hz. At 2 Hz, the effect was reversed by the α₂-adrenoceptor antagonist yohimbine (1 μM) (p < 0.01) with no further effect imposed by the muscarinic receptor antagonist atropine (1 μM). Additionally, high exogenous NE concentrations abolished NET reuptake (1 μM NE; p < 0.0001) and displaced terminal specific NTUA during washout (1–100 μM NE; p < 0.0001). We have also identified α₂-adrenoceptor-induced suppression of NET reuptake rate during resting stimulation frequencies, which could oppose the effect of autoinhibition-mediated suppression of exocytosis and thus amplify the effects of sympathetic drive on cardiac function.

结前去甲肾上腺素转运蛋白 (NET) 负责将释放的去甲肾上腺素 (NE) 清除回交感神经末梢。NET 调节必须受到严格控制，因为变化可能对神经传递产生重要影响。迄今为止，交感神经元活动对 NET 功能的影响尚不清楚。在这里，我们在体外光学监测单终端心脏 NET 活动，以响应广泛的交感神经节后动作电位 (AP) 发射频率。

分离的小鼠左心耳装有荧光 NET 底物 [神经递质转运蛋白摄取测定 (NTUA)]，并用共聚焦显微镜成像。通过 0.2-10 Hz（0.1-0.2 ms 脉冲宽度）的电场刺激诱导交感神经 AP。在 NTUA 吸收和冲洗阶段应用外源 NE 分别研究运输过程中的底物竞争和位移。

单端 NET 再摄取率以频率依赖的方式被迅速抑制，抑制性 EF ₅₀为 0.9 Hz。在 2 Hz 时，α _{2 -}肾上腺素受体拮抗剂育亨宾 (1 μM) ( p  < 0.01)逆转了这种作用，而毒蕈碱受体拮抗剂阿托品 (1 μM) 没有施加进一步的作用。此外，高外源性 NE 浓度消除了 NET 再摄取（1 μM NE；p  < 0.0001）并在冲洗过程中取代了末端特异性 NTUA（1-100 μM NE；p  < 0.0001）。我们还确定了α ₂-肾上腺素受体诱导的静息刺激频率期间 NET 再摄取率的抑制，这可以对抗自体抑制介导的胞吐作用抑制，从而放大交感神经驱动对心脏功能的影响。