Department of Biomedicine, Aarhus University , Aarhus , Denmark.
Department of Clinical Medicine, Aarhus University , Aarhus , Denmark.
Am J Physiol Heart Circ Physiol. 2018 Aug 1;315(2):H389-H401. doi: 10.1152/ajpheart.00551.2017. Epub 2018 Apr 6.
Human lymphatic vessels are myogenically active and respond to sympathetic stimulation. The role of various cations in this behavior has recently been investigated, but whether the anion Cl is essential is unclear. With ethical approval and informed consent, human thoracic duct and mesenteric lymphatic vessels were obtained from surgical patients. Spontaneous or norepinephrine-induced isometric force production from isolated vessels was measured by wire myography; the transmembrane Cl gradient and Cl channels were investigated by substitution of extracellular Cl with the impermeant anion aspartate and inhibition of Cl transport and channels with the clinical diuretics furosemide and bendroflumethiazide as well as DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid. The molecular expression of Ca-activated Cl channels was investigated by RT-PCR, and proteins were localized using immunoreactivity. Spontaneous and norepinephrine-induced contractility in human lymphatic vessels was highly abrogated after Cl substitution with aspartate. About 100-300 µM DIDS or 5-nitro-2-(3-phenylpropylamino)benzoic acid inhibited spontaneous contractile behavior. Norepinephrine-stimulated tone was furthermore markedly abrogated by 200 µM DIDS. Furosemide lowered only spontaneous constrictions, whereas bendroflumethiazide had nonspecific inhibitory effects. Consistent expression of transmembrane member 16A [TMEM16A (anoctamin-1)] was found in both the thoracic duct and mesenteric lymphatic vessels, and immunoreactivity with different antibodies localized TMEM16A to lymphatic smooth muscle cells and interstitial cells. The significant change in contractile function observed with inhibitors and anion substitution suggests that Cl movement over the plasma membrane of lymphatic myocytes is integral for spontaneous and α-adrenoceptor-evoked contractility in human collecting lymphatic vessels. Consistent detection and localization of TMEM16A to myocytes suggests that this channel could play a major functional role. NEW & NOTEWORTHY In this study, we report the first observations of Cl being a critical ionic component of spontaneous and agonist-evoked contractility in human lymphatics. The most consistently expressed Ca-activated Cl channel gene in the human thoracic duct and mesenteric lymphatic vessels appears to be transmembrane member 16A, suggesting that this channel plays a major role.
人类淋巴管具有肌源性活性，并对交感神经刺激产生反应。最近已经研究了各种阳离子在这种行为中的作用，但阴离子 Cl 是否是必需的尚不清楚。在获得伦理批准和知情同意的情况下，从手术患者中获得了胸导管和肠系膜淋巴管。通过wire myography 测量分离血管的自发性或去甲肾上腺素诱导的等长力产生；通过用不可渗透的阴离子天冬氨酸替代细胞外 Cl 以及用临床利尿剂呋塞米和苯并噻嗪抑制 Cl 转运和通道，以及 DIDS 和 5-硝基-2-(3- 苯丙基氨基)苯甲酸来研究跨膜 Cl 梯度和 Cl 通道。通过 RT-PCR 研究 Ca 激活 Cl 通道的分子表达，并使用免疫反应性定位蛋白质。用天冬氨酸替代 Cl 后，人类淋巴管的自发性和去甲肾上腺素诱导的收缩性大大减弱。约 100-300 μM DIDS 或 5-硝基-2-(3-苯丙基氨基)苯甲酸抑制自发性收缩行为。此外，200 μM DIDS 显著减弱去甲肾上腺素刺激的张力。呋塞米仅降低自发性收缩，而苯并噻嗪具有非特异性抑制作用。在胸导管和肠系膜淋巴管中均发现跨膜成员 16A[TMEM16A(anoctamin-1)]的一致表达，并且用不同抗体的免疫反应将 TMEM16A 定位到淋巴管平滑肌细胞和间质细胞。用抑制剂和阴离子替代观察到的收缩功能的显著变化表明，Cl 跨淋巴管肌细胞的质膜运动对于人集合淋巴管的自发性和α-肾上腺素能受体诱导的收缩性是必不可少的。TMEM16A 对肌细胞的一致检测和定位表明，该通道可能发挥主要功能作用。新的和值得注意的是，在这项研究中，我们首次观察到 Cl 是人类淋巴管自发性和激动剂诱导收缩性的关键离子组成部分。在人胸导管和肠系膜淋巴管中表达最一致的 Ca 激活 Cl 通道基因似乎是跨膜成员 16A，这表明该通道发挥主要作用。
