Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, One Hospital Drive, MA415 Medical Sciences Building, Columbia, MO, 65212, USA.
Department of Pharmacology, Tulane University School of Medicine, New Orleans, LA, 70112, USA.
Sci Rep. 2023 Sep 22;13(1):15862. doi: 10.1038/s41598-023-42877-6.
We previously identified two isoforms of T-type, voltage-gated calcium (Ca3) channels (Ca3.1, Ca3.2) that are functionally expressed in murine lymphatic muscle cells; however, contractile tests of lymphatic vessels from single and double Ca3 knock-out (DKO) mice, exhibited nearly identical parameters of spontaneous twitch contractions as wild-type (WT) vessels, suggesting that Ca3 channels play no significant role. Here, we considered the possibility that the contribution of Ca3 channels might be too subtle to detect in standard contraction analyses. We compared the sensitivity of lymphatic vessels from WT and Ca3 DKO mice to the L-type calcium channel (Ca1.2) inhibitor nifedipine and found that the latter vessels were significantly more sensitive to inhibition, suggesting that the contribution of Ca3 channels might normally be masked by Ca1.2 channel activity. We hypothesized that shifting the resting membrane potential (Vm) of lymphatic muscle to a more negative voltage might enhance the contribution of Ca3 channels. Because even slight hyperpolarization is known to completely silence spontaneous contractions, we devised a method to evoke nerve-independent, twitch contractions from mouse lymphatic vessels using single, short pulses of electric field stimulation (EFS). TTX was present throughout to block the potential contributions of voltage-gated Na channels in perivascular nerves and lymphatic muscle. In WT vessels, EFS evoked single contractions that were comparable in amplitude and degree of entrainment to those occurring spontaneously. When Ca1.2 channels were blocked or deleted, only small residual EFS-evoked contractions (~ 5% of normal amplitude) were present. These residual, EFS-evoked contractions were enhanced (to 10-15%) by the K channel activator pinacidil (PIN) but were absent in Ca3 DKO vessels. Our results point to a subtle contribution of Ca3 channels to lymphatic contractions that can be unmasked in the absence of Ca1.2 channel activity and when the resting Vm is more hyperpolarized than normal.
我们之前鉴定了两种 T 型电压门控钙（Ca3）通道（Ca3.1、Ca3.2）的同工型，它们在鼠淋巴管肌细胞中具有功能性表达；然而，对来自单个和双 Ca3 敲除（DKO）小鼠的淋巴管的收缩测试表明，自发抽搐收缩的参数与野生型（WT）血管几乎相同，表明 Ca3 通道没有发挥重要作用。在这里，我们考虑了 Ca3 通道的贡献可能过于细微，以至于无法在标准收缩分析中检测到的可能性。我们比较了 WT 和 Ca3 DKO 小鼠淋巴管对 L 型钙通道（Ca1.2）抑制剂硝苯地平的敏感性，发现后者的血管对抑制更为敏感，表明 Ca3 通道的贡献通常可能被 Ca1.2 通道活性所掩盖。我们假设将淋巴管肌的静息膜电位（Vm）转移到更负的电压可能会增强 Ca3 通道的贡献。由于即使是轻微的超极化也已知会完全沉默自发收缩，因此我们设计了一种方法，使用单个短电场刺激（EFS）脉冲从鼠淋巴管中引发神经独立的抽搐收缩。整个 TTX 存在以阻断血管周围神经和淋巴管肌中电压门控 Na 通道的潜在贡献。在 WT 血管中，EFS 引发的单次收缩在幅度和程度上与自发发生的收缩相当。当 Ca1.2 通道被阻断或缺失时，只有小的残余 EFS 诱发收缩（~正常幅度的 5%）存在。这些残余的 EFS 诱发收缩被 K 通道激活剂吡那地尔（PIN）增强（至 10-15%），但在 Ca3 DKO 血管中不存在。我们的结果表明 Ca3 通道对淋巴管收缩有细微的贡献，当 Ca1.2 通道活性缺失且静息 Vm 比正常更超极化时，这种贡献可以被揭示。
