Ca2+ sensitization pathways accessed by cholinergic neurotransmission in the murine gastric fundus
Ca2+ sensitization pathways accessed by cholinergic neurotransmission in the murine gastric fundus
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DOI：
10.1113/jphysiol.2013.255745
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发表时间：
2013-06-01
影响因子：
5.5
通讯作者：
Perrino, Brian A.
中科院分区：
文献类型：
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作者：
Bhetwal, Bhupal P.;Sanders, Kenton M.;Perrino, Brian A.
要点 中心点 在平滑肌中，CPI-17 和 MYPT1 磷酸化可增加收缩对 Ca2+ 的敏感性，从而抑制肌球蛋白轻链磷酸酶（MLCP）。通常通过将肌肉浸入含有收缩激动剂的溶液中来研究平滑肌收缩的中心点 Ca2+ 敏化。中心点 然而，通过沐浴施加的激动剂刺激肌肉可能并不等同于神经递质释放，因为不同的连接后受体可能会响应这些不同的刺激模式而被激活。中心点 在这项研究中，我们发现浴用胆碱能激动剂激活胃底平滑肌中的 Ca2+ 敏化机制，该机制与胆碱能神经传递不同。电场刺激 (EFS) 仅增加 CPI-17 磷酸化，而浴施加的卡巴胆碱则增加 CPI-17 和 MYPT1 磷酸化。中心点 在存在胆碱酯酶抑制剂新斯的明的情况下，EFS 增加了 CPI-17 和 MYPT1 磷酸化。中心点 在缺乏 Cajal 肌内间质细胞 (ICC-IM) 的 W/Wv 小鼠的眼底肌肉中，单独的 EFS 增加了 CPI-17 和 MYPT1 磷酸化。中心点 这些发现表明，ACh 的可用性决定了哪些 Ca2+ 敏化机制被激活，并且 ICC-IM 调节 ACh 进入平滑肌。摘要 通常通过将肌肉浸泡在含有激动剂的溶液中来研究 Ca2+ 收缩敏化。然而，尚不清楚浴用激动剂和肠神经传递是否激活类似的 Ca2+ 敏化机制。我们研究了通过浴用卡巴胆碱 (CCh) 或胆碱能运动神经传递刺激的小鼠胃底肌肉中蛋白激酶 C (PKC) 增强的 17 kDa (CPI-17) 磷酸酶抑制剂蛋白和肌球蛋白磷酸酶靶向亚基 1 (MYPT1) 的磷酸化。 CCh 增加 MYPT1 Thr696 (pT696) 和 Thr853 (pT853) 处的磷酸化、CPI-17 Thr38 (pT38) 处的磷酸化以及肌球蛋白轻链 Ser19 (pS19) 处的磷酸化。电场刺激 (EFS) 仅增加 pT38。在新斯的明存在的情况下，EFS 增加了 pT38、pT853 和 pS19。在 W/Wv 小鼠的眼底肌肉中，单独使用 EFS 会增加 pT38 和 pT853。阿托品阻断 pT696、pT853、pT38 和 pS19 的所有收缩和所有增加。 Rho 激酶 (ROCK) 抑制剂 SAR1x 阻断 pT853 和 pT696 的增加。 PKC 抑制剂 Go6976 和 Gf109203x 或尼卡地平阻断 pT38 和 pT696 的增加。这些发现表明胆碱能运动神经传递激活 PKC 依赖性 CPI-17 磷酸化。由于激动剂暴露于更广泛的毒蕈碱受体，浴液应用的 CCh 会招募额外的 ROCK 依赖性 MYPT1 磷酸化。肌内 Cajal 间质细胞 (ICC-IM) 和胆碱酯酶限制 ACh 对选定群体的毒蕈碱受体的可及性，可能仅限于 ICC-IM 表达的那些受体。这些结果为平滑肌组织中的聚焦（或突触样）神经传递而不是弥散体积神经传递提供了第一个生化证据。此外，这些发现表明，对胃肠道平滑肌应用收缩激动剂的浴并不能模拟对胆碱能神经传递的生理反应。
Key points center dot In smooth muscles, the sensitivity of contraction to Ca2+ can be increased by the phosphorylation of CPI-17 and MYPT1, resulting in the inhibition of myosin light chain phosphatase (MLCP). center dot Ca2+ sensitization of smooth muscle contraction has typically been studied by immersing muscles in solutions containing contractile agonists. center dot However, stimulating muscles by bath-applied agonists may not be equivalent to neurotransmitter release because different post-junctional receptors may be activated in response to these different modes of stimulation. center dot In this study we found that a bath-applied cholinergic agonist activates Ca2+ sensitization mechanisms in gastric fundus smooth muscles that are different than those of cholinergic neurotransmission. Electrical field stimulation (EFS) only increased CPI-17 phosphorylation, while bath-applied carbachol increased both CPI-17 and MYPT1 phosphorylation. center dot With the cholinesterase inhibitor neostigmine present, both CPI-17 and MYPT1 phosphorylation were increased by EFS. center dot In fundus muscles of W/Wv mice which lack intramuscular interstitial cells of Cajal (ICC-IMs), EFS alone increased both CPI-17 and MYPT1 phosphorylation. center dot These findings indicate that ACh availability determines which Ca2+ sensitization mechanisms are activated, and ICC-IMs regulate the access of ACh to smooth muscles. Abstract Ca2+ sensitization of contraction has typically been investigated by bathing muscles in solutions containing agonists. However, it is unknown whether bath-applied agonists and enteric neurotransmission activate similar Ca2+ sensitization mechanisms. We investigated protein kinase C (PKC)-potentiated phosphatase inhibitor protein of 17 kDa (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation in murine gastric fundus muscles stimulated by bath-applied carbachol (CCh) or cholinergic motor neurotransmission. CCh increased MYPT1 phosphorylation at Thr696 (pT696) and Thr853 (pT853), CPI-17 at Thr38 (pT38), and myosin light chain at Ser19 (pS19). Electrical field stimulation (EFS) only increased pT38. In the presence of neostigmine, EFS increased pT38, pT853 and pS19. In fundus muscles of W/Wv mice, EFS alone increased pT38 and pT853. Atropine blocked all contractions and all increases in pT696, pT853, pT38 and pS19. The Rho kinase (ROCK) inhibitor SAR1x blocked increases in pT853 and pT696. The PKC inhibitors Go6976 and Gf109203x or nicardipine blocked increases in pT38 and pT696. These findings suggest that cholinergic motor neurotransmission activates PKC-dependent CPI-17 phosphorylation. Bath-applied CCh recruits additional ROCK-dependent MYPT1 phosphorylation due to exposure of the agonist to a wider population of muscarinic receptors. Intramuscular interstitial cells of Cajal (ICC-IMs) and cholinesterases restrict ACh accessibility to a select population of muscarinic receptors, possibly only those expressed by ICC-IMs. These results provide the first biochemical evidence for focalized (or synaptic-like) neurotransmission, rather than diffuse volume' neurotransmission in a smooth muscle tissue. Furthermore, these findings demonstrate that bath application of contractile agonists to gastrointestinal smooth muscles does not mimic physiological responses to cholinergic neurotransmission.