Institute for Veterinary Physiology and Biochemistry, Justus-Liebig-University, Giessen, Germany.
Cell Tissue Res. 2013 Sep;353(3):355-66. doi: 10.1007/s00441-013-1643-5. Epub 2013 Jun 27.
We recently observed a bradykinin-induced increase in the cytosolic Ca2+ concentration in submucosal neurons of rat colon, an increase inhibited by blockers of voltage-dependent Ca2+ (Ca(v)) channels. As the types of Ca(v) channels used by this part of the enteric nervous system are unknown, the expression of various Ca(v) subunits has been investigated in whole-mount submucosal preparations by immunohistochemistry. Submucosal neurons, identified by a neuronal marker (microtubule-associated protein 2), are immunoreactive for Ca(v)1.2, Ca(v)1.3 and Ca(v)2.2, expression being confirmed by reverse transcription plus the polymerase chain reaction. These data agree with previous observations that the inhibition of L- and N-type Ca2+ currents strongly inhibits the response to bradykinin. However, whole-cell patch-clamp experiments have revealed that bradykinin does not enhance Ca2+ inward currents under voltage-clamp conditions. Consequently, bradykinin does not directly interact with Ca(v) channels. Instead, the kinin-induced Ca2+ influx is caused indirectly by the membrane depolarization evoked by this peptide. As intracellular Ca2+ channels on Ca(2+)-storing organelles can also contribute to Ca2+ signaling, their expression has been investigated by imaging experiments and immunohistochemistry. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) have been functionally demonstrated in submucosal neurons loaded with the Ca(2+)-sensitive fluorescent dye, fura-2. Histamine, a typical agonist coupled to the phospholipase C pathway, induces an increase in the fura-2 signal ratio, which is suppressed by 2-aminophenylborate, a blocker of IP3 receptors. The expression of IP3R1 has been confirmed by immunohistochemistry. In contrast, ryanodine, tested over a wide concentration range, evokes no increase in the cytosolic Ca2+ concentration nor is there immunohistochemical evidence for the expression of ryanodine receptors in these neurons. Thus, rat submucosal neurons are equipped with various types of high-voltage activated Ca(v) channels and with IP3 receptors for intracellular Ca2+ signaling.
我们最近观察到，在大鼠结肠的黏膜下神经元中，缓激肽诱导的细胞浆钙离子浓度增加，这种增加被电压依赖性钙通道（Ca(v)）阻滞剂抑制。由于肠神经系统的这一部分使用的 Ca(v)通道的类型未知，因此通过免疫组织化学方法在整个黏膜下制剂中研究了各种 Ca(v)亚基的表达。黏膜下神经元通过神经元标志物（微管相关蛋白 2）识别，对 Ca(v)1.2、Ca(v)1.3 和 Ca(v)2.2 具有免疫反应性，通过逆转录加聚合酶链反应确认表达。这些数据与先前的观察结果一致，即 L 和 N 型钙电流的抑制强烈抑制了对缓激肽的反应。然而，全细胞膜片钳实验表明，在电压钳条件下，缓激肽不会增强钙内流。因此，缓激肽不会直接与 Ca(v)通道相互作用。相反，激肽诱导的 Ca2+内流是由该肽引起的膜去极化间接引起的。由于细胞内 Ca2+通道在储存细胞器上也可以有助于 Ca2+信号转导，因此通过成像实验和免疫组织化学方法研究了它们的表达。在加载 Ca2+敏感荧光染料 fura-2 的黏膜下神经元中，已经证明了肌醇 1,4,5-三磷酸（IP3）受体（IP3R）具有功能。组胺，一种与磷脂酶 C 途径偶联的典型激动剂，诱导 fura-2 信号比的增加，该增加被 IP3 受体的阻滞剂 2-氨基苯硼酸抑制。通过免疫组织化学方法证实了 IP3R1 的表达。相比之下，在广泛的浓度范围内测试的瑞诺定不会引起细胞浆钙离子浓度的增加，也没有免疫组织化学证据表明这些神经元表达 Ryanodine 受体。因此，大鼠黏膜下神经元配备了各种类型的高电压激活的 Ca(v)通道和用于细胞内 Ca2+信号转导的 IP3 受体。
