Linaclotide activates guanylate cyclase-C/cGMP/protein kinase-II-dependent trafficking of CFTR in the intestine.
Linaclotide activates guanylate cyclase-C/cGMP/protein kinase-II-dependent trafficking of CFTR in the intestine.
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DOI：
10.14814/phy2.13299
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发表时间：
2017-06
影响因子：
2.5
通讯作者：
Ameen NA
中科院分区：
文献类型：
--
作者：
Ahsan MK;Tchernychev B;Kessler MM;Solinga RM;Arthur D;Linde CI;Silos-Santiago I;Hannig G;Ameen NA
跨膜受体鸟苷环化酶C(GC-C)表达于肠上皮细胞，是GC-C激动肽的分子靶点，FDA批准该药用于治疗患有便秘和慢性特发性便秘的成人肠易激综合征患者。以极化的人结肠癌细胞(T84，Caco-2BBe)大鼠和人肠组织为研究对象，采用共聚焦显微镜、活体表面生物素化和蛋白激酶-II(PKG-II)活性测定等方法，研究了利奈克肽激活的细胞信号转导和囊性纤维化跨膜传导调节因子(CFTR)转运途径。用聚合酶链式反应、免疫印迹和cGMP检测GC-C/cGMP途径组分的表达和活性。液体分泌作为CFTR细胞表面易位的标志，在活体大鼠肠环上进行了测定。在大鼠的肠环中，利那克肽处理30分钟可诱导强劲的液体分泌和CFTR从心尖下室到细胞表面的移位。类似地，利纳洛特处理T84和Caco-2BBe细胞(30min)后，细胞表面cftr水平增加。通过激活GC-C/cGMP/PKGII信号通路，导致细胞内cGMP和pVASP ser239磷酸化水平升高。抑制或沉默PKGII可显著减弱利纳氯肽诱导的CFTR向根尖膜的转运。蛋白激酶A(PKA)的抑制也减弱了利纳氯肽诱导的CFTR细胞表面转运，这意味着依赖于cGMP的PKA途径的交叉激活。综上所述，这些发现支持利纳氯肽诱导的GC-C/cGMP/PKG-II/CFTR途径的激活是利纳氯肽介导的肠液分泌的主要途径，并且依赖利纳氯肽的CFTR激活和CFTR从心尖下小泡到细胞表面的募集/运输是这一过程中的重要步骤。
The transmembrane receptor guanylyl cyclase‐C (GC‐C), expressed on enterocytes along the intestine, is the molecular target of the GC‐C agonist peptide linaclotide, an FDA‐approved drug for treatment of adult patients with Irritable Bowel Syndrome with Constipation and Chronic Idiopathic Constipation. Polarized human colonic intestinal cells (T84, CaCo‐2BBe) rat and human intestinal tissues were employed to examine cellular signaling and cystic fibrosis transmembrane conductance regulator (CFTR)‐trafficking pathways activated by linaclotide using confocal microscopy, in vivo surface biotinylation, and protein kinase‐II (PKG‐II) activity assays. Expression and activity of GC‐C/cGMP pathway components were determined by PCR, western blot, and cGMP assays. Fluid secretion as a marker of CFTR cell surface translocation was determined using in vivo rat intestinal loops. Linaclotide treatment (30 min) induced robust fluid secretion and translocation of CFTR from subapical compartments to the cell surface in rat intestinal loops. Similarly, linaclotide treatment (30 min) of T84 and CaCo‐2BBe cells increased cell surface CFTR levels. Linaclotide‐induced activation of the GC‐C/cGMP/PKGII signaling pathway resulted in elevated intracellular cGMP and pVASP ser239 phosphorylation. Inhibition or silencing of PKGII significantly attenuated linaclotide‐induced CFTR trafficking to the apical membrane. Inhibition of protein kinase‐A (PKA) also attenuated linaclotide‐induced CFTR cell surface trafficking, implying cGMP‐dependent cross‐activation of PKA pathway. Together, these findings support linaclotide‐induced activation of the GC‐C/cGMP/PKG‐II/CFTR pathway as the major pathway of linaclotide‐mediated intestinal fluid secretion, and that linaclotide‐dependent CFTR activation and recruitment/trafficking of CFTR from subapical vesicles to the cell surface is an important step in this process.