Knotek M, Jaksić O, Selmani R, Skorić B, Banfić H
Zavod za fiziologiju, Medicinski fakultet, Salata 3, POB 978, 41001 Zagreb, Croatia.
Pflugers Arch. 1996 Jun;432(2):165-73. doi: 10.1007/s004240050120.
Phospholipid signalling mediated by endothelin (ET) receptor subtypes was studied in the rat proximal tubule. In freshly isolated proximal tubule cells, ET-1, ET-2 and sarafotoxin S6c (S6c) evoked an increase in 1,2-diacylglycerol (DAG), inositol 1,4,5-trisphosphate (InsP3) and phosphocholine (PCho), suggesting stimulation of both phosphatidyl-inositol 4,5-bisphosphate- and phosphatidyl-choline-specific phospholipase C (PLC), while ET-3 increased only DAG and PCho, presumably via phosphatidyl-choline-dependent PLC. Renal cortical slices were also stimulated by the above-mentioned agonists, followed by isolation of either brush border (BBM) or basolateral (BLM) membranes for which mass measurements of inositol lipids and DAG were performed. In BBM, DAG increased in response to ET-1, ET-2 and ET-3, and was followed by protein kinase C (PKC) translocation to the BBM, while in BLM, DAG formation and translocation of PKC were observed only in response to ET-3, suggesting spatial segregation of signalling systems between two membane domains of proximal tubule cells. Tyrphostine, pertussis toxin (PTX) or cholera toxin (CTX) did not influence ET-mediated signalling in either of the membranes, suggesting involvement of PTX- and CTX-insensitive G-protein-mediated stimulation of PLCbeta by ET receptors. ET-dependent stimulation of PLC in BBM and BLM was used as a tool to examine the presence of different ET receptor subtypes in these two cell membrane domains. BQ123, an inhibitor of ETA receptors, did not prevent ET-1-mediated signalling in BBM, but an ETA,B antagonist, bosentan, inhibited ET-3-mediated signalling in BBM. In addition, an ETB agonist, S6c, stimulated PLC in BBM. Neither BQ123 nor bosentan inhibited ET-3 signalling in BLM. Therefore, these data strongly suggest the presence of ETB receptors coupled to phosphatidyl-inositol 4,5-bisphosphate- and phosphatidyl-choline-dependent PLC in BBM and ETC receptors linked to phosphatidyl-choline-dependent PLC in BLM.
在内皮素（ET）受体亚型介导的磷脂信号传导方面，研究人员以大鼠近端小管为对象展开了研究。在新鲜分离的近端小管细胞中，ET-1、ET-2和铃蟾肽S6c（S6c）可使1,2 - 二酰甘油（DAG）、肌醇1,4,5 - 三磷酸（InsP3）和磷酸胆碱（PCho）增加，这表明磷脂酰肌醇4,5 - 二磷酸特异性磷脂酶C（PLC）和磷脂酰胆碱特异性磷脂酶C均受到了刺激；而ET-3仅使DAG和PCho增加，推测是通过磷脂酰胆碱依赖性PLC起作用。上述激动剂同样也刺激了肾皮质切片，随后分离出刷状缘（BBM）或基底外侧膜（BLM），并对其中的肌醇脂质和DAG进行了质量测定。在BBM中，ET-1、ET-2和ET-3均可使DAG增加，随后蛋白激酶C（PKC）转位至BBM；而在BLM中，仅ET-3可使DAG形成并促使PKC转位，这表明近端小管细胞两个膜结构域之间的信号系统存在空间分隔。酪氨酸磷酸化抑制剂、百日咳毒素（PTX）或霍乱毒素（CTX）均未对任一膜中的ET介导信号传导产生影响，这表明ET受体通过对PTX和CTX不敏感的G蛋白介导刺激PLCβ。BBM和BLM中ET依赖性对PLC的刺激被用作一种工具，以检测这两个细胞膜结构域中不同ET受体亚型的存在情况。ETA受体抑制剂BQ123并不能阻止ET-1在BBM中介导的信号传导，但ETA、B拮抗剂波生坦可抑制ET-3在BBM中介导的信号传导。此外，ETB激动剂S6c可刺激BBM中的PLC。BQ123和波生坦均未抑制BLM中的ET-3信号传导。因此，这些数据有力地表明，BBM中存在与磷脂酰肌醇4,5 - 二磷酸和磷脂酰胆碱依赖性PLC偶联的ETB受体，而BLM中存在与磷脂酰胆碱依赖性PLC偶联的ETA受体。
