Rangel L B A, Lopes A G, Lara L S M, Carvalho T L G, Silva I V, Oliveira M M, Einicker-Lamas M, Vieyra A, Nogaroli L, Caruso-Neves C
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,CCS Bloco G, 21949-900, Rio de Janeiro, RJ, Brazil.
Regul Pept. 2005 Apr 15;127(1-3):177-82. doi: 10.1016/j.regpep.2004.12.007.
In previous papers we showed that Ang II increases the proximal tubule Na+-ATPase activity through AT1/PKC pathway [L.B. Rangel, C. Caruso-Neves, L.S. Lara, A.G. Lopes, Angiotensin II stimulates renal proximal tubule Na+-ATPase activity through the activation of protein kinase C. Biochim. Biophys. Acta 1564 (2002) 310-316, L.B.A. Rangel, A.G. Lopes, L.S. Lara, C. Caruso-Neves, Angiotensin II stimulates renal proximal tubule Na+)-ATPase activity through the activation of protein kinase C. Biochim. Biophys. Acta 1564 (2002) 310-316]. In the present paper, we study the involvement of PI-PLCbeta on the stimulatory effect of angiotensin II (Ang II) on the proximal tubule Na+-ATPase activity. Western blotting assays, using a polyclonal antibody for PI-PLCbeta, show a single band of about 150 KDa, which correspond to PI-PLCbeta isoforms. Ang II induces a rapid decrease in PIP2 levels, a PI-PLCbeta substrate, being the maximal effect observed after 30 s incubation. This effect of Ang II is completely abolished by 5 x 10(-8) M U73122, a specific inhibitor of PI-PLCbeta. In this way, the effect of 10(-8) M Ang II on the proximal tubule basolateral membrane (BLM) Na+-ATPase activity is completely abolished by 5 x 10(-8) M U73122. The increase in diacylglycerol (DAG) concentration, an product of PI-PLCbeta, from 0.1 to 10 nM raises the Na+-ATPase activity from 6.1+/-0.2 to 13.1+/-1.8 nmol Pi mg(-1) min(-1). This effect is similar and non-additive to that observed with Ang II. Furthermore, the stimulatory effect of 10 nM DAG is completely reversed by 10(-8) M calphostin C (Calph C), an inhibitor of PKC. Taken together these data indicate that Ang II stimulates the Na+-ATPase activity of proximal tubule BLM through a PI-PLCbeta/PKC pathway.
在之前的论文中，我们表明血管紧张素II（Ang II）通过AT1/PKC途径增加近端小管钠钾ATP酶活性[L.B. 兰热尔、C. 卡鲁索 - 内维斯、L.S. 拉腊、A.G. 洛佩斯，血管紧张素II通过蛋白激酶C的激活刺激肾近端小管钠钾ATP酶活性。生物化学与生物物理学报1564（2002）310 - 316，L.B.A. 兰热尔、A.G. 洛佩斯、L.S. 拉腊、C. 卡鲁索 - 内维斯，血管紧张素II通过蛋白激酶C的激活刺激肾近端小管钠钾ATP酶活性。生物化学与生物物理学报1564（2002）310 - 316]。在本文中，我们研究磷脂酰肌醇特异性磷脂酶Cβ（PI - PLCβ）在血管紧张素II（Ang II）对近端小管钠钾ATP酶活性的刺激作用中的参与情况。使用针对PI - PLCβ的多克隆抗体进行的蛋白质印迹分析显示一条约150 kDa的条带，其对应于PI - PLCβ同工型。Ang II诱导磷脂酰肌醇 - 4,5 - 二磷酸（PIP2）水平迅速下降，PIP2是PI - PLCβ的底物，在孵育30秒后观察到最大效应。Ang II的这种作用被5×10⁻⁸ M U73122（一种PI - PLCβ的特异性抑制剂）完全消除。这样，5×10⁻⁸ M U73122完全消除了10⁻⁸ M Ang II对近端小管基底外侧膜（BLM）钠钾ATP酶活性的作用。磷脂酰肌醇特异性磷脂酶Cβ的产物二酰基甘油（DAG）浓度从0.1 nM增加到10 nM，使钠钾ATP酶活性从6.1±0.2提高到13.1±1.8 nmol Pi mg⁻¹ min⁻¹。这种作用与用Ang II观察到的作用相似且无相加性。此外，10 nM DAG的刺激作用被10⁻⁸ M钙磷蛋白C（Calph C，一种PKC抑制剂）完全逆转。这些数据综合起来表明，Ang II通过PI - PLCβ/PKC途径刺激近端小管BLM的钠钾ATP酶活性。
