Cabral Lindsey M P, Wengert Mira, da Ressurreição Alexandre A A, Feres-Elias Pedro H P, Almeida Fernando G, Vieyra Adalberto, Caruso-Neves Celso, Einicker-Lamas Marcelo
Laboratório de Físico-Química Biológica Aída Hassón-Voloch, Universidade Federal do Rio de Janeiro, 21949-900, Rio de Janeiro, Brazil.
J Biol Chem. 2007 Aug 24;282(34):24599-606. doi: 10.1074/jbc.M701669200. Epub 2007 Jul 2.
The kidney-proximal tubules are involved in reabsorbing two-thirds of the glomerular ultrafiltrate, a key Ca(2+)-modulated process that is essential for maintaining homeostasis in body fluid compartments. The basolateral membranes of these cells have a Ca(2+)-ATPase, which is thought to be responsible for the fine regulation of intracellular Ca(2+) levels. In this paper we show that nanomolar concentrations of ceramide (Cer(50) = 3.5 nm), a natural product derived from sphingomyelinase activity in biological membranes, promotes a 50% increase of Ca(2+)-ATPase activity in purified basolateral membranes. The stimulatory effect of ceramide occurs through specific and direct (cAMP-independent) activation of a protein kinase A (blocked by 10 nm of the specific inhibitor of protein kinase A (PKA), the 5-22 peptide). The activation of PKA by ceramide results in phosphorylation of the Ca(2+)-ATPase, as detected by an anti-Ser/Thr specific PKA substrate antibody. It is observed a straight correlation between increase of Ca(2+)-ATPase activity and PKA-mediated phosphorylation of the Ca(2+) pump molecule. Ceramide also stimulates phosphorylation of renal Ca(2+)-ATPase via protein kinase C, but stimulation of this pathway, which inhibits the Ca(2+) pump in kidney cells, is counteracted by the ceramide-triggered PKA-mediated phosphorylation. The potent effect of ceramide reveals a new physiological activator of the plasma membrane Ca(2+)-ATPase, which integrates the regulatory network of glycerolipids and sphingolipids present in the basolateral membranes of kidney cells.
肾近端小管参与重吸收三分之二的肾小球超滤液，这是一个关键的Ca(2+)调节过程，对维持体液 compartments 中的体内平衡至关重要。这些细胞的基底外侧膜有一个Ca(2+)-ATP酶，被认为负责细胞内Ca(2+)水平的精细调节。在本文中，我们表明纳摩尔浓度的神经酰胺（Cer(50)=3.5纳米），一种源自生物膜中鞘磷脂酶活性的天然产物，可使纯化的基底外侧膜中的Ca(2+)-ATP酶活性提高50%。神经酰胺的刺激作用是通过蛋白激酶A的特异性和直接（不依赖cAMP）激活而发生的（被10纳米的蛋白激酶A（PKA）特异性抑制剂5-22肽阻断）。神经酰胺对PKA的激活导致Ca(2+)-ATP酶的磷酸化，这可通过抗Ser/Thr特异性PKA底物抗体检测到。观察到Ca(2+)-ATP酶活性的增加与PKA介导的Ca(2+)泵分子磷酸化之间存在直接相关性。神经酰胺还通过蛋白激酶C刺激肾Ca(2+)-ATP酶的磷酸化，但这条抑制肾细胞中Ca(2+)泵的途径的刺激作用被神经酰胺触发的PKA介导的磷酸化所抵消。神经酰胺的强大作用揭示了质膜Ca(2+)-ATP酶的一种新的生理激活剂，它整合了肾细胞基底外侧膜中存在的甘油脂质和鞘脂的调节网络。
